J Virol, 1991 Mar, 65(3), 1149 - 59 The conserved DNA-binding domains encoded by the herpes simplex virus type 1 ICP4, pseudorabies virus IE180, and varicella-zoster virus ORF62 genes recognize similar sites in the corresponding promoters; Wu CL et al.; Herpes simplex virus types 1 and 2 (HSV-1 and HSV-2), pseudorabies virus (PRV), varicella-zoster virus (VZV), and equine herpesvirus 1 (EHV-1) are all classified as Alphaherpesvirinae . Each of these five viruses encodes an essential immediate-early (IE) regulatory protein referred to as HSV-1 ICP4, HSV-2 ICP4, PRV IE180, VZV ORF62 protein, and EHV-1 IE1, respectively . These five proteins share extensive homology with each other in domains referred to as regions 2 and 4 . The HSV-1 ICP4 region 2 domain contains residues that are required for the DNA-binding capability of ICP4 . In this report, we describe the expression of region 2 domains from the ICP4, IE180, and ORF62 genes as fusion proteins in Escherichia coli . DNA-binding assays revealed that each of these region 2 fusion proteins binds to a sequence that overlaps the transcription start site in the promoter for the gene encoding the corresponding protein . Each of the sites with high affinity for one or more of these fusion proteins contains the sequence 5'-ATCGT-3' . This sequence spans the mRNA cap site in the HSV-2 ICP4 gene promoter and is immediately upstream from the transcription start site in the EHV-1 IE1 gene . These results suggest that formation of a specific complex between an IE protein and its own gene promoter may be a common mechanism used by Alphaherpesvirinae to autoregulate transcription of an essential IE gene. Virology, 1991 Mar, 181(1), 78 - 90 Simian virus 40 Vp2/3 small structural proteins harbor their own nuclear transport signal; Clever J et al.; We have used a microinjection approach to identify a domain of the simian virus 40 (SV40) structural proteins Vp2 and Vp3(Vp2/3) responsible for their nuclear transport . By using both synthetic peptides, containing small regions of Vp2/3 conjugated to bovine serum albumin (BSA), and beta-galactosidase-Vp3 fusion proteins, we have narrowed this nuclear transport signal (NTS) to 9 amino acids (198 to 206 of Vp3 or 316 to 324 of Vp2), Gly-Pro-Asn-Lys-Lys-Lys-Arg-Lys-Leu . The porter proteins carrying the NTS or mutant NTS were microinjected into the cytoplasm of TC7 cells and their subcellular localization following the subsequent incubation period was determined immunologically using anti-BSA IgG or anti-beta-galactosidase . The 9-residue NTS peptide localized BSA into the nucleus of injected cells, changing lysine-202 to threonine or valine abolished this accumulation while changing arginine-204 to lysine did not grossly affect transport . A peptide containing the carboxyl-terminal 13 residues of Vp3 failed to localize BSA to the nucleus . Several single or double point mutations at Vp3 residues 202 and 204 have been introduced by site-directed mutagenesis . Vp3 residues 194-234, containing either a wild-type or mutated sequence at 202 and/or 204, were expressed in Escherichia coli as Vp3-beta-galactosidase fusion proteins . Addition of the carboxyl-terminal 40 residues, but not an internal 150 residues, to otherwise cytoplasmic beta-galactosidase promoted entry of the fusion protein into the nucleus . Changing lysine-202 into threonine, valine, or methionine abolished this nuclear accumulation as did changing arginine-204 into lysine . A double mutant at both positions was also blocked . We have also observed that the lectin wheat germ agglutinin inhibits the nuclear accumulation of BSA carrying the Vp2/3 NTS while the lectin concanavalin A had no effect . These data indicate that even small nuclear proteins can contain NTS's which most likely utilize a mechanism for nuclear import similar to that described for other larger proteins. Gig Sanit, 1991 Mar, (3), 26 - 8 {Standardization of miral levels in the soil}; Motuzinskii NF et al.; On the basis of results of studies on experimental substantiation of MAC for miral in soil it has been found out, that the sub-threshold miral amounts according to the water-migrational, air-migrational, translocational and general sanitary indices of harmfulness equal 0.03, 3, 0.03 and 0.15 mg/kg respectively . Therefore, water-migrational and translocational indices are the limiting indices of harmfulness for miral . 0.03 mg per 1 kg of absolutely dry soil is recommended for miral MAC according to the acting substance. Khirurgiia (Mosk), 1991 Mar, (3), 103 - 6 {Causes of suppurative complications after appendectomy}; Deviatov VA et al.; The causes of purulent complications in 9,518 patients after appendectomy were studied . The total percentage of purulent complications was 8.0 (6.4 among males and 10.1 among females) . It is shown that the form of appendicitis (phlegmonous in 2.0% of males and in 2.0% of females and perforating in 17.7% and 44.9%, respectively) and the time from the onset of the disease to the operation (less than 6 hours in 4.0% of males and in 6.1% of females; more than 48 hours in 11.7% and 18.8%, respectively) have an effect on the frequency of purulent complications . The authors revealed a correlation between the frequency of purulent complications and some factors (the time spent by the patients in the clinic before the operation, surgical team with an incomplete staff, insufficient anesthesia, insufficient operative approach, irrational use of gauze tampons and antibiotics. Gene, 1991 Mar 1, 99(1), 9 - 14 Genomic hsd-Mu(lac) operon fusion mutants of Escherichia coli K-12; Prakash A et al.; Genomic (chromosomal)hsd-Mu(lac) operon fusions have been constructed in two strains of Escherichia coli K-12 for the three hsd genes, hsdRK, hsdMK and hsdSK, using MudX and lambda placMu53 . Expression of hsdK mutants ranged from 16 to 74 units (u) (with a mean of 52 u) for fusions to promoter pres and ranged from 26-75 u (also with a mean of 52 u) for fusions to promoter pmod . The expression of the two hsdK promoters was measured in different stages of growth . The pres fusion mutant showed a lag in beta-galactosidase (beta Gal) production, as compared to the pmod fusion mutant . One r-Km-K mutant (JR205) showed more than ten times the beta Gal activity of other insertion mutants . The activity of this mutant decreased by 20-fold upon the transfer of F101-102, which includes the wild-type hsd region . Positive gene-dosage effect was observed using F' plasmids containing the hsd-lacZ region. Mutat Res, 1991 Mar, 254(2), 107 - 17 Introduction of a UV-damaged replicon into a recipient cell is not a sufficient condition to produce an SOS-inducing signal; Sommer S et al.; Three models have been proposed for the nature of the SOS-inducing signal in E . coli . One model postulates that degradation products of damaged DNA generate an SOS-inducing signal; another model surmises that the very lesions produced by UV damage constitute the SOS-inducing signal in vivo; a third model proposes that DNA damage is processed upon DNA replication to form single-stranded DNA (the SOS signal) that activates RecA protein . We tested the models by measuring SOS induction produced by introducing into recipient cells the UV-damaged DNA of 2 constructed phagemids . We used phagemids since they transferred DNA to the recipients with 100% efficiency . The origin of replication of the phagemids was either oriC from the E . coli chromosome, or oriF from F plasmid . Replication of the oriC phagemid was dependent on methylation . A UV-damaged oriC phagemid failed to induce SOS functions in a recipient cell whereas an oriF phagemid did induce them . Our results disprove the first and the second model proposed for the nature of the SOS-inducing signal . The failure of a UV-damaged oriC replicon to induce SOS can be explained by the third model if one assumes that replication of a UV-damaged oriC plasmid does not generate single-stranded DNA as does the E . coli chromosome after UV damage. Proc Natl Acad Sci U S A, 1991 Mar 1, 88(5), 1938 - 42 Mutational analysis of yeast vacuolar H(+)-ATPase; Noumi T et al.; Yeast mutants in which genes encoding subunits of the vacuolar H(+)-ATPase were interrupted were assayed for their vacuolar ATPase and proton-uptake activities . The vacuoles from the mutants lacking subunits A (72 kDa), B (57 kDa), or c (proteolipid, 16 kDa) were completely inactive in these reactions . Immunological studies revealed that in the absence of each one of those subunits the catalytic sector was not assembled . Labeling with N,N'-{14C}dicyclohexylcarbodiimide showed the presence of the proteolipid in vacuoles of mutants in which genes encoding subunits of the catalytic sectors were interrupted . No labeling was detected in the mutant in which the gene encoding the proteolipid was interrupted . We conclude that of all the ATPase subunits only the proteolipid is assembled independently and it serves as a template for the assembly of the other subunits . Site-specific mutations were generated in the gene encoding the proteolipid . All of the drastic changes and replacements gave inactive proteins . About half of the single amino acid replacements gave active proteins . Replacing glutamic acid-137 by any of several amino acids, except for aspartic acid, abolished the activity of the enzyme . Other amino acids that may function in proton conductance were changed . It was found that glycine residues may replace amino acids with exchangeable protons. Glycobiology, 1991 Mar, 1(2), 187 - 91 CMP-N-acetylneuraminic acid synthetase of Escherichia coli: high level expression, purification and use in the enzymatic synthesis of CMP-N-acetylneuraminic acid and CMP-neuraminic acid derivatives; Shames SL et al.; The gene encoding CMP-N-acetylneuraminic acid (CMP-NeuAc) synthetase (EC 2.7.7.43) in Escherichia coli serotype O7 K1 was isolated and overexpressed in E.coli W3110 . Maximum expression of 8-10% of the soluble E.coli protein was achieved by placing the gene with an engineered 5'-terminus and Shine-Dalgarno sequence into a pKK223 vector derivative behind the tac promoter . The overexpressed synthetase was purified to greater than 95% homogeneity in a single step by chromatography on high titre Orange A Matrex dye resin . Enzyme purified by this method was used directly for the synthesis of CMP-NeuAc and derivatives . The enzymatic synthesis of CMP-NeuAc was carried out on a multigram scale using equimolar CTP and N-acetylneuraminic acid as substrates . The resultant CMP-NeuAc, isolated as its disodium salt by ethanol precipitation, was prepared in an overall yield of 94% and was judged to be greater than 95% pure by 1H NMR analysis . N-Carbomethoxyneuraminic acid and N-carbobenzyloxyneuraminic acid were also found to be substrates of the enzyme; 5-azidoneuraminic acid was not a substrate of the enzyme . N-Carbomethoxyneuraminic acid was coupled to CMP at a rate similar to that observed with NeuAc, whereas N-carbobenzyloxyneuraminic acid was coupled greater than 100-fold more slowly . The high level of expression achieved with the E.coli synthetase, together with the high degree of purity readily obtainable from crude cell extracts, make the recombinant bacterial enzyme the preferred catalyst for the enzymatic synthesis of CMP-N-acetylneuraminic acid. J Mol Recognit, 1991 Mar-Jun, 4(2-3), 77 - 83 Immunological mapping of fine molecular surface structures of citrate synthase enzymes from different cell types; Nemeth P et al.; Citrate synthase (EC 4.1.3.7), which is present in all living organisms as a key enzyme in aerobic energy metabolism, is one of the most highly phylogenetically conserved enzymes known in terms of its primary and active site structure . However, in terms of other parameters such as in vitro stability, tolerance to changes in pH, degree of self-polymerization, etc., citrate synthases from different sources are markedly different . These divergences can be observed even between isoforms of the enzyme within the same species . Data documenting these diversities suggest that a high degree of difference in tertiary structures may occur . Therefore, the surface profiles of citrate synthase enzymes from yeast, pig, rat, tomato and Escherichia coli were investigated with immunological methods using monoclonal antibody families generated against either pig citrate synthase (alpha-PCS) or yeast citrate synthase-2 (alpha-YCS-2) . A high degree of homology of enzyme epitopes was detected on the mitochondrial citrate synthases originating from yeast, tomato, pig and rat cells . Major differences were found between the hexameric citrate synthase originating from E . coli compared with those dimeric forms prepared from eukaryotic cells . Only modest similarities were detected between the highly homologous peroxisomal and mitochondrial yeast citrate synthases . Furthermore, a point mutation of one of the catalytic residues (H274R on recombinant pig and H313R on yeast enzyme) of mitochondrial citrate synthase (CS-1) resulted in a significant increase in immunological similarity with the peroxisomal isoenzyme (CS-2) . These findings are discussed in terms of the possible mechanism of evolution of CS-2 in yeast. Zhongguo Yao Li Xue Bao, 1991 Mar, 12(2), 135 - 40 Influences of a novel immunopotentiator polyactin A on interleukin 1 production and responsiveness in mice; Hu QL et al.; Effects of a novel immunopotentiator Polyactin A (PAA), developed in China, on production and responsiveness of murine interleukin 1(IL-1) were investigated . The results demonstrated that: (1) PAA 0.01-100 micrograms.ml-1 directly induced IL-1 synthesis and secretion from murine peritoneal macrophages (PMO) and markedly enhanced IL-1 production of the mouse PMO stimulated by lipopolysaccharides (LPS) of E coli; (2) IL-1 release from the PMO cultured in PAA 0.1 micrograms.ml-1 was detectable as early as 2 h after the incubation, peaked at 24 h, and then decreased gradually; (3) PAA stimulated and enhanced both IL-1 synthesis and release, but its effect on IL-1 release was stronger; (4) PMO from the mice given po PAA 200 mg.kg-1.d-1 for 7 d produced a higher level of IL-1 than