J Biochem (Tokyo), 1988 Jul, 104(1), 30 - 4 High-level expression of human BSF-2/IL-6 cDNA in Escherichia coli using a new type of expression-preparation system; Tonouchi N et al.; BSF-2 (B cell stimulatory factor-2/IL-6) is a member of the lymphokine family and responsible for B cell differentiation . Expression plasmids of human BSF-2 cDNA were constructed using a trp promotor/operator and a trpA terminator . In an extract of Escherichia coli HB101 holding "direct" expression plasmid pBSF-2D, activity of BSF-2 was detected, but overproduction was not observed . A "fused" expression system was therefore developed to prepare the recombinant protein . In this system, cDNA was expressed as a fused protein with human IL-2 N-terminal peptide . In the case of the fused BSF-2 expression plasmid, pBSF-2F, inclusion bodies were observed and overproduction of the protein occurred . As this fused protein had a Phe-Arg-Ala sequence at the junction of hIL-2 and BSF-2, it was possible to process mature BSF-2 from the fused BSF-2 by treatment with kallikrein and aminopeptidase P . From 1 liter of E . coli culture, 45 mg of mature BSF-2 was purified; it had a relative biological activity equal to that of natural BSF-2 purified from T cells. Can J Microbiol, 1988 Jul, 34(7), 905 - 7 Thymineless death in Escherichia coli mutants deficient in the RecF recombination pathway; Nakayama K et al.; Like recF and recQ mutants studied earlier, two other classes of Escherichia coli mutants defective in the RecF conjugal recombination pathway, recJ and recO, were found to be partially resistant to thymineless death . In contrast, a recN mutant, also belonging to the pathway, was indistinguishable from the wild type with respect to thymineless death. Infection, 1988 Jul-Aug, 16(4), 215 - 21 Polymorphonuclear leucocyte dysfunction during short term metabolic changes from normo- to hyperglycemia in type 1 (insulin dependent) diabetic patients; Kjersem H et al.; Polymorphonuclear leucocyte (PMN) ingestion of particles coated with lipopolysaccharide (LPS) from Escherichia coli was compared to other PMN functions in seven patients with insulin dependent diabetes mellitus (IDDM) during short-term controlled metabolic changes from normo- to hyperglycemia without ketoacidosis . Factors known to interfere with PMN functions were excluded . PMN ingestion of particles coated with both LPS and bovine serum albumin became reduced from normo- to hyperglycemia . PMN motility was impaired in IDDM, but did not seem to be affected by short-term changes in metabolic control . PMN metabolism did not change from normo-to hyperglycemia . Particle-uptake by diabetic PMN is impaired after short term hyperglycemia in the range normally occurring in diabetics in every-day life. Mol Microbiol, 1988 Jul, 2(4), 531 - 8 The structure of a plasmid of Chlamydia trachomatis believed to be required for growth within mammalian cells; Comanducci M et al.; Sequence analysis of a 7.5 kb DNA plasmid isolated from Chlamydia trachomatis shows 8 open reading frames (ORFs) regularly spaced along most of the sequence . One of these ORFs encodes a 451-amino-acid polypeptide highly homologous to the DnaB protein of Escherichia coli . A region between ORFs 6 and 7 contains a cluster of alternating ATs and a 22 bp sequence tandemly repeated 4 times, suggesting a replication control region . Several ORFs correspond to plasmid-specific polypeptides that have been described . Codons ending with A or T are more frequent, as might be expected from the high A/T content (64%) of the plasmid, and codon usage is similar to that of the C . trachomatis chromosomal gene, omp1L2. Mol Microbiol, 1988 Jul, 2(4), 497 - 505 The integral membrane protein from a virulent isolate of transmissible gastroenteritis virus: molecular characterization, sequence and expression in Escherichia coli; Britton P et al.; Subgenomic mRNA from a virulent isolate of porcine transmissible gastroenteritis virus (TGEV) was used to produce cDNA clones . Part of a new clone and a previously reported clone were sequenced and used to construct the viral gene for integral membrane protein . A single open reading frame (ORF) encoding a polypeptide of 262 amino acids, relative molecular mass (Mr) 29,459, was identified . The positive identification of the polypeptide as the integral membrane protein was demonstrated by the production in E . coli of a chimaeric protein comprising most of the ORF encoding the Mr 29,459 polypeptide and beta-galactosidase . The chimaeric protein reacted with a specific monoclonal antibody to viral integral membrane protein and antibodies raised against the chimaeric protein immune precipitated the viral protein . Comparison with the sequence of an avirulent isolate indicates amino acid residues that may be important in pathogenicity. Mol Microbiol, 1988 Jul, 2(4), 473 - 9 Molecular characterization of malQ, the structural gene for the Escherichia coli enzyme amylomaltase; Pugsley AP et al.; The structural gene for the Escherichia coli enzyme amylomaltase, malQ, is the second gene in the malPQ operon . The nucleotide sequence of malQ shows that the gene encodes an Mr 78360 protein close to the experimentally determined Mr of purified amylomaltase (72000-74000) . The malQ initiation codon was identified by sequence analysis of clustered deletions around the 5' end of the gene . One of these deletions removed the first 5 bases from the malQ coding sequence . Strains carrying a plasmid with this truncated malQ gene under lacZ promoter control and out-of-frame with the first four codons of lacZ were Mal- . The Mal+ phenotype could be restored by inserting small, random fragments of E . coli chromosomal DNA into the unique EcoRI site . Nucleotide sequencing showed that the inserts either joined the lacZ and malQ sequences in frame, or contained a new translation start signal and coding sequence in frame with malQ . These results indicate that amylomaltase could be useful as a reporter protein in gene fusion studies. J Med Virol, 1988 Jul, 25(3), 329 - 37 Low incidence and high titers of antibodies to hepatitis B virus X-protein in sera of Chinese patients with hepatocellular carcinoma; Liang XH et al.; Sera of patients from China with hepatocellular carcinoma (HCC) were tested for the presence of HBc/HBe- and HBx antibodies by immunoblotting using recombinant MS2 or beta gal fusion proteins as substrate . Antibodies against HBx were detected in four out of 68 HBsAg positive and in one out of three HBsAg negative sera, antibodies against HBc/HBe in 52 and two serum samples, respectively . Competition experiments in which sera were preincubated with individual viral proteins synthesized in E . coli were carried out to demonstrate the specificity of signals obtained in immunoblot analyses . In the five anti-HBx positive sera, the antibody titer against X fusion protein was higher than against core fusion protein and in one of these sera anti-x activity could be demonstrated even at a serum dilution of 1:50,000 . These data indicate that X antibodies occur rarely in Chinese patients and are not serodiagnostic for HCC . The high titer of X antibodies in some patients shows that the X protein can be highly immunogenic in vivo . Induction of antibody formation may be triggered by X protein expressed from integrated viral DNA. Circ Shock, 1988 Jul, 25(3), 197 - 207 Contractile function of aortic smooth muscle in guinea pig endotoxin shock; Kutsky P et al.; Contractile responsiveness of aortic rings from control and endotoxin-shocked guinea pigs was examined in vitro . Aortae were removed 16 h following intraperitoneal administration of 4 mg/kg of Escherichia coli endotoxin or sterile saline . Although no shift in the dose-response curve or maximal contraction to norepinephrine (10(-9)-10(-4) M) was observed, there was an enhanced contractile response to KCl in shock rings . Calcium dose-response curves in both 60 mM and normal (5.4 mM) KCl were shifted upward in shock rings . This upward shift was abolished in normal KCl in the presence of the Ca2+ blocker D 600 (50 microM), suggesting enhanced Ca2+ influx via voltage-dependent Ca2+ channels in shock vascular smooth muscle . A deleterious effect of high cytoplasmic Ca2+ on vascular smooth muscle mitochondrial function may play a role in endotoxin shock. Mol Cell Biol, 1988 Jul, 8(7), 2837 - 47 Epstein-Barr virus shuttle vector for stable episomal replication of cDNA expression libraries in human cells; Margolskee RF et al.; Efficient transfection and expression of cDNA libraries in human cells has been achieved with an Epstein-Barr virus-based subcloning vector (EBO-pcD) . The plasmid vector contains a resistance marker for hygromycin B to permit selection for transformed cells . The Epstein-Barr virus origin for plasmid replication (oriP) and the Epstein-Barr virus nuclear antigen gene have also been incorporated into the vector to ensure that the plasmids are maintained stably and extrachromosomally . Human lymphoblastoid cells can be stably transformed at high efficiency (10 to 15%) by such plasmids, thereby permitting the ready isolation of 10(6) to 10(7) independent transformants . Consequently, entire high-complexity EBO-pcD expression libraries can be introduced into these cells . Furthermore, since EBO-pcD plasmids are maintained as episomes at two to eight copies per cell, intact cDNA clones can be readily isolated from transformants and recovered by propagation in Escherichia coli . By using such vectors, human cells have been stably transformed with EBO-pcD-hprt to express hypoxanthine-guanine phosphoribosyltransferase and with EBO-pcD-Leu-2 to express the human T-cell surface marker Leu-2 (CD8) . Reconstruction experiments with mixtures of EBO-pcD plasmids demonstrated that one clone of EBO-pcD-hprt per 10(6) total clones or one clone of EBO-pcD-Leu-2 per 2 x 10(4) total clones can be recovered intact from the transformed cells . The ability to directly select for expression of very rare EBO-pcD clones and to then recover these episomes should make it possible to clone certain genes where hybridization and immunological screening methods are not applicable but where a phenotype can be scored or selected in human cell lines. Proc Natl Acad Sci U S A, 1988 Jul, 85(14), 5269 - 73 Alterations of amino acid repeats in the Escherichia coli hemolysin affect cytolytic activity and secretion; Felmlee T et al.; The primary structure of the Escherichia coli hemolysin polypeptide (HlyA) is used to predict intramolecular structures involved in the secretion and cytolytic activity of the molecule . The C-terminal region of HlyA contains a repeated, 8-amino acid chain represented by the consensus sequence Leu-Xaa-Gly-Gly-Xaa-Gly-Asn-Asp . Three in vitro derived mutations of hlyA are described that encode molecules missing various portions of the C-terminal region, including the repeat region . The wild-type and mutated HlyA molecules were analyzed for the ability to be secreted and to lyse erythrocytes . Hemolytic activity absolutely requires the presence of the repeats . The ability of the mutated HlyA molecules to initiate membrane translocation and be secreted required the presence of the C terminus and, to a degree, the repeated amino acid octets. Proc Natl Acad Sci U S A, 1988 Jul, 85(14), 5146 - 50 Human p53 oncogene contains one promoter upstream of exon 1 and a second, stronger promoter within intron 1; Reisman D et al.; To gain insight into how transcription of the human p53 oncogene is controlled, we characterized the regulatory regions of the gene . A 3.8-kilobase-pair (kbp) EcoRI restriction fragment encompassing the 5' end of the human p53 gene, as well as subfragments generated by restriction digests, was cloned upstream of the Escherichia coli chloramphenicol acetyltransferase (CAT) gene and CAT activity was assayed in extracts of transfected cells . Two types of CAT vectors were used: Epstein-Barr virus oriP-derived constructs that were stably introduced into the human cell lines K562, Raji, and HL-60, and pSV0-CAT-derived constructs that were transiently introduced into the monkey cell line COS . By this approach we have identified two promoters for the human p53 gene . One promoter, p53P1, is located 100-250 bp upstream of the 218-bp noncoding first exon; a second, stronger promoter, p53P2, maps within the first intron . CAT activity and expression of CAT RNA indicate that p53P2 functions up to 50-fold more efficiently than p53P1 . We conclude that the expression of the human p53 gene may be controlled by two promoters and that differential regulation of these promoters may play an important role in the altered expression of the gene in both normal and transformed cells. Virology, 1988 Jul, 165(1), 262 - 7 Identification of human papillomavirus type 11 E4 gene products in human tissue implants from athymic mice; Brown DR et al.; A new method has recently been described for the growth of human papillomavirus type 11 (HPV11), an agent associated with genital warts, in human tissue xenografts implanted under the renal capsules of athymic nude mice (J . W . Kreider et al., 1986, J . Virol . 59, 369-376; 1987, J . Virol . 