bluuebunny/crossref_metadata_2025 huggingface dataset:
Molecular cloning of integrated simian sarcoma virus: genome organization of infectious DNA clones.
Authors: Robbins K C , Devare S G , Aaronson S A
Published: May 2006

The integrated form of simian sarcoma virus (SSV) was molecularly cloned in the Charon 16A strain of bacteriophage lambda. In transfection analysis, the recombinant viral DNAs demonstrated the ability to transform cells in tissue culture at high efficiency. Such transformants possessed typical SSV morphology, expressed simian sarcoma associated virus (SSAV) gag gene products in the absence of virus release, and released SSV after superinfection with a type C helper virus. A physical map of the 5.8-kilobase-pair (kbp) recombinant viral DNA clone, deduced from restriction endonuclease analysis, revealed a 5.1-kbp SSV genome containing 0.55-kbp-long terminal repeats flanked by 0.45 and 0.25 kbp of contiguous host cell sequences. By R-loop analysis, the viral DNA molecule contained two regions of homology to SSAV, separated by a 1.0-kbp nonhomologous region. This SSV-specific sequence was shown to be uniquely represented within the normal cellular DNA of diverse mammalian species, including human. Our results demonstrate that this primate transforming retrovirus arose in nature by recombination of a type C helper virus and a host cellular gene.
Oscillations and control features in glycolysis: numerical analysis of a comprehensive model.
Authors: Termonia Y , Ross J
Published: May 2006

We present an analysis of glycolysis based on experimental findings and an interpretation based on concepts of efficiency, resonance response, and control features available in highly nonlinear reaction kinetics. We begin with a model for the glycolytic mechanism that is comprehensive, includes a large number of known activations and inhibitions of enzymes by metabolites, and couples the phosphofructokinase (PFKase) and the pyruvate kinase (PKase) reactions. The PFKase and PKase reactions and the coupling between them are modeled according to experimental information, but we do not attempt to model the glyceraldehyde-3-phosphate dehydrogenase-3-phosphoglycerate kinase reaction. We use experimental data to obtain the best estimates for the kinetic parameters and test the model by calculating the concentration variations of the intermediate metabolites. We confirm oscillatory behavior and calculate the ATP/ADP ratio and the free-energy dissipation for an extended range of the kinetic parameters as a function of the driving force for the glycolytic pathway, a measure of which is the total adenine nucleotide concentration. We find agreement of the calculated results with experimental findings except for the insufficiently represented reactions. Our model shows that the average ATP/ADP ratio is increased and the average free-energy dissipation is decreased in an oscillatory compared with a steady state mode of operation. The average values of the ATP/ADP ratio and of the free energy dissipation change abruptly past the onset of sustained oscillations.
Proton NMR studies of the Zn(II)--bleomycin-A2--poly(dA-dT) ternary complex.
Authors: Glickson J D , Pillai R P , Sakai T T
Published: May 2006

Proton NMR spectra demonstrate the formation of a ternary complex, Zn(II)--bleomycin-A2--poly(dA-dT), which serves as an analog of the putative active complex, Fe(II)--bleomycin-A2--DNA. Specific sites of metal--drug and drug--nucleic acid interaction have been delineated on the basis of chemical shift perturbations. On the basis of this criterion there appear to be three distinct regions of the drug: (i) the NH2 terminus up to and including the disaccharide and hydroxyhistidine residues, whose resonances are perturbed only by metal interactions; (ii) the COOH-terminal dipeptide, whose resonances are displaced only by nucleic acid interactions; and (iii) the methylvaleric acid-threonine dipeptide, which links these domains and whose resonances are sensitive to both types of interactions. The spectral perturbations in the first and second domains are very similar to changes observed in the corresponding binary complexes--i.e., Zn(II)--bleomycin-A2 and bleomycin-A2--poly(dA-dT), respectively.
Differential staining of actin in metaphase spindles with 7-nitrobenz-2-oxa-1,3-diazole-phallacidin and fluorescent DNase: is actin involved in chromosomal movement?
Authors: Barak L S , Nothnagel E A , DeMarco E F , Webb W W
Published: May 2006

The distribution and polymerization state of actin in metaphase rat kangaroo cells was studied by fluorescence microscopy. Formaldehyde-fixed, acetone-extracted cells were labeled with either of two types of actin probes. The first, 7-nitrobenz-2-oxa-1,3-diazole-phallacidin, has high affinity for F actin and does not bind monomeric G actin. The second was a conjugate of DNase I labeled with either tetramethylrhodamine or fluorescein. DNase binds with high affinity to G actin and with lesser affinity to F actin. The polymerization state of actin was deduced by comparing the fluorescence distribution of the phallacidin derivative with that of the fluorescent DNase. The results indicate that the pole-to-chromosome region of the metaphase spindle contains G actin but little if any conventional F actin. F actin is found concentrated in a diffuse distribution outside the spindle region in metaphase cells and returns to the interzone area between the chromosomes by early telophase. These results exclude spindle models for chromosomal movement that require more than about five F actin filaments per chromosome, support the hypothesis that F actin is involved in force generation for cell cleavage, and are not inconsistent with the possibility that actin outside the spindle may be involved in chromosomal movement.
Angiotensin alters 45Ca2+ fluxes in bovine adrenal glomerulosa cells.
Authors: Elliott M E , Goodfriend T L
Published: May 2006

