bluuebunny/crossref_metadata_2025 huggingface dataset:
Tubulins from different higher plant species are immunologically nonidentical and bind colchicine differentially
Authors: Morejohn Louis C. , Bureau Thomas E. , Tocchi Lee P. , Fosket Donald E.
Published: May 2006

We have initiated immunological and drug-binding studies on the tubulins from different higher plant species. Antibodies were raised against electrophoretically separated rose (
Rosa
sp.) tubulin α- and β-subunits and characterized by immunoblot autoradiographic assays. Each IgG preparation bound to its antigen and cross-reacted differentially with the respective tubulin subunits from an alga, sea urchin, rabbit, and cow. Antigenic determinants were shared more among the β-subunits than among the α-subunits from these organisms. Tubulins were isolated from cultured cells of carrot (
Daucus carota
) and hibiscus (
Hibiscus rosa-senensis
). Immunoautoradiography and quantitation of cross-reactivity on blots showed nonidentity among homologous subunits from rose, carrot, hibiscus, and alga tubulins, with more antigenic differences among α-subunits than among β-subunits. Comparative colchicine-binding assays showed that rose and hibiscus tubulins bound 33% and 65%, respectively, of the colchicine bound by carrot tubulin and that higher plant tubulins bound much less colchicine than bovine brain tubulin under identical conditions.
Balbiani ring induction in phosphate metabolism
Authors: Galler R. , Rydlander L. , Riedel N. , Kluding H. , Edström J.-E.
Published: May 2006

Balbiani rings (BR), giant puffs in
Chironomus
larval salivary glands, code for giant secretory proteins. As shown earlier, the normally dominant BR2 is turned off with its putative translation product during exposure of larvae to compounds that diminish the stores of P
i
. A BR6 develops from a compact chromosome band, and a new giant protein appears in the secretion as the major component. We have determined the sequence of cloned DNA fragments representative for large parts of BR1 and BR2 (normally active) and the inducible BR6. There is an excess of positive charges and high contents of serine/threonine in the coded amino acid composition for the BR1 and BR2 sequences. The coded amino acid sequence for the BR6 clone shares homologies with the others but has an excess of negative charges and lacks serine/threonine. This suggested that the P
i
effects observed earlier could be related to differences in phosphorylation between the normal proteins and the BR6 product. This could be confirmed by measurements of phosphorylation, which occurs in the normal giant proteins mainly at seryl residues.
P
export with giant secretory protein is normally quantitatively important. Thus, BR6 activation should decrease
P
loss when P
i
pools are lowered because of inducer action.
Human dihydrofolate reductase gene is located in chromosome 5 and is unlinked to the related pseudogenes.
Authors: Maurer B J , Barker P E , Masters J N , Ruddle F H , Attardi G
Published: May 2006

The chromosomal location of the human dihydrofolate reductase (DHFR; EC 1.5.1.3) gene that is amplified in a methotrexate-resistant human cell line has been investigated by screening a large number of human-mouse cell hybrids containing overlapping subsets of human chromosomes. A correlation of genomic blotting data with the chromosome constitution of the individual cell hybrids has allowed the assignment of the human DHFR gene to chromosome 5. This chromosome assignment has been confirmed by the observation of a concomitant loss of the human DHFR gene and of sensitivity to diphtheria toxin, a marker associated with chromosome 5, in two human-mouse cell hybrids selected for resistance to the toxin. Six EcoRI fragments of human DNA containing DHFR pseudogenes or other DHFR-related sequences have been assigned to chromosomes other than chromosome 5.
Coding nucleotide sequence of 3-methylcholanthrene-inducible cytochrome P-450d cDNA from rat liver.
Authors: Kawajiri K , Gotoh O , Sogawa K , Tagashira Y , Muramatsu M , Fujii-Kuriyama Y
Published: May 2006

We determined the coding nucleotide sequence of the mRNA for a 3-methylcholanthrene-inducible cytochrome P-450, P-450d, of rat liver by sequence analysis of cloned cDNAs. The predicted amino acid sequence of the cytochrome is composed of 513 amino acids, and its NH2-terminal sequence of 30 amino acids completely coincides with that reported from analysis of the purified cytochrome P-450d. The amino acid composition of the deduced sequence also agrees well with that determined from the purified protein. Computer-aided analysis was carried out to compare the complete primary structures of five species of cytochrome P-450, two molecular species of phenobarbital-inducible rat liver cytochrome P-450 (P-450b and P-450e), phenobarbital-inducible rabbit liver cytochrome P-450LM2, 3-methylcholanthrene-inducible rat liver cytochrome P-450d, and camphor-hydroxylating P-450 of Pseudomonas putida (P-450CAM). It is concluded therefrom that the time of divergence between cytochrome P-450b (P-450e) and P-450d is much earlier than that of branching between phenobarbital-inducible cytochromes P-450 of rat and rabbit. One highly conserved cysteine-containing region that is close to the COOH terminus is found in all of these cytochrome P-450 sequences, indicating that the heme-binding site is the cysteine residue in this region.
Biogenesis of 3-hydroxy-3-methylglutaryl-coenzyme A reductase, an integral glycoprotein of the endoplasmic reticulum.
Authors: Brown D A , Simoni R D
Published: May 2006