61, 590-593) . With this model it is now possible to study productive HPV11 infection under controlled laboratory conditions . To identify proteins encoded by the HPV11 E4 open reading frame in infected implants, we have cloned an HPV11 E4 genomic DNA fragment representing all of the E4 region thought to be expressed in vivo, as evidenced by cDNA cloning and R loop mapping . The cloned HPV11 fragment was expressed in Escherichia coli as a cro-beta-galactosidase E4 fusion protein (Gal-E4 fusion) . Rabbit antibodies raised against the Gal-E4 fusion protein were affinity purified using an HPV11 E1 E4 fusion protein . The E1--E4 protein was synthesized independently by expressing an HPV11 E1--E4 cDNA in E . coli using a second expression vector . Affinity-purified anti-E4 antibodies identified putative E4 proteins of 10 and 11 kDa in both the condylomatous cyst walls and in the desquamated cells in the cavities of HPV11-infected human skin implants from athymic mice . Similar proteins were not detected in uninfected controls . Implications for use of the athymic mouse system are discussed. Virology, 1988 Jul, 165(1), 200 - 8 Proteins encoded by bovine viral diarrhea virus: the genomic organization of a pestivirus; Collett MS et al.; The genome of bovine viral diarrhea virus (BVDV) contains a single large open reading frame capable of encoding 449 kDa of protein . Short segments from along the length of the molecularly cloned BVDV genome were engineered so as to be expressed as bacterial fusion polypeptides in Escherichia coli . These BVDV analog fusion proteins were used as immunogens to generate a panel of sequence-specific antisera . These antiserum reagents were in turn employed in immunoprecipitation analyses to identify the authentic BVDV protein to which they were directed . The results allowed for the identification and positioning along the genome of BVDV gene products accounting for approximately 83% of the coding capacity of the virus . A preliminary map of the genetic organization of BVDV is presented and discussed. Proc Natl Acad Sci U S A, 1988 Jul, 85(13), 4799 - 803 Isolation of gene fusions (soi::lacZ) inducible by oxidative stress in Escherichia coli; Kogoma T et al.; Mu dX phage was used to isolate three gene fusions to the lacZ gene (soi::lacZ; soi for superoxide radical inducible) that were induced by treatment with superoxide radical anion generators such as paraquat and plumbagin . The induction of beta-galactosidase in these fusion strains with the superoxide radical generating agents required aerobic metabolism . Hyperoxygenation (i.e., bubbling of cultures with oxygen gas) also induced the fusions . On the other hand, hydrogen peroxide did not induce the fusions at concentrations that are known to invoke an adaptive response . Introduction of oxyR, htpR, or recA mutations did not affect the induction . Two of the fusion strains exhibited increased sensitivity to paraquat but not to hydrogen peroxide . The third fusion strain showed no increased sensitivity to either agent . All three fusions were located in the 45- to 61-min region of the Escherichia coli chromosome. Proc Natl Acad Sci U S A, 1988 Jul, 85(13), 4677 - 81 Model for how type I restriction enzymes select cleavage sites in DNA; Studier FW et al.; Under appropriate conditions, digestion of phage T7 DNA by the type I restriction enzyme EcoK produces an orderly progression of discrete DNA fragments . All details of the fragmentation pattern can be explained on the basis of the known properties of type I enzymes, together with two further assumptions: (i) in the ATP-stimulated translocation reaction, the enzyme bound at the recognition sequence translocates DNA toward itself from both directions simultaneously; and (ii) when translocation causes neighboring enzymes to meet, they cut the DNA between them . The kinetics of digestion at 37 degrees C indicates that the rate of translocation of DNA from each side of a bound enzyme is about 200 base pairs per second, and the cuts are completed within 15-25 sec of the time neighboring enzymes meet . The resulting DNA fragments each contain a single recognition site with an enzyme (or subunit) remaining bound to it . At high enzyme concentrations, such fragments can be further degraded, apparently by cooperation between the specifically bound and excess enzymes . This model is consistent with a substantial body of previous work on the nuclease activity of EcoB and EcoK, and it explains in a simple way how cleavage sites are selected. J Bacteriol, 1988 Jul, 170(7), 3281 - 2 Regulation of the Escherichia coli secA gene by protein secretion defects: analysis of secA, secB, secD, and secY mutants; Rollo EE et al.; SecA protein synthesis levels were elevated 10- to 20-fold when protein secretion was blocked in secA, secD, and secY mutants or in a malE-lacZ fusion-containing strain but not in a secB null mutant . An active secB gene product was not required to derepress secA, since SecA levels were elevated during protein export blocks in secB secY and secB malE-lacZ double mutants. J Bacteriol, 1988 Jul, 170(7), 3262 - 8 Promoter of the Mycoplasma pneumoniae rRNA operon; Hyman HC et al.; RNA transcripts starting from the 5' end of the single Mycoplasma pneumoniae rRNA operon were analyzed by several methods . By primer extension analysis a start site was found 62 nucleotides upstream from the start site of the 16S rRNA . This site was preceded by a putative Pribnow box; however, a defined -35 recognition region was absent . The cloned rRNA operon was transcribed in vitro by using purified RNA polymerase of Escherichia coli . A single start site could be demonstrated within a few nucleotides of the start site found by primer extension analysis of M . pneumoniae transcripts . When fragments from the cloned operon were used as hybridization probes, S1 nuclease mapping yielded a single transcript extending approximately 193 nucleotides upstream from the 16S rRNA start site . The region surrounding this endpoint did not resemble any known promoter sequence . Dot blot hybridization of M . pneumoniae RNA to three oligonucleotides consisting of nucleotides -5 to -21, -38 to -54, and -112 to -132 (from the start of the 16S rRNA gene) indicated that most rRNA transcripts were processed at the stem site preceding the 16S rRNA gene . The majority of the longer precursor transcripts, extending beyond this point, did not extend further upstream to an oligonucleotide consisting of nucleotides -112 to -132 . It was concluded that transcription of the rRNA operon of M . pneumoniae is initiated by a single promoter . The nucleotide sequence of the region is presented. J Bacteriol, 1988 Jul, 170(7), 3094 - 101 Minicell-forming mutants of Escherichia coli: production of minicells and anucleate rods; Jaffe A et al.; The Escherichia coli minB mutant originally isolated is known to septate at cell poles to form spherical anucleate minicells . Three new minicell-producing mutants were isolated during a screening by autoradiography for chromosome partition mutants giving rise spontaneously to normal-sized anucleate cells . These min mutants were affected close to or in the minB locus . Autoradiography analysis as well as fluorescent staining of DNA showed that in addition to minicells, these strains and the original minB mutant also spontaneously produced anucleate rods of normal size and had an abnormal DNA distribution in filaments . These aberrations were not associated with spontaneous induction of the SOS response . Inhibition of DNA synthesis in these mutants gave rise to anucleate cells whose size was longer than unit cell length, suggesting that the min defect allows septation to take place at normally forbidden sites not only at cell poles but also far from poles . Abnormal DNA distribution and production of anucleate rods suggest that the Min product(s) could be involved in DNA distribution. J Bacteriol, 1988 Jul, 170(7), 3040 - 5 Construction of a dihydrofolate reductase-deficient mutant of Escherichia coli by gene replacement; Howell EE et al.; The dihydrofolate reductase (fol) gene in Escherichia coli has been deleted and replaced by a selectable marker . Verification of the delta fol::kan strain has been accomplished using genetic and biochemical criteria, including Southern analysis of the chromosomal DNA . The delta fol::kan mutation is stable in E . coli K549 {thyA polA12 (Ts)} and can be successfully transduced to other E . coli strains providing they have mutations in their thymidylate synthetase (thyA) genes . A preliminary investigation of the relationship between fol and thyA gene expression suggests that a Fol- cell (i.e., a dihydrofolate reductase deficiency phenotype) is not viable unless thymidylate synthetase activity is concurrently eliminated . This observation indicates that either the nonproductive accumulation of dihydrofolate or the depletion of tetrahydrofolate cofactor pools is lethal in a Fol- ThyA+ strain . Strains containing the thyA delta fol::kan lesions require the presence of Fol end products for growth, and these lesions typically increase the doubling time of the strain by a factor of 2.5 in rich medium. J Virol, 1988 Jul, 62(7), 2474 - 82 The product of the bovine papillomavirus type 1 modulator gene (M) is a phosphoprotein; Thorner L et al.; The M gene of bovine papillomavirus type 1 has been genetically defined as encoding a trans-acting product which negatively regulates bovine papillomavirus type 1 replication and is important for establishment of stable plasmids in transformed cells . The gene for this regulatory protein has been mapped in part to the 5' portion of the largest open reading frame (E1) in the virus . We constructed a trpE-E1 fusion gene and expressed this gene in Escherichia coli . Rabbits were immunized with purified fusion protein, and antisera directed against the product were used to identify the M gene product in virus-transformed cells . In this way a polypeptide with an apparent molecular mass of 23 kilodaltons was detected . The virus-encoded product is phosphorylated and can be readily detected by immunoprecipitation assays from cells transformed by the virus . Cells that harbor viral DNA without M as integrated copies do not produce this protein, whereas cells that harbor integrated viral genomes which are defective for another E1 viral gene important for plasmid replication, R, do produce this protein . The protein has an anomalously low electrophoretic mobility . An in vitro translation product of an SP6 RNA product of a sequenced cDNA predicts a molecular mass of 16 kilodaltons for the protein, and this in vitro translation product has an electrophoretic mobility identical to that of the in vivo immunoprecipitated protein . The results of these studies confirm our previous genetic studies which indicated that part of the E1 open reading frame defined a discrete gene product distinct from other putative products which may be encoded by this open reading frame. J Virol, 1988 Jul, 62(7), 2358 - 65 Properties of avian sarcoma-leukosis virus pp32-related pol-endonucleases produced in Escherichia coli; Terry R et al.; The gag-pol precursor protein of the avian sarcoma-leukosis virus is processed into three known pol-encoded mature polypeptides; the 95- and 63-kilodalton (kDa) beta and alpha subunits, respectively, of reverse transcriptase and the 32-kDa pp32 protein . The pp32 protein possesses DNA endonuclease activity and is produced from the precursor by two proteolytic cleavage events, one of which removes 4.1 kDa of protein from the C terminus . A 36-kDa protein (p36pol) which retains this C-terminal segment is detectable in small quantities in virions . We have constructed Escherichia coli plasmid clones that express the C-terminal domains of pol corresponding to pp32 and p36 . These proteins have been purified by column chromatographic methods to near homogeneity . No significant differences could be detected in the enzymatic properties of the bacterially produced p32pol and p36pol proteins . Both possess DNA endonuclease activity and, like the pp32 protein isolated from virions, can cleave near the junction of two tandem avian sarcoma-leukosis virus long terminal repeats in double-stranded supercoiled DNA substrates . In the presence of Mg2+, both p32pol and viral pp32 cleave either strand of DNA 2 nucleotides 5' to the junction. J Virol, 1988 Jul, 62(7), 2243 - 50 Identification and procaryotic expression of the gene coding for the highly immunogenic 28-kilodalton structural phosphoprotein (pp28) of human cytomegalovirus; Meyer H et al.; Human cytomegalovirus contains a structural polypeptide that is 28 kilodaltons in apparent molecular size and is reactive in Western blot (immunoblot) analysis with the majority of human sera . The gene coding for this polypeptide was mapped on the genome of human cytomegalovirus strain AD169 . A monoclonal antibody specific for the 28-kilodalton polypeptide was used to screen a cDNA library constructed from poly(A)+ RNA of human cytomegalovirus-infected cells in the procaryotic expression vector lambda gt11 . Hybridization of cDNA with cosmid and plasmid clones mapped the gene to the HindIII R fragment . The gene was transcribed into a late 1.3-kilobase RNA . The nucleotide sequence of the coding region was determined . Parts of the 28-kilodalton polypeptide were expressed in Escherichia coli as hybrid proteins fused to beta-galactosidase . In Western blots these proteins were recognized by human sera . Antibodies raised against the hybrid proteins reacted specifically with the viral antigen in immunoprecipitations and Western blots . In vitro phosphorylation of HCMV virions and immunoprecipitation showed that the 28-kilodalton polypeptide was phosphorylated. FEMS Microbiol Immunol, 1988 Jul, 1(2), 109 - 14 A new method for induction of specific polyclonal antibodies using immunostained proteins on nitrocellulose membrane, and HIV 1 core antigen and Escherichia coli verotoxin as examples; Sandkamp O et al.; Two polyclonal antibodies against verotoxin 1 and the core protein p24 of HIV 1 were raised in mice by a new immunization procedure . Both proteins were transferred to nitrocellulose, reacted with polyspecific antisera and the antigen-antibody complexes were then visualized by immunostaining . For preparation of antisera the stained protein bands were cut from the nitrocellulose sheets and implanted subcutaneously into the backs of BALB/c mice, without any adjuvant . A single booster was given 4 weeks later by implanting a second strip . All mice produced high titers of antibody directed against the antigen used for immunization . Thus, antibodies of high specificity can be elicited against protein bands in stained immunoblots. J Gen Microbiol, 1988 Jul, 134 ( Pt 7), 1795 - 1805 Stability of plasmid sequences in an acute Q-fever strain of Coxiella burnetii; Samuel JE et al.; The rickettsial pathogen Coxiella burnetii undergoes a variation in which virulent isolates (phase 1) become avirulent (phase 2) after repeated passage