those from control group, and the increase in extracellular IL-1 was more significant than that in intracellular one; (5) in vivo, PAA had no effect on IL-1 receptor expression and IL-1 responsiveness of murine lymphocytes, but eliminated the suppressing effects of cyclophosphamide (Cyc) on IL-1 receptor expression and IL-1 responsiveness of mouse lymphocytes . The above findings provide new explanation for action of PAA and new basis for wider clinical applications of PAA. Mol Gen Mikrobiol Virusol, 1991 Mar, (3), 22 - 4 {Polymerase and immunologic activity of reverse transcriptase of the HIV-1 virus, isolated from Escherichia coli}; Shvetsov IuP et al.; The recombinant reverse transcriptase of HIV-1 virus has been isolated from Escherichia coli cells transformed by the plasmid pRT40 DNA . The 103 Kd protein produced by these cells is shown to be processed to proteins with lower molecular masses by the reverse transcriptases own protease activity as well as Escherichia coli proteases . The resulting 103-66 Kd proteins possess the polymerase activity while 51 Kd and smaller proteins are lacking the activity . The 66 and 51 Kd reverse transcriptase fragments demonstrate the positive immunological reaction with the human blood serum from the people possessing antibodies to HIV-1 virus . The recombinant reverse transcriptase of HIV-1 produced by Escherichia coli cells is shown to be useful in AIDS diagnosis in humans. Anticancer Res, 1991 Mar-Apr, 11(2), 793 - 800 Establishment, characterization and determination of cell surface sugar receptor (lectin) expression by neoglycoenzymes of a human myeloid marker-expressing B lymphoblastoid cell line; Gabius S et al.; Mediation of cellular interactions by protein (lectin)-carbohydrate recognition presupposes the expression of respective surface determinants . Due to the importance of cellular contacts between bone marrow stromal cells, recently shown to express cell surface lectins, and tumor or normal progenitor cells for biosignaling and marrow egress, quantitation of cell surface sugar receptor expression by a panel of chemically glycosylated enzymes (tetrameric E . coli beta-galactosidase) for human leukemia/lymphoma cells was initiated . Cells of the new B lymphoblastoid line Croco II that are partially positive for the CD15-specific epitope expressed receptors for various sugar specificities on their surface, fulfilling an indispensable prerequisite for establishment of glycobiological interactions . Binding studies with increasing neoglycoenzyme concentrations up to saturation in four cases disclosed values for apparent affinity constants in the range of 25-200 nM with 0.25-3 x 10(5) bound probes per cell . The presence of receptors for constituents of carbohydrate chains of cellular glycoconjugates was also ascertained biochemically, namely for beta-galactosides, alpha-mannosides, alpha-fucosides and N-acetylgalactosaminides . Expression of this property was modulated by changes in the culture conditions, as revealed by binding studies with cells, derived from growth in medium containing different serum concentrations . These findings indicate that cell surface sugar receptors of tumor cells warrant further attention with respect to recognitive interactions. Mol Biochem Parasitol, 1991 Mar, 45(1), 101 - 7 Molecular characterisation of a protective, 11-kDa excretory-secretory protein from the parasitic stages of Trichostrongylus colubriformis; Dopheide TA et al.; An 11-kDa protein occurring as a major component of the non-glycosylated fraction of 4th larval stage (L4) and adult Trichostrongylus colubriformis excretory-secretory (ES) fluid has been found to be highly protective in guinea pigs, an alternate host for T . colubriformis . The protein has been purified, characterised and partly sequenced . With a reverse-complement oligonucleotide based on the carboxy-terminal sequence of the protein, recombinant lambda gt11 clones were detected in an L4 cDNA library . The DNA sequence from one clone has a single extended open reading frame coding for a highly charged 11-kDa protein which lacks a leader sequence and contains a potential N-glycosylation site . Expression of the cloned DNA in Escherichia coli was detected with an antibody, raised in rabbits against gel-purified 11-kDa protein. Biophys J, 1991 Mar, 59(3), 516 - 22 Fourier transform infrared evidence for a predominantly alpha-helical structure of the membrane bound channel forming COOH-terminal peptide of colicin E1; Rath P et al.; The structure of the membrane bound state of the 178-residue thermolytic COOH-terminal channel forming peptide of colicin E1 was studied by polarized Fourier transform infrared (FTIR) spectroscopy . This fragment was reconstituted into DMPC liposomes at varying peptide/lipid ratios ranging from 1/25-1/500 . The amide I band frequency of the protein indicated a dominant alpha-helical secondary structure with limited beta- and random structures . The amide I and II frequencies are at 1,656 and 1,546 cm-1, close to the frequency of the amide I and II bands of rhodopsin, bacteriorhodopsin and other alpha-helical proteins . Polarized FTIR of oriented membranes revealed that the alpha-helices have an average orientation less than the magic angle, 54.6 degrees, relative to the membrane normal . Almost all of the peptide groups in the membrane-bound channel protein undergo rapid hydrogen/deuterium (H/D) exchange . These results are contrasted to the alpha-helical membrane proteins, bacteriorhodopsin, and rhodopsin. Mol Microbiol, 1991 Mar, 5(3), 747 - 55 Osmotic induction of the periplasmic trehalase in Escherichia coli K12: characterization of the treA gene promoter; Repoila F et al.; The Escherichia coli treA gene encodes an osmotically inducible periplasmic trehalase . A strain carrying a treA-lacZ transcriptional fusion was constructed . The beta-galactosidase activity produced in this strain growing exponentially in a medium of high osmotic pressure was 10-fold higher than that produced in a medium of low osmotic pressure, demonstrating that treA transcription is osmotically inducible . treA transcriptional induction depends neither on the presence of trehalase itself nor on the synthesis of cytoplasmic trehalose which occurs in response to osmotic stress in wild-type E . coli strains . The treA promoter was identified by S1 nuclease protection . Deletion analysis demonstrated that sequences sufficient for the osmotic induction lie downstream from nucleotide -40 with respect to the transcription start . Transcription initiation at treAp required the presence of a functional sigma 70 subunit of RNA polymerase . treA expression was increased in the presence of a mutation in osmZ, which was previously identified as leading to a partially constitutive expression of the osmotically inducible proU operon. Mol Microbiol, 1991 Mar, 5(3), 631 - 40 A copy-number mutant of plasmid pSC101; Xia GX et al.; Copy-number mutants of plasmid pSC101 were isolated by u.v . mutagenesis and selection for elevated expression of ampicillin resistance . Three independent mutations were identical and mapped in codon 93 of the initiation protein RepA . The mutated plasmids were maintained at a level four to five times higher than that of the wild type . For one of them, it was determined that: (i) the mRNA of the autoregulated repA gene, cloned onto a pUC19 plasmid under the control of its own promoter, was expressed at a level 1.7 times higher than that of the wild type; (ii) the RepA protein, under the same conditions, was expressed at a similarly higher level; (iii) the affinity of the mutated protein for three repeated sequences in the origin region of the plasmid was, on average, 3.4 times higher than that of the wild-type protein . We postulate that the copy-number effect is due to a combination of these two effects, i.e . higher protein concentration and increased affinity of the protein for the repeated sequences. J Gen Microbiol, 1991 Mar, 137 ( Pt 3), 679 - 84 Role of ribosome release in the basal level of expression of the Escherichia coli gene pheA; Gavini N et al.; In Escherichia coli, the expression of the phenylalanine biosynthetic operon pheA is regulated by an attenuation mechanism . In the presence of excess phenylalanine, gene expression was decreased to 10% of the fully deattenuated level . To understand the factors that determine the basal level of pheA expression, we examined the role of ribosome release from the leader peptide stop codon UGA . The transcriptional readthrough from the pheA attenuator decreased by over 2-fold in the presence of the defective release factor 2 . However, a release factor 1 (UAG and UAA specific) mutation did not influence the basal level of pheA expression . These results support the proposal that the release of translating ribosomes from the leader peptide stop codon in stem 2 of the pheA attenuator plays a crucial role in determining the basal level of expression of this gene. Sci China B, 1991 Mar, 34(3), 297 - 305 Arginyl-tRNA synthetase from Escherichia coli affinity labeling with 3'-oxidized tRNA(Arg); Cheng XD et al.; The covalent modification of E . coli arginyl-tRNA synthetase by the 2',3'-dialdehyde derivative of tRNA(Arg) (tRNA(oxArg)) resulted in the complete inactivation of the ATP-PPi exchange and aminoacylation activities of the enzyme . Sodium dodecyl sulfate polyacrylamide gel electrophoresis of the ArgRS-tRNA(oxArg) covalent complexes indicated that two bands simultaneously appeared on the gel parallel with inactivation corresponding to different higher molecular weights . This result was different from that of the other aminoacyl-tRNA synthetase labeling systems as previously reported . Upon the ribonuclease treatment of the modified ArgRS, less than 15% of both the initial ATP-PPi exchange and aminocylation activities were recovered . During the whole process of labeling and RNase treatment, the two activities of the enzyme were closely associated. Mol Gen Genet, 1991 Mar, 225(3), 435 - 42 Role of DnaA protein during replication of plasmid pBR322 in Escherichia coli; Chiang CS et al.; The in vivo role of the Escherichia coli protein DnaA in the replication of plasmid pBR322 was investigated, using a plasmid derivative carrying an inducible dnaA+ gene . In LB medium without inducer, the replication of this plasmid, like that of pBR322, was inhibited by heat inactivation of chromosomal DnaA46 protein so that plasmid accumulation ceased 1 to 2 h after the temperature shift . This inhibition did not occur when the plasmid dnaA+ gene was expressed in the presence of the inducer isopropyl-1-thio-beta-D-galactopyranoside (IPTG) . Inhibition was also not observed in glycerol minimal medium or in the presence of low concentrations of rifampicin or chloramphenicol . Deletion of the DnaA binding site and the primosome assembly sites (pas, rri) downstream of the replication origin did not affect the plasmid copy number during exponential growth at 30 degrees C, or after inactivation of DnaA by a shift to 42 degrees C in a dnaA46 host, or after oversupply of DnaA, indicating that these sites are not involved in a rate-limiting step for replication in vivo . The accumulation of the replication inhibitor, RNAI, was independent of DnaA activity, ruling out the possibility that DnaA acts as a repressor of RNAI synthesis, as has been suggested in the literature . Changes in the rate of plasmid replication in response to changes in DnaA activity (in LB medium) could be resolved into an early, rom-dependent, and a late, rom-independent component . Rom- plasmids show only the late effect . After heat inactivation of DnaC, plasmid replication ceased immediately.(ABSTRACT TRUNCATED AT 250 WORDS) Mol Cell Biol, 1991 Mar, 11(3), 1607 - 13 Identification of domains of the v-crk oncogene product sufficient for association with phosphotyrosine-containing proteins; Matsuda M et al.; The oncogene product of the avian sarcoma virus CT10, P47gag-crk, contains the SH2, SH2', and SH3 domains and binds proteins in a phosphotyrosine (ptyr)-dependent manner . In this study, we have determined the region of P47gag-crk essential for binding to ptyr-containing proteins . Mutant P47gag-crk proteins expressed in Escherichia coli that have the intact SH2 and SH2' regions retained the capacity to bind ptyr-containing proteins obtained from cells transformed by crk and src . The deletion of SH2 resulted in the loss of binding activity . Other mutants that have altered SH2 or SH2' bound few, if any, of the ptyr-containing proteins . Those mutants that bound ptyr-containing proteins associated with tyrosine kinase activity . We also found that polypeptides containing SH2, SH2', and SH3 of p60v-src and p60c-src associated with ptyr-containing proteins from crk-transformed cells . Thus, the SH2 and SH2' domains of P47gag-crk are responsible for their binding to ptyr-containing proteins. J Virol, 1991 Mar, 65(3), 1611 - 5 Epitope mapping and characterization of the infectious hematopoietic necrosis virus glycoprotein, using fusion proteins synthesized in Escherichia coli; Xu L et al.; A characterization of the antigenic determinants (epitopes) of the glycoprotein (G) of infectious hematopoietic necrosis virus was made by expressing different regions of the G gene in Escherichia coli . A cDNA copy of the G gene was divided into four fragments by TaqI digestion, and the fragments were subcloned into pATH vectors, placing the expression of each G gene fragment under control of the trpE promoter . The resulting plasmids, pXL2, pXL3, and pXL7, encoded trpE-G fusion proteins subsequently detected with anti-infectious hematopoietic necrosis virus sera by Western immunoblots . A comparison of reactivities of the fusion proteins encoded by these plasmids was made by Western immunoblot and radioimmunoassay with a number of anti-G specific monoclonal antibodies (MAbs) . The nonneutralizing MAb 136J reacted with the trpE-G fusion protein encoded by pXL3 and fusion proteins encoded by plasmids p52G and p618G, which were described in previous studies (R . D . Gilmore, Jr., H . M . Engelking, D . S . Manning, and J . C . Leong . Bio/Technology 