Angiotensin II stimulated 45Ca2+ release from bovine adrenal glomerulosa cells. It also decreased the influx of 45Ca2+ into glomerulosa cells. The effects were observed within 2 min of hormone addition and were blocked by Saralasin a competitive inhibitor of angiotensin. Des-Phe8-angiotensin II, a biologically inert analog, was inactive in this system. Angiotensin II also inhibited the influx of 133Ba2+ and 54Mn2+, whereas 51Cr6+ and 57Co2+ were unaffected. Alterations in 45Ca2+ fluxes were seen with concentrations of angiotensin that stimulate aldosterone biosynthesis in bovine glomerulosa cell preparations. These results suggest that calcium plays a key role in angiotensin-stimulated aldosteronogenesis.
Replication of alpha and beta globin DNA sequences occurs during early S phase in murine erythroleukemia cells.
Authors: Epner E , Rifkind R A , Marks P A
Published: May 2006

Murine erythroleukemia cells (MELC) can be induced to express the characteristics of erythroid differentiation by a variety of agents. Previous studies indicate that an action of inducer, occurring during early S phase, may be critical to the expression of differentiated characteristics such as initiation of accumulation of newly synthesized alpha and beta globin mRNAs. In this investigation, the time of replication of globin genes in MELC was studied. DNA was isolated from synchronous populations of cells obtained by centrifugal elutriation. Newly replicated DNA sequences were prepared from synchronized cells cultured for 1 1/2 hr with 5-bromodeoxyuridine; bromodeoxyuridine-containing DNA was isolated by CsCl gradient centrifugation. By employing cloned probes for hybridization to newly synthesized DNA, it was found that alpha and beta globin gene sequences are replicated early in S phase, while ribosomal RNA gene sequences are replicated to about the same extent in early, middle, and late S phases.
Differential activity of DNA methyltransferase in the life cycle of Chlamydomonas reinhardi.
Authors: Sano H , Grabowy C , Sager R
Published: May 2006

Two molecular weight forms of DNA (cytosine-5-)-methyltransferase [S-adenosyl-L-methionine:DNA (cytosine-5-)- methyltransferase, EC 2.1.1.37], both active in assays in vitro, were isolated from the green alga Chlamydomonas reinhardi at various stages of the life cycle. The enzyme with Mr 60,000 was found in vegetative cells and gametes of both male (mt-) and female (mt+) mating types. The enzyme with Mr 200,000 was specific to gametic cells and zygotes, which are the only stages at which methylation of chloroplast DNA occurs in vivo. Chloroplast DNA from gametes was shown to be methylated on both strands at most if not all methylation sites and the Mr 200,000 enzyme was shown to methylate both unmethylated and hemimethylated sites, the latter at an elevated rate. Micrococcus luteus DNA showed the same nearest-neighbor frequencies of methylation after methylation by each molecular weight component. The data suggest strongly that the Mr 200,000 enzyme is the active multimeric form of the Mr 60,000 enzyme and that it acts as both initiation and maintenance methylase. It is proposed that methylation of chloroplast DNA in female gametes and zygotes is regulated by assembly of the multimeric Mr 200,000 active enzyme, which in turm determines the maternal inheritance of chloroplast DNA.
Hybridoma-derived antibody with immunodiagnostic potential for schistosomiasis japonica.
Authors: Mitchell G F , Cruise K M , Garcia E G , Anders R F
Published: May 2006

A murine hybridoma-derived antibody (IPH.134) has been produced which has apparent high binding specificity for an extract of Schistosoma japonicum adult worms. No binding was detected to extracts of S. japonicum eggs or to extracts from other adult trematodes (Fasciola hepatica, Paragonimus westermanii, Clonorchis sinensis, and Schistosoma mansoni) and several other helminths and protozoa. When sera from two series of S. japonicum-infected Philippine patients (19 and 20 patients, respectively) were tested for inhibition of binding of 125I-labeled IPH.134 to S. japonicum adult worm antigen, a low (10%) false-negative rate was obtained. In the 19-patient series, the 4 patients with highest fecal egg counts had high inhibitory activity in their sera. In the 20-patient series, the 3 patients with prominent disease had high inhibitory activity in their sera. Evidence was obtained that IgG anti-S. japonicum antibodies (rather than circulating antigens, immune complexes, or anti-idiotypic antibodies) were most likely to be responsible for serum inhibitory activity in the test. No false-positive reactions have been obtained with pooled or individual sera from patients infected with numerous parasites other than S. japonicum, although no information is yet available on the inhibitory activities of sera fro S. mansoni- or S. hematobium-infected individuals. On the basis of the data obtained to date, it is a reasonable prediction that the molecule or determinant to which this hybridoma antibody is directed will be a useful immunodiagnostic antigen for schistosomiasis japonica in the Philippines. A test based on detection of serum antibodies to this antigen should have high specificity and may provide additional information on the level of infection or disease status in patients.
Expression in Escherichia coli of biologically active enzyme by a DNA sequence coding for the human plasminogen activator urokinase.
Authors: Ratzkin B , Lee S G , Schrenk W J , Roychoudhury R , Chen M , Hamilton T A , Hung P P
Published: May 2006

We have isolated clones of Escherichia coli strain K-12 that contain a hybrid pBR322 plasmid having a 4.2-kilobase insert of a DNA transcript of the mRNA of human plasminogen activator, urokinase. The bacterially produced enzyme has properties similar to those of urokinase from human fetal kidney cells. Both enzymes occur in discrete forms ranging from 32,000 to 150,000 daltons in size. They react with antibody to purified urokinase from human kidney cells, bind to a benzamidine-Sepharose column, and induce plasminogen-dependent lysis of a fibrin clot.
Hydrogen bonding, overlap geometry, and sequence specificity in anthracycline antitumor antibiotic.DNA complexes in solution.
Authors: Patel D J , Kozlowski S A , Rice J A
Published: May 2006