in a non-immunologically competent host . Biochemically, this variation is associated with a lipopolysaccharide modification and possibly other factors . Genetically, the regions of DNA responsible for phase variation have not been identified . We have sought to determine whether the plasmid identified in acute disease isolates, QpH1, which represents approximately 5% of the coding capacity of this organism is involved in phase variation . Plasmids from phase 1 and phase 2 variants (designated QpH1 and QpH2, respectively) were compared by restriction endonuclease digestion and Southern blot hybridization to determine whether sequence changes in the phase 2 plasmid might account for changes in the virulence of phase 2 organisms compared with that of phase 1 cells . Using over 20 different restriction enzymes, no changes in DNA restriction fragment patterns were detected regardless of whether the phase change occurred during egg or tissue culture passage . The plasmid-specific mRNAs produced from metabolically active, purified cells were identical for each phase type . Using QpH1 or QpH2 DNA as a template, the mRNA produced by an E . coli extract was also identical . Finally, the proteins encoded by either plasmid in an in vitro transcription/translation reaction were identical . These data indicate that within the limits of our analysis, the plasmid DNA from C . burnetii phase variants is structurally and functionally the same and is therefore unlikely to be involved in phase variation. J Gen Microbiol, 1988 Jul, 134 ( Pt 7), 1747 - 53 Serotype-specific monoclonal antibodies against the H12 flagellar antigen of Escherichia coli; Whitfield C et al.; The flagellar filaments of morphotype E isolates of Escherichia coli characteristically possess an apparent helically arranged sheath structure, surrounding the central core of the filament . Re-examination of the type strains of H-serotypes belonging to morphotype E showed that all but serotype H34 possessed the expected morphology . Heterogeneity was observed in both the diameter of filaments from individual morphotype E strains and in the Mr of individual flagellins . There was no apparent correlation between these two features . Monoclonal antibodies (MAbs) of the IgM class were raised against serotype H12 flagella . In Western immunoblotting and agglutination tests, the MAbs recognized the H12 antigen of six isolates with different O:K antigen combinations . The MAbs were H-serotype-specific, with no significant reaction with the H-antigens of other morphotype E strains . The location of the serotype-specific H12 epitope(s) was studied by immunolabelling with colloidal gold markers . The epitope was surface-exposed and appeared to be helically arranged on the flagellar filament . The pattern of colloidal gold labelling was consistent with the possibility that the H12 serotype-specific epitope resides in the apparent sheath structure. J Chromatogr, 1988 Jul 1, 444, 47 - 65 High-resolution chromatography of nucleic acids on the Gen-Pak FAX column; Stowers DJ et al.; High-performance liquid chromatography (HPLC) on a Gen-Pak FAX column has been used to separate and purify microgram amounts of single- and double-stranded DNA and RNA molecules . HPLC of mixtures of DNA restriction fragments showed that fragments within the size range 0.125-23.1 kilobase were easily resolved . Supercoiled (form I) plasmid DNA molecules were readily separated from single-stranded circular DNA of the same length and from various DNA conformational isomers including nicked (form II) and linear (form III) species . Topological isomers generated from supercoiled plasmid DNA molecules by DNA topoisomerase I exhibited different retention times than supercoiled molecules . Supercoiled (form I) DNA molecules were resolved from fully relaxed (form IV) molecules . Synthetic oligonucleotides of 74 and 128 nucleotides in length were separated from failure sequences, as well as from other contaminating synthesis products . Single-stranded circular M13mp18 DNA molecules sufficiently pure for use in automated DNA sequencing systems were prepared by HPLC on a Gen-Pak FAX column . HPLC was also used to fractionate linear double-stranded porcine rotavirus genomic RNA fragments into size classes between 0.3 and 3 kilobase . Finally, HPLC of unfractionated Escherichia coli tRNA molecules resolved multiple species . In all cases, HPLC on Gen-Pak FAX was carried out in phosphate or Tris buffers at neutral pH in the presence of sodium chloride . Columns were not damaged by repeated exposure to impure samples, provided they were cleaned frequently with sodium hydroxide and acetic acid . Although procedures for resolution of the various size ranges for each class of DNA and RNA molecules require further optimization, our preliminary data on the separations obtained, the moderate salt concentrations employed, and the durability of the matrix suggest that this column merits further study. Zentralbl Bakteriol Mikrobiol Hyg {A}, 1988 Jul, 269(1), 7 - 14 Modes of action of colicins E4-E7: rates of basic biosyntheses inhibition; Smarda J et al.; The progress of the inhibition of prominent biopolymer syntheses (namely of DNA, RNA and protein synthesis) in cultures of susceptible bacteria Escherichia coli treated with colicins E4, E5, E6 or E7 was followed . The method used was the measurement of incorporation of specific radioactive precursors into an instantly precipitable fraction . All the three syntheses being inhibited successively, progressively and with different urgency during unfolding of the lethal effect of each colicin, emphasis was put on a comparative mathematical analysis of the development of the biosyntheses inhibition rates . In bacteria treated with colicins E4, E5 or E6, protein synthesis was blocked preferentially and most heavily, followed by cessation of DNA synthesis and finally also by a rather slowly proceeding impairment of RNA synthesis . (In E6-treated cells, damage to DNA synthesis started prior to that to protein synthesis.) Thus, the effect of colicins E4, E5 and E6 follows the general pattern of colicin E3 action . Bacteria treated with colicin E7 developed an immediate block of DNA synthesis, soon followed by protein and (again after a significant delay) by RNA synthesis switch-off . Thus the action of colicin E7 is closely related to that of colicin E2 . The presumptive direct targets of these colicins remain to be elucidated. Cytometry, 1988 Jul, 9(4), 316 - 24 Phagocytosis, intracellular pH, and cell volume in the multifunctional analysis of granulocytes by flow cytometry; Rothe G et al.; Phagocytosis of Escherichia coli K12 strain bacteria was used to measure by flow cytometry the functional activities of human granulocytes in whole blood or buffy coat preparations . In a first measurement, the increase in electric cell volume and acridine orange (AO) green and red fluorescence were used to quantify the degree of phagocytosis . In a second measurement, the intracellular pH and esterase activity of each cell were determined with 1,4-diacetoxy-2,3-dicyanobenzene to obtain information on the metabolic activities during phagocytosis and degradation of bacteria . The DNA of dead cells was simultaneously counterstained with propidium iodide in both assays . The volume, the AO green and red fluorescence, the internal pH, and esterase activity were automatically averaged for all granulocytes or lymphocytes of a measurement . The calculated mean values were transferred into the self-learning database of the DIAGNOS1-program system . The functional granulocyte parameters of normal healthy individuals can be used as reference values for the automated diagnosis of abnormal granulocytes in various infectious disease states . The assays require 1 ml of heparinized whole blood and the results are available within 1 hour. Mol Biochem Parasitol, 1988 Jul, 30(1), 19 - 26 Schistosoma mansoni: localisation of antigenic regions on the 31 kilodalton diagnostic protein; Felleisen R et al.; Antigenic sites on the 31 kDa diagnostic protein of Schistosoma mansoni (Sm31) were identified using the cDNA fragment H3 cloned in the expression vector pEx34b . This fragment encodes approximately two-thirds of the polypeptide . A set of deletion mutants was generated by the exonuclease Bal31 and the resulting shortened proteins synthesised in Escherichia coli as fusions to MS2 polymerase were tested in Western blots with human schistosomiasis patient sera . Three antigenic regions were identified, one of which was narrowed down by appropriate restriction sites to a sequence specifying only 27 amino acid residues . Examination of a longer MS2 fusion product extending into the N-terminus of the protein, corresponding to the nearly full length Sm31 sequence, revealed that its reactivity in immunoblots is comparable with that of the H3 clone . This suggests that additional antigenic sites, which might be more reactive than those already identified, are absent from the remaining part of the protein. Eur J Biochem, 1988 Jul 1, 174(4), 585 - 92 Cloning and expression of cDNA for human vascular anticoagulant, a Ca2+-dependent phospholipid-binding protein; Maurer-Fogy I et al.; Based on sequence information from tryptic peptides an almost full-size cDNA coding for the human vascular anticoagulant was isolated from a placental cDNA library and sequenced . The coding region was cloned into an Escherichia coli expression vector and the protein expressed at high levels . The recombinant protein was purified and found to be indistinguishable from its natural counterpart in several biological assays. Science, 1988 Jul 1, 241(4861), 74 - 9 Aminoacylation of synthetic DNAs corresponding to Escherichia coli phenylalanine and lysine tRNAs; Khan AS et al.; Synthetic DNA oligomers (tDNAs) corresponding to Escherichia coli tRNA(Phe) or tRNA(Lys) have been synthesized with either deoxythymidine (dT) or deoxyuridine (dU) substituted in the positions occupied by ribouridine or its derivatives . The tDNAs inhibited the aminoacylation of their respective tRNAs with their cognate amino acids, but not the aminoacylation of tRNA(Leu) with Leu . In the presence of aminoacyl-tRNA synthetase, species of both a tDNA(Phe) synthesized with a 3' terminal riboadenosine and a tDNA(Lys) containing only deoxynucleotides could be aminoacylated with the appropriate amino acids, although the Michaelis constant Km and observed maximal rate Vmax values for aminoacylation were increased by three- to fourfold and decreased by two- to threefold, respectively . The aminoacylation of synthetic tDNAs demonstrates that the ribose backbone of a tRNA is not absolutely required for tRNA aminoacylation. Proc Natl Acad Sci U S A, 1988 Jul, 85(13), 4710 - 4 RNase PH: an Escherichia coli phosphate-dependent nuclease distinct from polynucleotide phosphorylase; Deutscher MP et al.; Final trimming of the 3' terminus of tRNA precursors in Escherichia coli is thought to proceed by an exonucleolytic mechanism . However, mutant strains lacking as many as four exoribonucleases known to act on tRNA still grow normally and process tRNA normally . Extracts from such a multiple-RNase-deficient strain accurately mature tRNA precursors exonucleolytically in vitro in a reaction that requires inorganic phosphate . Here we show that this reaction is not due to polynucleotide phosphorylase (PNPase) but, rather, that it is mediated by a phosphate-requiring exonuclease that we have named RNase PH . Purified PNPase is incapable of completely processing tRNA precursors, and extracts from a PNPase- strain retain full activity for phosphorolytic processing . Although both PNPase and RNase PH act in a phosphorolytic manner, they differ substantially in size and substrate specificity . RNase PH has a molecular mass of 45-50 kDa and favors tRNA precursors as substrates . The possible physiological role of RNase PH and the advantages of phosphorolytic processing are discussed. Proc Natl Acad Sci U S A, 1988 Jul, 85(13), 4667 - 71 Identification of a region in the S1 subunit of pertussis toxin that is required for enzymatic activity and that contributes to the formation of a neutralizing antigenic determinant; Cieplak W et al.; The S1 subunit of pertussis toxin possesses two regions (homology boxes), each spanning 8 residues, that are nearly identical in sequence to similarly located regions in the enzymatically active A fragments of two other ADP-ribosylating toxins: cholera toxin and Escherichia coli heat-labile toxin . This observation suggests a functional role for one or both of these regions in enzymatic activity . We have examined the role of one of these regions, located near the amino terminus of the S1 subunit, by using a high-level recombinant expression system and progressive truncation of the gene sequence encoding the amino terminus of the molecule . A series of six truncated, recombinant proteins were produced at high levels in E . coli and examined for their enzymatic and antigenic properties . The three molecules that lacked most or all of the homology box delimited by amino acid residues 8 and 15 lacked detectable enzymatic activity . All of the three molecules in which the box was retained exhibited detectable activity . Only those recombinant molecules that possessed the homology box reacted with a neutralizing and passively protective monoclonal anti-S1 antibody . These findings identify the region of homology located near the amino terminus of S1 as an apparent enzymatic subsite and a potentially important antigenic determinant. J Bacteriol, 1988 Jul, 170(7), 3110 - 4 Cyclic AMP-cyclic AMP receptor protein as a repressor of transcription of the spf gene of Escherichia coli; Polayes DA et al.; The spf gene of Escherichia coli encodes an unstable 109-nucleotide RNA, spot 42 RNA; the level of this RNA was reduced three- to fivefold when cells were grown in the presence of 3',5'-cyclic AMP (cAMP) . We show that this regulation occurs through reduction in transcription and depends on both cAMP and the cAMP receptor protein (CRP) but is independent of the de novo protein synthesis . Through deletion analysis of the spf gene promoter, we have identified sequences that are important in the synthesis of spot 42 RNA . Deletion of sequences upstream of -77 completely eliminated the negative control of cAMP-CRP and resulted in high constitutive levels of transcription . This region contained a sequence that both conformed to the consensus binding site for cAMP-CRP in positively regulated promoters and acted as a cAMP-CRP binding site in a gel retardation assay . Deletion of sequences between positions -77 and -60 greatly reduced the level of transcription in the presence or absence of cAMP-CRP, indicating that at least part of this region is a binding site for a positive-acting transcription factor (or RNA polymerase itself) . We propose that the proximity of the two sites defined here allows for the negative control of spf gene transcription by cAMP-CRP . In particular, if only one site at a time can be occupied, the binding of cAMP-CRP would interfere with the binding of a transcription factor. J Virol, 1988 Jul, 62(7), 2525 - 9 A single 66-kilodalton polypeptide processed from the human immunodeficiency virus type 2 pol polyprotein in Escherichia coli displays reverse transcriptase activity; Le Grice SF et al.; We have cloned the entire pol gene of human immunodeficiency virus type 2 into a high-level Escherichia coli expression system . Induction of cultures containing the recombinant plasmid, p2RTL1, leads to rapid accumulation of polypeptides of 66, 54, and 34 kilodaltons . We have designated the larger polypeptides reverse transcriptase, and we have designated the smaller polypeptide endonuclease . Purification of reverse transcriptase via ion-exchange and affinity chromatography yields the 66-kilodalton polypeptide, with which reverse transcriptase activity is associated . Purified enzyme furthermore displays a higher apparent molecular weight than its counterpart from human immunodeficiency virus type 1. Nature, 1988 Jun 30, 333(6176), 869 - 71 The structure of trp pseudorepressor at 1.65A shows why indole propionate acts as a trp 'inducer'; Lawson CL et al.; The trp repressor is a small dimeric regulatory protein which controls the expression of three operons in Escherichia coli . The inactive aporepressor protein must bind two molecules of L-tryptophan to form the active repressor . If desamino analogues of L-tryptophan such as indole propionate (IPA) are substituted for L-tryptophan, an inactive pseudorepressor is formed . Because the desamino analogues thus cause derepression of operons under control of the trp repressor, they appear to be 'inducers' . We have determined the crystal structure of the pseudorepressor and refined it to 1.65 A . The molecular structure was compared to that of the nearly isomorphous orthorhombic form of the repressor . Surprisingly, the indole ring of IPA is in the same position as the indole ring of L-tryptophan in the repressor, but is 'flipped over' . As a result, the carboxyl group of IPA is oriented toward the DNA-binding surface of the protein and is in a position where it sterically and electrostatically repels the phosphate backbone of both operator and non-operator DNA . This explains why IPA acts as an apparent trp inducer. Gene, 1988 Jun 30, 66(2), 295 - 300 Expression and excretion of human pancreatic secretory trypsin inhibitor in lipoprotein-deletion mutant of Escherichia coli; Kanamori T et al.; We constructed a gene coding for the 56-amino acid human pancreatic secretory trypsin inhibitor (PSTI), and ligated it on a plasmid downstream from the trp promoter and the signal peptide sequence of alkaline phosphatase . The resulting plasmid was transfected into a lipoprotein deletion mutant (Escherichia coli JE5505) and the plasmid-carrying cells were induced with 3-indoleacrylic acid . A considerable amount (50 micrograms/ml culture) of the mature PSTI protein was detected in the culture supernatant . The excreted PSTI was identical to the natural PSTI protein with respect to the trypsin-inhibiting activity, the N-terminal and the C-terminal amino acid sequences and the amino acid composition. Gene, 1988 Jun 30, 66(2), 269 - 78 Highly efficient positive selection of recombinant plasmids using a novel rglB-based Escherichia coli K-12 vector system; Noyer-Weidner M et al.; We have developed pBR328-derived vectors which allow highly efficient positive selection of recombinant plasmids . The system is based on the rglB-coded restriction activity of Escherichia coli K-12 directed against 5-methylcytosine (5mC)-containing DNA . The vectors code for cytosine-specific, temperature-sensitive DNA methyltransferases (ts-Mtases), whose specificity elicits RglB restriction . 5mC-free vector DNA - a prerequisite to allow establishment of such plasmids in cells expressing the RglB nuclease activity - can be prepared from cultures grown at 42 degrees C . At 30 degrees C the vector plasmids are vulnerable to RglB restriction due to the expression of suicidal Mtase activity . Cloning a DNA fragment into the ts-Mtase-coding gene disrupts the lethal methylation and thus permits selection of such recombinant plasmids at 30 degrees C . The standard vector used, pBN73, contains unique recognition sites for nine restriction enzymes within the ts-Mtase-coding gene, which can be used independently or in combination for the construction of recombinant plasmids selectable by the rglB-coded activity . Plasmid pBN74, which carries the determinants for both the ts-Mtase and the RglB nuclease, contains seven unique sites within the ts-Mtase-coding gene . While selection of recombinant plasmids derived from pBN73 obligatorily requires the employment of rglB+ strains, selection of pBN74 derivatives can be performed independent of the E . coli-host genotype . It remains to be elucidated whether positive selection of pBN74-derived recombinant plasmids can also be achieved in hosts other than E . coli . Plasmids pBN73, pBN74 and the recombinants are structurally stable . Generally applicable procedures, as developed during the establishment of this vector system, are described; they allow the isolation of ts-Mtases and facilitate the cloning of genes coding for nucleases directed against 5mC-containing DNA. Gene, 1988 Jun 30, 66(2), 259 - 68 Nucleotide sequence and transcriptional analysis of a third function (Flm) involved in F-plasmid maintenance; Loh SM et al.; The leading region of the conjugative F plasmid encodes for a function, Flm, capable of extending the maintenance of normally unstable plasmids . Nucleotide sequencing and functional studies of flm locus have shown that it consists of at least two genes, flmA and flmB, which are physically and functionally homologous to hok and sok of parB in plasmid R1 . The 52-amino acid flmA-coded polypeptide is almost identical to the hok product which has been shown to be a membrane-associated lethal protein {Gerdes et al., EMBO J . 5 (1986) 2023-2029} . Gene flmB codes for a 100 nucleotide, non-translated, complementary RNA which overlaps the 5' leader sequence of the flmA RNA . The flmA RNA also encodes an open reading frame (ORF70) which overlaps the flmA-coding sequence and may be a third gene involved in the Flm function . S1 analysis and functional studies suggest that the antisense flmB RNA binds to the flmA RNA and suppresses the expression of the lethal product, presumably by blocking coupled translation of ORF70 and flmA . Secondary structure analysis predicts that the flmA RNA is extremely stable compared to the regulatory flmB RNA . We suggest that when these RNA species are retained by cells which have lost the F plasmid, the more stable flmA RNA will eventually be translated thus leading to cell death . This phenomenon provides a third mechanism, additional to ParFIA and Ccd functions, to ensure maintenance of the F plasmid in a growing bacterial population. Gene, 1988 Jun 30, 66(2), 245 - 58 The nucleotide sequence of a plasmid determinant for resistance to tellurium anions; Jobling MG et al.; The plasmid pMJ606 contains a 5-kb insert specifying resistance to tellurium salts (TeR) which was cloned from the large conjugative plasmid pMER610 . Nucleotide sequence analysis of this insert has identified five open reading frames (ORFs) . ORFs 1, 2, 4 and 5 correspond in terms of their predicted polypeptide products to the 41, 15.5, 22 and 23-kDa polypeptides, respectively, which are synthesised by the resistance determinant in maxicells . An additional ORF, ORF3, whose product has not been identified is predicted by the sequence data . The sequence of the presumptive polypeptide specified by ORF3 indicates a membrane location . The nucleotide and predicted amino acid sequences of ORF4 and ORF5 show considerable homology which is consistent with the duplication and minor divergence of an ancestral gene . ORFs 4 and 5 appear to retain the same function and while each can function separately and contribute to the resistance mechanism, the full level of wild-type resistance requires both genes to function . The transcriptional signals for the TeR determinant may be located beyond and 5' of the sequences cloned in pMJ606. Gene, 1988 Jun 30, 66(2), 279 - 94 Total chemical synthesis of a gene for hepatitis B virus core protein and its functional characterization; Nassal M; We have chemically synthesized a DNA duplex of 560 nucleotides that codes for the hepatitis B virus (HBV) core protein . The synthetic gene contains 27 unique internal restriction sites . Thereby, it can easily be mutagenized by replacement of rather short restriction fragments . A number of restriction recognition sequences are in common between the synthetic and the authentic gene, thus allowing for the transfer of synthetic segments into the cloned viral genome . Several unexpected mutations in the synthetic gene were readily corrected utilizing the multiple unique restriction sites . In Escherichia coli, the expression level of the synthetic gene product amounts to about 4% of the total soluble protein . It forms particles closely resembling native HBV cores . After transfer of the synthetic gene into the viral genome, transient expression in a hepatoma cell line yields proteins indistinguishable from the native gene products . The synthetic gene thus provides a useful tool for studies on the structure and function of the isolated HBV core protein as well as the gene and its various products in the viral life-cycle. Gene, 1988 Jun 30, 66(2), 163 - 81 High-level transient expression of influenza virus proteins from a series of SV40 late and early replacement vectors; Huylebroeck D et al.; We have constructed a collection of simian virus 40 (SV40) plasmid vectors useful for transient or constitutive expression of cDNA or genomic DNA in animal cells . Most vectors contain several unique restriction sites downstream from the SV40 late or early promoter, and are available with or without the virus-specific splicing signals . The use of these vectors for transient expression in monkey cells of X47 (H3N2) influenza hemagglutinin (HA) and matrix protein (M1) was demonstrated . Membrane-bound (HAm) as well as secreted forms of the HA glycoprotein lacking the sequence of the C-terminal anchor (HA-) have been obtained . Depending on the insert, the type of vector and the amount of transfected DNA, HA levels in COS cells {Gething and Sambrook, Nature 293 (1981) 620-625} transfected with late replacement SV40 vectors vary from 10(9) (HAm) to 10(8) (HA-) molecules per transfected cell . The maximum expression levels with early replacement vectors in COS cells are at least 50 times lower . In addition to the optimalization and the characterization of the expression of each vector-coded influenza protein, cotransfections, including vectors expressing HAm, neuraminidase (NA) and M1, were undertaken . The latter experiments did not result in a measureable amount of HAm or NA in the cell culture medium, suggesting that expression of these three structural viral proteins does not result in budding of (empty) influenza particles from the cell surface. Biochem Biophys Res Commun, 1988 Jun 30, 153(3), 1244 - 50 Site-directed mutagenesis of the cAMP-binding sites of the recombinant type I regulatory subunit of cAMP-dependent protein kinase; Kuno T et al.; The type I regulatory subunit (R-I) of rat brain cAMP-dependent protein kinase was expressed in E . coli and site-directed mutagenesis was used to substitute amino acids in the putative cAMP-binding sites . The wild-type recombinant R-I bound 2 mol of cAMP/mol subunit, while two mutant R-Is with a single amino acid substitution in one of the two intrachain cAMP-binding sites (clone N153:a glutamate for Gly-200, and clone C254:an aspartate for Gly-324) bound 1 mol of cAMP/mol subunit . When these two substitutions were made in one mutant, cAMP did not bind to this mutant, indicating that binding of cAMP to N153 or C254 was to their nonmutated sites . Competition experiments with site-selective analogs and dissociation of bound cAMP from mutant R-Is provided evidence for strong intrachain interactions between the two classes of cAMP-binding sites in R-I. Nature, 1988 Jun 30, 333(6176), 824 - 9 DNA sequence determinants of CAP-induced bending and protein binding affinity; Gartenberg MR et al.; The sites of DNA bending induced by binding catabolite activator protein are identified and shown to coincide with positions where DNA grooves face the protein . The bendability of DNA with different sequences at these bend centres parallels the bending preference of the sequences in nucleosomal DNA . Anisotropic DNA bendability significantly affects the structure and strength of regulatory protein-DNA complexes. Gene, 1988 Jun 30, 66(2), 319 - 23 Expression in Escherichia coli of a Moloney murine leukemia virus reverse transcriptase whose structure closely resembles the viral enzyme; Hizi A et al.; We have constructed an expression plasmid containing the portion of the Moloney murine leukemia virus genome encoding the reverse transcriptase (RT) . When introduced into Escherichia coli this plasmid induces the synthesis of a 70-kDa