Using a cell line, C100, that overproduces 3-hydroxy-3-methylglutaryl-coenzyme A reductase (HMG-CoA reductase; EC 1.1.1.34) 100-fold, we have studied the synthesis and insertion of this protein into the endoplasmic reticulum. The enzyme is synthesized on membrane-bound polysomes. It is cotranslationally but not post-translationally inserted into dog pancreatic microsomes. This cotranslational insertion is dependent upon signal recognition particle. HMG-CoA reductase is glycosylated with an oligosaccharide(s) of the "high-mannose" type sensitive to endo-beta-D-N-acetylglucosaminidase H. Partial determination of the NH2-terminal amino acid sequence of the in vitro translation product and the mature polypeptide indicate they are the same and demonstrate there is no cleavage of an NH2-terminal signal sequence.
Evidence for rotational contribution to protein-facilitated proton transport.
Authors: Gros G , Lavalette D , Moll W , Gros H , Amand B , Pochon F
Published: May 2006

Two modes of molecular motion of carrier molecules can, in principle, lead to a facilitated transport of a substrate: translational and rotational diffusion. In the present study, which deals with the mechanism of the facilitated diffusion of H+ and O2 in solutions of earthworm hemoglobin, examples for both types of facilitation are presented. Only translational, not rotational, diffusion of earthworm hemoglobin appears to lead to a facilitated O2 flux. In contrast, substantial facilitated H+ fluxes of comparable size arise from rotational diffusion as well as from translational diffusion of this large protein. This is derived from measurements of facilitated H+ and O2 fluxes in earthworm hemoglobin solutions and determinations of the rotational and translational diffusion coefficients of earthworm hemoglobin with the help of a theoretical treatment of facilitated diffusion by rotational carrier diffusion. H+ transport by rotational protein diffusion appears to be a case where the often-postulated mechanism of facilitated transport by rotation of a carrier lends itself to experimental verification.
A model for the origin of biological catalysis.
Authors: Stein D L , Anderson P W
Published: May 2006

We propose a mathematical model for the next stage in the origin of life after that treated in our earlier work. At this stage we introduce the possibility of the modification of the environment by the information-containing entities and feedback between the environment and the population of macromolecules and hence provide a model for the development of the Eigen hypercycle.
Stimulation of human granulocyte function by monoclonal antibody WEM-G1.
Authors: López A F , Vadas M A
Published: May 2006

From a panel of monoclonal antibodies (MAb) produced against purified human neutrophils, MAb WEM-G1 was selected for its ability to stimulate granulocyte function as assessed by the capacity to kill antibody-coated tumor target cells. MAb WEM-G1 bound to neutrophils and eosinophils and not to monocytes, lymphocytes, or erythrocytes, and thus identified a granulocyte differentiation antigen. It enhanced killing by both neutrophils and eosinophils in a dose-dependent fashion, with a range of effector-to-target ratios and dilutions of anti-target cell antibody. The effect of MAb WEM-G1 was additive with that of colony-stimulating factor (CSF-alpha). Similarly, the ability of neutrophils to exhibit enhanced cytotoxicity in the presence of WEM-G1 was increased by preincubation of neutrophils with CSF-alpha. Immunoprecipitation analysis showed that WEM-G1 identified a cell-surface protein on human neutrophils of approximate molecular weight 110,000. It is suggested that this structure is involved in regulation of the function of human granulocytes.
Differential effects of the mottled yellow and pseudoagouti phenotypes on immunocompetence in Avy/a mice.
Authors: Roberts D W , Wolff G L , Campbell W L
Published: May 2006

Whereas genetic factors are known to influence both susceptibility to carcinogens and the capacity to respond to specific antigenic stimuli, little is known regarding the influence of phenotype per se. With this in mind, we utilized (YS x VY)F1 hybrid mice consisting of genetically identical (Avy/a) but phenotypically distinct (mottled yellow and pseudoagouti) mice as well as congeneic (a/a) black mice to evaluate immunocompetence as a function of genotype and phenotype. Mottled yellow Avy/a mice, which are fat and have increased susceptibility to neoplasia, presented an immunologic profile characterized by decreased antibody response to the T-cell-dependent immunogen tetanus toxoid, enhanced antibody response to the T-cell-independent immunogen type III pneumococcal polysaccharide, decreased (unadjusted) rates of carbon clearance, and increased levels of serum IgA. In contrast, pseudoagouti Avy/a mice were immunologically comparable to black a/a mice. The data demonstrate that differences in phenotypic expression between genetically identical mice may significantly alter immune function.
Mouse primase initiation sites in the origin region of simian virus 40.
Authors: Tseng B Y , Ahlem C N
Published: May 2006

The sites of initiation of DNA synthesis by purified mouse DNA primase in the origin-of-replication region of simian virus 40 (SV40) were examined. Using as template the separated strands of a cloned fragment of SV40 approximately equal to 300 base pairs (bp) long that includes the origin, we observed specific sites of initiation on the two strands. On the early strand that is the template for early mRNA synthesis, the primary starts are at four positions within 10 nucleotides of each other around nucleotide 5215 and an additional site around nucleotide 5147 that is used at one-sixth the frequency of the major sites. The major start sites on the early strand are within the 65-bp minimal origin of replication and lie between tumor antigen binding sites I and II. On the late strand that is the template for late mRNA synthesis, six major initiation sites were observed, each within the 3' C-C-C-G-C-C 5' sequence in the template that is repeated twice within each of the three 21-bp repeats that lie adjacent to the minimal origin, on its late side. A 6-bp deletion in the 65-bp minimal origin that eliminates its function as an origin reduced the major initiations around nucleotide 5215 on the early strand by 90% but did not affect initiations at the minor start site on the early strand or initiations on the late strand. Mouse DNA primase is able to recognize specific regions on the SV40 DNA. Those on the early strand are within the minimal origin of replication and those on the late strand are within the 21-bp repeat region necessary for maximum replication.
Birth of a unique enzyme from an alternative reading frame of the preexisted, internally repetitious coding sequence.
Authors: Ohno S
Published: May 2006