We have deduced structural aspects of the intercalation complex of the anthracycline antitumor antibiotic daunomycin and its analogs with the synthetic DNA poly(dA-dT) by 1H and 31P NMR in high-salt solution. We demonstrate that the base pairs are intact at the antibiotic binding site and that the anthracycline phenolic hydroxyls form intramolecular hydrogen bonds with the quinone carbonyls and are shielded from solvent in the intercalation complex. The complexation shifts of the exchangeable phenolic and nonexchangeable aromatic protons demonstrate that rings B and C of the anthracycline chromophore overlap with adjacent base pairs, while anthracycline ring D passes right through the intercalation site in the complex. We observe two resolved 31P resonances attributable to the dA-dT and dT-dA phosphodiester linkages in the phosphorus spectra of the neighbor-exclusion daunomycin.poly(dA-dT) complex. This suggests that the anthracycline antitumor antibiotic exhibits a sequence specificity in its intercalation complex with alternating purine-pyrimidine synthetic DNAs in solution. These conclusions on hydrogen bonding and overlap geometry at the intercalation site and sequence specificity for the daunomycin.poly(dA-dT) complex in solution are in agreement with the structure of the daunomycin.dC-dG-dT-dA-dC-dG hexanucleotide duplex crystalline complex at atomic resolution published recently [Quigley, G. J., Wang, A. H.-J., Ughetto, G., van der Marel, G., van Boom, J. H. & Rich, A. (1980) Proc. Natl. Acad. Sci. USA 77, 7204-7208].
Hepatic calcium efflux during cytochrome P-450-dependent drug oxidations at the endoplasmic reticulum in intact liver.
Authors: Sies H , Graf P , Estrela J M
Published: May 2006

During metabolism of (type I) drugs by cytochrome P-450-dependent monooxygenase of the endoplasmic reticulum, the NADPH/NADP+ ratio in rat liver selectively decreases to approximately one-half of the control values, whereas the NADH/NAD+ ratio remains practically unaffected [Sies, H. & Brauser, B. (1970) Eur. J. Biochem. 15, 521-540]. In view of the observations with isolated mitochondria [Lehninger, A. L., Vercesi, A. & Bababunmi, E. A. (1978) Proc. Natl. Acad. Sci. USA 75, 1690-1694] of stimulated Ca2+ efflux upon nicotinamide nucleotide oxidation, the selective oxidation of NADPH in cytosol and mitochondria during drug oxidations was considered a useful experimental tool for the determination of whether the oxidation of NADPH or of NADH is responsible for Ca2+ efflux. With perfused livers from phenobarbital-treated rats, Ca2+ efflux was demonstrated, amounting to 8 nmol/min per gram of liver (wet weight), with aminopyrine, ethylmorphine, or hexobarbital as drug substrates. Drug-associated Ca2+ release was diminished when the inhibitor metyrapone was also present, or when drug oxidation was suppressed during N2 anoxia or in the presence of antimycin A in livers from fasted rats. Ca2+ efflux was elicited also by infusion of the thiol oxidant diamide, and by t-butyl hydroperoxide. However whereas Ca2+ efflux elicited by these compounds was restricted upon addition of the thiol dithioerythritol, there was little, if any, sensitivity of the drug-associated Ca2+ efflux to the thiol. Further mitochondrial oxidation of NADPH by addition of ammonium chloride had no effect on drug-associated Ca2+ efflux. Prior addition of the alpha-agonist phenylephrine suppressed the Ca2+ release by drug addition. While the molecular mechanism involved in Ca2+ efflux from liver mitochondria and from hepatocytes as well as the regulatory significance are not yet known, it is concluded from the present experiments that in case of nicotinamide nucleotide-linked Ca2+ efflux the oxidation of NADPH may suffice, with oxidation of NADH not being a requirement.
recA protein of Escherichia coli promotes branch migration, a kinetically distinct phase of DNA strand exchange.
Authors: Cox M M , Lehman I R
Published: May 2006

The recA protein of Escherichia coli promotes the complete exchange of strands between full-length linear duplex and single-stranded circular DNA molecules of bacteriophage phi X-174, converting more than 50% of the single-stranded DNA into heteroduplex replicative form II-like structures. Kinetically, the reaction can be divided into two phases, formation of short heteroduplex regions (D loops) and extension of the D loops via branch migration. recA protein participates directly in both phases. D loops are formed efficiently in the presence of ATP or the nonhydrolyzable ATP analog adenosine 5'-[gamma-thio]triphosphate, whereas D-loop extension requires continuous ATP hydrolysis. Complete strand exchange requires a stoichiometric amount of recA protein and is strongly stimulated by the single-stranded-DNA-binding protein of E. coli.
Cloning, mapping, and
in vitro
transcription-translation of the gene for the large subunit of ribulose-1,5-bisphosphate carboxylase from spinach chloroplasts
Authors: Erion Jack L. , Tarnowski Joseph , Weissbach Herbert , Brot Nathan
Published: May 2006

An 11.2-kilobase pair (kbp)
Bam
HI restriction nuclease fragment from spinach chloroplast DNA has been found to contain the gene for the large subunit (LS) of ribulose-1,5-bisphosphate carboxylase [Ru
P
2
carboxylase; 3-phospho-D-glycerate carboxy-lyase (dimerizing), EC 4.1.1.39]. The gene was located by hybridization of cloned chloroplast DNA fragments containing the maize LS gene (Bedbrook, J. R., Coen, D. M., Beaton, A. R., Bogorad, L. & Rich, A. (1979)
J. Biol. Chem.
254, 905-910) to spinach chloroplast DNA cleaved with restriction nucleases. The 11.2-kbp
Bam
HI fragment has been inserted into the
Bam
HI site of the plasmid pBR322. The resulting recombinant plasmid, pSoe3101, was used to direct the synthesis of a protein, which was immunoprecipitable with antibody to Ru
P
2
carboxylase, in a partially defined
in vitro
transcription-translation system derived from
Escherichia coli
. The product synthesized
in vitro
has a molecular weight identical to that of authentic spinach LS. By using pSoe3101 DNA cleaved at various positions with restriction nucleases, and the
in vitro
transcription-translation system, the LS gene has been mapped to a 1.5-kbp region located at one end of the 11.2-kbp
Bam
HI fragment. The direction of transcription of the LS gene on the plasmid as well as on the chloroplast chromosome has also been determined. The position of the LS gene on circular spinach chloroplast DNA is approximately 27 kbp from the start of one of the inverted repeat regions and 180° from one of the rRNA-coding regions.
Partial characterization of gamma (immune) interferon mRNA extracted from human lymphocytes.
Authors: Taniguchi T , Pang R H , Yip Y K , Henriksen D , Vilcek J
Published: May 2006