protein . The RT made in E . coli differs from the viral protein only in that there are two new amino acids, methionine and glycine, substituted for the threonine found at the N terminus of the viral enzyme . Approximately half of the E . coli synthesized RT enzyme is soluble in cell extracts . This protein is active in an RT assay, and like the enzyme purified from virions, is more active in the presence of Mn2+ than Mg2+ . We have also constructed a plasmid that induces the synthesis of an RT-integration protein fusion. J Chromatogr, 1988 Jun 29, 443, 381 - 97 Separation of proteins by reversed-phase high-performance liquid chromatography . II . Optimizing sample pretreatment and mobile phase conditions; Nugent KD et al.; The effects of separation variables such as temperature, pH and composition of the mobile phase (including additives such as chaotropes, ion-pairing agents and surfactants), sample size and sample pretreatment for reversed-phase high-performance liquid chromatography (RP-HPLC) of proteins is examined . Experimental optimization of these parameters using the preferred instrumental and column conditions described previously lead to well behaved chromatographic performance for most proteins . This allowed us to achieve the required level of performance for the first dimension (RP-HPLC) separation of most protein samples by the chromatophoresis process. J Immunol Methods, 1988 Jun 28, 111(1), 1 - 9 Erythropoietin-beta-D-galactosidase . The generation, purification and use of a fusion protein; Nielsen OJ et al.; A human erythropoietin (Epo) cDNA fragment encoding the complete erythropoietin peptide sequence was fused to the 3'-end of the lacZ gene in the polylinker region of the high expression vector, pUR 278 . Escherichia coli bacteria were transformed with the recombinant plasmid harboring the hybrid Epo-beta-D-galactosidase gene . After induction with isopropyl-thiogalactoside large amounts of the fusion protein, Epo-beta-D-galactosidase were synthesized in the transformed bacteria . The fusion protein was partially purified and shown to exhibit intact galactosidase enzymatic activity . Although no biological activity of the Epo counterpart of the fusion protein was detected both in an in vivo and in an in vitro bioassay, the fusion protein served as an effective antigen for the production of anti-erythropoietin antibodies . Antifusion protein antibodies raised in rabbits were shown to react with the intact human Epo molecule from erythropoietin producing culture supernatants . The affinity of these anti-fusion protein antibodies was sufficiently high to permit the development of a sensitive radioimmunoassay for human Epo . This fusion protein approach is a relatively straightforward and rapid method of generating antibodies with specificity for any protein encoded by a cloned eukaryotic gene. Biochemistry, 1988 Jun 28, 27(13), 4952 - 6 Direct photoaffinity labeling of ribonucleotide reductase from Escherichia coli using dTTP: characterization of the photoproducts; Kierdaszuk B et al.; Subunit B1 of Escherichia coli ribonucleotide reductase contains one type of allosteric binding site that controls the substrate specificity of the enzyme . This site binds the allosteric effector dTTP as well as other nucleoside triphosphates . Cross-linking of dTTP to protein B1 by direct photoaffinity labeling, as well as the isolation and sequence determination of the labeled tryptic peptide, has recently been reported {Eriksson, S., Sjoberg, B.-M., Jornwall, H., & Carlquist, M . (1986) J . Biol . Chem . 261, 1878-1882} . In this study, we have further purified the dTTP-labeled peptide and characterized it using UV spectroscopy . Two types of dTTP-cross-linked peptide were found: one having an absorbance maximum at 261 nm typical for a dTTP spectrum, i.e., containing an intact 5,6 double bond, and one minor form with low absorbance at 261 nm . In both cases, the same amino acid composition was found, corresponding to the peptide Ser291-X-Ser-Gln-Gly-Gly-Val-Arg299 in the B1 sequence with X being Cys-292 cross-linked to dTTP . Isotope labeling experiments revealed that one proton in the 5-methyl group of thymine was lost during photoincorporation . Therefore, the cross-linking occurs via the 5-methyl group to Cys-292 in a majority of incorporated dTTPs, but a second, possibly 5,6-saturated form of incorporated nucleotide was also detected . The reasons for the high stereospecificity of the reaction and the possible structure of the allosteric site of protein B1 are discussed. Biochemistry, 1988 Jun 28, 27(13), 4800 - 4 Ligand-induced structural constraints in human dihydrofolate reductase revealed by peptide-specific antibodies; Ratnam M et al.; Peptides from human dihydrofolate reductase (DHFR) generated by cyanogen bromide cleavage and corresponding to residues 15-52, 53-111, 112-125, and 140-186 (carboxyl terminus) were purified and used to immunize rats . Titration of the immune sera against denatured human DHFR by solid-phase immunoassay showed that peptides 15-52 and 140-186 were relatively highly immunogenic, unlike the native enzyme which is most immunogenic in the sequence 53-111 . The antisera were specific for the corresponding peptides used for immunization . Antibodies to peptides 15-52, 53-111, and 140-186 cross-reacted with native human DHFR in solution in competition assays . However, the binding of nicotinamide adenine dinucleotide phosphate (reduced) (NADPH) and the inhibitors folate and methotrexate, both in binary and in ternary complexes with the enzyme, caused a striking reduction in binding of antibody . Using a sensitive radioactive assay, it was found that antisera to peptides 15-52 and 140-186, both of which exhibited a high antibody titer, caused significant inhibition of DHFR . Because peptide 140-186 does not include any active-site residues, it is concluded that at least in this case all the antibodies bound to regions outside the active site . Since comparison of the X-ray structures of the chicken liver DHFR holoenzyme with the apoenzyme reveals no changes in secondary structural elements (alpha-helices and beta-sheets), the reduction in antibody binding to DHFR-ligand complexes must not involve epitopes within these structures.(ABSTRACT TRUNCATED AT 250 WORDS) Biochemistry, 1988 Jun 28, 27(13), 4777 - 80 Stereochemistry of phospho group transfer catalyzed by a mutant alkaline phosphatase; Butler-Ransohoff JE et al.; The stereochemical course of the phospho group transfer catalyzed by mutant (S102C) alkaline phosphatase from Escherichia coli was investigated by using 31P nuclear magnetic resonance spectroscopy . Transphosphorylation from 4-nitrophenyl (Rp)-{16O, 17O, 18O}phosphate to (S)-propane-1,2-diol occurs with overall retention of configuration at phosphorus . This result is consistent with the view that the hydrolysis of substrates by this mutant enzyme proceeds by way of a covalent phosphoenzyme intermediate in the same manner as the wild-type alkaline phosphatase. Biochemistry, 1988 Jun 28, 27(13), 4735 - 40 Spectroscopic and hydrodynamic studies reveal structural differences in normal and transforming H-ras gene products; Pingoud A et al.; We have recorded the circular dichroism spectra of the cellular and the viral H-ras gene products both in the absence and in the presence of guanine nucleotides and analyzed these spectra in terms of the secondary structure composition of these proteins . It is shown that the GTP complex of the ras proteins has a different secondary structure composition than the GDP complex and, furthermore, that there are differences in the secondary structure of the viral ras protein and the cellular ras protein . We have also recorded and analyzed the circular dichroism spectrum of the isolated guanine nucleotide binding domain of the Escherichia coli elongation factor Tu (EF-Tu), which has been considered as a model for the tertiary structure of the ras proteins {McCormick, F., Clark, B . F . C., LaCour, T . F . M., Kjeldgaard, M., Norskov-Lauritsen, L., & Nyborg, J . (1985) Science (Washington, D.C.) 230, 78-82} . Our data show that the guanine nucleotide binding domain of EF-Tu (30% alpha-helix and 16% beta-pleated sheet for the GDP complex) has quite a different secondary structure composition than the ras proteins (e.g., the cellular ras protein has 47% alpha-helix and 22% beta-pleated sheet for the GDP complex), indicating that the protein core comprising the guanine nucleotide binding site might be similar but that major structural differences must exist at the portion outside this core . Normal and transforming ras proteins also differ slightly in their hydrodynamic properties as shown by sedimentation velocity runs in the analytical ultracentrifuge.(ABSTRACT TRUNCATED AT 250 WORDS) Biochemistry, 1988 Jun 28, 27(13), 4687 - 95 Complementary oligodeoxynucleotide probes of RNA conformation within the Escherichia coli small ribosomal subunit; Lasater LS et al.; The large RNA molecule within each ribosomal subunit is folded in a specific and compact form . The availability of specific 16S RNA sequences on the surface of the small ribosomal subunit has been probed by using complementary oligodeoxynucleotides . The hybridization of 8-15-nucleotide-long oligomers to their RNA complements within the subunit was quantitated by using a nitrocellulose membrane filter binding assay . The probes have been grouped into classes on the basis of sequence-specific binding ability under different conditions of ionic environment, incubation temperature, and subunit activation state {as defined by the ability to bind phenylalanyl-tRNA in response to a poly(uridylic acid) message} . Oligodeoxynucleotides complementary to nucleotides flanking 7-methylguanosine residue 527 and to the 3'-terminal sequence bound 30S subunits regardless of the activation state . Oligodeoxynucleotides that complement 16S ribosomal RNA residues 1-16, 60-70, 685-696, and 1330-1339 and the sequence adjacent to the colicin E3 cleavage site at residue 1502 all bound efficiently only to subunits in an inactivated conformation . Probes complementary to residues 1-11 and 446-455 bound only inactivated subunits, and then with low efficiency . Sequences complementary to nucleotides 6-16, 99-109, 1273-1281, and 1373-1383 bound 30S subunits poorly regardless of the activation state . With one exception, each probe was bound by native or heat-denatured 16S ribosomal RNA (as determined by size-exclusion chromatography) . We conclude that complementary oligodeoxynucleotide binding efficiency is a sensitive measure of the availability of specific RNA sequences under easily definable conditions. Biochemistry, 1988 Jun 28, 27(13), 4680 - 6 Isolation, nucleotide sequence, and expression of a cDNA encoding pig citrate synthase; Evans CT et al.; Citrate synthase is a key enzyme of the Krebs tricarboxylic acid cycle and catalyzes the stereospecific synthesis of citrate from acetyl coenzyme A and oxalacetate . The amino acid sequence and three-dimensional structure of pig citrate synthase dimers are known, and regions of the enzyme involved in substrate binding and catalysis have been identified . A cloned complementary DNA sequence encoding pig citrate synthase has been isolated from a pig kidney lambda gt11 cDNA library after screening with a synthetic oligonucleotide probe . The complete nucleotide sequence of the 1.5-kilobase cDNA was determined . The coding region consists of 1395 base pairs and confirms the amino acid sequence of purified pig citrate synthase . The derived amino acid sequence of pig citrate synthase predicts the presence of a 27 amino acid N-terminal leader peptide whose sequence is consistent with the sequences of other mitochondrial signal peptides . A conserved amino acid sequence in the mitochondrial leader peptides of pig citrate synthase and yeast mitochondrial citrate synthase was identified . To express the pig citrate synthase cDNA in Escherichia coli, we employed the inducible T7 RNA polymerase/promoter double plasmid expression vectors pGP1-2 and pT7-7 {Tabor, S., & Richardson, C . C . (1985) Proc . Natl . Acad . Sci . U.S.A . 82, 1074-1078} . The pig citrate synthase cDNA was modified to delete the N-terminal leader sequence; then by use of a synthetic oligonucleotide linker, the modified cDNA was cloned into pT7-7 immediately following the initiator Met . A glutamate-requiring (citrate synthase deficient), recA- E . coli mutant, DEK15, was transformed with pGP1-2 and then pT7-7PCS . pT7-7PCS complemented the E . coli gltA mutation.