The mechanism of gene duplication as the means to acquire new genes with previously nonexistent functions is inherently self limiting in that the function possessed by a new protein, in reality, is but a mere variation of the preexisted theme. As the source of a truly unique protein, I suggest an unused open reading frame of the existing coding sequence. Only those coding sequences that started from oligomeric repeats are likely to retain alternative long open reading frames. Analysis of the published base sequence residing in the pOAD2 plasmid of Flavobacterium Sp. K172 indicated that the 392-amino acid-residue-long bacterial enzyme 6-aminohexanoic acid linear oligomer hydrolase involved in degradation of nylon oligomers is specified by an alternative open reading frame of the preexisted coding sequence that originally specified a 472-residue-long arginine-rich protein.
Human T-cell growth factor: partial amino acid sequence, cDNA cloning, and organization and expression in normal and leukemic cells.
Authors: Clark S C , Arya S K , Wong-Staal F , Matsumoto-Kobayashi M , Kay R M , Kaufman R J , Brown E L , Shoemaker C , Copeland T , Oroszlan S
Published: May 2006

The partial amino acid sequences of human T-cell growth factors (TCGFs) isolated from normal peripheral blood lymphocytes and from a leukemia T-cell line (Jurkat) show that the amino-terminal sequences of the two proteins (15 residues) are identical. Oligonucleotides based on the published Jurkat TCGF DNA sequence were used to isolate six cDNA clones of TCGF mRNA from normal lymphocytes. The predicted amino acid sequence of normal lymphocyte TCGF was identical to the sequence of the Jurkat protein, showing that the differences in biochemical properties of the two proteins result from post-translational events. Amino acid and nucleotide sequence data suggest that TCGF is derived from a precursor polypeptide that is cleaved at the amino terminus but not at the carboxyl terminus. Hybridization of the cloned lymphocyte TCGF cDNA to cellular DNA and RNA strongly suggested that the TCGF gene is expressed as a single mRNA species from a single-copy gene. No differences in the organization of the TCGF gene in normal, leukemic, and human T-cell leukemia/lymphoma virus-infected cells was detected regardless of whether they produce TCGF or not.
Control of biologically active interleukin 2 messenger RNA formation in induced human lymphocytes.
Authors: Efrat S , Kaempfer R
Published: May 2006

The regulation of human interleukin 2 (IL-2) mRNA production in induced normal lymphocytes was studied by following the expression of isolated mRNA in microinjected oocytes of Xenopus laevis. Mitogenic stimulation results in the appearance of greatly increased levels of IL-2 mRNA activity. This process requires de novo transcription. Induction is followed promptly by a shutoff of active IL-2 mRNA formation. This shutoff requires the synthesis of a protein repressor and can be prevented by cycloheximide, an inhibitor of translation. The presence of cycloheximide leads to extensive superinduction of IL-2, concomitant with an increase in active IL-2 mRNA formation up to 30-fold over normal levels. The repressor appears to be short-lived, as the addition of cycloheximide after shutoff leads to an immediate resumption of active IL-2 mRNA formation. The shutoff mechanism is restored rapidly upon removal of cycloheximide. The repressed state is readily reversed also by reinduction of the cells, even soon after shutoff has occurred, without a refractory period. The accumulated active IL-2 mRNA decays with a half-life of about 20 hr. The net result is the generation of a relatively short wave of IL-2 mRNA activity, demonstrating the tight control of IL-2 gene expression.
Measurement of the repulsive force between polyelectrolyte molecules in ionic solution: hydration forces between parallel DNA double helices.
Authors: Rau D C , Lee B , Parsegian V A
Published: May 2006

We have measured the repulsive force between B-form double helices in parallel packed arrays of polymer-condensed DNA in the presence of 0.005-1.0 M ionic solutions. Molecular repulsion is consistently exponential with a 2.5-3.5 A decay distance, when the separation between DNA surfaces is 5-15 A. Only weakly dependent on ionic strength and independent of molecular size, this intermolecular repulsion does not obey the predictions of electrostatic double-layer theory. Rather, it strongly resembles the "hydration force" first recognized and quantified between phospholipid bilayers. Only beyond 15 A separation between molecules is there evidence of electrostatic double-layer forces. The quantitative failure of electrostatic double-layer theory seen here must gravely affect accepted analyses of other polyelectrolyte systems. Because the packing of condensed DNA resembles the spacings of DNA in many bacteriophages, our results permit estimation of the "DNA pressure" in phage heads.
Mechanism of the concerted action of recA protein and helix-destabilizing proteins in homologous recombination.
Authors: Muniyappa K , Shaner S L , Tsang S S , Radding C M
Published: May 2006

Secondary structure in single-stranded DNA impedes the presynaptic association of recA protein and consequently blocks the formation of joint molecules as evidenced by effects of temperature, nucleotide sequence, and ionic conditions. Escherichia coli single-strand-binding protein eliminates sequence-specific "cold spots" by removing folds even from sites of strong secondary structure. Thus, destabilization of secondary structure in single-stranded DNA is critical for the action of recA protein, whereas specific interactions directly between helix-destabilizing proteins and recA protein are unimportant.
Stimulation of recombination between homologous sequences on plasmid DNA and chromosomal DNA in Escherichia coli by N-acetoxy-2-acetylaminofluorene.
Authors: Luisi-DeLuca C , Porter R D , Taylor W D
Published: May 2006