gamma (immune) interferon (IFN-gamma) was induced in cultures of fresh human lymphocytes by combined treatment with a phorbol ester (12-O-tetradecanoylphorbol 13-acetate, TPA) and the T cell mitogen phytohemagglutinin (PHA). Compared to the IFN-gamma yields obtained with PHA induction alone, the inclusion of TPA caused a significant enhancement of IFN-gamma production. Poly(A)-containing mRNA was isolated from mononuclear cells induced with TPA and PHA. Injected into Xenopus laevis oocytes, this mRNA preparation gave rise to IFN activity with characteristic properties of human IFN-gamma. Sucrose density gradient centrifugation analysis showed that IFN-gamma mRNA sedimented at 15 S, suggesting that it contains a total of about 1400 nucleotides.
Novel role of calcium in exocytosis: mechanism of nematocyst discharge as shown by x-ray microanalysis.
Authors: Lubbock R , Gupta B L , Hall T A
Published: May 2006

Mature nematocysts of the sea anemones Rhodactis rhodostoma and Anthopleura elegantissima contain a fluid that has a high concentration of solutes and is extraordinarily rich in calcium (ca. 500-600 mmol/kg wet weight); this contrasts with the surrounding cytoplasm which is rich in potassium but poor in calcium. The undischarged capsule is surrounded by a membrane that probably acts as a selective permeability barrier between the cytoplasm and the nematocyst fluid. During discharge the nematocyst moves to the surface of the nematocyte and comes into contact with the external sea water medium. Calcium, which may be bound to proteins in the undischarged state, is rapidly lost from the fluid; at the same time, sea water enters the capsule. In vitro experiments have already shown that calcium loss increases the osmotic pressure of the capsular fluid, causing an influx of water from the external medium; this influx appears to increase the hydrostatic pressure inside the capsule to the point that the thread everts explosively.
Microsomal acetyl-CoA carboxylase: evidence for association of enzyme polymer with liver microsomes.
Authors: Witters L A , Friedman S A , Bacon G W
Published: May 2006

Fatty acid synthesis is traditionally viewed as being confined to the cytosolic cellular fraction, although a substantial body of data indicates that both microsomes and mitochondria are capable of initiating fatty acid synthesis and may contain acetyl-CoA carboxylase [acetyl-CoA:carbon-doxide ligase (ADP-forming), EC 6.4.1.2], fatty acid synthetase, and ATP-citrate lyase [ATP citrate (pro-3S)-lyase; ATP:citrate oxaloacetate-lyase (pro-3S-CH2COO- leads to acetyl-CoA; ATP-dephosphorylating), EC 4.1.3.8] activities. We have identified 32P-labeled acetyl-CoA carboxylase and 32P-labeled ATP-citrate lyase by immunoprecipitation of a rat hepatocyte microsomal preparation. In the transition between the fasting state (low rates of lipogenesis) and fasting/re-feeding (high rates), the fraction of total cytosolic plus microsomal acetyl-CoA carboxylase in the microsomes increases from 6% to 43%, whereas the microsomal proportion of total fatty acid synthetase and ATP-citrate lyase remains approximately 10%. Microsome isolation conditions favoring carboxylase polymerization (presence of citrate) promote microsomal association, whereas conditions favoring enzyme protomerization (malonyl-CoA, preincubation with cyclic AMP/ATP/Mg2+) diminish this association. The microsomal enzyme has a 5-fold higher specific activity than the cytosolic enzyme as determined by immunotitration. Sucrose density gradient analysis of the microsomal fraction indicates that a substantial portion of carboxylase activity sediments with marker enzymes for endoplasmic reticulum, plasma membrane, Golgi apparatus, and outer mitochondrial membrane, while cytosolic enzyme or isolated enzyme incubated under polymerizing conditions does not penetrate the gradient. These data suggest that the microsomes may be a significant locus of fatty acid synthesis initiated with association of acetyl-CoA carboxylase polymer with this fraction.
Augmentation of human monocyte opsonin-independent phagocytosis by fragments of human plasma fibronectin.
Authors: Czop J K , Kadish J L , Austen K F
Published: May 2006

Human plasma fibronectin isolated by gelatin-affinity chromatography increases in a dose-dependent fashion the number of human monocytes that ingest particulate activators of the human alternative complement pathway in a fully synthetic medium. The fibronectin effect is selective for these particulate activators, does not extend to particles whose ingestion is dependent upon opsonization with IgG, and is not observed with pretreatment of the monocytes. Affinity chromatography with monoclonal antibody to plasma fibronectin of 440,000 daltons reveals that only 12-53% of the protein in a phagocytically active gelatin-affinity-purified fibronectin preparations is bound to the antibody. The protein eluted after affinity chromatography with monoclonal antibody of active preparations, which represented 10-43% of the protein applied, exhibits a 2- to 10-fold increment of activity per microgram of protein above the starting gelatin-affinity-purified material. Thus, the activity that augments the percent of human monocytes ingesting particulate activators of the alternative pathway is antigenically defined as plasma fibronectin. Preparations containing only intact 440,000-dalton fibronectin are also bound to and eluted from the monoclonal antibody, but they fail to augment phagocytosis. When inactive 440,000-dalton plasma fibronectin is subjected to limited trypsin cleavage, phagocytosis-enhancing activity develops that is bound to and elutes from the affinity column prepared with monoclonal antibody, thereby indicating that the enhancing activity of plasma fibronectin resides in cleavage fragments.
Assignment of the human gene for liver-type 6-phosphofructokinase isozyme (PFKL) to chromosome 21 by using somatic cell hybrids and monoclonal anti-L antibody.
Authors: Vora S , Francke U
Published: May 2006