(ABSTRACT TRUNCATED AT 250 WORDS) Biochemistry, 1988 Jun 28, 27(13), 4712 - 20 Mutations in the nucleotide binding loop of adenylate kinase of Escherichia coli; Reinstein J et al.; The adk gene of Escherichia coli has been used to overexpress the adenylate kinase protein in two ways: (1) by cloning the adk gene with its own promoter into pEMBL plasmids, which have an increased copy number, and (2) by deleting the adk promoter and cloning the gene behind the regulatable tac promoter . Adenylate kinase comprises up to 40% of the soluble cellular extracts from E . coli strains containing these plasmids . Mutations have been introduced into the gene by site-directed mutagenesis to exchange amino acids in the nucleotide binding loop, which is highly conserved in many mononucleotide binding proteins . The mutation of Lys13----Gln is nearly inactive, whereas the Pro9----Leu and the Gly10----Val mutant proteins have an increased Km for both substrates and a Vmax that is similar to wild type . Proton NMR measurements of the proteins show that a major structural change seems to have taken place for the Pro9----Leu and Gly10----Val mutants . The results are discussed in the light of the kinetic mechanism for adenylate kinase and the three-dimensional structure of the protein. Biochemistry, 1988 Jun 28, 27(13), 4698 - 705 Functional consequences of the arabinosylcytosine structural lesion in DNA; Mikita T et al.; Cytosine arabinoside (araC) is a potent antileukemic agent that is misincorporated into DNA in the course of its action . We have developed a chemical synthetic method that allows site-specific introduction of araC into synthetic DNA oligomers . We describe here the utilization of these oligomers as primer/template substrates for in vitro DNA synthesis reactions and as fragments for DNA ligation . These studies were undertaken to investigate the manner in which sites of araC misincorporation constitute sites of DNA dysfunction . AraCMP at the primer terminus dramatically reduced the rate of next nucleotide addition for Escherichia coli polymerase I (Klenow fragment) (Pol I), T4 polymerase, HeLa cell polymerase alpha 2 (Pol alpha 2), and AMV reverse transcriptase . Polymerases with associated 3'-5' exonuclease activity preferentially excised araCMP from the primer terminus prior to chain elongation . AraCMP-terminated fragments were ligated more slowly than control fragments by T4 DNA ligase . AraCMP located at an internucleotide site in the template markedly slowed replicative bypass for Pol I, T4 polymerase, and Pol alpha 2, but not for reverse transcriptase . Synthesis was partially arrested after insertion of the correct nucleotide opposite the lesion site . These results suggest a complex mechanism for the inhibition of DNA replication by araC when it is misincorporated into DNA. J Biol Chem, 1988 Jun 25, 263(18), 9015 - 9 Sequence of thioredoxin reductase from Escherichia coli . Relationship to other flavoprotein disulfide oxidoreductases; Russel M et al.; The DNA sequence of the Escherichia coli gene encoding thioredoxin reductase has been determined . The predicted protein sequence agrees with an earlier determination of the 17 amino-terminal amino acids and with a fragment of the protein containing the redox-active half-cystines . Similarity between E . coli thioredoxin reductase and other flavoprotein disulfide oxidoreductases is quite limited, but three short segments, two of which are probably involved in FAD and NADPH binding, are highly conserved between thioredoxin reductase, glutathione reductase, dihydrolipoamide dehydrogenase, and mercuric reductase. J Biol Chem, 1988 Jun 25, 263(18), 8953 - 7 Studies on the mutator gene, mutT of Escherichia coli . Molecular cloning of the gene, purification of the gene product, and identification of a novel nucleoside triphosphatase; Bhatnagar SK et al.; The mutator gene, mutT, has been cloned into an expression vector and overproduced in Escherichia coli . The gene product has been purified to over 90% homogeneity as judged by gel electrophoresis and amino acid analysis . The amino acid composition of the protein and the sequence of the 20 amino acids of the N-terminal region agree well with the nucleotide sequence of the gene reported by Akiyama et al . (Akiyama, M., Horiuchi, T., and Sekiguchi, M . (1987) Mol . Gen . Genet . 206, 9-16) and indicate that the first of the potential initiation codons (position 164) of the open reading frame in the PvuII fragment carrying the mutT gene is the site of initiation of translation of the 15,000-Da polypeptide . A novel nucleoside triphosphatase activity which has a preference for dGTP is associated with the purified protein, and preliminary experiments are consistent with the notion that the mutT gene product is the enzyme responsible for this activity. J Biol Chem, 1988 Jun 25, 263(18), 8735 - 9 The RNA N-glycosidase activity of ricin A-chain . The characteristics of the enzymatic activity of ricin A-chain with ribosomes and with rRNA; Endo Y et al.; Ricin A-chain cleaves the N-glycosidic bond at A-4324 in 28 S rRNA when intact rat ribosomes are the substrate . Cleavage occurs at a concentration of the toxin of 1 X 10(-10) M, and specificity for this single residue is retained when the concentration is as high as 3 X 10(-7) M . The apparent Michaelis constant (Km) for the reaction is 2.6 microM, and the turnover number (Kcat) is 1777 min-1 . The same N-glycosidic bond is cleaved by ricin A-chain in naked 28 S rRNA, but at a greatly reduced rate . The Km value for this reaction is 5.8 microM . The results suggest that the A-chain has a similar affinity for 28 S rRNA in ribosomes and in the absence of ribosomal proteins . Ricin A-chain has no effect on 23 S rRNA in Escherichia coli ribosomes, however, the N-glycosidic bond at A-2600 in naked 23 S rRNA is cleaved by the toxin; this corresponds to the ricin site in eukaryotic 28 S rRNA . Since the Km value (3.3 microM) for the reaction with E . coli 23 S rRNA approximates that obtained with rat liver ribosomes, it is possible that E . coli ribosomal protein(s) protect this site against ricin attack in intact ribosomes . Ricin A-chain also acted on naked 16 S rRNA cleaving the N-glycosidic bond of adenine at position 1014 . The results suggest that ricin A-chain recognizes a specific structure in rRNA, perhaps a loop and stem having the sequence GAGA in the loop. J Biol Chem, 1988 Jun 25, 263(18), 8710 - 5 The expression in Escherichia coli of recombinant human platelet factor 4, a protein with immunoregulatory activity; Barone AD et al.; In order to establish more firmly the immunoregulatory effect of platelet factor 4 (PF4) and develop a means to provide material for possible clinical use, the nucleotide sequence for PF4 was synthesized utilizing a ligation strategy of six duplexes ranging from 27 to 43 base pairs in length . The individual oligodeoxynucleotides were synthesized on an automated system . The resultant gene segment (226 base pairs), which incorporated convenient HindIII and BamHI overhangs at the 5' and 3' ends, respectively, was cloned into the pIN-III-ompA-2-expression vector in Escherichia coli, affording a fusion protein of Mr = 8900 with 7 additional amino acids at the amino terminus and 4 at the carboxyl terminus and with aspartic acid rather than asparagine in position 47 . The recombinant PF4 (rPF4) was purified by heparin-agarose affinity chromatography and reverse-phase high performance liquid chromatography . It reacted with a monoclonal mouse anti-human PF4 antibody on a Western blot and in an enzyme-linked immunosorbent assay . The rPF4 protein exhibited an immunoregulatory effect like that of human PF4 in its ability to reverse concanavalin A-induced immunosuppression in BALB/c mice. J Biol Chem, 1988 Jun 25, 263(18), 8666 - 70 Identification of the reactive sulfhydryl groups of S-adenosylmethionine synthetase; Markham GD et al.; S-Adenosylmethionine synthetase from Escherichia coli is rapidly inactivated by N-ethylmaleimide . In the presence of excess N-ethylmaleimide inactivation follows pseudo first-order kinetics, and loss of enzyme activity correlates with the incorporation of 2 eq of N-{ethyl-2-3H}maleimide/subunit . Preincubation of the enzyme with methionine and the ATP analog adenylylimidodiphosphate reduced the rate of N-ethylmaleimide incorporation more than 30-fold . Two N-{ethyl-2-3H}maleimide-labeled tryptic peptides were purified from the modified enzyme by reverse phase high performance liquid chromatography . The modified residues were identified as cysteine 90 and cysteine 240 by comparison of the amino acid compositions of these peptides with the protein sequence . These are the first residues to be implicated in the activity and/or structure of the enzyme . N-Ethylmaleimide-modified S-adenosylmethionine synthetase exists mainly as a dimer in conditions where the native enzyme is a tetramer . Accumulation of the dimer parallels the loss of the enzyme activity . When an enzyme sample was partially inactivated, separation of tetrameric and dimeric enzyme forms by gel filtration revealed that the residual enzyme activity was solely present in the tetramer and N-{ethyl-2-3H} maleimide was present predominantly in the dimer . Gel filtration studies of the tetramer-dimer equilibrium for the native enzyme indicated that the dissociation constant between the tetramer and dimers is less than 6 x 10(-11) M . Similar studies for the N-ethylmaleimide-modified protein indicated that the dissociation constant of the tetramer is approximately 4 x 10(-4) M . Upon modification the strength of dimer-dimer interactions is diminished by at least 9 kcal/mol. J Biol Chem, 1988 Jun 25, 263(18), 8727 - 34 Protease Ti, a new ATP-dependent protease in Escherichia coli, contains protein-activated ATPase and proteolytic functions in distinct subunits; Hwang BJ et al.; In addition to protease La (the lon gene product), Escherichia coli contains another ATP-dependent protease, Ti . This enzyme (approximately 340 kDa) is composed of two components, both of which are required for proteolysis . Both have been purified to homogeneity by conventional procedures using {3H}casein as the substrate . The ATP-stabilized component, A, has a subunit molecular weight of 80,000 upon gel electrophoresis in the presence of sodium dodecyl sulfate, but it behaves as a dimer (140 kDa) upon gel filtration . Component P, which is relatively heat stable, is inactivated by diisopropyl fluorophosphate and can be labeled with {3H} diisopropyl fluorophosphate . It has a subunit size of 23 kDa, but the isolated component behaves as a complex (260 kDa) of 10-12 subunits . The isoelectric point of component A is 7.0 and that of P is 8.2, and their amino acid compositions differ considerably . The purified enzyme has an ATPase activity that is stimulated 2-4-fold by casein and other protein substrates but not by nonhydrolyzed proteins . Component A also shows ATPase activity which can be stimulated by casein . Addition of component P (which lacks ATPase activity) inhibits basal ATP hydrolysis by A and makes this ATPase more responsive to casein . Although component P contains the serine active site for proteolysis, it shows no proteolytic activity in the absence of component A, Mg2+, and ATP or dATP . Other nucleoside triphosphates are not hydrolyzed and do not support proteolysis . Protease Ti has a Km for ATP of 210 microM for hydrolysis of both casein and ATP . Casein increases the Vmax for ATP without affecting the Km . A Mg2+ concentration of 5 mM is necessary for half-maximal rates of ATP and casein hydrolysis . Ca2+ and Mn2+ partially support these activities . Thus, protease Ti shares many unusual properties with protease La (e.g . coupled ATP and protein hydrolysis and protein-activated ATPase), but these functions in protease Ti are associated with distinct subunits that modify each other's activities. J Biol Chem, 1988 Jun 25, 263(18), 8716 - 23 Construction of a recombinase-deficient mutant recA protein that retains single-stranded DNA-dependent ATPase activity; Bryant FR; The recA1 mutation is a single point mutation that replaces glycine 160 of the recA polypeptide with an aspartic acid residue . The mutant recA1 protein has a greatly reduced single-stranded DNA-dependent ATPase activity at pH 7.5 compared to the wild-type protein . Interestingly, the recA1 protein does exhibit a vigorous ATPase activity at pH 6.2 . To explore the molecular basis of this pH effect, we used site-directed mutagenesis to replace aspartic acid 160 of the recA1 polypeptide with an isosteric, but nonionizing, asparagine residue . The new {Asn160}recA protein catalyzes ATP hydrolysis at pH 7.5 with the same turnover number as the wild-type protein . This result suggests that the activation of the recA1 protein ATPase activity that occurs at pH 6.2 may be due, in part, to neutralization of the negatively charged aspartic acid 160 side chain . Although it is an active single-stranded DNA-dependent ATPase, the {Asn160}recA protein is unable to complement a recA deletion in vivo and is unable to carry out the three-strand exchange reaction in vitro . Further examination of ATP hydrolysis (under strand exchange conditions) revealed that the ATPase activity of the {Asn160}recA protein is strongly suppressed in the presence of Escherichia coli single-stranded DNA-binding protein (a component of the strand exchange assay), whereas the ATPase activity of the wild-type recA protein is stimulated by the E . coli protein . To account for these results, we speculate that ATP may induce specific conformational changes in the wild-type recA protein that are essential to the DNA pairing process and that these conformational changes may not occur with the {Asn160}recA protein. J Biol Chem, 1988 Jun 25, 263(18), 8872 - 8 Acetyl-coenzyme A:polysialic acid O-acetyltransferase from K1-positive Escherichia coli . The enzyme responsible for the O-acetyl plus phenotype and for O-acetyl form variation; Higa HH et al.; The capsular polysaccharide of Escherichia coli K1 is a linear polymer of N-acetylneuraminic acid in alpha-2,8 linkage . Certain substrains of E . coli K1 (designated OAc+) modify the polysaccharide by O-acetylation of the sialic acids . We demonstrate here an acetyl-coenzyme A: polysialosyl O-acetyltransferase activity that is found only in E . coli K1 OAc+ substrains . When form variation between the O-acetyl-positive and -negative states occurred in strain D698:K1, the fluctuations were accompanied by appropriate changes in the expression of enzyme activity . Thus, expression of this enzyme can account for the OAc+ phenotype and for the form variation between OAc+ and OAc- . The enzyme was solubilized in nonionic detergent and freed of endogenous acceptor activity by DEAE-cellulose chromatography, and its general properties were determined . Analysis of the reaction product showed a highly preferential acetylation reaction that was confined to polysialosyl units of greater than 14 residues . Acetyl groups were shown to be transferred to both the 7- and the 9-positions of the sialic acid residues . The partially purified enzyme was stable even after prolonged incubation at 57 degrees C . In contrast, any further purification resulted in loss of activity, even at 4 degrees C . Treatment of the stable enzyme with a polysialic acid-specific endoneuraminidase caused a similar loss of enzyme stability . This effect of the endoneuraminidase could be protected against by the addition of exogenous polysialic acid . This indicates that the partially purified enzyme contains traces of endogenous polysialic acid substrate that are