A plasmid containing a wild-type lac operon and a tetracycline-resistance gene was covalently modified by N-acetoxy-2-acetylaminofluorene and used to transform two series of Lac- Escherichia coli cell types. Each set contained wild-type and repair-deficient mutants. One set of cells contained a lacY mutation and the other a deletion of the entire lac operon. Survival and mutagenesis of the plasmid were measured as a function of the N-acetoxy-2-acetylaminofluorene concentration. The results indicate that when no homologous sequences are present in the chromosomal DNA, mutations occur at a low frequency: at 10% survival the frequency was 1-2 X 10(-4) mutants per transformant. When homologous sequences, the lacY allele, are present in the chromosomal DNA, Lac- plasmids are found at a high frequency in a recA-dependent, lexA-independent fashion: at 10% survival the frequency was 5-10 X 10(-2) mutants per transformant. Southern blot analysis of the restriction enzyme profiles of the resulting plasmid and host-cell DNA sequences showed recombinational transfer of host sequences to the N-acetoxy-2-acetylamino-fluorene-treated plasmid had occurred. When the host chromosomes contained Lac+ homologous sequences no mutants were found, indicating that the results were not caused by error-prone recombination.
Pseudotypes of human T-cell leukemia virus types 1 and 2: neutralization by patients' sera.
Authors: Clapham P , Nagy K , Weiss R A
Published: May 2006

Pseudotypes of vesicular stomatitis virus (VSV) bearing envelope antigens of human T-cell leukemia virus (HTLV) types 1 and 2 were prepared by propagating VSV in cells lines productively infected with HTLV. Plaque assays of VSV (HTLV) pseudotypes were employed to determine the presence of (i) HTLV receptors on cells and (ii) neutralizing antibodies in the serum of patients with adult T-cell leukemia-lymphoma (ATLL). Cell surface receptors for HTLV-1 and HTLV-2 were found on nonlymphoid cells of human and mammalian origin. Neutralizing antibodies specific to VSV(HTLV-1) were found in sera of ATLL patients in titers varying from 1:50 to 1:30,000 and did not correlate closely with antibody titers for internal viral antigens. Sera from ATLL patients in the United Kingdom (Caribbean immigrants), United States, and Japan completely neutralized VSV (HTLV-1), indicating that the HTLV isolates from these distinct geographic regions represent a single envelope serotype. Neutralization of VSV (HTLV-1) was more specific and more sensitive than assays of syncytium inhibition. No cross-neutralization was observed between bovine leukosis virus and HTLV, and only limited cross-reaction was found for envelope antigens of HTLV-1 and HTLV-2. These studies show that VSV (HTLV) pseudotypes can be readily used to screen for neutralizing antibodies in patients' sera and to distinguish HTLV envelope serotypes.
Mechanism of the inhibition of the gamma-carboxylation of glutamic acid by N-methylthiotetrazole-containing antibiotics.
Authors: Lipsky J J
Published: May 2006

Antibiotics that contain a 1-N-methyl-5-thiotetrazole (MTT) side group have been associated with hypoprothrombinemia. In a detergent-treated rat liver microsomal system, MTT inhibited the carboxylation of the gamma carbon of glutamic acid, a necessary reaction in the synthesis of four of the clotting factors. In the present work, the inhibition by MTT was found to be slow in onset, with a lag time of 15 min before significant inhibition occurred. A preincubation of MTT with the microsomes decreased the lag time and increased the extent of inhibition. Glutathione at 1 mM was found to markedly decrease the ability of MTT to inhibit this reaction. The disulfide dimer of MTT was a more potent inhibitor of the system than was MTT, with inhibition detected as low as 1 microM dimer. Disulfiram also inhibited the carboxylation system. These results indicate that the sulfhydryl group of MTT is important for the inhibitory effect of MTT and suggest that a slowly formed metabolite of MTT may be directly responsible for the observed inhibition. The inhibitory mechanism of MTT may be analogous to that of disulfiram, which would explain some pharmacologic effects in common with disulfiram. In addition, the in vitro observations presented here and a closer examination of the clinical evidence raise the possibility that MTT-containing antibiotic-induced hypoprothrombinemia may not be a vitamin K reversible phenomenon.
The structure of bovine brain myelin proteolipid and its organization in myelin.
Authors: Laursen R A , Samiullah M , Lees M B
Published: May 2006

A model, based on amino acid sequence data, is proposed for the organization of the myelin proteolipid in myelin membrane. The model has three distinctive features: three trans-membrane segments that traverse the lipid bilayer, two cis-membrane domains that enter and exit the same side of the membrane, and a highly charged segment resembling myelin basic protein on the cytoplasmic side of the membrane. It is proposed that the cis-membrane domain(s) can promote the formation and stabilization of the multilamellar myelin structure by hydrophobic interaction with the apposite bilayer across the extracellular space.
Molybdate reduction by Escherichia coli K-12 and its chl mutants.
Authors: Campbell A M , del Campillo-Campbell A , Villaret D B
Published: May 2006