Human 6-phosphofructokinase (PFK; ATP:D-fructose-6-phosphate 1-phosphotransferase, EC 2.7.1.11) is under the control of structural loci that code for muscle (M), liver (L), and platelet (P) subunits, which are variably expressed in different tissues; human diploid fibroblasts and leukocytes express all three genes. Random tetramerization of these subunits produces various isozymes, which can be distinguished from one another by ion exchange chromatography or by subunit-specific monoclonal antibodies. We have examined 17 somatic cell hybrids established between Chinese hamster cells and human diploid fibroblasts or leukocytes for the expression of L-type subunits of human PFK. As electrophoresis does not distinguish between Chinese hamster PFKs and human PFKs, we used an anti-human L-subunit-specific monoclonal antibody, which does not react with chinese hamster PFKs. The expression of human L subunits in the hybrids was detected by the enzyme-immunoprecipitation technique using staphylococci bearing protein A as an immunoadsorbent. Twelve out of 17 hybrids expressed human L subunits and retained chromosome 21, as determined by chromosome and isozyme marker analysis, whereas 5 did not express human PFKL and lacked chromosome 21. The mean erythrocyte PFK of seven individuals with trisomy 21 was found to be elevated (147% of normal). A specific increase in L subunits in trisomic erythrocytes was evident chromatographically by a striking increase in L4 species (50%; normal 10%) and immunologically by decreased precipitation with anti-M monoclonal antibody (50%; normal 80%). We conclude from these data that PFKL is located on chromosome 21 and that the previously noted elevation of erythrocyte PFK activity in individuals with trisomy 21 is due to a gene-dosage effect.
Direct selection procedure for the isolation of functional centromeric DNA.
Authors: Hsiao C L , Carbon J
Published: May 2006

Several plasmids, each containing a segment of DNA capable of conferring mitotic stability on the ars1 vector YRp7 replicating in yeast (Saccharomyces cerevisiae), have been isolated from a yeast genomic library by direct selection for stable plasmid maintenance. The mitotic-stabilizing sequences fall into two classes: (i) segments of the yeast "2-micrometers" plasmid and (ii) DNA segments containing yeast centromeric DNA. Plasmids of the first class segregate 4+:0- through meiosis whereas the centromere plasmids segregate predominantly 2+:2-, as typical chromosomes. Previously identified centromeres from chromosomes II (CEN3) and XI (CEN11), along with the CEN11-linked gene MET14, can be isolated by this direct selection procedure. In addition, several other unidentified centromere DNAs were obtained. There are no detectable sequence homologies between CEN3 and CEN11 DNAs nor do these DNAs hybridize to the other yeast centromere regions.
Retrovirus-encoded transformation-specific polyproteins: expression coordinated with malignant phenotype in cells from different germ layers.
Authors: Chen A P , Essex M , Shadduck J A , Niederkorn J Y , Albert D
Published: May 2006

A transformation-associated polyprotein designated "gag-x" was previously shown to be induced by the feline sarcoma virus (FeSV) after the nonproductive transformation of rat or mink cells. We found that this protein was also expressed in cells derived from the native species (cat) with or without the production of feline leukemia helper virus (FeLV) and that cats could mount a humoral antibody response to the transformation-specific (x) portion of the molecule. Such antisera also reacted with the feline oncornavirus-associated cell membrane antigen (FOCMA) by membrane immunofluorescence. Expression of the gag-x protein was coordinated with malignant phenotype in that both transformed cat fibroblasts and cultured cells from a FeSV-induced melanoma expressed antigenically indistinguishable proteins of the same size. These cells are derived from different embryonic germ layers, suggesting that such transformation-related proteins may function in a pleiotropic manner when introduced by a virus.
Semiclassical model for atoms
Authors: Pearson Ralph G.
Published: May 2006

The energies of several two- and three-electron atoms, in both ground states and excited states, are calculated by a very simple semiclassical model. The only change from Bohr's original method is to replace definite orbits by probability distribution functions based on classical dynamics. The energies are better than Hartree-Fock values. There is still a need for an exchange-energy correction.
New segment-coupling method for peptide synthesis in aqueous solution: application to synthesis of human [Gly17]-beta-endorphin.
Authors: Blake J , Li C H
Published: May 2006

The thiolcarboxyl peptide [17-thiolglycine]-beta-endorphin-(1-17) (I) was synthesized by the solid-phase method. Reaction of peptide I with citraconic anhydride gave citraconyl-[Lys(Cit9,GlyS17]-beta-endorphin-(1-17) (Ia). Peptide Ia was coupled to another synthetic peptide, [Lys(Cit)19,24,28,29]-beta-endorphin-(18-31), by reaction with silver nitrate--N-hydroxysuccinimide in water. All citraconyl groups were removed in aqueous acetic acid, and [Gly17]-beta-endorphin was isolated in 30-40% yield. The synthetic analog had 10% analgesic potency and 59% opiate--receptor binding activity when compared with human beta-endorphin.
Elimination of cannibalistic denaturation by enzyme immobilization or inhibition
Authors: Wu Hua-Lin , Lace Daniel A. , Bender Myron L.
Published: May 2006