6:295-300, 1988) . Another nonneutralizing MAb, 2F, bound to the pXL3 fusion protein, and the neutralizing MAb RB/B5 recognized the pXL7 fusion protein . All fusion proteins were tested as vaccines in rainbow trout fry . Although significant protection was induced by all fusion proteins, the pXL3 fusion protein was most effective as a vaccine. J Virol, 1991 Mar, 65(3), 1507 - 15 Retrovirus promoter-trap vector to induce lacZ gene fusions in mammalian cells; Reddy S et al.; A retrovirus promoter-trap vector (U3LacZ) has been developed in which Escherichia coli lacZ coding sequences were inserted into the 3' long terminal repeat (LTR) of an enhancerless Moloney murine leukemia virus . The U3LacZ virus contains the longest reported LTR (3.4 kbp); nevertheless, lacZ sequences did not interfere with the ability of the virus to transduce a neomycin resistance gene expressed from an internal promoter . Duplication of the LTR placed lacZ sequences in the 5' LTR just 30 nucleotides from the flanking cellular DNA . Approximately 0.4% of integrated proviruses expressed beta-galactosidase as judged by 5-bromo-4-chloro-3-indolyl-beta-D-galactopyranoside (X-Gal) staining, and individual clones expressing lacZ were isolated by fluorescence-activated cell sorting . In all clones examined, beta-galactosidase expression resulted from the fusion of lacZ sequences to transcriptional promoters located in the flanking cellular DNA . Furthermore, by differential sorting of neomycin-resistant cell populations, clones were isolated in which lacZ expression was induced and repressed in growth-arrested and log phase cells, respectively. Virology, 1991 Mar, 181(1), 14 - 21 Peptide mapping of neutralizing and nonneutralizing epitopes of duck hepatitis B virus pre-S polypeptide; Yuasa S et al.; Antibodies to the envelope proteins of duck hepatitis B virus neutralize viral infection in vitro . Using a library of murine monoclonal antibodies (Mabs) against the envelope proteins, we previously identified four neutralizing and two non-neutralizing epitopes on the pre-S region of the large envelope proteins . In this study we report the localization of all but one of these epitopes at the amino acid level . All but 28 nucleotides of the pre-S and S genes were cloned in pUC vectors and expressed in Escherichia coli . All Mabs in this study reacted with the expressed gene products in Western blots . Deletion mutants of the pre-S region were generated and their expressed products tested on Western blots for reactivity with the Mabs . Of the three epitopes involved in neutralization, the epitope found to be immunodominant in convalescent ducks was localized to nine amino acids of the middle portion of the pre-S gene product, while a second epitope was mapped to nine amino acids upstream of the immunodominant epitope and the third epitope to seven amino acids adjacent to the S gene . One of the two non-neutralizing epitopes was located between the two groups of neutralizing epitopes while the other mapped to the same region as one of the neutralizing epitopes . Our data indicate that several regions of the pre-S polypeptide may play a role in neutralization of hepadnaviruses. Int J Radiat Biol, 1991 Mar, 59(3), 717 - 27 The relationship between the anoxic sensitivity and the extent of sensitization by nitrous oxide; Ewing D et al.; Nitrous oxide reacts during irradiation to increase the yield of .OH, a radical many believe to be a major cause of lethality . Logically, one would expect N2O to be a radiation sensitizer . In some instances it is, while in others it is not . In some cases we can explain why N2O fails to sensitize; factors such as dose rate, cell concentration, buffer composition and ionic strength all influence when N2O will sensitize and, if it sensitizes, by what magnitude . Based on the results presented here with multiple strains of procaryotic and eucaryotic cells, we believe the anoxic sensitivity is another critical factor that governs whether N2O will sensitize . Our data, with data from the literature, show a relationship between the anoxic sensitivity and the N2O enhancement ratio . N2O does not sensitize in vitro unless the anoxic sensitivity (inactivation constant, k) is less than approximately 0.2 daGy-1. J Bacteriol, 1991 Mar, 173(6), 1894 - 901 Purification and properties of tryptophan-sensitive 3-deoxy-D-arabino-heptulosonate-7-phosphate synthase from Escherichia coli; Ray JM et al.; The aroH gene of Escherichia coli, which encodes the tryptophan-sensitive 3-deoxy-D-arabino-heptulosonate-7-phosphate synthase isoenzyme of the common aromatic biosynthetic pathway, was cloned behind the tac promoter in expression plasmid pKK223-3 . The enzyme was overexpressed, purified to homogeneity, and characterized . The native enzyme was found to be a dimeric metalloprotein containing 0.3 mol of iron per mol of subunit and variable amounts of zinc . The activity of the native enzyme was stimulated two- to threefold when assayed in the presence of Fe2+ ions . Pretreatment of the enzyme with Fe2+ also resulted in activation, accompanied by an equivalent increase in iron content . Treatment of the enzyme with chelating agents led to inactivation, which was fully reversed by the presence of Fe2+ in the assay mixture . The native enzyme exhibited a unique absorption profile, having a shoulder of absorbance on the aromatic band with a maximum around 350 nm and a broad, weak band with a maximum around 500 nm . Treatment of the enzyme with Fe2+ enhanced the absorbance at 350 nm and eliminated the band at 500 nm . Treatment with reducing agents caused the disappearance of both bands and destabilized the enzyme . Feedback regulation of the activity of the enzyme was specific for tryptophan, with maximum inhibition at about 70%. J Bacteriol, 1991 Mar, 173(5), 1789 - 800 Evidence for a methylation-blocking factor (mbf) locus involved in pap pilus expression and phase variation in Escherichia coli; Braaten BA et al.; Transcription of the pyelonephritis-associated pilus (pap) operon of Escherichia coli is subject to regulation by a phase variation control mechanism in which the pap pilin gene alternates between transcriptionally active (phase-on) and inactive (phase-off) states . Pap phase variation appears to involve differential inhibition of deoxyadenosine methylase (Dam) methylation of two pap GATC sites, GATC1028 and GATC1130, located in the regulatory region upstream of the papBA promoter . DNA from phase-on cells contains an unmethylated adenosine in the GATC1028 site, whereas DNA from phase-off cells contains an unmethylated adenosine in the GATC1130 site . papI and papB are two regulatory genes in the pap operon . Analysis of pap deletion mutants suggests that papI is required for methylation inhibition at the GATC1028 site; however, neither papI nor papB is required for inhibition of methylation at the GATC1130 site . We have identified a chromosomal locus, mbf (methylation-blocking factor), that is required for methylation protection of both the pap GATC1028 and GATC1130 sites . The mbf locus was identified after transposon mTn10 mutagenesis and mapped to 19.6 min on the E . coli chromosome . The effect of transposon mutations within mbf on pap pilin transcription was determined by using a papBAp-lac operon fusion which places lacZ under control of the papBA promoter . E . coli containing mbf::mTn10 and phase-off mbf+ E . coli cells both expressed beta-galactosidase levels about 30-fold lower than the beta-galactosidase level measured for phase-on mbf+ E . coli cells . These results indicated that mbf was necessary for pap pilin transcription and were supported by Northern (RNA) blotting and primer extension analyses . Moreover, transposon insertion within mbf greatly reduced Pap pilus expression . The mbf locus was isolated on a low-copy-number cosmid, pMBF1 . Complementation analysis indicated that each of seven mbf::mTn10 mutants isolated contained a transposon insertion within the same gene or operon . The identification of the mbf locus, required for pap transcription, supports the hypothesis that pap phase variation is controlled by a mechanism involving alternation between different methylation states. Infect Immun, 1991 Mar, 59(3), 971 - 6 The msp1 beta multigene family of Anaplasma marginale: nucleotide sequence analysis of an expressed copy; Barbet AF et al.; A gene for the beta subunit of the immunoprotective surface antigen MSP-1 of Anaplasma marginale was previously cloned and expressed in Escherichia coli . A nucleic acid probe based on this gene detects A . marginale infection in carrier cattle and in the tick vector . We report here the sequence and structural features of the cloned msp1 beta gene and expressed polypeptide . The gene codes for a polypeptide of 756 amino acids that contains domains of tandemly repeated sequence and glutamine-rich regions at the N and C termini . The cloned copy is a member of a multigene family with multiple restriction fragment length polymorphisms in isolates of this rickettsia from different geographical regions . The availability of the sequence will allow use of the polymerase chain reaction in diagnostic assays and the preparation and testing of different vaccine constructs in cattle. Infect Immun, 1991 Mar, 59(3), 1015 - 23 Mucin isolated from rabbit colon inhibits in vitro binding of Escherichia coli RDEC-1; Mack DR et al.; The rabbit enteric pathogen Escherichia coli RDEC-1 (serotype O15:H-) mediates attaching and effacing binding to colonic epithelium in a manner morphologically identical to that observed in both human enteropathogenic E . coli and enterohemorrhagic E . coli infections . The aim of this study was to determine if colonic mucus and its constituents, including mucin derived from goblet cells, inhibited RDEC-1 adherence in vitro . Crude mucus was prepared from mucosal scrapings of rabbit colon and separated by buoyant density into eight fractions . Purified mucin was characterized by gel electrophoresis, dot immunoblotting, indirect immunofluorescence, and amino acid composition . RDEC-1 bacteria were grown to promote and suppress the expression of mannose-resistant, hydrophobic pili . A nonpiliated mutant, strain M34, was also used as a negative control . Binding of radiolabeled RDEC-1 expressing pili was quantitated in the presence of crude mucus, purified mucin, and nonmucin fractions . Binding of piliated RDEC-1 to hydrophobic polystyrene wells was greater than for both nonpiliated RDEC-1 and strain M34 (P less than 0.05) . Both crude mucus and purified mucin mediated a concentration-dependent inhibition of piliated-RDEC-1 binding . Fractions of mucus without immunoreactive mucin did not inhibit the binding of RDEC-1 expressing hydrophobic pili . We conclude that colonic goblet cell-derived mucin mediates inhibition of piliated RDEC-1 attachment in vitro . Inhibition of bacterial adherence could prevent access of attaching and effacing E . coli enteric pathogens to the colonic mucosa in vivo. Mol Biol (Mosk), 1991 Mar-Apr, 25(2), 515 - 24 {Construction and expression of hybrid genes based on a deletion mutant of the herpes virus thymidine kinase gene in Escherichia coli}; Zemskova MIu et al.; The plasmids with the deletion mutant of thymidine kinase gene (tk') of Herpes simplex virus were constructed . The promoter, transcription initiation site and first 35 codons of tk gene were removed and replaced with a series of DNA restriction sites . The DNA fragments, containing the gene regulatory sequences specific for bacteria, were cloned into these sites and shown to express enzymatically active proteins . The obtained ene fusions were able to complement in E . coli strain deficient in thymidine kinase function . Such plasmid vectors carrying tk' are useful for construction and selection of hybrid gene fusions. Plasmid, 1991 Mar, 25(2), 145 - 8 A sequence at the inside end of IS50 down regulates transposition; Dodson KW et al.; Deletion analysis has shown that the segment at the IS50 inside (I) end that is needed for efficient transposition is approximately 19 bp long . Dam methylation at two 5' GATC sequences within this segment decreases I-end transposition activity . A third 5' GATC sequence is present at bp 21-24 of the I end . The comparisons presented here show that extension of the I end from 19 to 24 bp decreases its transposition activity in dam cells 5- to 50-fold, depending on the overall transposon structure. Bioorg Khim, 1991 Mar, 17(3), 379 - 86 {Site-directed mutagenesis with the use of addressed fragmentation of single-stranded DNA for obtaining hybrids of homologous proteins}; Shekhter II et al.; Site-directed multiple mutagenesis to obtain hybrids of homologous proteins was carried out by means of the oligonucleotide-targeting digestion of ss DNA . The procedure is more convenient, rapid and simple than the step-by-step approach . To demonstrate different approaches to targeting digestion of ss DNA, NcoI, HinfI, FokI endonucleases were used for DNA cleavage . A hydride of the human and porcine IFN-alpha-2-genes has been constructed. J Electron Microsc Tech, 1991 Mar, 17(3), 336 - 43 Suitability of different silver enhancement methods applied to 1 nm colloidal gold particles: an immunoelectron microscopic study; Stierhof YD et al.; In order to exploit the recently introduced 1 nm gold colloids in routine electron microscopic labeling experiments, an efficient enhancement step for a better visualization of this small marker is a prerequisite . Efficiency and reproducibility of enhancement as well as growth homogeneity of gold particles were evaluated for three different silver intensifying solutions: silver lactate/hydroquinone/gum arabic (Danscher, 1981), Ilford L4/Metol (Bienz et al., 1986), and the commercially available IntenSE M silver enhancement kit (Janssen Pharmaceutica) . The best results were obtained by using the silver lactate/hydroquinone/gum arabic mixture . The quality of enhancement of the IntenSE M kit was considerably increased by the addition of the protective colloid gum arabic. Brain Res Mol Brain Res, 1991 Mar, 9(4), 347 - 51 A phospholipase A2-stimulating