During anaerobic growth, Escherichia coli can reduce phosphomolybdate. The reduction can also be carried out by washed cells suspended in buffer at pH 5.7. Phosphate, molybdate, glucose, cells, and anaerobic conditions are required. Reduction is inhibited by 200 microM chromate, 290 microM nitrite, 10 mM tungstate, or 20 mM cysteine. Wild-type (chl+) cells are inhibited by addition of 200 microM nitrate, but chlA, chlB, and chlE mutants are not. The inhibition of chl+ cells results from reduction of nitrate to nitrite. This nitrate reduction is not catalyzed by nitrate reductase. Wild-type cells are more sensitive than chl mutants to inhibition by nitrite and cysteine but more resistant to chromate. Pregrowth of chlD cells in 1 mM Na2MoO4 increases their sensitivity to nitrite and cysteine, and pregrowth of chl+ cells in 1 mM Na2MoO4 increases their resistance to these agents. Assays of biotin sulfoxide reductase show that the tightness of the chlD block depends on growth conditions; chlD cells grown aerobically in tryptone broth make about 50% as much active enzyme as chl+ cells, whereas chlD cells grown anaerobically with tryptone plus glucose make less than 10%. The effect of anaerobic pregrowth on the inhibition of molybdate reduction by added nitrate indicates that in vivo nitrate reduction responds to growth conditions in the same manner as biotin sulfoxide reductase does.
Conformationally restricted analogs of somatostatin with high mu-opiate receptor specificity.
Authors: Pelton J T , Gulya K , Hruby V J , Duckles S P , Yamamura H I
Published: May 2006

A series of cyclic, conformationally restricted analogs of somatostatin have been prepared and tested for their ability to inhibit the binding of [3H]naloxone and [D-Ala2, D-Leu5] [3H]enkephalin to rat brain membranes. The most potent analog, D-Phe-Cys-Tyr-D-Trp-Lys-Thr-Pen-Thr-NH2 where Pen is penicillamine in [D-Phe5, Cys6, Tyr7, D-Trp8, Pen11]somatostatin-(5-12)-octapeptide amide, exhibited high affinity for mu-opiate receptors (IC50 value of [3H]naloxone = 3.5 nM), being 7800 times more potent than somatostatin. The cyclic octapeptide also displayed high mu-opiate receptor selectivity with an IC50 [( D-Ala2,D-Leu5]enkephalin)/IC50 (naloxone) ratio of 271. The high affinity and selectivity of the somatostatin analog for mu-opiate receptors may be of use in examining the physiological role(s) of the mu-opiate receptor.
Isolation of tyrosine-O-sulfate by Pronase hydrolysis from fibronectin secreted by Fujinami sarcoma virus-infected rat fibroblasts.
Authors: Liu M C , Lipmann F
Published: May 2006

In a recent paper, we reported the loss of large amounts of protein-bound tyrosine sulfate after infection of rat fibroblasts by avian sarcoma viruses. The analogy to the reported loss of surface fibronectin on malignant transformation, which contained sulfate of unknown location, called our attention to this compound. In a previous paper, we briefly reported on isolation from the supernatant fraction of rat fibroblasts infected by Fujinami sarcoma virus fibronectin that yielded tyrosine-O-sulfate on Pronase hydrolysis. In this paper, we confirm and enlarge on this observation. Highly purified fibronectin was obtained from the supernatant fraction secreted by Fujinami sarcoma virus infected rat fibroblasts that contained 1.52 residues of sulfated tyrosine per protein molecule after exhaustive Pronase hydrolysis. Assuming some loss during work up, this probably indicates 2 residues of the tyrosine sulfated per fibronectin molecule.
Transcription of three c-myc exons is enhanced in chicken bursal lymphoma cell lines.
Authors: Linial M , Groudine M
Published: May 2006

The chicken c-myc gene, as defined by its homology to the v-myc gene of MC29 virus, is comprised of two exons. Using the techniques of runoff transcription, primer extension, and S1 nuclease protection, we demonstrate that there is a third c-myc exon of approximately equal to 345 base pairs (bp) located 0.7 kbp upstream of the 5' end of the v-myc homology. This first exon is transcribed and present in myc mRNA in normal chicken cells. We also examined RNA from five cell lines derived from avian leukosis virus-induced bursal lymphomas. In all these lines, the level of transcription of the 2.2- to 2.5-kbp myc mRNA is increased 30- to 60-fold over normal cells. The myc mRNA in four of these lines also contains increased levels of the first noncoding exon, and evidence is presented that the long terminal repeat (LTR) in the vicinity of c-myc is functioning as an enhancer of c-myc transcription rather than as a promoter in several of these cell lines. In two cell lines in which the viral LTR has integrated between the first and second exons in the proper orientation for downstream promotion of myc, the LTR does not exhibit promoter function. The pattern of c-myc transcription observed by others in a vast majority of avian leukosis virus-induced neoplasms is not observed in any of the five cell lines examined.
Castanospermine inhibits alpha-glucosidase activities and alters glycogen distribution in animals.
Authors: Saul R , Ghidoni J J , Molyneux R J , Elbein A D
Published: May 2006

Castanospermine, an inhibitor of alpha-glucosidase activity, was injected into rats to determine its effects in vivo. Daily injections of alkaloid, at levels of 0.5 mg/g of body weight, or higher, for 3 days decreased hepatic alpha-glucosidase to 40% of control values, whereas alpha-glucosidase in brain was reduced to 25% of control values and that in spleen and kidney was reduced to about 40%. In liver, both the neutral (pH 6.5) and the acidic (pH 4.5) alpha-glucosidase activities were inhibited, but the former was more susceptible. On the other hand, beta-N-acetylhexosaminidase activity was elevated in the livers of treated animals, whereas beta-galactosidase activity was unchanged and alpha-mannosidase activity was somewhat inhibited. Livers of treated animals were examined by light and electron microscopy and compared to control animals to determine whether changes in morphology had occurred. In treated animals fed normal rat chow, the hepatocytes were smaller in size and simplified in structure, whereas the high-glucose diet lessened these alterations. Furthermore, in those animals receiving castanospermine at 1.0 mg or higher per g of body weight for 3 days, there was a marked decrease in the amount of glycogen in the cytoplasm, while a large number of lysosomes were observed that were full of dense, granular material. That this dense material was indeed glycogen was shown by the fact that it disappeared when blocks of fixed tissue were pretreated with alpha-amylase. Glycogen levels in liver, as measured either colorimetrically or enzymatically, were somewhat depressed at the higher levels of castanospermine.
Modulation of stoichiometry of the sarcoplasmic reticulum calcium pump may enhance thermodynamic efficiency.
Authors: Gafni A , Boyer P D
Published: May 2006