required for the stability of the enzyme . Finally, the enzyme can O-acetylate the polysialic acid chains on the eucaryotic protein neural cell adhesion molecule, suggesting that enzymatic recognition of the substrate requires only the polysialic acid sequence. J Biol Chem, 1988 Jun 25, 263(18), 8765 - 70 A homologous sequence between H+-ATPase (F0F1) and cation-transporting ATPases . Thr-285----Asp replacement in the beta subunit of Escherichia coli F1 changes its catalytic properties; Noumi T et al.; A sequence of 10 amino acids (I-C-S-D-K-T-G-T-L-T) of ion motive ATPases such as Na+/K+-ATPase is similar to the sequence of the beta subunit of H+-ATPases, including that of Escherichia coli (I-T-S-T-K-T-G-S-I-T) (residues 282-291) . The Asp (D) residue phosphorylated in ion motive ATPase corresponds to Thr (T) of the beta subunit . This substitution may be reasonable because there is no phosphoenzyme intermediate in the catalytic cycle of F1-ATPase . We replaced Thr-285 of the beta subunit by an Asp residue by in vitro mutagenesis and reconstituted the alpha beta gamma complex from the mutant (or wild-type) beta and wild-type alpha and gamma subunits . The uni- and multisite ATPase activities of the alpha beta gamma complex with mutant beta subunits were about 20 and 30% of those with the wild-type subunit . The rate of ATP binding (k1) of the mutant complex under uni-site conditions was about 10-fold less than that of the wild-type complex . These results suggest that Thr-285, or the region in its vicinity, is essential for normal catalysis of the H+-ATPase . The mutant complex could not form a phosphoenzyme under the conditions where the H+/K+-ATPase is phosphorylated, suggesting that another residue(s) may also be involved in formation of the intermediate in ion motive ATPase . The wild-type alpha beta gamma complex had slightly different kinetic properties from the wild-type F1, possibly because it did not contain the epsilon subunit. J Biol Chem, 1988 Jun 25, 263(18), 8611 - 4 Evidence that glutamic acid 49 of tryptophan synthase alpha subunit is a catalytic residue . Inactive mutant proteins substituted at position 49 bind ligands and transmit ligand-dependent to the beta subunit; Miles EW et al.; Glutamic acid 49 of the alpha subunit of tryptophan synthase from Escherichia coli is an essential residue since 19 mutant proteins substituted at position 49 were found previously to be inactive . Our present findings that five mutants of the alpha subunit, substituted with Asp, Lys, Ala, Phe, or Gly at position 49, bind a substrate analog normally are further evidence that glutamic acid 49 is a catalytic base . Ligands of the alpha subunit also have similar effects on site-site interactions between the beta subunit and the wild type or mutant alpha subunits . These effects include inhibition of the activity of the beta subunit, reduction of the dissociation constant for D-tryptophan, and increase of the equilibrium concentration of a quinonoid intermediate formed with L-tryptophan. J Biol Chem, 1988 Jun 25, 263(18), 8943 - 52 Synthesis, antiviral activity, and conformational characterization of mouse-human alpha-interferon hybrids; Raj NB et al.; Reciprocal hybrids were constructed between human and mouse interferons (IFNs), and their antiviral activity was examined on different target cells and compared to the activity of the parental molecules . In addition, we used a number of predictive algorithms on a data base of the available alpha-interferon sequences to propose a working model for the overall conformation of the alpha-interferon molecule that is consistent with the structural predictions . Remarkable conservation within the predicted alpha-helical segments of the interferon molecule was observed . We propose that the observed changes in the activity and specificity of the hybrids obtained are largely due to the sequences present in the loops at the ends of the major helical structures; these are less conserved, contain beta-bends, and are generally hydrophilic and flexible . The data on the constructed mouse-human hybrids have shown that the activity on human cells is contributed by determinants present in the N-terminal 122 amino acids of human IFN, thus implicating one or more loops within this region (e.g . loops 1-12, 25-38, 70-74, and 103-113) . The activity on bovine cells appears to be localized mainly in sequence 60-121, implicating the role of loops 70-74 and/or 103-113 of the human IFN molecule . The specificity of mouse IFN for mouse cells is in some or all of the loops (70-74, 103-113, 134-139, and 163-166) in the C-terminal sequence . The proposed working model should provide guidelines for the study of the specificity of action in molecular terms. Nucleic Acids Res, 1988 Jun 24, 16(12), 5391 - 406 The core region of human glutaminyl-tRNA synthetase homologies with the Escherichia coli and yeast enzymes; Thommes P et al.; We have isolated from a Lambda-gt 11 library a human cDNA clone with one open reading frame of about 2400 bases . A stretch of about 350 amino acids in the deduced amino acid sequence is up to 40 percent identical with parts of the known amino acid sequences of E . coli and yeast glutaminyl (Gln)-tRNA synthetase . The isolated cDNA sequence corresponds to an internal section of a 5500 bases long mRNA that codes for a 170 kDa polypeptide associated with Gln-tRNA synthetase . Thus, the human enzyme is about three times larger than the E . coli and two times larger than the yeast Gln-tRNA synthetase . The three enzymes share an evolutionarily conserved core but differ in amino acid sequences linked to the N-terminal and C-terminal side of the core. Nucleic Acids Res, 1988 Jun 24, 16(12), 5305 - 22 Evolution and mutagenesis of the mammalian excision repair gene ERCC-1; van Duin M et al.; The human DNA excision repair protein ERCC-1 exhibits homology to the yeast RAD10 repair protein and its longer C-terminus displays similarity to parts of the E . coli repair proteins uvrA and uvrC . To study the evolution of this 'mosaic' ERCC-1 gene we have isolated the mouse homologue . Mouse ERCC-1 harbors the same pattern of homology with RAD10 and has a comparable C-terminal extension as its human equivalent . Mutation studies show that the strongly conserved C-terminus is essential in contrast to the less conserved N-terminus which is even dispensible . The mouse ERCC-1 amino acid sequence is compatible with a previously postulated nuclear location signal and DNA-binding domain . The ERCC-1 promoter harbors a region which is highly conserved in mouse and man . Since the ERCC-1 promoter is devoid of all classical promoter elements this region may be responsible for the low constitutive level of expression in all mouse tissues and stages of embryogenesis examined. Nucleic Acids Res, 1988 Jun 24, 16(12), 5557 - 68 Expression of glutathione peroxidase I gene in selenium-deficient rats; Reddy AP et al.; We have characterized a cDNA pGPX1211 encoding rat glutathione peroxidase I . The selenocysteine in the protein corresponded to a TGA codon in the coding region of the cDNA, similar to earlier findings in mouse and human genes, and a gene encoding the formate dehydrogenase from E . coli, another selenoenzyme . The rat GSH peroxidase I has a calculated subunit molecular weight of 22,155 daltons and shares 95% and 86% sequence homology with the mouse and human subunits, respectively . The 3'-noncoding sequence (greater than 930 bp) in pGPX1211 is much longer than that of the human sequences . We found that glutathione peroxidase I mRNA, but not the polypeptide, was expressed under nutritional stress of selenium deficiency where no glutathione peroxidase I activity can be detected . The failure of detecting any apoprotein for the glutathione peroxidase I under selenium deficiency and results published from other laboratories supports the proposal that selenium may be incorporated into the glutathione peroxidase I co-translationally. Nucleic Acids Res, 1988 Jun 24, 16(12), 5439 - 58 Upstream and downstream transcriptional control signals in the yeast retrotransposon, TY; Fulton AM et al.; The yeast retrotransposon, Ty, shares many structural and functional features with retroviral proviruses . These include production of a terminally redundant major transcript . There are also two less abundant transcripts of 5.0 kb and 2.2 kb . Ty transcription is regulated by cell-type, that is it is reduced 5-20 fold in a/alpha diploids as compared to haploids . However control of expression of Ty is not well understood . By deletion analysis we have identified regions of the element which are involved in the activation and regulation of transcription . These signals are found both upstream and downstream of the mRNA start site . The downstream signals are within the region encoding the major Ty proteins . This organisation of transcriptional control signals is discussed with reference to the organisation of control signals in other yeast genes and in retroviral proviruses and other retro-elements. Nucleic Acids Res, 1988 Jun 24, 16(12), 5345 - 59 Gene regulation on broad host range plasmid RK2: identification of three novel operons whose transcription is repressed by both KorA and KorC; Thomas CM et al.; The product of the korA gene of broad host range plasmid RK2 is a key transcriptional repressor which regulates not only the expression of the essential replication gene trfA but also its own expression and that of the kilA operon . It has previously been proposed that korA also encodes a positive activator of transcription of the korC gene, which may act as a transcriptional antiterminator . Here we show that the action of korA in relation to korC can be explained entirely through the korA protein's property as a transcriptional repressor . The limited ability of the previously cloned korC gene to suppress kilC on its own is shown to be due to the fact that korC in RK2 is transcribed from the bla promoter of Tn1 which was deleted in the original korC clones . We demonstrate that korA is a second repressor along with korC of three operons, one of which encodes kilC, the other two not having been described previously and serving an as yet unknown function . We have designated these operons kcrA, B and C for KorC-regulated . Putative kilC is designated kcrC . The homology between the expression signals of these operons suggests that they have arisen by duplication . This is confirmed in the case of kcrA and B by the existence of considerable homology between the products of the first ORFs in each of these operons. Nucleic Acids Res, 1988 Jun 24, 16(12), 5277 - 90 A second RNA-polymerase can bind specifically to the bla promoter of Tn3, repressing transcription initiation; Duval-Valentin G et al.; We showed earlier that the region of the bla promoter of Tn3 protected by the RNA-polymerase (RNAP), has the normal size (about 60bp) at RNAP/promoter molar ratio r less than or equal to 2, but rises to about twice this extent as r increases . We confirm here that the species corresponding to normal and extended footprint distinguish by their electrophoretic mobilities . Furthermore, inspection of the complexes by electron microscopy confirms that at r greater than 2, the bla promoter can bind specifically a second RNAP particle, as compared to the 1:1 complex observed at r less than or equal to 2 . At r greater than 2, the ability of the bla promoter to initiate transcription in vitro is repressed when compared to the complex 1:1 obtained at r less than or equal to 2 . The unexpected decrease in initiation efficiency as the concentration of RNAP particles is increased, together with the striking sequence homology of the bla promoter with promoters of stable RNA, suggest that in vivo, this promoter could be regulated by growth rate. FEBS Lett, 1988 Jun 20, 233(2), 432 - 6 Automated Sanger dideoxy sequencing reaction protocol; Zimmermann J et al.; The protocol for Sanger dideoxy chain termination reactions in DNA sequencing is tedious and prone to errors due to the repetitive character of the pipetting steps . An industrial robot, with the addition of a few simple parts, was programmed to automate the dideoxy sequencing reactions . The system is set up in a short time for routine operation and it is faster and more reliable than a human operator . It is flexible and allows variations and optimization of the standard procedure . Disposable microtiter plates at a controlled temperature are used . In one reaction cycle (about 50 min) up to 48 templates are processed . Up to 450 bases were resolved in automated DNA sequencing on samples prepared by the robot . The protocol is applicable to fluorescent as well as to radioactive labeling. FEBS Lett, 1988 Jun 20, 233(2), 367 - 70 Degradation of epidermal growth factor receptors by cathepsin L-like protease: inhibition of the degradation by c-Ha-ras gene products; Hiwasa T et al.; Extract of NIH3T3 mouse fibroblasts contains a protease which can cleave epidermal growth factor receptor (EGF receptor) . This protease was tentatively named cathepsin X and purified to near homogeneity . The characteristics of cathepsin X were similar to those of cathepsin L and the proteolytic activity of cathepsin X was inhibited by c-Ha-ras gene products. FEBS Lett, 1988 Jun 20, 233(2), 326 - 30 Protein NMR resonance assignment by isotropic mixing experiments on random fractionally deuterated samples; LeMaster DM; The 108-residue protein E . coli thioredoxin has been uniformly enriched to 50% with deuterium at all carbon-bound hydrogen positions . Isotropic mixing (i.e . TOCSY) experiments have been conducted for both the deuterated and natural-abundance samples . Using a 54 ms mixing time correlation peaks can be seen for all four protons on the benzenoid ring of tryptophan in both samples . The deuteration results in an average decrease in cross-sectional area of a factor of 2-3 for the TOCSY cross-peaks . The cross-peak intensities for the deuterated sample systematically decrease as a function of the number of protons involved in the transfer process thus overcoming a common ambiguity in the TOCSY experiment. FEBS Lett, 1988 Jun 20, 233(2), 347 - 51 Identification of alpha-subunit Lys201 and beta-subunit Lys155 at the ATP-binding sites in Escherichia coli F1-ATPase; Tagaya M et al.; Binding of about 1 mol of adenosine triphosphopyridoxal to Escherichia coli F1-ATPase resulted in the nearly complete inactivation of the enzyme {(1987) J . Biol . Chem . 