protein regulated by protein kinase C in Aplysia neurons; Calignano A et al.; We describe some properties on an Mr 30,000 thermolabile and trypsin-sensitive protein that activates phospholipase A2 (PLA2) and which was isolated from nervous tissue of the marine mollusk, Aplysia californica . A similar protein is present in rat cerebral cortex . This protein was partially purified from crude homogenates of nervous tissue by ion exchange chromatography on DEAE-Sephadex followed by size-exclusion high performance liquid chromatography (HPLC) . It is loosely associated with membrane fractions, and is extracted by 0.05% Tween 20 . Although similar in size to several previously described PLA2-stimulating proteins from non-neural mammalian cells and tissues, it differs from them in some aspects of biological activity . The protein promotes the release of eicosanoids from the membranes of intact Aplysia neurons prelabeled with {3H}arachidonic acid and appears to be an in vitro substrate for protein kinase C (PKC) . PLA2-stimulating activity is greatly enhanced after exposing isolated ganglia to phorbol dibutyrate (PDBu) and is reduced by treatment with immobilized E . coli alkaline phosphatase . These observations suggest that phosphorylation of this stimulatory protein by PKC regulates PLA2 in neurons. Biotechnology (N Y), 1991 Mar, 9(3), 273 - 8 The functional expression of antibody Fv fragments in Escherichia coli: improved vectors and a generally applicable purification technique; Skerra A et al.; We have previously demonstrated that the expression of fully functional Fv and Fab fragments in E . coli is possible by the simultaneous secretion of both chains to the periplasm . To increase production levels and facilitate engineering and random mutagenesis, we improved our previous vectors by introducing a resident repressor gene and a filamentous phage origin . We also developed a new purification strategy based on immobilized metal ion chromatography, with which a single-chain Fv fragment can be purified to homogeneity in a single step . We investigated the most efficient tail constructions and found that only a minimal structural change of three additional C-terminal amino acids is necessary . This modification has no deleterious effect on in vivo transport and folding or antigen affinity. Biochem Biophys Res Commun, 1991 Feb 28, 175(1), 250 - 5 Zn+2 induces reversible cross-linking of human placental thyroid hormone nuclear receptor with no effect on hormone binding; Lin KH et al.; Zn+2 is required for specific binding of c-erbA proteins to the hormone response elements of target genes . It is unclear whether Zn+2 is important for the binding of ligand to c-erbA proteins . The present study evaluated the effect of Zn+2 and other divalent cations on the binding of 3,3',5-triiodo-L-thyronine(T3) to the purified human placental c-erbA protein (h-TR beta 1) . Zn+2 induced cross-linking of h-TR beta 1 to form aggregates in a dose-dependent manner with an apparent half-maximal concentration of approximately 200 microM at 22 degrees C . Cross-linking was reversible by the addition of 5 microM EDTA or 10 mM dithiothreitol . The cross-linked h-TR beta 1 bound T3 . These results indicated Zn+2 had no effect on T3 binding and suggested that the cysteines and histidines involved in cross-linking are not essential for T3 binding. Eur J Biochem, 1991 Feb 26, 196(1), 87 - 93 Amino acid 55 plays a central role in tetramerization and function of Escherichia coli single-stranded DNA binding protein; Curth U et al.; The histidine at position 55 of the amino acid sequence of the Escherichia coli single-stranded DNA binding protein was replaced by tyrosine, glutamic acid, lysine, phenylalanine, and isoleucine . The properties of the mutant proteins were determined using analytical ultracentrifugation, NMR spectroscopy, gel filtration, and fluorimetric detection of their single-stranded DNA binding ability . While the phenylalanine and isoleucine substitutions did not change the properties of the protein measurably, tyrosine and lysine mutants dissociate into subunits and loose some of their binding affinity for poly(dT) . For the lysine mutant we show by electron microscopy that the protein, although fully dissociated and possibly denatured in the free state, binds to poly(dT) as a tetramer indistinguishable from the wild-type protein . The process of tetramerization as observed via single-stranded DNA binding ability is composed of a variety of steps ranging in time from some milliseconds to several hours; it probably involves several forms of dissociated and non-native protein. Biochemistry, 1991 Feb 26, 30(8), 2273 - 80 Deduced amino acid sequence of Escherichia coli adenosine deaminase reveals evolutionarily conserved amino acid residues: implications for catalytic function; Chang ZY et al.; The goal of the research reported here is to identify evolutionarily conserved amino acid residues associated with enzymatic deamination of adenosine . To do this, we isolated molecular clones of the Escherichia coli adenosine deaminase gene by functional complementation of adenosine deaminase deficient bacteria and deduced the amino acid sequence of the enzyme from the nucleotide sequence of the gene . Nucleotide sequence analysis revealed the presence of a 996-nucleotide open reading frame encoding a protein of 332 amino acids having a molecular weight of 36,345 . The deduced amino acid sequence of the E . coli enzyme has approximately 33% identity with those of the mammalian adenosine deaminases . With conservative amino acid substitutions the overall sequence homology approaches 50%, suggesting that the structures and functions of the mammalian and bacterial enzymes are similar . Additional amino acid sequence analysis revealed specific residues that are conserved among all three adenosine deaminases and four AMP deaminases for which sequence information is currently available . In view of previously published enzymological data and the conserved amino acid residues identified in this study, we propose a model to account for the enzyme-catalyzed hydrolytic deamination of adenosine . Potential catalytic roles are assigned to the conserved His 214, Cys 262, Asp 295, and Asp 296 residues of mammalian adenosine deaminases and the corresponding conserved amino acid residues in bacterial adenosine deaminase and the eukaryotic AMP deaminases. Biochemistry, 1991 Feb 26, 30(8), 2227 - 39 Crystal structure of unliganded Escherichia coli dihydrofolate reductase . Ligand-induced conformational changes and cooperativity in binding; Bystroff C et al.; The crystal structure of unliganded dihydrofolate reductase (DHFR) from Escherichia coli has been solved and refined to an R factor of 19% at 2.3-A resolution in a crystal form that is nonisomorphous with each of the previously reported E . coli DHFR crystal structures {Bolin, J . T., Filman, D . J., Matthews, D . A., Hamlin, B . C., & Kraut, J . (1982) J . Biol . Chem . 257, 13650-13662; Bystroff, C., Oatley, S . J., & Kraut, J . (1990) Biochemistry 29, 3263-3277} . Significant conformational changes occur between the apoenzyme and each of the complexes: the NADP+ holoenzyme, the folate-NADP+ ternary complex, and the methotrexate (MTX) binary complex . The changes are small, with the largest about 3 A and most of them less than 1 A . For simplicity a two-domain description is adopted in which one domain contains the NADP+ 2'-phosphate binding site and the binding sites for the rest of the coenzyme and for the substrate lie between the two domains . Binding of either NADP+ or MTX induces a closing of the PABG-binding cleft and realignment of alpha-helices C and F which bind the pyrophosphate of the coenzyme . Formation of the ternary complex from the holoenzyme does not involve further relative domain shifts but does involve a shift of alpha-helix B and a floppy loop (the Met-20 loop) that precedes alpha B . These observations suggest a mechanism for cooperativity in binding between substrate and coenzyme wherein the greatest degree of cooperativity is expressed in the transition-state complex . We explore the idea that the MTX binary complex in some ways resembles the transition-state complex. Biochemistry, 1991 Feb 26, 30(8), 2192 - 5 Kinetics of electron transfer from thioredoxin reductase to thioredoxin; Navarro JA et al.; The reduction of Escherichia coli thioredoxin by thioredoxin reductase was studied by stopped-flow spectrophotometry . The reaction showed no dependence on thioredoxin concentration, indicating that complex formation was rapid and occurred during the dead time of the instrument . The kobs for the reaction of approximately 20 s-1 probably reflects the rate of electron transfer from thioredoxin reductase to thioredoxin and agrees with the kcat observed by steady-state kinetics . The reaction rate was unaffected by increasing the ionic strength, suggesting a lack of electrostatic stabilization in the interaction of the two proteins . A mutant thioredoxin in which a positively charged lysine in the active-site region was changed to a glutamic acid residue resulted in an electrostatic destabilization . Thioredoxin K36E was still a substrate for the reductase, but binding was impaired so that the rate could be measured by stopped-flow techniques as reflected by a dependence on protein concentration . Raising the ionic strength in this reaction served to shield the negative charge and increased the rate of binding to the reductase. Biochemistry, 1991 Feb 26, 30(8), 2184 - 91 Evidence for two interconverting protein isomers in the methotrexate complex of dihydrofolate reductase from Escherichia coli; Falzone CJ et al.; Two-dimensional 1H NMR methods and a knowledge of the X-ray crystal structure have been used to make resonance assignments for the amino acid side chains of dihydrofolate reductase from Escherichia coli complexed with methotrexate . The H7 proton on the pteridine ring of methotrexate was found to have NOEs to the methyl protons of Leu-28 which were assigned by using the L28F mutant . These NOEs indicated that the orientation of the methotrexate pteridine ring is similar in both solution and crystal structures . During the initial assignment process, it became evident that many of the resonances in this complex, unlike those of the folate complex, are severely broadened or doubled . The observation of two distinct sets of resonances in a ratio of approximately 2:1 was attributed to the presence of two protein isomers . At 303 K, NOESY spectra with mixing times of 100 ms did not show interconversion between these isomers . However, exchange cross-peaks were observed in a 700-ms NOESY spectrum at 323 K which demonstrated that these isomers are interconverting slowly on the NMR time scale . Many of the side chains with clearly doubled resonances were located in the beta-sheet and the active site . Preliminary studies on the apoprotein also revealed doubled resonances in the absence of the inhibitor, indicating the existence of the protein isomers prior to methotrexate binding . In contrast to the methotrexate complex, the binary complex with folate and the ternary MTX-NADPH-DHFR complex presented a single enzyme form . These results are proposed to reflect the ability of folate and NADPH to bind predominantly to one protein isomer. Biochemistry, 1991 Feb 26, 30(8), 2156 - 65 1H NMR study of the solution molecular and electronic structure of Escherichia coli ferricytochrome b562: evidence for S = 1/2 in equilibrium S = 5/2 spin equilibrium for intact His/Met ligation; Wu JZ et al.; The solution 500-MHz 1H NMR spectral parameters for ferricytochrome b562, a soluble 12-kDa electron carrier from Escherichia coli with axial His/Met coordination, are shown to be strongly influenced by protein concentration and ionic strength at low pH and 25 degrees C in a manner consistent with significant aggregation at low ionic strength . At high ionic strength a well-resolved 1H NMR spectrum reveals over 40 hyperfine-shifted resonances which arise from two isomeric species in the ratio 2:1 . 2D COSY and NOESY maps at 25 degrees C for the hyperfine-shifted resonances allow the assignment of a number of axial His resonances and all heme peripheral substituent peaks . The resulting asymmetric heme contact shift patterns, together with the halving of the number of lines when reconstituting with 2-fold symmetric hemin, demonstrate the molecular basis of the solution heterogeneity to be heme orientational disorder . The strongly upfield-shifted axial Met-7 resonances, characteristic of low-spin ferricytochromes c with His/Met ligation, appear upfield only at very low temperatures . At elevated temperatures, all resonances, in particular those of the axial Met, move strongly downfield . Detailed analysis of the deviation from Curie behavior for different functional groups demonstrates the presence of a low spin in equilibrium high spin equilibrium with an intact His-Fe-Met coordination . The weaker axial field in ferricytochrome b562, relative to the purely low-spin ferricytochromes c, is attributed to a perturbed iron-Met bond . The contact shifts for a coordinated Met in the high-spin state are estimated . A link between equatorial hemin and axial ligand interactions is indicated by a differential population of the high-spin form for the two hemin orientations. Biochemistry, 1991 Feb 26, 30(8), 2012 - 7 Toward a simplification of the protein folding problem: a stabilizing polyalanine alpha-helix engineered in T4 lysozyme; Zhang XJ et al.; In an attempt to simplify the protein folding problem, and also to further investigate the role of alanine as a helix-stabilizing residue, a series of alanines was introduced within the alpha-helix that includes residues 126-134 of T4 lysozyme . In wild-type lysozyme this alpha-helix contains alanine residues at positions 129, 130, and 134 . Mutant lysozymes with alanines substituted at positions 128, 131, 132, and 133, either as single substitutions or in selected combinations, were constructed by oligonucleotide-directed mutagenesis . With the exception of the replacement of Leu 133, which is buried within the hydrophobic core of the protein, all the variants were more stable than wild-type lysozyme . The variant with alanines substituted at positions 128, 131, and 132 (E128A/V131A/N132A), which incorporates the sequence Ala 128-Ala 129-Ala 130-Ala 131-Ala 132-Leu 133-Ala 134, has a melting temperature 3.3 degrees C above that of wild-type lysozyme . Determination of the crystal structure of this mutant lysozyme shows that the replacement of Glu 128, Val 131, and Asn 132 with alanine causes alpha-helix 126-134 to rotate 3.4 degrees about an axis parallel to its own axis . This rotation seems to be triggered primarily by the loss of a hydrogen bond between Asn 132 and Ser 117 and is associated with the repacking of several side chains at the interface between alpha-helix 126-134 and the adjacent alpha-helix 115-122.