The cannibalistic denaturation of α-chymotrypsin (EC 3.4.21.1) around neutral pH can be eliminated by immobilization (insolubilization) of the enzyme or by inhibition by specific reversible inhibitors, but the high-pH denaturation cannot be. The denaturation of the immobilized enzyme at high pH follows first-order kinetics, just as the denaturation of the soluble enzyme does. These results lend credence to the description of the denaturation of chymotrypsin as cannibalistic around neutrality and due to a hydroxide ion reaction at high pH; this interpretation followed from kinetic arguments given in the previous article [Wu, H.-L., Wastell, A. & Bender, M. L. (1981)
Proc. Natl. Acad. Sci. USA
78, 4116-4117]. Elimination of denaturation around neutrality by immobilization may be the reason why membrane-bound enzymes are so common
in vivo
.
Isolation and characterization of adult bovine heart muscle chalones.
Authors: Kriek J A , van der Walt B J , Bester A J
Published: May 2006

A three-step purification procedure for the isolation of heart muscle chalones is described. The method uses ultrafiltration followed by concanavalin A-Sepharose affinity chromatography and subsequent sucrose density gradient centrifugation. Analytical ultracentrifugation of the highly purified chalone fraction indicated a sedimentation coefficient of 16.7 S and an average Mr of 715,527. Chemical analyses have confirmed this protein to be a glycoprotein, and polypeptide analysis indicated the involvement of identical subunits in its composition. In vivo studies confirmed that the chalone isolated from adult bovine hearts inhibits DNA synthesis of newborn hamster hearts. In addition, it was found to be tissue and not species specific and had no cytotoxic effects on the target cells.
Radioimmunochemical measurement of the transferrin receptor in human trophoblast and reticulocyte membranes with a specific anti-receptor antibody.
Authors: Enns C A , Shindelman J E , Tonik S E , Sussman H H
Published: May 2006

A radioimmunoassay was developed to directly assay the presence of transferrin receptors in human tissues. Antisera developed in a goat against purified human placental transferrin binding protein was purified by fractional sodium sulfate precipitation and adsorption against Sepharose-bound transferrin to remove trace anti-transferrin activity. The antisera immunoprecipitates a Mr 94,000 peptide on 125I-iodinated syncytial trophoblast membranes from placentae. This polypeptide has been identified previously as the transferrin binding protein of the placenta [Wada, H. G., Hass, P. E. & Sussman, H. H. (1979) J. Biol. Chem. 254, 12629-12635]. A standard curve using purified 125I-iodinated placental transferrin receptor and various amounts of the purified noniodinated receptor is sensitive from 5 to 900 ng. A reticulocyte-enriched membrane ghost preparation (5% reticulocyte) gives a value of 9.5 micrograms of receptor per mg of protein. Normal erythrocyte membrane ghosts show binding (0.57 micrograms of receptor per mg of protein) proportional to the amount of reticulocytes normally present in blood (0.5-1.0%). In other tissues in which the transferrin receptor binding has been reported, purified syncytial trophoblastic membranes are found to have 34.5 micrograms of receptor per mg of protein, and BeWo cells, a choriocarcinoma cell line, are found to have 15.7 micrograms of receptor per mg of protein. In contrast, normal breast tissue, which has no demonstrated transferrin binding, contains only 0.18 micrograms of receptor per mg of protein by this method.
Mitogenic effect of alkaline pH on quiescent, serum-starved cells.
Authors: Zetterberg A , Engström W
Published: May 2006

The effect of environmental pH on the proliferative activity of sparse 3T3 cell cultures was investigated. When quiescent (serum-starved) cells were transferred to a serum-depleted medium in which pH had been elevated, cell proliferation was stimulated during the first 24 hr after the medium change, as reflected by an increase in cell number and in the proportion of cells synthesizing DNA (measured by autoradiography and flow cytophotometry). The growth-stimulatory effect was greater with increasing pH. At pH 8.2 the effect was approximately 50% of that seen in serum-stimulated cells (i.e., upon the addition of 10% serum). Above pH 8.2 toxic effects and cell death were observed. However, the stimulatory effect could be performed without interference of toxic effects by exposing the cells to the high alkaline environment for a short period. A maximum stimulatory effect on the quiescent cells--without any observed toxic effects--was seen after exposure to an alkaline pulse with a duration of 2-10 min at a pH between 8.5 and 10. More than 80% of the cells synthesized DNA during the first 24 hr after the alkaline-pulse stimulation, which is an almost similar response to that seen after stimulation with serum. The growth-stimulatory effect of the alkaline-pulse treatment could not be prevented by a subsequent treatment at acid pH. Kinetic analysis revealed that DNA synthesis was not initiated until 9-12 hr after the alkaline-pulse treatment. This lag period was of the same length as that seen after stimulation with serum. Alkaline-pulse treatment only resulted in stimulation of DNA synthesis and mitosis during one cell cycle. When the alkaline treatment was repeated, only a small proportion of cells could proceed through a second cell cycle.
Gut bacteria recycle uric acid nitrogen in termites: A strategy for nutrient conservation
Authors: Potrikus Catherine J. , Breznak John A.
Published: May 2006

Reticulitermes flavipes
termites synthesize uric acid via purine-nucleoside phosphorylase (purine-nucleoside: orthophosphate ribosyltransferase, EC 2.4.2.1) and xanthine dehydrogenase (xanthine:NAD
+
oxidoreductase, EC 1.2.1.37), but their tissues lack uricase (urate:oxygen oxidoreductase, EC 1.7.3.3) or any other enzyme that degrades uric acid. Nevertheless, uricolysis occurs in termites, but as an anaerobic process mediated by hindgut bacteria.
14
C-Tracer experiments showed that termites transport uric acid from the site of synthesis and storage (fat body tissue) to the site of degradation (hindgut microbiota) via Malpighian tubules. Moveover, [1,3-
15
N]uric acid dissimilated by gut bacteria
in vivo
leads to assimilation of
15
N into termite tissues. NH
3
, a product of uricolysis, is a potential N source for termites, either directly via glutamine synthetase [L-glutamate:ammonia ligase (ADP-forming), EC 6.3.1.2] activity of fat body tissue or indirectly through microbe assimilation. Symbiotic recycling of uric acid N appears to be important to N conservation in these oligonitrotrophic insects.
Molecular cloning and amplification of the adenylate cyclase gene.
Authors: Wang J Y , Clegg D O , Koshland D E
Published: May 2006