262, 7686-7692} . About two thirds of the label was bound to the alpha-subunit, and the rest to the beta-subunit . The present study revealed that Lys201 in the alpha-subunit and Lys155 in the glycine-rich region of the beta-subunit are the major sites labeled with this reagent . Thus, these two residues might be located close to the gamma-phosphate of the bound ATP. J Mol Biol, 1988 Jun 20, 201(4), 697 - 716 Probing the assembly of the 3' major domain of 16 S rRNA . Interactions involving ribosomal proteins S2, S3, S10, S13 and S14; Powers T et al.; We have used rapid probing methods to follow the changes in reactivity of residues in 16 S rRNA to chemical and enzymatic probes as ribosomal proteins S2, S3, S10, S13 and S14 are assembled into 30 S subunits . Effects observed are confined to the 3' major domain of the RNA and comprise three general classes . (1) Monospecific effects, which are attributable to a single protein . Proteins S13 and S14 each affect the reactivities of different residues which are adjacent to regions previously found protected by S19 . S10 effects are located in two separate regions of the domain, the 1120/1150 stem and the 1280 loop; both of these regions are near nucleotides previously found protected by S9 . Both S2 and S3 protect different nucleotides between positions 1070 and 1112 . In addition, S2 protects residues in the 1160/1170 stem-loop . (2) Co-operative effects, which include residues dependent on the simultaneous presence of both proteins S2 and S3 for their reactivities to appear similar to those observed in native 30 S subunits . (3) Polyspecific effects, where proteins S3 and S2 independently afford the same protection and enhancement pattern in three distal regions of the domain: the 960 stem-loop, the 1050/1200 stem and in the upper part of the domain (nucleotides 1070 to 1190) . Proteins S14 and S10 also weakly affect the reactivities of several residues in these regions . We believe that several of the protected residues of the first class are likely sites for protein-RNA contact while the third class is indicative of conformational rearrangement in the RNA during assembly . These results, in combination with the results from our previous study of proteins S7, S9 and S19, are discussed in terms of the assembly, topography and involvement in ribosomal function of the 3' major domain. Biochem Biophys Res Commun, 1988 Jun 16, 153(2), 875 - 80 In vivo phosphorylation of isocitrate lyase from Escherichia coli D5H3G7; Hoyt JC et al.; This report describes the in vivo phosphorylation of isocitrate lyase and examines the possible consequences to the control of the Kreb's cycle and glyoxylate bypass . NADP-specific isocitrate dehydrogenase from E . coli was the first bacterial protein whose enzymic activity was shown to be modulated by reversible phosphorylation . This enzyme has been thought to be solely responsible for the partitioning of isocitrate between the Kreb's cycle and glyoxylate bypass . No studies to date have examined the possible role of isocitrate lyase in controlling this flux. Biochem Biophys Res Commun, 1988 Jun 16, 153(2), 767 - 72 L-leucine and its analogue: specific inhibitors for S-benzyl-L-cysteine-p-nitroanilide-hydrolyzing enzyme in Escherichia coli B; Murata K et al.; An enzyme that catalyzes hydrolysis of S-benzyl-L-cysteine-p-nitroanilide was purified from E . coli B . The enzyme was a monomer with a molecular weight of 82,000 . In addition to L-cysteinylglycine, the enzyme hydrolyzed various glycine-containing dipeptides most efficiently at pH 7.0 . The enzyme required no metal ions for activity and was specifically inhibited by L-leucine and its analogue with free carboxyl group at the physiological concentrations. Biochem Biophys Res Commun, 1988 Jun 16, 153(2), 753 - 9 Mutant isolation and cloning of the gene encoding protease VII from Escherichia coli; Sugimura K; A mutant of Escherichia coli lacking protease VII, the outer membrane-associated protease which specifically cleaves paired basic residues (1), was isolated by using N-methyl-N'-nitro-N-nitrosoguanidine treatment . The mutant exhibited no significant change as for its growth rate and microscopic feature compared with wild cells . The gene encoding protease VII was cloned by using complementation analysis of protease VII (-) mutation . The minicell experiment showed that the gene encoded a putative precursor protein of 38,000 Mr which was processed into a protein of 36,000 Mr suggesting the presence of a signal peptide on the putative precursor. Thromb Haemost, 1988 Jun 16, 59(3), 378 - 82 Endotoxin-induced platelet activation in human whole blood in vitro; Csako G et al.; The effect of purified bacterial endotoxin was studied on human platelets in vitro . In adding up to 1 microgram/mL of a highly purified endotoxin, we found neither aggregation nor ATP release in heparinized or citrated human platelet-rich plasma . On the other hand; endotoxin at concentrations as low as a few ng/mL (as may be found in septic patients) caused platelet aggregation in both heparinized and citrated human whole blood, as monitored by change in impedance, free platelet count, and size . Unlike collagen, the platelet aggregation with endotoxin occurred after a long lag phase, developed slowly, and was rarely coupled with measurable release of ATP . The platelet aggregating effect of endotoxin was dose-dependent and modified by exposure of the endotoxin to ionizing radiation . Thus, the activation of human platelets by "solubilized" endotoxin in plasma requires the presence of other blood cells . We propose that the platelet effect is mediated by monocytes and/or neutrophils stimulated by endotoxin. Biochem Biophys Res Commun, 1988 Jun 16, 153(2), 760 - 6 Mechanism of the EPSP synthase catalyzed reaction: evidence for the lack of a covalent carboxyvinyl intermediate in catalysis; Wibbenmeyer J et al.; In order to detect covalent reaction intermediates in the 5-enolpyruvylshikimate-3-phosphate (EPSP) synthase reaction, we have investigated the interaction of EPSP synthase with the reaction product EPSP . An exchange of EPSP-methylene protons could be demonstrated by incubating EPSPS with EPSP in D2O . Since trace amounts of contaminating Pi would lead to reversal of EPSPS reaction and hence methylene proton exchange, we added pyruvate kinase, ADP, Mg++ and K+ . Under these conditions, any contaminating Pi that is converted to PEP is trapped as ATP . No exchange of EPSP protons with those of the solvent could be detected in the presence of this trap system, suggesting that enzyme-bound EPSP is unable to form a covalent tetrahedral complex . Incorporation of {14C} from {14C}-S3P and {14C}-PEP into EPSP could be detected, but only in the absence of a PEP (or Pi) trap system . This indicates that for the exchange reaction, Pi is required, and also indicates the absence of a covalent intermediate, unless the carboxyvinyl-enzyme-bound S3P is completely restricted from exchange. Biochem J, 1988 Jun 15, 252(3), 909 - 12 Evidence that the pyrromethane cofactor of hydroxymethylbilane synthase (porphobilinogen deaminase) is bound through the sulphur atom of a cysteine residue; Hart GJ et al.; Hydroxymethylbilane synthase (porphobilinogen deaminase) from Escherichia coli uses a novel pyrromethane cofactor to bind the growing pyrrolic chain for hydroxymethylbilane biosynthesis {Hart, Miller, Leeper & Battersby (1987) J . Chem . Soc . Chem . Commun . 1762-1765} . We show that this cofactor is bound to the protein through the sulphur atom of a cysteine residue. J Biol Chem, 1988 Jun 15, 263(17), 7929 - 32 2-Deoxy-2-fluoro-D-glycosyl fluorides . A new class of specific mechanism-based glycosidase inhibitors; Withers SG et al.; Mechanism-based glycosidase inhibitors are of considerable use in studies of enzyme mechanism, in studies of glycoprotein processing, and possibly therapeutically in control of sugar uptake . This paper describes a new general approach to mechanism-based inactivation of glycosidases which involves trapping a covalent glycosyl enzyme intermediate . This is achieved by use of 2-deoxy-2-fluoro-D-glycosyl fluorides, for which the rate of hydrolysis of the fluoroglycosyl enzyme intermediate is extremely slow, resulting in accumulation of the intermediate . Eleven different glycosidases were tested with their corresponding 2-deoxy-2-fluoro-D-glycosyl fluorides . Eight of the eleven were inactivated, four of them according to pseudo first-order kinetics and four according to a more complex kinetic scheme . The specificity of these inhibitors was investigated by assaying for inhibition of one enzyme with four different 2-deoxy-2-fluoro-D-glycosyl fluorides . Large differences in inactivation rate were observed which paralleled previously observed substrate specificities. J Biol Chem, 1988 Jun 15, 263(17), 8282 - 7 Chemical and immunological characterization of the 21-kDa ADP-ribosylation factor of adenylate cyclase; Kahn RA et al.; The ADP-ribosylation factor (ARF) is a 21-kDa GTP-binding protein cofactor in the cholera toxin-catalyzed ADP-ribosylation of the stimulatory regulatory subunit of adenylate cyclase . Purified bovine brain ARF was digested with cyanogen bromide, and peptides were purified and sequenced . Approximately 25-30% of the protein was sequenced in this manner . Peptides contained consensus sequences for GTP-binding proteins but were distinct from any of the previously published GTP-binding proteins . Antibodies were raised in rabbits against both protein and synthetic peptide fragments of ARF . Specific ARF immunoreactivity was detected in every eukaryotic tissue or cell examined, including yeast, slime mold, and man . No ARF immunoreactivity was observed when Escherichia coli proteins were tested . Immunoblotting revealed the majority of ARF to be present in the 100,000 x g supernatant . Immunological cross-reactivity with the cytosolic factor indicate that it and ARF are likely to be the same protein . ARF is shown to be myristylated at the amino terminus . The potential role of myristylation in cellular localization is discussed. Gene, 1988 Jun 15, 66(1), 77 - 85 cDNA cloning and sequence analysis of a chicken gene expressed during the gonadal development and homologous to mammalian cytochrome P-450c17; Ono H et al.; A cDNA clone, pLOA0511, was isolated from the cDNA library prepared from the left ovaries of one- to three-day-old chickens . The transcript corresponding to this cDNA clone is approx . 1.9 kb in length and is present in steroidogenic tissues (ovary, testis and adrenal gland) but is undetectable in non-steroidogenic tissues . Relative abundance of the transcript in the ovary and testis is high and developmentally regulated but is much lower in the adrenal gland . Relative levels of this transcript in the developing left ovary and the regressing right ovary of the one- to three-day-old chicken are similar . The nucleotide sequence of this cDNA clone shows significant homology to some mammalian cytochromes P-450 . Out of the 508 amino acids (aa) coded, 244 and 243 aa residues are identical with those of the bovine and human cytochrome P-450c17 (steroid 17 alpha-hydroxylase/17,20 lyase), respectively . There are three highly conserved regions among the bovine, human and chicken sequences, one of which is unique to P-450c17 . These data suggest strongly that this cDNA corresponds to the mRNA of a chicken cytochrome P-450c17. Gene, 1988 Jun 15, 66(1), 97 - 106 Structure and characterisation of a duplicated human alpha 1 acid glycoprotein gene; Merritt CM et al.; Human alpha 1-acid glycoprotein (AGP), also known as orosomucoid, is a major acute-phase plasma protein . The amino acid sequence of AGP, which was determined by sequencing from protein isolated from pooled plasma, contained amino acid substitutions in 21 different positions . Genomic and cDNA clones which correspond to one of the possible amino acid sequences have been previously reported . In this paper we present the complete nucleotide sequence of a second gene, AGP2 which is located approx . 3.3 kb downstream from AGP1 . The derived amino acid sequence of AGP2 contains 19 of the possible alternative amino acid substitutions as well as two additional differences . It is clear from the results presented here that the AGP in human plasma is the product of two separate gene loci. Gene, 1988 Jun 15, 66(1), 87 - 96 Isolation and characterization of the Bos taurus beta-casein gene; Gorodetsky SI et al.; The expression of casein genes in the mammary cells is regulated by peptide and steroid hormones . To investigate the controlling mechanisms we have isolated and characterized the bovine beta-casein gene . The gene has the size of 8.6 kb, which is 7.8 times longer than the corresponding mRNA composed of nine exons . The genomic clones include additional 8.5-kb and 4.5-kb sequences of the 5'- and 3'-flanking regions . We have determined the sequences of the 5' and 3' ends of the gene and compared them with the respective sequences of the rat beta-casein gene . Conserved sequences are identical or homologous to the potential binding sites for nuclear factors and for glucocorticoid and progesterone receptors . The regulatory region contains two different TATA signals and a repeat sequence between them. J Biol Chem, 1988 Jun 15, 263(17), 8037 - 43 Cloning and expression of cDNA for human endonexin II, a Ca2+ and phospholipid binding protein; Kaplan R et al.; Endonexin II is a member of the family of Ca2+-dependent phospholipid binding proteins known as annexins . We cloned human endonexin II cDNA and expressed it in Escherichia coli . The apparent size and Ca2+-dependent phospholipid binding properties of purified recombinant endonexin II were indisti