(ABSTRACT TRUNCATED AT 250 WORDS) Eur J Biochem, 1991 Feb 26, 196(1), 123 - 8 A characterization of copper/zinc superoxide dismutase mutants at position 124 . Zinc-deficient proteins; Banci L et al.; Substitution of the completely conserved aspartic acid residue at position 124 of Cu,Zn superoxide dismutase with asparagine and glycine has been performed through site-directed mutagenesis on the human enzyme . Asp124 is H-bonded to the NH of two histidines, one of which is bound to copper and the other to zinc . The mutant proteins, as expressed in Escherichia coli, result in an essential zinc-free enzyme which is similar to that obtained from the wild-type derivative through chemical manipulation . Only by extensive dialysis against 0.5 M ZnCl2 or CoCl2 at pH 5.4 was it possible to reconstitute approximately 50% of the molecules in the Cu2Zn2 or Cu2Co2 form . The new derivatives have been characterized through EPR, CD and nuclear magnetic relaxation dispersion techniques . The Cu2Cox derivatives (x approximately 1) were used to monitor, through electronic and 1H-NMR spectroscopies, the metal sites which are found to be similar to those of the wild type . In addition, a double substitution with asparagine has been made, replacing the invariant aspartate at position 124 and the highly conserved aspartate at position 125 . The behavior is similar to that of the other mutants in most respects . The Cu2E2 (E = empty) derivatives of the mutants are stable, even in the pH range 8-10, whereas in the case of the Cu2E2 derivative of the wild type, copper migration occurs at high pH, producing both Cu2Cu2 and apo derivatives . The activity measurements indicate that the various Cu2E2 derivatives have the same activity at low pH and similar to that of the holoenzyme . A full profile up to pH 10.5 was obtained for the mutants. Biochemistry, 1991 Feb 26, 30(8), 2022 - 6 A novel dipyridodiazepinone inhibitor of HIV-1 reverse transcriptase acts through a nonsubstrate binding site; Wu JC et al.; A novel dipyridodiazepinone, 6,11-dihydro-11-cyclopropyl-4-methyldipyrido{2,3-b:2',3'-e}- {1,4}diazepin-6-one (BI-RG-587), is a selective noncompetitive inhibitor of HIV-1 reverse transcriptase (RT-1) . An azido photoaffinity analogue of BI-RG-587 was synthesized and found to irreversibly inhibit the enzyme upon UV irradiation . BI-RG-587 and close structural analogues competitively protected RT-1 from inactivation by the photoaffinity label . A thiobenzimidazolone (TIBO) derivative, a nonnucleoside inhibitor of RT-1, also protected the enzyme from photoinactivation, which suggests a common binding site for these compounds . Substrates dGTP, template-primer, and tRNA afforded no protection from enzyme inactivation . A tritiated photoaffinity probe was found to stoichiometrically and selectively label p66 such that 1 mol of probe inactivates 1 mol of RT-1. Nucleic Acids Res, 1991 Feb 25, 19(4), 867 - 9 Suppression of a double missense mutation by a mutant tRNA(Phe) in Escherichia coli; Pages D et al.; We report here the isolation of a mutant tRNAPhe that suppresses a double missense auxotrophic mutation in trpA of Escherichia coli, trpA218 . The doubly mutant protein product differs from wild-type TrpA by the replacements of Phe22 by Leu and Gly211 by Ser . A partial revertant TrpA phenotype can be obtained from trpA218 by changing either Leu22 back to Phe or Ser211 back to Gly . Translational suppressors were previously obtained that act at codon 211, replacing the Ser211 in the TrpA218 protein, presumably with Gly . In the present study, we selected for trpA218 suppressors caused by mutation of a cloned tRNAPhe gene, pheV . DNA sequence analysis of the suppressor isolated reveals a singular structural alteration, changing the anticodon from 5'-GAA-3' to 5'-GAG-3' . Sequencing of trpA218 confirmed the likely identity of Leu22 as CUC . The new missense suppressor, designated pheV(SuCUC), is lethal to the cell when highly expressed, as from a high copy number plasmid . This may be due to efficient replacement of Leu by Phe at CUC (and, probably, CUU) codons throughout the genome . We anticipate that pheV(SuCUC) will prove, like other missense suppressors, to be extremely useful in studies on the specificity and accuracy of decoding. Nucleic Acids Res, 1991 Feb 25, 19(4), 809 - 13 A novel DNA joining activity catalyzed by T4 DNA ligase; Western LM et al.; The use of T4 and E . coli DNA ligases in genetic engineering technology is usually associated with nick-closing activity in double stranded DNA or ligation of 'sticky-ends' to produce recombinant DNA molecules . We describe in this communication the ability of T4 DNA ligase to catalyze intramolecular loop formation between annealed oligodeoxyribonucleotides wherein Watson-Crick base pairing is absent on one side of the ligation site . Enzyme concentration, loop size, substrate specificity, and base composition were explored in an effort to maximize yield . Amounts of T4 DNA ligase in large molar excess to DNA template and ligated product are necessary to achieve high yields. Nucleic Acids Res, 1991 Feb 25, 19(4), 727 - 31 Gene disruption and gene replacement in Streptomyces via single stranded DNA transformation of integration vectors; Hillemann D et al.; For the isolation of single stranded plasmid DNA, various E . coli and E . coli-Streptomyces shuttle plasmids were equipped with the phage f1 replication origin . The transformation of some representative Streptomyces species with plasmid vectors occurred irrespective of whether single or double stranded DNA was used . In contrast, the transformation of Streptomyces was 10 to 100 times more efficient when an integration vector was in the single stranded form as opposed to the double stranded form . Streptomyces viridochromogenes was transformed by single stranded DNA integration vectors in order to replace the pat by the tsr gene and generate mutants unable to synthesize phosphinothricin-tripeptide (PTT). FEBS Lett, 1991 Feb 25, 279(2), 233 - 6 Purification of SecE and reconstitution of SecE-dependent protein translocation activity; Tokuda H et al.; SecE was solubilized from SecE-overproducing E . coli cells and purified through ion exchange and size exclusion chromatographies . When the solubilized membrane containing overproduced amounts of SecY and SecE was fractionated by means of size exclusion chromatography, the two proteins were eluted in different fractions with slight overlapping . Proteoliposomes active in protein translocation were reconstituted from these fractions only when both SecE and SecY were present . When reconstitution was carried out with the purified SecE and fractions containing SecY but only a small amount of SecE, the resultant proteoliposomes exhibited appreciable translocation activity, indicating that SecE is essential for protein translocation . The translocation activity of proteoliposomes was proportional to the amount of purified SecE used for reconstitution . SecE-dependent protein translocation absolutely required ATP and SecA. J Biol Chem, 1991 Feb 25, 266(6), 3752 - 9 The 1-aminocyclopropane-1-carboxylate synthase of Cucurbita . Purification, properties, expression in Escherichia coli, and primary structure determination by DNA sequence analysis; Sato T et al.; The key regulatory enzyme in the biosynthetic pathway of the plant hormone ethylene is 1-aminocyclopropane-1-carboxylic acid (ACC) synthase (EC 4.4.1.14) . We have partially purified ACC synthase 6,000-fold from Cucurbita fruit tissue treated with indoleacetic acid + benzyladenine + aminooxyacetic acid + LiCl . The enzyme has a specific activity of 35,000 nmol/h/mg protein, a pH optimum of 9.5, an isoelectric point of 5.0, a Km of 17 microM with respect to S-adenosylmethionine, and is a dimer of two identical subunits of approximately 46,000 Da each . The subunit exists in vivo as a 55,000-Da species similar in size to the primary in vitro translation product . DNA sequence analysis of the cDNA clone pACC1 revealed that the coding region of the ACC synthase mRNA spans 493 amino acids corresponding to a 55,779-Da polypeptide; and expression of the coding sequence (pACC1) in Escherichia coli as a COOH terminus hybrid of beta-galactosidase or as a nonhybrid polypeptide catalyzed the conversion of S-adenosylmethionine to ACC (Sato, T., and Theologis, A . (1989) Proc . Natl . Acad . Sci . U.S.A . 86, 6621-6625) . Immunoblotting experiments herein show that the molecular mass of the beta-galactosidase hybrid polypeptide is 170,000 Da, and the size of the largest nonhybrid polypeptide is 53,000 Da . The data suggest that the enzyme is post-translationally processed during protein purification. J Biol Chem, 1991 Feb 25, 266(6), 3744 - 51 Heteroduplex repair in extracts of human HeLa cells; Thomas DC et al.; A general repair process for DNA heteroduplexes has been detected in HeLa cell extracts . Using a variety of M13mp2 DNA substrates containing single-base mismatches and extra nucleotides, extensive repair is observed after incubation with HeLa cell cytoplasmic extracts and subsequent transfection of bacterial cells with the treated DNA . Most, but not all, mispairs as well as two frameshift heteroduplexes are repaired efficiently . Parallel measurements of repair in HeLa extracts and in Escherichia coli suggest that repair specificities are similar for the two systems . The presence of a nick in the molecule is required for efficient repair in HeLa cell extracts, and the strand containing the nick is the predominantly repaired strand . Mismatch-dependent DNA synthesis is observed when radiolabeled restriction fragments, produced by reaction of the extract with heteroduplex and homoduplex molecules, are compared . Specific labeling of fragments, representing a region of approximately 1,000 base pairs and containing the nick and the mismatch, is detected for the heteroduplex substrate but not the homoduplex . The repair reaction is complete after 20 min and requires added Mg2+, ATP, and an ATP-regenerating system, but not dNTPs, which are present at sufficient levels in the extract . An inhibitor of DNA polymerase beta, dideoxythimidine 5'-triphosphate, does not inhibit mismatch-specific DNA synthesis . Aphidicolin, an inhibitor of DNA polymerases alpha, delta, and epsilom, inhibits both semiconservative replication and repair synthesis in the extract . Butylphenyl-dGTP also inhibits both replicative and repair synthesis but at a concentration known to inhibit DNA polymerase alpha preferentially rather than delta or epsilon . This suggests that DNA polymerase alpha may function in mismatch repair. J Biol Chem, 1991 Feb 25, 266(6), 3408 - 10 A de novo designed signal peptide cleavage cassette functions in vivo; Nilsson I et al.; Leader peptidase (Lep) is a membrane-bound enzyme of the Escherichia coli inner membrane that serves to remove signal peptides from exported proteins . Statistical and experimental studies of known signal peptides have defined a short C-terminal region that seems to provide the information for correct cleavage by Lep . Based on the patterns of conserved amino acids found in this region, we have designed a signal peptide "cleavage cassette." This cassette is processed at the expected site when introduced after an uncleaved signal peptide . Furthermore, processing is blocked in the predicted manner when the (-3, -1)-rule for signal peptide cleavage is violated . This suggests that current understanding of the sequence requirements for signal peptide cleavage is sufficiently advanced to be used in, e.g . protein engineering applications. Nucleic Acids Res, 1991 Feb 25, 19(4), 841 - 50 Characterization of the cloned BamHI restriction modification system: its nucleotide sequence, properties of the methylase, and expression in heterologous hosts; Brooks JE et al.; The BamHI restriction modification system was previously cloned into E . coli and maintained with an extra copy of the methylase gene on a high copy vector (Brooks et al., (1989) Nucl . Acids Res . 