The coupling of calcium transport to ATP hydrolysis in rabbit muscle sarcoplasmic reticulum vesicles was determined under steady-state conditions in the presence of 5 mM oxalate and using various concentrations of vesicles to modulate the concentration of free Ca2+ in the medium. This experimental approach takes advantage of the fact that at high concentrations of vesicles the slow rate of liberation of Ca2+ from its oxalate complex becomes rate limiting for pumping, therefore pushing the steady-state levels of this cation to very low values. A reduction in the number of calcium ions transported per ATP cleaved from a value near 2 at a low concentration of vesicles (high medium Ca2+ concentration) to a limiting value of about 1 at a very high concentration of vesicles (low medium Ca2+ concentration) was observed. A marked decrease in the specific ATPase activity was also found to take place as the concentration of the sarcoplasmic reticulum vesicles was increased to high levels and the concentration of medium Ca2+ declined. The data presented indicate that binding of 1 Ca2+ to the sarcoplasmic reticulum ATPase is sufficient to activate the pump. Furthermore, these findings support the existence of a control mechanism for the calcium pump that helps to avoid a futile cycle of ATP cleavage with no net transport of calcium and that increases the pumping capability at low concentrations of free Ca2+.
A cosmid vector that facilitates restriction enzyme mapping.
Authors: Little P F , Cross S H
Published: May 2006

We describe the construction and use of a cosmid vector, loric, which is derived from the phage lambda origin of replication and appears to be more stable than ColE1-derived cosmids. Loric recombinants can be efficiently packaged in vivo to yield 100-300 micrograms of DNA per liter that is linear and has single-stranded cos ends. We call such molecules "phosmids." Phosmid restriction maps can be rapidly generated by labeling either the left or right cos site by annealing on a 32P-labeled oligonucleotide complementary to either cos-L or cos-R. Partial restriction enzyme digestion, agarose gel electrophoresis, and autoradiography are used to size restriction fragments of increasing length, all of which terminate at the labeled cos site. The procedures have been tested by isolating and mapping a region of the H-2 locus of mouse chromosome 17.
Evidence for conduction of protons along the interface between water and a polar lipid monolayer.
Authors: Teissié J , Prats M , Soucaille P , Tocanne J F
Published: May 2006

Movements of H+ along the polar heads of phospholipids spread in monolayers were compared to movements of H+ in the aqueous subphase. The probe for detecting H+ movement along the monolayer was a pH-sensitive fluorescein chromophore covalently bound to the head group of phosphatidylethanolamine. The behavior of this probe was not affected by the electrical properties of the lipid/water interface. Lateral diffusion of H+ along the phospholipid/water interface was then studied by acid-jump experiments in which advantage was taken of the large size of the monolayer. H+ was injected a few centimeters away from the probe observation area. The time needed for H+ diffusion to the probe was monitored by the change in the fluorescence signal, fluorescein being nonfluorescent in an acid medium. Diffusion of H+ in the bulk phase was monitored by the fluorescence change of water-soluble fluorescein isothiocyanate. Diffusion along the lipid monolayer was found to be 20 times faster than in the bulk water phase and required a structured monolayer in order to occur, as revealed by variation of the molecular area occupied by the lipid molecules. The molecular basis of rapid H+ transfer along the lipid monolayer may be the existence of a hydrogen-bond network along the polar heads, capable of supporting a rapid "hop and turn" of H+.
Stochastic free energy transduction.
Authors: Westerhoff H V , Chen Y
Published: May 2006

Theoretical free-energy coupling systems in which the free energy coupling intermediate (e.g., the proton) occurs only in small numbers of molecules per coupling unit are shown to exhibit a number of peculiar properties: (i) the reactions involving the intermediates do not follow conventional kinetic (or nonequilibrium thermodynamic) rate laws in terms of the average concentration or chemical potential of the intermediate, (ii) the variation of the output reaction rate with the average intermediate concentration (or apparent chemical potential) is not unequivocal but depends on whether the input reaction or the leak is varied to alter that concentration, and (iii) when the apparent free energy contained in the average concentration of the intermediate is compared with the average free energy recovered in the output reaction, apparent violations of the second law of thermodynamics can occur. These phenomena are reminiscent of experimental observations in proton-linked free-energy transducing systems that suggest a more direct coupling between electron transfer chains and H+-ATPases than only through a bulk proton gradient, delta muH. Consequently, the chemiosmotic coupling theory can account for those observations if it limits the number of free energy coupling protons per chemiosmotic coupling unit to small values.
Defective macromolecule biosynthesis and cell-cycle progression in a mammalian cell starved for mevalonate.
Authors: Sinensky M , Logel J
Published: May 2006