A segment of DNA containing cya, the gene for adenylate cyclase [ATP pyrophosphate-lyase (cyclizing), EC 4.6.1.1], has been isolated from Salmonella typhimurium. The phage lambda gt4 was used as a cloning vector and adenylate cyclase-positive hybrid phages were isolated that complemented adenylate cyclase-negative bacteria. The cloned DNA fragment encodes a polypeptide of molecular weight 81,000 that gives rise to adenylate cyclase activity. This protein represents a functional mutant of the bacterial adenylate cyclase. When the cya gene was amplified by inserting into a multicopy plasmid, the enzyme activity was overproduced 20-fold, but the cyclic AMP level increased only 60%, suggesting several probable regulatory mechanisms. Overproduction of enzymes by recombinant DNA techniques can be a useful probe of relationships in the metabolizing organism in vivo.
Biosynthesis of phosphorylated forms of corticotropin-related peptides.
Authors: Bennett H P , Browne C A , Solomon S
Published: May 2006

Phosphorylated forms of corticotropin[ACTH (1-39)], corticotropin-like intermediary lobe peptide[CLIP, ACTH (18-39)], and the common precursor for ACTH and beta-lipotropin (beta-LPH) have been identified in extracts of rat pituitaries, 32P-Labeled inorganic phosphate was successfully incorporated into ACTH (1-39), CLIP, and the ACTH/beta-LPH precursor in rat neurointermediary lobe explants and into ACTH (1-39) in isolated rat anterior pituitary cells. After peptidase digestion of the labeled CLIP and ACTH, the radioactive phosphate was recoverable as O-phosphoserine. The serine residue at position 31 was the only amino acid found to be phosphorylated in CLIP and ACTH (1-39). The unphosphorylated forms of both peptides were also synthesized. The demonstration of he incorporation of [32P]phosphate into CLIP, ACTH (1-39), and the ACTH/beta-LPH precursor is consistent with the hypothesis that, within the rat intermediary lobe, phosphorylated CLIP is derived from a phosphorylated form of the common precursor, with phosphorylated ACTH (1-39) acting as a biosynthetic intermediate.
A gene coding for tRNA
Val
is located near 5′ terminus of 16S rRNA gene in
Zea mays
chloroplast genome
Authors: Schwarz Z. , Kössel H. , Schwarz E. , Bogorad L.
Published: May 2006

A region of 635 base pairs preceding a gene for 16S rRNA in the
Zea mays
chloroplast genome has been mapped and its sequence has been determined. Screening for structural elements common to tRNAs reveals a gene coding for tRNA
Val
(GU
U
C
) positioned 303 base pairs proximal to the 5′ end of the 16S rRNA gene. Both the tRNA
Val
and the 16S rRNA are coded in the same DNA strand. The tRNA nucleotide sequence predicted from the DNA sequence meets all structural characteristics common to tRNA primary and secondary structures. In a quantitative comparison with primary structures of the 14 known tRNA
Val
species the chloroplast isoaceptor shows much higher homology with that from prokaryotic than that from eukaryotic species. Regions that
Escherichia coli
RNA polymerase protects from nuclease attack are observed 25 and 100 base pairs upstream of the tRNA
Val
gene and 105 base pairs upstream of the 16S rRNA gene. Within these regions are short sequences that are very similar to those in the -35 region of
E. coli rrn
and that may therefore represent all or parts of transcription initiation signals of the respective genes.
Z-DNA: vacuum ultraviolet circular dichroism.
Authors: Sutherland J C , Griffin K P , Keck P C , Takacs P Z
Published: May 2006

In concentrated salt or ethanolic solutions, the self-complementary copolymer poly(dG-dC).poly(dG-dC) forms a left-handed double-helical structure that has been termed "Z-DNA." The first evidence for this structure came from changes observed in the circular dichroism (CD) spectrum between 230 and 300 nm for low- and high-salt solutions (Pohl, F. M. & Jovin, T. M. (1972) J. Mol. Biol. 67, 675-696). In 3 M NaCl, the CD spectrum is approximately inverted compared to the B-form spectrum observed in low-salt solution. We measured the vacuum ultraviolet CD spectrum of poly(dG-dC).poly(dG-dC) down to 180 nm under conditions in which the 230- to 300-nm spectrum is inverted. Below 200 nm, where the B form exhibits the large positive peak at 187 nm that is characteristic of right-handed double-helical DNAs, the Z form exhibits a large negative peak at 194 nm and a positive band below 186 nm. Therefore, the Z-form vacuum ultraviolet CD spectrum resembles an inverted and red-shifted B-form spectrum. The magnitudes of the differences observed between the B and Z forms in the CD spectrum below 200 nm are about 10 times greater than those observed between 230 and 300 nm. The vacuum ultraviolet CD spectrum of poly(dG-dC).poly(dG-dC) in 3 M Cs2SO4 also is inverted compared to the B-form spectrum; however, between 230 and 300 nm, it is nonconservative with a negative maximum at 290 nm and a weak positive CD signal above 300 nm, presumably reflecting differential light scattering and indicating the existence of molecular aggregates. Our results suggest that the vacuum ultraviolet CD spectrum is sensitive to the handedness of double-helical DNA structures. The CD spectrum in this region should complement other spectroscopic methods in relating the structures of poly(dG-dC).poly(dG-dC) existing in solution to those determined in the solid state by x-ray crystallography.
Promoter sequence of fibroin gene assigned by in vitro transcription system.
Authors: Tsujimoto Y , Hirose S , Tsuda M , Suzuki Y
Published: May 2006