17, 979-997) . The nucleotide sequence of a 3014 bp region containing the endonuclease (R) and methylase (M) genes has now been determined . The sequence predicts a methylase protein of 423 amino acids, Mr 49,527, and an endonuclease protein of 213 amino acids, Mr 24,570 . Between the two genes is a small open reading frame capable of encoding a 102 amino acid protein, Mr 13,351 . The M . BamHI enzyme has been purified from a high expression clone, its amino terminal sequence determined, and the nature of its substrate modification studied . The BamHI methylase modifies the internal C within its recognition sequence at the N4 position . Comparisons of the deduced amino acid sequence of M . BamHI have been made with those available for other DNA methylases: among them, several contain five distinct regions, 12 to 22 amino acids in length, of pronounced sequence similarity . Finally, stability and expression of the BamHI system in both E . coli and B . subtilis have been studied . The results suggest R and M expression are carefully regulated in a 'natural' host like B . subtilis. J Biol Chem, 1991 Feb 25, 266(6), 3760 - 7 Overexpression of the relA gene in Escherichia coli; Schreiber G et al.; Intracellular levels of guanosine 3',5'-bispyrophosphate (ppGpp) governed by the relA gene are normally regulated by aminoacyl-tRNA availability for protein synthesis . An experimental system is described in which cellular levels of ppGpp are controlled instead by induction of plasmid pKK223-3 derivatives with the relA structural gene, or portions thereof, under control of the Ptac promoter . In amino acid-rich media, isopropyl-1-thio-beta-D-galactopyranoside induction of transcription of the wild type relA gene in pSM10 yields about a 100-fold overexpression of a metabolically stable, full length (743 amino acid) RelA protein to levels approximating the number of cellular ribosomes . This overexpression is accompanied by a roughly parallel and relC-dependent elevation of ppGpp levels . Induction of a relA gene deletion mutant in pSM11 containing 455 amino-terminal amino acids results in much lower levels of expression of a metabolically unstable 55-kDa protein and elevated ppGpp levels that are almost equivalent to induced pSM10 and are relC-independent . Induction of a larger deletion in pSM12 containing 331 amino-terminal amino acids does not provoke ppGpp accumulation . We are able to elicit high levels of ppGpp without changing nutritional abundance and without massive overexpression of the RelA protein by inducing the metabolically unstable, truncated RelA protein . We find the effects of elevated ppGpp levels to include a slowing of growth, an inhibition of stable RNA accumulation, an inhibition of cellular rrn P1 promoter activities as measured by primer extension, and changes in the pattern of gene expression viewed by two-dimensional electrophoresis of cellular proteins. Biochim Biophys Acta, 1991 Feb 25, 1062(2), 177 - 86 The interaction between aspartic acid 237 and lysine 358 in the lactose carrier of Escherichia coli; King SC et al.; The lacY from Escherichia coli strains 020 and AE43 have been cloned on plasmids which were designated p020-K358T and pAE43-D237N . These lacY mutants contain amino acid substitutions changing Lys-358 to Thr or Asp-237 to Asn, respectively . The charge neutralizing effect of each mutation is associated with a functional defect in melibiose transport which we exploited in order to isolate second site revertants to the melibiose-positive phenotype . Eleven melibiose-positive revertants of p020-K358T were isolated . All contained a second-site mutation converting Asp-237 to a neutral amino acid (8 to Asn, 1 to Gly, and 2 to Tyr) . Twelve melibiose-positive revertants of pAE43-D237N were isolated . Two were second-site revertants converting Lys-358 to a neutrally Gln residue, while the remainder directly reverted Asn-237 to the wild-type Asp-237 . We conclude that the functional intimate relationship between Asp-237 and Lys-358 suggests that these residues may be closely juxtaposed in three-dimensional space, possibly forming a 'charge-neutralizing' salt bridge. FEBS Lett, 1991 Feb 25, 279(2), 285 - 8 Glycine-144 is required for efficient folding of outer membrane protein PhoE of Escherichia coli K12; de Cock H et al.; Short stretches of similar sequences have been detected in unrelated bacterial outer membrane proteins (Nikaido and Wu (1984) Proc . Natl . Acad . Sci . USA 81, 1048-1052) . In the most pronounced similarity region, only a glycine residue is absolutely conserved . To investigate whether this glycine residue is essential for outer membrane incorporation, oligonucleotide-directed mutagenesis was applied to replace this residue, i.e . Gly-144, as well as two other Gly-residues in pore protein PhoE . Substitution of Gly-52 and Gly-258 by Ala and Val, respectively, did not influence outer membrane incorporation . However, the substitution of Gly-144 by Leu affected the efficiency of outer membrane incorporation . After in vitro synthesis this mutant protein was less efficiently precipitated with monoclonal antibodies that recognize conformational epitopes than wild-type PhoE, showing that the mutation interferes with correct folding of the protein. J Biol Chem, 1991 Feb 25, 266(6), 3863 - 9 Efficient purification of recombinant human tumor necrosis factor beta from Escherichia coli yields biologically active protein with a trimeric structure that binds to both tumor necrosis factor receptors; Schoenfeld HJ et al.; A fast and efficient method for medium scale purification of recombinant human tumor necrosis factor beta (rTNF-beta) from Escherichia coli cells is described . The purified rTNF-beta displayed biological activity similar to rTNF-alpha in a WEHI 164 cell cytotoxicity assay . The titration curve of rTNF-beta and elution profiles of rTNF-beta in gel filtration experiments were different from those of rTNF-alpha . However, light scattering and ultra-centrifugation studies showed that both cytokines have trimeric structures in solution at 0.5 mg/ml, with minor differences in the distribution of nontrimeric species . rTNF-beta bound to purified 55- and 75-kDa TNF receptors with high affinity . The binding of rTNF-beta to either receptor was analyzed on Scatchard plots and compared with that of rTNF-alpha. J Biol Chem, 1991 Feb 25, 266(6), 3654 - 60 Analysis of an Escherichia coli dnaB temperature-sensitive insertion mutation and its cold-sensitive extragenic suppressor; Chang SF et al.; An Escherichia coli mutant, ts121, was isolated following random insertional mutagenesis using phage lambda Mu transposition . The mutant phenotype includes inability to form colonies at temperatures above 38 degrees C and inability to propagate phage lambda at all temperatures . A lambda i434 cI- (ts121)+ transducing phage was isolated on the basis of its ability to form plaques on ts121 mutant bacteria . Using this transducing phage, it was shown through complementation and protein analyses, that the ts121 mutation is located in the dnaB gene . The exact insertion event was identified by polymerase chain reaction amplification of the DNA sequences containing the insertion junction . The mutational insertion event in ts121 was mapped precisely between base pairs 1514 and 1515 of the dnaB gene . This result predicts that the mutant dnaB protein has lost its six terminal amino acids . The reading frame shifts into Mu-specific DNA sequences resulting in an additional 20 amino acid residues . The E . coli wild type dnaB protein participates in host replication and interacts with lambda P protein to initiate phage lambda DNA replication . Our results demonstrate that the extreme carboxyl end of the dnaB protein is required for productive interaction with the lambda P replication protein at all temperatures, and is important for dnaB function at temperatures above 38 degrees C . Cold-sensitive extragenic suppressors of the ts121 mutation were isolated on the basis of their ability to restore colony formation at 42 degrees C . One of these extragenic suppressors was mapped at 54 min on the E . coli genetic map and localized to the suhB gene, whose product may affect the expression of a number of genes at the translational level. J Biol Chem, 1991 Feb 25, 266(6), 3547 - 53 Mutations that alter the charge of type I regulatory subunit and modify activation properties of cyclic AMP-dependent protein kinase from S49 mouse lymphoma cells; Steinberg RA et al.; Mutations in regulatory (R) subunit of cAMP-dependent protein kinase were analyzed from cAMP-resistant mutants of S49 mouse lymphoma cells by direct sequencing of amplified regions of mutant R subunit cDNAs . Eight distinct single base-change lesions were identified in 24 independent mutants that were hemizygous for expression of mutant R subunits with altered protein charge . CG----TA transitions predominated, but AT----GC transitions and GC----TA transversions were also observed . Four of five spontaneous mutants had identical C----T transitions at CG causing substitution of Trp for Arg-334 . Sites mutated in isolates obtained after mutagenesis with ethyl methanesulfonate or N-methyl-N'-nitro-N-nitrosoguanidine were more varied . Six of the lesions (two in binding site A and four in site B) were at amino acid residues that are highly conserved among cAMP-binding sites of R subunits and the Escherichia coli catabolite activator protein . These mutations all either prevented or strongly hindered binding of cyclic nucleotides to the mutated site . One of the remaining lesions (at Arg-242) also prevented cyclic nucleotide binding to the mutated binding site; the other (at Gly-170) had only minimal effects on binding of cyclic nucleotides but, nevertheless, increased the apparent constant for cAMP-dependent kinase activation . These results are discussed with reference to a model for the cAMP-binding sites of R subunit based on the crystal structure of the E . coli catabolite activator protein. J Biol Chem, 1991 Feb 25, 266(6), 3540 - 6 Probing the limits of the DNA breakage-reunion domain of the Escherichia coli DNA gyrase A protein; Reece RJ et al.; In a previous report (Reece, R . J., and Maxwell, A . (1989) J . Biol . Chem . 264, 19648-19653) we showed that treatment of the Escherichia coli DNA gyrase A protein with trypsin generates two stable fragments . The N-terminal 64-kDa fragment supports DNA supercoiling, while the C-terminal 33-kDa fragment shows no enzymic activity . We proposed that the 64-kDa fragment represents the DNA breakage-reunion domain of the A protein . We have now engineered the gyrA gene such that the 64-kDa protein is generated as a gene product . The properties of this protein confirm the findings of the experiments with the 64-kDa tryptic fragment . We have also generated a series of deletions of the gyrA gene such that C-terminal and N-terminal truncated versions of the A protein are produced . The smallest of the N-terminal fragments found to be able to carry out the DNA breakage-reunion reaction is GyrA(1-523) . The cleavage reaction mediated by this protein occurs with equal efficacy as that performed by the intact GyrA protein . Deletion of the N-terminal 6 amino acids from either the A protein or these deletion derivatives has no effect on enzymic activity, while deletion of the N-terminal 69 amino acids completely abolishes the DNA breakage-reunion reaction . Therefore the smallest GyrA protein we have found that will perform DNA breakage and reunion is GyrA(7-523) . A model is proposed for the domain organization of the gyrase A protein. J Biol Chem, 1991 Feb 25, 266(6), 3361 - 4 Expression of hepatitis B virus surface antigen in adult rat liver . Co-introduction of DNA and nuclear protein by a simplified liposome method; Kato K et al.; We established a simple and efficient method for gene transfer in vitro (to cultured cells) and in vivo (to an adult organ) using liposomes . Plasmid DNA and proteins were efficiently co-encapsulated in liposomes by agitation and sonication, and were co-introduced into cells by hemagglutinating virus of Japan (HVJ)-mediated membrane fusion . Introduction of the Escherichia coli beta-galactosidase gene with non-histone chromosomal protein high mobility group 1 (HMG1) into LLCMK2 cells resulted in about 3 times higher beta-galactosidase activity than that on introduction of the gene alone . Two days after injection of HVJ-liposomes containing the beta-galactosidase gene and HMG1 under the perisplanchnic membrane of adult rat liver, hepatic cells near the injection site were found by 5-bromo-4-chloro-3-indolyl beta-D-galactoside staining to have beta-galactosidase activity . After similar injection of HVJ-liposomes containing the hepatitis B virus surface antigen (HBsAg) gene and HMG1, HBsAg was detected in the serum for 9 days with a maximum of 25-45 ng/ml on day 2 after the injection. FEBS Lett, 1991 Feb 25, 279(2), 193 - 7 A heptapeptide repeat contributes to the unusual length of chloroplast ribosomal protein S18 . Nucleotide sequence and map position of the rpl33-rps18 gene cluster in maize; Weglohner W et al.; The rpl33-rps18 gene cluster of the maize chloroplast genome has been mapped and sequenced . The derived amino acid sequence of the S18 protein shows a 7-fold repeat of a hydrophilic heptapeptide domain, S K Q P F R K, in the N-terminal region . Such a sequence is absent in the E . coli S18 and in the chloroplast S18 of the lower plant liverwort . In tobacco and rice chloroplast S18 it is present 2 and 6 times, respectively . Thus a long N-terminal repeat (resembling in composition the large C-terminal heptapeptide repeat in the eukaryotic pol II) appears to be characteristic of monocot cereal S18. J Biol Chem, 1991 Feb 25, 266(6), 3630 - 5 Catalysis of protein folding by cyclophilins from different species; Schonbrunner ER et al.; Cyclophilins are a class of ubiquitous proteins with yet unknown function . They were originally discovered as the major binding proteins for the immunosuppressant cyclosporin A . The only known catalytic function of these proteins in vitro is the cis/trans isomerization of Xaa-Pro bonds in oligopeptides . This became clear after the discovery that bovine cyclophilin is identical with porcine prolyl isomerase . This enzyme accelerates slow, proline-limited steps in the refolding of several proteins . Here we demonstrate that the cyclophilins from man, pig, Neurospora crassa, Saccharomyces cerevisiae, and Escherichia coli are all active as prolyl isomerases and as catalysts of protein folding . This evolutionary conservation suggests that catalysis of prolyl peptide bond isomerization may be an important function of the cyclophilins . It could be related with de novo protein folding or be involved in regulatory processes . Catalysis of folding is very efficient in the presence of the high cellular concentrations of prolyl isomerase. J Biol Chem, 1991 Feb 25, 266(6), 3387 - 95 The catalytic mechanism of the amidotransferase domain of the Syrian hamster multifunctional protein CAD . Evidence for a CAD-glutamyl covalent intermediate in the formation of carbamyl phosphate; Chaparian MG et al.; The multifunctional protein CAD catalyzes the first three steps in pyrimidine biosynthesis in mammalian cells, including the synthesis of carbamyl phosphate from bicarbonate, MgATP and glutamine . The Syrian hamster CAD glutaminase (GLNase) domain, a trpG-type amidotransferase, catalyzes glutamine hydrolysis in the absence of MgATP and bicarbonate (Km = 95 microM and kcat = 0.14 s-1) . Unlike E . coli carbamyl phosphate synthetase (Wellner, V.P., Anderson, P.M., and Meister, A . (1973) Biochemistry 12, 2061-2066), a stable thioester intermediate did not accumulate when the mammalian enzyme was incubated with glutamine . However, a covalent adduct could be isolated when the protein was denatured in acid . The steady state concentration of the intermediate increased with increasing glutamine concentration to nearly one mole per mole of enzyme with half saturation at 105 microM, close to the Km value for glutamine . The adduct formed at the active site of the glutaminase domain . The rate of breakdown of the intermediate (k4), determined directly, was 0.17 s-1 and the rate of formation (k3) was estimated as 0.52 s-1 . In the absence of MgATP and bicarbonate, k4 = kcat indicating that the decomposition of the intermediate is the rate-limiting step . The intermediate was chemically and kinetically competent, and the glutamine dissociation constant (330 microM) and rate constants were consistent with steady state kinetics and accurately predicted the steady state concentration of the intermediate . These studies suggest a mechanism similar to the cysteine proteases such as recently proposed by Mei and Zalkin (Mei, B., and Zalkin, H . (1989) J . Biol . Chem . 