The isolation of a somatic cell mutant (Mev-1) with a block in one of the mevalonate-biosynthesizing enzymes (3-hydroxy-3-methylglutaryl-coenzyme A synthase, EC 4.1.3.5) has afforded us the opportunity to test and to extend the hypothesis that a product of mevalonate biosynthesis other than cholesterol is required for cellular proliferation. We present evidence here that both DNA synthesis and protein synthesis are inhibited in this mutant by mevalonate starvation, although RNA synthesis appears to be unaffected. The loss of DNA synthesis and the loss of protein synthesis in this mutant appear to be due to independent processes. DNA synthesis is reversibly inhibited by mevalonate starvation at a unique point in the cell cycle. Resumption of DNA synthesis after readdition of mevalonate exhibits a long lag; the peak of S-phase DNA synthesis occurs approximately 17 hr after mevalonate readdition, suggesting that mevalonate starvation puts cells into a quiescent (G0) state owing to their failure to transit a restriction point. The loss of DNA biosynthesis in the Mev-1 cell is well correlated with the rate of turnover of mevalonate label of certain terpenylated polypeptides.
Directed elongation model for microtubule GTP hydrolysis.
Authors: Caplow M , Reid R
Published: May 2006

We propose a role for GTP hydrolysis in microtubule assembly in which the GTPase reaction serves to stabilize tubulin subunits in the microtubule. The GTPase reaction in tubulin subunits containing GTP at microtubule ends is presumed to occur predominately in subunits at one of the interfaces between a cap of GTP-containing tubulin subunit and a core of GDP-containing tubulin subunit in the microtubule, resulting in elongation of the core. The proposed model interprets the effects of GDP on microtubule assembly, using a reaction scheme in which GDP-containing tubulin subunits are able to add to microtubule ends. The model can account for the GTP requirement for microtubule assembly, the GDP inhibition of the rate for microtubule elongation, and the fact that a metastable state exists after the enzymic conversion of GTP to GDP, with microtubules which are at steady state. To account for the fact that the microtubule assembly and disassembly rates are nonlinearly dependent upon the tubulin subunit concentration and for the effects of GDP-containing tubulin subunits on the kinetic properties of microtubules, our scheme includes nonproductive as well as productive binding of GTP- and GDP-containing tubulin subunits. We compare our model with an alternative scheme [Hill, T. L. & Carlier, M. F. (1983) Proc. Natl. Acad. Sci. USA 80, 7234-7238], which interprets the effects of GDP on microtubule assembly using a reaction scheme in which GDP is able to exchange with GTP in GTP-containing tubulin subunits in the microtubule and in which the principal GTPase occurs in GTP-containing tubulin subunits at the microtubule/solution interface.
Biphasic pattern of histone gene expression during Drosophila oogenesis.
Authors: Ruddell A , Jacobs-Lorena M
Published: May 2006

The expression of histone genes during Drosophila oogenesis was compared to periods of DNA synthesis as well as to the pattern of actin gene expression. Accumulation of histone mRNAs was measured by RNA blot hybridization. Relatively low levels of histone mRNAs are present in egg chambers prior to stage 10, during the period of nurse and follicle cell polyploidization. Surprisingly, histone mRNAs accumulate rapidly and selectively after stage 10, coinciding with the onset of nurse cell degeneration and well after DNA synthesis and actin mRNA accumulation have ceased. A large proportion of the histone mRNAs is associated with polysomes at all times, indicating that expression of histone genes is not strictly coupled to DNA synthesis. The burst of histone mRNA accumulation near the end of oogenesis may provide a store of maternal histone mRNA to support the rapid cleavages that occur during early embryogenesis. These and previous results suggest that genes are independently regulated during differentiation of the Drosophila egg chamber.
Genes for respiratory chain proteins and ribosomal RNAs are present on a 16-kilobase-pair DNA species from Chlamydomonas reinhardtii mitochondria.
Authors: Boer P H , Bonen L , Lee R W , Gray M W
Published: May 2006

We have used heterologous hybridization and DNA sequence analysis to determine whether the 16-kilobase-pair (kbp) DNA from Chlamydomonas reinhardtii mitochondria is the functional equivalent of mtDNA in other eukaryotes. Restriction fragments corresponding to a continuous internal stretch spanning 75% of the 16-kbp DNA have been cloned and mapped, and regions hybridizing with probes specific for the cytochrome oxidase subunit I [CytOx I (acronym COI)] and apocytochrome b (Cyt b) genes of yeast and the mitochondrial 26S and 18S rRNA genes of wheat have been identified. Sequence analysis has verified the presence of CytOx I and the large and small subunit rRNA genes in the C. reinhardtii 16-kbp DNA. In the region of the 16-kbp DNA corresponding to exon 4 in the yeast CytOx I gene, the derived amino acid sequence is 61% and 63% identical with the CytOx I amino acid sequences of yeast and human mitochondria, respectively. Notably, tryptophan is specified by TGG rather than by TGA in this section of the C. reinhardtii CytOx I gene. A probe from the CytOx I region of the 16-kbp DNA hybridizes only with this 16-kbp DNA in Southern blots of total cellular DNA from C. reinhardtii but with a larger DNA species in the total cellular DNA of C. moewusii and C. eugametos--two species that lack a 16-kbp DNA. These observations provide evidence that C. reinhardtii 16-kbp DNA comprises at least part of the mitochondrial genome of this organism and that a homologous DNA exists in other species of Chlamydomonas.
Human alpha 1(III) and alpha 2(V) procollagen genes are located on the long arm of chromosome 2.
Authors: Emanuel B S , Cannizzaro L A , Seyer J M , Myers J C
Published: May 2006