We have shown that the silk fibroin gene from Bombyx mori is faithfully transcribed in an in vitro transcription system of the HeLa cell extract prepared by the method of Manley et al. [Manley, J. L., Fire, A., Cano, A., Sharp, P. A. & Gefter, M. L. (1980) Proc. Natl. Acad. Sci. USA 77, 3855-3859]. Using this system and a series of deletion mutants of fibroin gene, we have assigned the promoter sequence of fibroin gene. The 5' boundary of the promoter is around nucleotide position -29, indicating that most of the T-A-T-A-A-A-A sequence (-30 to -24) is essential for the promoter function, where the transcription initiation point of fibroin gene is assigned as nucleotide position +1 [Tsuda, M., Ohshima, Y. & Suzuki, Y. (1979) Proc. Natl. Acad. Sci. USA 76, 4872-4876]. The 3' boundary is around nucleotide position +6. However, to support the efficient, faithful transcription, some additional (more than 26 but less than 41) nucleotides of nonspecific origin are required at the 5' side of -29. Functions ascribed to the promoter region are discussed.
Selective hormonal control of myo-inositol biosynthesis in reproductive organs and liver of the male rat.
Authors: Hasegawa R , Eisenberg F
Published: May 2006

myo-Inositol biosynthesis has been examined in hypophysectomized and thyroidectomized male rats. After hypophysectomy, inositol-1-phosphate synthase [1L-myo-inositol-1-phosphate lyase (isomerizing), EC 5.5.1.4] in the reproductive organs and liver decreased markedly. At the same time, testicular acid phosphatase [orthophosphoric-monoester, phosphohydrolase (acid optimum), EC 3.1.3.2] and beta-glucuronidase (beta-D-glucuronide glucuronosohydrolase, EC 3.2.1.31) increased. Thyroidectomy caused a similar decrease in inositol-1-phosphate synthase in the liver but not in the reproductive organs. Follicle-stimulating in the liver but not in the reproductive organs. Follicle-stimulating hormone (follitropin) and luteinizing hormone (lutropin) restored the activity to at least normal levels in the testis, prostate, and seminal vesicle but not in the liver of hypophysectomized animals. Triiodothyronine and thyroxine stimulated liver synthase 30-fold in hypophysectomized animals. We conclude that inositol-1-phosphate synthase in the reproductive organs is under more or less direct control of the pituitary; in the liver, the control is mediated through the thyroid.
Structural variation among human beta-tubulin genes.
Authors: Cowan N J , Wilde C D , Chow L T , Wefald F C
Published: May 2006

A chicken beta-tubulin cDNA probe has been used to screen two independently generated human genomic libraries. Of 13 EcoRI fragments detectable in a human genomic Southern blot experiment, 7 correspond in size to EcoRI fragments isolated from recombinant bacteriophage. The location of beta-tubulin-specific regions and the direction of transcription were determined within each cloned fragment. One clone (5 beta) contained a beta-tubulin-specific region of 6.8 kilobase pairs (kbp) that included three intervening sequences as well as a number of inverted repeat structures. The remaining clones contained beta-tubulin-specific sequences that were close to or, in two cases, substantially less than 1.9 kbp long. Because mature human beta-tubulin mRNA is approximately 1.9 kbp long, these short DNA regions cannot on their own encode a functional beta-tubulin mRNA. Analysis using 3'- and 5'-specific probes derived from the chicken cDNA clone showed the presence of both of these end regions within one truncated tubulin-like sequence. A second short tubulin-specific region failed to hybridize with a 3'-specific probe. These short sequences are therefore likely to be examples of pseudogenes that have arisen by loss of a portion of DNA essential to the production of functional human beta-tubulin mRNA.
Identification and cloning of endogenous retroviral sequences present in human DNA.
Authors: Martin M A , Bryan T , Rasheed S , Khan A S
Published: May 2006

Using nonstringent annealing conditions and a 2.75-kilobase segment of cloned African green monkey DNA that specifically hybridizes to the proviruses of AKR ecotropic murine leukemia virus (MuLV) and baboon endogenous virus (BaEV) as a probe, we detected related sequences in three different preparations of human brain DNA fragments. The blot-hybridization pattern obtained with cleaved human DNA was similar to that previously reported for the interaction of MuLV cDNA and cleaved mouse DNA and suggested the presence of numerous copies of retrovirus-related sequences in the human genome. The labeled 2.75-kilobase fragment derived from cloned monkey DNA was used to screen a human DNA library in Charon 4A. One clone obtained hybridized to three contiguous MuLV-and BaEV-reactive fragments of the cloned monkey DNA and to multiple fragments of human DNA including a prominent 1.0-kilobase EcoRI fragment also present in the clone.
Human--rat muscle somatic cell hybrids form myotubes and express human muscle gene products.
Authors: Quinn C A , Goodfellow P N , Povey S , Walsh F S
Published: May 2006

Somatic cell hybrids have been prepared at high frequency between the rat muscle cell line L6 and human fetal muscle cells. The hybrid cells express several human gene products including an antigen, 12E7, controlled by the human X chromosome, Thy-1, and several human isoenzymes. In addition, one clone (37-11) expresses a human muscle-specific surface antigen (5.1 H11) and, under appropriate conditions, can be induced to form myotubes. Upon myotube formation, this clone switches on the production of the human muscle-specific creatine kinase (ATP:creatine N-phosphotransferase, EC 2.7.3.2) M subunit. This is an example of developmental regulation of human muscle-specific genes in somatic cell hybrids.