264, 16613-16619) who identified a catalytic triad in glutamine phosphoribosyl-5'-pyrophosphate amidotransferase, a purF-type enzyme . MgATP and bicarbonate increased kcat of the glutaminase reaction 14-fold by accelerating both the rate of formation and the rate of breakdown of the intermediate, and prevented the accumulation of the intermediate; however, the Km value for glutamine was not significantly altered . The instability of the thioester intermediate leads to appreciable hydrolysis of glutamine in the absence of the other substrates . However, bicarbonate alone spares glutamine by increasing the Km and Ks of glutamine to 600 and 8960 microM, respectively, thus reducing kcat/Km 3-fold when MgATP is limiting . In the absence of MgATP and bicarbonate, ammonia decreased the rate of hydrolysis and the accumulation of the thioester intermediate indicating that ammonia had direct access to the thioester at the GLNase domain active site.(ABSTRACT TRUNCATED AT 400 WORDS) J Chromatogr, 1991 Feb 22, 539(2), 501 - 5 Purification of cloned trypanosomal calmodulin and preliminary NMR studies; Sweeney PJ et al.; Cloned trypanosomal calmodulin was expressed in Escherichia coli and purified to homogeneity using hydrophobic interaction chromatography on phenyl-Sepharose . The purified protein was subjected to NMR analysis which allows detailed changes to be observed when, firstly, calcium, and secondly, the drug calmidazolium bind . These spectral changes are the result of conformational changes in the protein and proximity effects due to the drug. J Chromatogr, 1991 Feb 22, 539(2), 465 - 73 Purification and characterization of recombinant tropomyosins; Ferraz C et al.; The cloning of a cDNA coding for the skeletal human beta-tropomyosin in the bacterial expression vector pKK233-2 is reported . Deletion mutants were also constructed . pCF-T1088 was obtained by elimination of exon 9 and pCF-T1089 was built by deleting 2/3 of the first exon . The recombinant tropomyosins were synthesized in E . coli after induction by IPTG . The mutant proteins were characterized by western blot using antibodies raised against native tropomyosin . The amount of the human protein synthesized in E . coli varies with each mutant, suggesting the involvement of the structure of the protein or of the mRNA on the synthesis or the stability of the recombinant protein . After precipitation of most of the bacterial proteins at 100 degrees C, purification was achieved by high-performance liquid chromatography (HPLC) using TSK-DEAE, hydroxyapatite and reversed-phase columns . The chromatographic behaviour of the mutants were compared . Characterization of the mutated tropomyosins was achieved by tryptic digestion and analysis of the peptide composition by reversed-phase HPLC . A computer program for predicting the retention times of the peptides generated was written . It is shown that it is possible to identify the mutations solely by comparing the chromatogram of the tryptic digest with the profile obtained by computer simulation. J Chromatogr, 1991 Feb 22, 539(2), 383 - 91 Unfolding of truncated and wild type aspartate aminotransferase studied by size-exclusion chromatography; Herold M et al.; The reversible unfolding of globular proteins with increasing concentration of guanidinium chloride (GuCl) can be analysed by size-exclusion chromatography, because the hydrodynamic volume of the proteins increases during unfolding . The dimeric enzyme aspartate aminotransferase (AAT) shows an uncoupled dissociation of the identical subunits followed by the unfolding of the monomers . During the monomer unfolding formation of an intermediate is observed . A monomeric mutant of AAT unfolds with a similar shape of the unfolding transition phase, but is less stable, as shown by a shift of the transition mid-point from 1.7 M GuCl for the wild type to 1.3 M GuCl for the mutant. Proc R Soc Lond B Biol Sci, 1991 Feb 22, 243(1307), 155 - 60 Site-directed mutagenesis of the lipoate acetyltransferase of Escherichia coli; Russell GC et al.; Remote but significant similarities between the primary and predicted secondary structures of the chloramphenicol acetyltransferases (CAT) and lipoate acyltransferase subunits (LAT, E2) of the 2-oxo acid dehydrogenase complexes, have suggested that both types of enzyme may use similar catalytic mechanisms . Multiple sequence alignments for CAT and LAT have highlighted two conserved motifs that contain the active-site histidine and serine residues of CAT . Site-directed replacement of Ser550 in the E2p subunit (LAT) of the pyruvate dehydrogenase complex of Escherichia coli, deemed to be equivalent to the active-site Ser148 of CAT, supported the CAT-based model of LAT catalysis . The effects of other substitutions were also consistent with the predicted similarity in catalytic mechanism although specific details of active-site geometry may not be conserved. Nature, 1991 Feb 21, 349(6311), 713 - 5 A dynamin-like protein encoded by the yeast sporulation gene SPO15; Yeh E et al.; The tightly centromere-linked gene SPO15 is essential for meiotic cell division in the yeast Saccharomyces cerevisiae . Diploid cells without the intact SPO15 gene product are able to complete premeiotic DNA synthesis and genetic recombination, but are unable to traverse the division cycles . Electron microscopy of blocked cells reveals a duplicated but unseparated spindle-pole body . Thus cells are unable to form a bipolar spindle . Sequence analysis of SPO15 DNA reveals an open reading frame that predicts a protein of 704 amino acids . This protein is identical to VPS1, a gene involved in vacuolar protein sorting in yeast which has significant sequence homology (45% overall, 66% over 300 amino acids) to the microtubule bundling-protein, dynamin . The SPO15 gene product expressed in Escherichia coli can be affinity-purified with microtubules . SPO15 encodes a protein that is likely to be involved in a microtubule-dependent process required for the timely separation of spindle-pole bodies in meiosis. J Mol Biol, 1991 Feb 20, 217(4), 661 - 79 CAP and Nag repressor binding to the regulatory regions of the nagE-B and manX genes of Escherichia coli; Plumbridge J et al.; The divergent nagE-BACD operons located at 15.5 min on the Escherichia coli chromosome encode genes involved in the uptake and metabolism of N-acetylglucosamine . The start sites of the divergent transcripts are separated by 133 base-pairs (bp) . A repressor protein for the regulon is encoded by the gene nagC, one of the genes of the nagBACD operon . Strains overproducing the NagC protein have been used to investigate the binding of repressor to the intergenic nagE-B regulatory region . Two binding sites have been detected, overlapping the promoters of the nagE and nagB genes . NagC binding produces a series of DNase I hypersensitive sites separated by 9 to 11 bp in the region between the two NagC binding sites, supporting a model where the NagC proteins bind co-operatively to these two sites on the DNA and interact to form a DNA loop . A strong CAP binding site exists between the two operator sites . It is located at -61.5 and -71.5 relative to the nagE and nagB transcription start sites . CAP and NagC can bind simultaneously and produce a complex more stable than the binary NagC-DNA complex . In addition NagC and CAP binding sites have been found upstream from the manXYZ operon . Although the sites exhibit a similar organization there is no evidence for formation of a DNA loop in this operon. Biochemistry, 1991 Feb 19, 30(7), 1980 - 5 Activity and structure of the active-site mutants R386Y and R386F of Escherichia coli aspartate aminotransferase; Danishefsky AT et al.; Arginine-386, the active-site residue of Escherichia coli aspartate aminotransferase (EC 2.6.1.1) that binds the substrate alpha-carboxylate, was replaced with tyrosine and phenylalanine by site-directed mutagenesis . This experiment was undertaken to elucidate the roles of particular enzyme-substrate interactions in triggering the substrate-induced conformational change in the enzyme . The activity and crystal structure of the resulting mutants were examined . The apparent second-order rate constants of both of these mutants are reduced by more than 5 orders of magnitude as compared to that of wild-type enzyme, though R386Y is slightly more active than R386F . The 2.5-A resolution structure of R386F in its native state was determined by using difference Fourier methods . The overall structure is very similar to that of the wild-type enzyme in the open conformation . The position of the Phe-386 side chain, however, appears to shift with respect to that of Arg-386 in the wild-type enzyme and to form new contacts with neighboring residues. Biochemistry, 1991 Feb 19, 30(7), 1948 - 57 Construction, expression, and purification of recombinant kringle 1 of human plasminogen and analysis of its interaction with omega-amino acids; Menhart N et al.; An Escherichia coli expression vector, containing the alkaline phosphatase promoter and the stII heat-stable enterotoxin signal sequence, along with the cDNA of the kringle 1 (K1) region of human plasminogen (HPg), has been employed to express into the periplasmic space amino acid residues 82-163 (E163----D) of HPg . This region of the molecule contains the entire K1 domain (residues C84-C162) of HPg, as well as two non-kringle amino-terminal amino acids (S82-E83) that are present in their normal locations in HPg and a carboxyl-terminal amino acid, D163, that results from mutation of the E163, normally present at this location in the HPg amino acid sequence . After purification of r-K1 by chromatographic techniques, we have investigated its omega-amino acid binding properties by titration calorimetry, intrinsic fluorescence, and differential scanning microcalorimetry (DSC) . The antifibrinolytic agent, epsilon-aminocaproic acid (EACA), possesses a single binding site for r-K1 . The thermodynamic properties of this interaction, studied by calorimetric titrations of the heats of binding with this ligand, reveal a Kd of 12 +/- 2 microM at 25 degrees C and pH 7.4, a corresponding delta G of -6.7 +/- 0.1 kcal/mol, a delta H of -3.6 +/- 0.1 kcal/mol, and a delta S of 10.5 +/- 0.8 eu . The intrinsic fluorescence of r-K1 decreases by approximately 44% when its binding site is saturated with EACA, and titrations of this perturbation with EACA lead to calculation of a Kd of approximately 13 microM, a value in good agreement with that obtained from titration calorimetric analysis . EACA represents the strongest binding ligand of a variety of simple aliphatic omega-amino acids examined . A cyclic analogue of EACA, trans-4-(aminomethyl)cyclohexanecarboxylic acid, interacts with r-K1 with an approximate 12-fold tighter Kd (1.0 +/- 0.2 microM) . Investigations by DSC, at pH 7.4, demonstrate that a significant stabilization of the r-K1 structure occurs when EACA binds to this domain . The temperature of maximum heat capacity change (Tm) in the thermal denaturation of r-K1 increases from approximately 340.8 to 359.1 K as a consequence of EACA binding . These studies demonstrate that a fully functional EACA-binding kringle from HPg can be expressed and secreted in E . coli, purified by techniques that do not require refolding, and investigated as an independent structural unit. Biochemistry, 1991 Feb 19, 30(7), 1845 - 51 Structural microheterogeneity of a tryptophan residue required for efficient biological electron transfer between putidaredoxin and cytochrome P-450cam; Stayton PS et al.; The carboxy-terminal tryptophan of putidaredoxin, the Fe2S2.Cys4 iron-sulfur physiological redox partner of cytochrome P-450cam, is essential for maximal biological activity {Davies, M . D., Qin, L., Beck, J . L., Suslick, K . S., Koga, H., Horiuchi, T., & Sligar, S . G . (1990) J . Am . Chem . Soc . 112, 7396-7398} . This single tryptophan-containing protein thus represents an excellent system for studying the solution dynamics of a residue directly implicated in an electron-transfer pathway . Steady-state and time-resolved measurements of the tryptophan fluorescence have been conducted across the emission spectrum as a function of redox state to probe potential structural changes which might be candidates for structural gating phenomena . The steady-state emission spectrum (lambda max = 358 nm) and anisotropy (alpha = 0.04) suggest that Trp-106 is very solvent-exposed and rotating partially free of global protein constraints . The time-resolved fluorescence kinetics for both oxidized and reduced putidaredoxin are fit best with three discrete components of approximately 5, 2, and 0.3 ns . The lifetime components were assigned to physical species with iodide ion quenching experiments, where differential quenching of the longer components was observed (k tau = 2 = 5.9 X 10(8) M-1 s-1, k tau = 5 = 1.3 X 10(8) M-1 s-1) . These findings suggest that the multiexponential fluorescence decay results from ground-state conformational microheterogeneity and thus demonstrate that the essential tryptophan exists in at least two distinguishable conformations . Small differences in the relative proportions of the components between redox states were observed but not cleanly resolved.