The multigene procollagen family encodes probably greater than 20 genetically distinct but structurally related polypeptide chains. Recent characterization of human procollagen clones has allowed determination of functional domains within the proteins, genomic organization, and chromosomal location. Previously, we assigned the coordinately expressed type I genes (alpha 1 and alpha 2) to chromosomes 17 and 7, respectively, and now other investigators have mapped the type II gene to chromosome 12 [Strom, C. M., Eddy, R. L. & Shows, T. B. (1984) Somatic Cell Genet. 10, 651-655]. Recently, we isolated cDNA clones encoding the fourth interstitial procollagen, type III, and the alpha 2 chain of the type V cytoskeletal components. To determine whether these genes were clustered with alpha 1(I), alpha 2(I), or alpha 1(II) or were further dispersed in the genome, in situ hybridization of the alpha 1(III) and alpha 2(V) probes to metaphase chromosomes was carried out. Here we report a fourth autosome with procollagen gene loci but the first cytological evidence for linkage. By using normal and translocated cell lines, our results show that both the alpha 1(III) and alpha 2(V) procollagen genes map to the q24.3----q31 region of chromosome 2.
A frameshift mutation in the pre-S region of the human hepatitis B virus genome allows production of surface antigen particles but eliminates binding to polymerized albumin.
Authors: Persing D H , Varmus H E , Ganem D
Published: May 2006

The coding region for the major polypeptide (p24S) of hepatitis B surface antigen (HBsAg) is preceded by an in-phase open reading frame termed pre-S. The coding potential of the pre-S region was examined in mouse L cells transformed with cloned hepatitis B virus DNA. Such cells produce three HBsAg-related polypeptides of Mr 24,000, 27,000, and 35,000 organized into complex particles of 22 nm diameter. These HBsAg particles bind to polymerized human albumin, but not to polyalbumins of several other species. In contrast, cells transformed with hepatitis B virus DNA bearing a frameshift mutation near the 3' end of the pre-S region secrete immunoreactive HBsAg particles containing only the 24,000 and 27,000 Mr species. These mutant particles, which lack the 35,000 Mr species, are unable to bind polymerized human albumin. These studies indicate that the pre-S region encodes the 35,000 Mr species, that this product accounts for the known polyalbumin-binding activity of HBsAg but is not required for assembly and secretion of HBsAg 22-nm particles, and that the major polypeptide of HBsAg is not derived primarily by cleavage of larger precursors encoded by the pre-S region.
Glycolysis and methylaminoisobutyrate uptake in rat-1 cells transfected with ras or myc oncogenes.
Authors: Racker E , Resnick R J , Feldman R
Published: May 2006

A high rate of aerobic glycolysis was catalyzed by rat-1 cells transfected with a ras oncogene (ras cells); rat-1 cells and rat-1 cells transfected with myc oncogene (myc cells) showed a low rate of glycolysis that was increased after exposure of the cells to type B transforming growth factor (TGF-beta). The uptake of radioactive methylaminoisobutyric acid or L-methionine via system A of amino acid transport also was accelerated after exposure of these cells to TGF-beta, with the myc cells being most sensitive and the ras cells least sensitive. Methionine was found to be a potent inhibitor of glycolysis in ras cells as well as in rat-1 or myc cells that were exposed to TGF-beta. We propose a relationship between the product of the ras oncogene (p21) and the protein(s) induced by exposure to TGF-beta.
Coding sequences for vasoactive intestinal peptide and PHM-27 peptide are located on two adjacent exons in the human genome.
Authors: Bodner M , Fridkin M , Gozes I
Published: May 2006

The human precursor gene for vasoactive intestinal peptide (VIP) and PHM-27, a peptide that has an NH2-terminal histidine and COOH-terminal methionine amide and is closely related in sequence and activity to VIP, was detected with synthetic oligodeoxynucleotide probes. These specific hybridization segments were constructed according to the neuroblastoma VIP cDNA sequence and contained up to 39 bases. The gene structure was partly deduced by hybridization to synthetic oligodeoxynucleotide probes and partly by direct chemical nucleotide sequencing. Four exons were discovered thus far; among them are two short exons separated by a 0.75-kilobase DNA stretch, one encoding PHM-27 and the second encoding VIP (exons 1 and 2). Each of these two exons encodes both the hormone amino acid residues as well as the post-translational processing signal sequences. The 3' splice sites of the two exons contain an identical stretch of nine nucleotides. At the cDNA level, the 3' splice sites contain the same stretch of six nucleotides, which are identically spliced. The occurrence of VIP and PHM-27 coding sequences on two separate exons of the human genome and the homology of their 3' splice site may allow alternative RNA processing as discussed below.
Tobacco chloroplast tRNA
Lys
(UUU) gene contains a 2.5-kilobase-pair intron: An open reading frame and a conserved boundary sequence in the intron
Authors: Sugita Mamoru , Shinozaki Kazuo , Sugiura Masahiro
Published: May 2006

The nucleotide sequence of a tRNA
Lys
(UUU) gene on tobacco (
Nicotiana tabacum
) chloroplast DNA has been determined. This gene is located 215 base pairs upstream from the gene for the 32,000-dalton thylakoid membrane protein on the same DNA strand and has a 2526-base-pair intron in the anticodon loop. The intron boundary sequence does not follow the G-U/A-G rule but is similar to those of tobacco chloroplast split genes for tRNA
Gly
(UCC) and ribosomal proteins L2 and S12. The intron contains one major open reading frame of 509 codons. The codon usage in the open reading frame resembles those observed in the genes for tobacco chloroplast proteins so far analyzed. The primary transcript of this tRNA gene is 2.7 kilobases long.