(ABSTRACT TRUNCATED AT 250 WORDS) Biochemistry, 1991 Feb 19, 30(7), 1788 - 95 Sites of contact of mRNA with 16S rRNA and 23S rRNA in the Escherichia coli ribosome; Wollenzien P et al.; The locations of close encounter between ribosomal RNA (rRNA) and messenger RNA (mRNA) were determined by photochemical cross-linking experiments that employ an artificial mRNA, 51 nucleotides long, containing 14 U residues that were randomly substituted by 1-4 4-thiouridine (s4U) residues . The mRNA was bound to 70S ribosomes or 30S subunits and then was irradiated at 366 nm to activate cross-linking between the s4U residues and rRNA . Cross-linking occurred to both 16S rRNA and 23S RNA . The rRNA was then analyzed by a series of reverse transcriptase experiments to determine the locations of cross-linking . Twelve sites in the 16S rRNA and two sites in the 23S rRNA have been detected . In the 16S rRNA, two of the sites (U1381, C1395) are in the middle part of the secondary structure close to position C1400, and the remaining sites (G413, U421, G424; A532; G693; U723; A845; G1131/C1132; G1300; G1338) are distributed between six regions that are peripheral in the secondary structure . In the 23S rRNA, one site (U1065) is located in the GTPase center close to A1067, the site of thiostrepton-resistance methylation in domain II, and the other site (U887) is located a short distance away also in domain II . The distribution of these rRNA sites in the ribosome specifies an mRNA track that is consistent with other information . In addition, some of the contact points represent new constraints for the three-dimensional folding of the rRNA. Biochemistry, 1991 Feb 19, 30(7), 1768 - 73 Details of mannitol transport in Escherichia coli elucidated by site-specific mutagenesis and complementation of phosphorylation site mutants of the phosphoenolpyruvate-dependent mannitol-specific phosphotransferase system; van Weeghel RP et al.; The mannitol transport protein (EIImtl) carries out translocation with concomitant phosphorylation of mannitol from the periplasm to the cytoplasm, at the expense of phosphoenolpyruvate (PEP) . The phosphoryl group which is needed for this group translocation is sequentially transferred from PEP via two phosphorylation sites, located exclusively on the C-terminal cytoplasmic domain, to mannitol . Oligonucleotide-directed mutagenesis was used to investigate the precise role of these sites in phosphoryl group transfer, by producing specific amino acid substitutions . The first phosphorylation site, His-554 (P1), was replaced by Ala, which renders the EII-H554A completely inactive in PEP-dependent mannitol phosphorylation, but not in mannitol/mannitol 1-phosphate exchange . The P2 site mutant, EII-C384S, was inactive both in the mannitol phosphorylation reaction and in the exchange reaction, due to replacement of the essential Cys-384 by Ser . Although EII-H554A and EII-C384S were both catalytically inactive in the PEP-dependent phosphorylation, EII-C384S was able to restore up to 55% of the wild-type mannitol phosphorylation activity with the EII-H554A mutant, indicating a direct phosphotransfer between two subunits . These phosphorylation data together with the data obtained from mannitol/mannitol phosphate exchange kinetics, after mixing EII-H554A and EII-C384S, indicated the formation of functionally stable heterodimers, which consist of an EII-H554A and an EII-C384S monomer. Biochemistry, 1991 Feb 19, 30(7), 1939 - 47 Purification and characterization of recombinant mouse and herpes simplex virus ribonucleotide reductase R2 subunit; Mann GJ et al.; Overexpression of recombinant mouse and herpes simplex virus ribonucleotide reductase small subunit (protein R2) has been obtained by using the T7 RNA polymerase expression system . Both proteins, which constitute about 30% of the soluble Escherichia coli proteins, have been purified to homogeneity by a rapid and simple procedure . At this stage, few of the molecules contain the iron-tyrosyl free-radical center necessary for activity; however, addition of ferrous iron and oxygen under controlled conditions resulted in a mouse R2 protein containing 0.8 radical and 2 irons per polypeptide chain . In this reaction, one oxygen molecule was needed to generate each tyrosyl radical . Both proteins had full enzymatic activity . EPR spectroscopy showed that iron-center/radical interactions are considerably stronger in both mouse and viral proteins than in E . coli protein R2 . CD spectra showed that the bacterial protein contains 70% alpha-helical structure compared to only about 50% in the mouse and viral proteins . Light absorption spectra between 310 and 600 nm indicate close similarity of the mu-oxo-bridged binuclear iron centers in all three R2 proteins . Furthermore, the paramagnetically shifted iron ligand proton NMR resonances show that the antiferromagnetic coupling and ligand arrangement in the iron center are nearly identical in all three species. Biochemistry, 1991 Feb 19, 30(7), 1801 - 7 Requirement for the beta,gamma-pyrophosphate bond of ATP in a stage between transcription initiation and elongation by Escherichia coli RNA polymerase; Fujioka M et al.; A linear fragment of DNA was fixed to acrylamide or agarose beads by its ends . When a fragment containing the lambda PR promoter is immobilized and transcribed, the RNA products are unchanged from those obtained on the unfixed DNA . Transcription from the immobilized fragment can be interrupted by diluting the reaction mixture into a large volume of the same buffer . Brief centrifugation allows isolation of the transcription complex with the immobilized DNA . If interruption occurs during elongation, the elongation can be resumed upon a second addition of substrates . If ATP is replaced by a beta, gamma-unhydrolyzable analogue in the second addition, the elongated products are similar to those obtained when the substrate contain ATP . When ATP is replaced by the analogue at the initiation step, however, the yield of elongated products is decreased to less than one-sixth and that of short abortive products is increased . Thus the ATP analogues are good substrates once elongation has been established in the presence of ATP, but not good enough to get past a stage just after initiation in the absence of ATP . We conclude that the beta, gamma-pyrophosphate bond of ATP is important for preparation of efficient elongation. Biochim Biophys Acta, 1991 Feb 16, 1088(2), 323 - 4 Nucleotide sequence of the lipase gene lip3 from the antarctic psychotroph Moraxella TA144; Feller G et al.; A lipase gene (lip3) from the psychotrophic strain Moraxella TA144 has been cloned and sequenced . The deduced primary structure of the lipase preprotein is composed of 315 amino acids with a predicted Mr of 34,772 . This enzyme contains two consensus peptides showing cluster of glycine residues that may be involved in domain flexibility . The cloned gene product conserves the low temperature activity and the thermolability properties of the wild enzyme. Biochim Biophys Acta, 1991 Feb 16, 1088(2), 292 - 300 Cloning and sequencing of the gene cluster encoding two subunits of membrane-bound alcohol dehydrogenase from Acetobacter polyoxogenes; Tamaki T et al.; The membrane-bound alcohol dehydrogenase (ADH) from Acetobacter polyoxogenes NBI1028 is composed of a 72 kDa subunit and a 44 kDa cytochrome c subunit . The amino acid sequences of the two regions of the 72 kDa subunit were determined to prepare oligonucleotides for the purpose of amplification of a DNA fragment corresponding to the intermediate region by the polymerase chain reaction . A 0.5 kb DNA fragment thus amplified was used as the probe to clone a 7.0 kb PstI fragment coding for the whole 72 kDa subunit . Nucleotide sequencing and immunoblot analysis revealed that the cloned fragment contained the full structural genes for the 72 kDa and the 44 kDa subunits and they were clustered with the same transcription polarity . The predicted amino acid sequence of the gene for the 72 kDa subunit showed homology with that of the 72 kDa subunit from ADH of A . aceti and those of methanol dehydrogenase from methylotrophic bacteria . The 72 and 44 kDa subunits contained one and three typical haem binding sequences, respectively. Biochim Biophys Acta, 1991 Feb 16, 1088(2), 197 - 207 Isolation and characterization of a human cDNA encoding uracil-DNA glycosylase; Muller SJ et al.; DNA repair of genetic information is an essential defense mechanism, which protects cells against mutation and transformation . The biochemistry of human DNA repair is in its beginning stages . Our research has concentrated on the enzymes involved in the removal of atypical bases from DNA . We present information on the identification and characterization of a cDNA isolate encoding uracil-DNA glycosylase . Uracil-DNA glycosylase was purified to homogeneity from HeLa S3 cells and used to generate polyclonal antibodies . These antibodies were in turn used to isolate a uracil-DNA glycosylase specific cDNA from a human T cell (Jurkat) lambda-gt11 library . The identity of this 1.25 kb cDNA was verified using in vitro transcription and translation systems to generate specific uracil-DNA glycosylase activity . Sequence data revealed a 327 amino acid open reading frame, which encodes a protein with a predicted molecular weight of 35351 . No significant amino acid homology was found between this human uracil-DNA glycosylase and the glycosylases of yeast, Escherichia coli, herpes simplex virus, or a recently identified 26,000 Da species of human uracil-DNA glycosylase . This apparent lack of homology prompted an investigation of uracil-DNA glycosylase in a variety of eukaryotic species . Western analysis demonstrated the presence of a 36 kDa uracil-DNA glycosylase protein in human fibroblast, human placental and Vero cell extracts . Interestingly, these antibodies did not detect glycosylase protein in Chinese hamster ovary (CHO) or mouse NIH3T3 fibroblast cells . Under conditions of reduced stringency, Southern blot analysis of BamHI digested DNA from human fibroblasts, human placental cells and Vero cells revealed common 12 kb and 3 kb fragments . In contrast, using the same reduced stringency protocol, 6 and 8 kb fragments for CHO and NIH3T3 DNA were seen, respectively, as well as a common 3 kb fragment . Under more stringent wash conditions, the common 3 kb band was absent in all samples analyzed, and no hybridization signal was detected from DNA of hamster or mouse origin . The lack of immunological reactivity between the human uracil-DNA glycosylase and the rodent forms is therefore reflected at the genetic level as well . This distinction in human and CHO hybridization patterns enabled us to localize this human uracil-DNA glycosylase cDNA to chromosome 5 by somatic cell hybridization. Gene, 1991 Feb 15, 98(2), 289 - 93 Cloning and expression in Escherichia coli of a synthetic gene encoding the extracellular domain of the human muscle acetylcholine receptor alpha-subunit; Talib S et al.; To better define the antigenic sites on the human muscle acetylcholine receptor (AChR) that are involved in stimulating the production of pathogenic antibodies in myasthenia gravis (MG), the nucleotide sequence encoding the major extracellular domain of the AChR alpha subunit was chemically synthesized . The gene cassettes encoding amino acids (aa) 1-85 (AChR-I) and 86-210 (AChR-II), were cloned individually, and the coding sequence representing the complete major extracellular domain (aa 1-210; AChR-C) was obtained by subsequent fusion of cassettes encoding AChR-I and AChR-II . The genes were inserted into the inducible expression plasmid, pKK-223-3, and expressed in vitro and in vivo in Escherichia coli . Biological activity was demonstrated by immunoprecipitation of in vitro-synthesized AChR-C by sera from MG patients and by the alpha-bungarotoxin-binding activity of E . coli-synthesized AChR-II and AChR-C . The availability of the recombinant AChR polypeptides should facilitate studies on the molecular basis of the autoimmune response in MG. Gene, 1991 Feb 15, 98(2), 259 - 63 The rodent B2 sequence can affect expression when present in the transcribed region of a reporter gene; Bladon TS et al.; The mouse B2 element is a moderately repetitive nt sequence of 180 bp transcribed by RNA polymerase III (Pol III) at high levels in embryonic and transformed cells . The B2 sequence is present in either orientation within the noncoding regions of a number of genes transcribed by RNA polymerase II (Pol II) . We sought to determine if the small B2 transcripts generated by Pol III are natural antisense RNA molecules which might hybridize to complementary sequences present within Pol II transcripts . Chimaeric reporter genes encoding Escherichia coli gpt were constructed containing a B2 repeat in either orientation within the 5'- or 3'-untranslated regions . These constructs were transfected into embryonal carcinoma (EC) cells and expression of the reporter gene was analysed in EC cells and retinoic acid-treated EC cells, which contain high and low levels of small B2 RNAs, respectively . Although the B2 sequences affected expression of the reporter gene, these effects did not appear to be due to hybridization of the small B2 RNA to the reporter transcripts . The presence of B2 sequences near a Pol II-transcribed gene can alter expression of that gene in a position- and orientation-dependent manner, suggesting these repetitive elements may be cis-acting regulators of gene expression. Gene, 1991 Feb 15, 98(2), 243 - 8 Rapid isolation of a rice waxy sequence: a simple PCR method for the analysis of recombinant plasmids from intact