bluuebunny/crossref_metadata_2025 huggingface dataset:
Cell-division control in Escherichia coli: specific induction of the SOS function SfiA protein is sufficient to block septation.
Authors: Huisman O , D'Ari R , Gottesman S
Published: May 2006

Blocks in DNA replication cause a rapid arrest of cell division in Escherichia coli. We have previously established that the function SfiA (SulA), induced under these conditions as part of the SOS response, is involved in this inhibition of division. To separate the effects of SfiA from those of other SOS functions, we have constructed a plac-sfiA operon fusion, permitting specific induction of SfiA protein by addition of the lac operon inducer isopropyl beta-D-thiogalactopyranoside (IPTG). In lon mutants, in which the unstable SfiA protein has a longer half-life, IPTG caused a rapid arrest of cell division. Under these conditions, there is no concomitant induction of the SOS response. IPTG also caused a rapid arrest of cell division in lon+ strains. These results demonstrate that induction of the SfiA protein is sufficient to cause inhibition of division. Mutations in the sfiB gene can suppress IPTG-induced SfiA-mediated inhibition of division. At higher SfiA concentrations, however, even sfiB mutants cease division; an additional mutation genetically inseparable from sfiB restores normal division. These observations reinforce the hypothesis that the SfiB protein, probably required for cell septation, is the target of action of the SfiA division inhibitor.
Molecular cloning and nucleotide sequence of cDNA for mRNA of mitochondrial cytochrome P-450(SCC) of bovine adrenal cortex.
Authors: Morohashi K , Fujii-Kuriyama Y , Okada Y , Sogawa K , Hirose T , Inayama S , Omura T
Published: May 2006

We have isolated cDNA clones of the mRNA for cytochrome P-450(SCC), which catalyzes the side-chain cleavage reaction of cholesterol in bovine adrenal cortex mitochondria, by using synthetic oligonucleotides as probes. Sequence analysis of the cloned cDNAs enabled us to deduce the primary structure of the precursor form of P-450(SCC), which consisted of 520 amino acids and contained an extrapeptide of 39 amino acids at the NH2 terminus. The amino acid sequence from the 40th to 55th amino acid residue in the predicted structure completely coincided with the sequence of the NH2-terminal portion of purified P-450(SCC). The amino acid composition calculated from the predicted structure showed an excellent agreement with that determined with the purified protein. The extrapeptide of the precursor molecule resembles those of a few nuclear-encoded yeast mitochondrial proteins reported so far. Although P-450(SCC) is a component of mitochondria, comparison of its primary structure with those of other forms of cytochrome P-450 shows that P-450(SCC) is structurally more related to microsomal cytochrome P-450s than to a bacterial cytochrome P-450, P-450cam. A homologous sequence observed with various forms of cytochrome P-450 is also highly conserved in the P-450(SCC) molecule. Only two cysteinyl residues are present in the mature form of P-450(SCC), one of which is located in the middle of the conserved sequence, confirming the function of this cysteinyl residue as the fifth ligand of the heme.
Two nuclear mutations that block mitochondrial protein import in yeast.
Authors: Yaffe M P , Schatz G
Published: May 2006

We isolated two yeast mutants that are temperature-sensitive for import of mitochondrial proteins. Each strain contains a single mutation that results in arrest of growth and accumulation of precursor to the beta subunit of the mitochondrial F1-ATPase after incubation at 37 degrees C. These lesions (mas1 and mas2) are nonallelic and recessive. Cells harboring either mutation stop growing only after 2-3 generations at 37 degrees C. Import of the F1 beta subunit at 37 degrees C is more than 250 times slower in mas1 and 15 times slower in mas2 than in wild-type cells. At 23 degrees C, import occurs with similar rates in mutant and wild-type cells. The two mutations also reduce the rate of import of other proteins; however, import of different precursors is affected to different degrees in the two strains. The temperature-sensitive step in import in both mas1 and mas2 occurs before arrival of precursors in the mitochondrial matrix.
A specific mutation abolishing Na+/H+ antiport activity in hamster fibroblasts precludes growth at neutral and acidic pH.
Authors: Pouysségur J , Sardet C , Franchi A , L'Allemain G , Paris S
Published: May 2006

A H+-suicide technique based on the reversibility of Na+/H+ antiport was developed for the selection of mutants deficient in this membrane-bound activity. The strategy was to use the Na+/H+ antiporter as a H+-vector killing device. Chinese hamster lung fibroblasts (CCL39) were loaded with LiCl and incubated in Na+-, Li+-free choline Cl saline solution (pH 5.5). Under these conditions, intracellular pH dropped in 5 min from 7.1 to 4.8, leading to a rapid loss of cell viability (less than 0.1% survival after 30 min). Cytoplasmic acidification and cell death were prevented by treatment with 5-N,N-dimethylamiloride, a potent inhibitor of Na+/H+ antiport. Of the H+-suicide resistant clones that survived two cycles of selection, 90% were found deficient in Na+/H+ antiport activity. One class of mutants (PS10, PS12) fully resistant to the H+-suicide test, does not acidify the cell interior in response to an outward-directed Li+ gradient and has no detectable amiloride-sensitive Na+ influx measured either in Li+- or H+-loaded cells. Growth of these fibroblast clones lacking Na+/H+ antiport was found to be pH conditional in HCO3(-)-free medium. Whereas wild-type cells can grow over a wide range of external pHs (6.6-8.2), PS mutants cannot grow at neutral and acidic pHs (pH less than 7.2); their optimal growth occurs at alkaline pH values (pH 8-8.3). These findings strongly suggest that the Na+/H+ antiport activity through regulation of intracellular pH plays a crucial role in growth control.
Guatemalan forest synthesis after Pleistocene aridity
Authors: Leyden Barbara W.
Published: May 2006

Sediments from two lakes in the Peten Department, Guatemala, provide palynological evidence from Central America of late Pleistocene aridity and subsequent synthesis of mesic forests. Late Glacial vegetation consisted of marsh, savanna, and juniper scrub. An early Holocene temperate forest preceded a mesic tropical forest with
Brosimum
(ramon). Thus “primeval” rain forests of Guatemala are no older than 10,000 to 11,000 years and are considerably younger in the Peten due to Mayan disturbances. Among dated Neotropical sites, the Peten has the most mesic vegetation yet shown to have supplanted xeric vegetation present during the Pleistocene. The arid late Glacial-humid early Holocene transition appears to have been pantropical in the lowlands. The Peten was not a Pleistocene refugium for mesophytic taxa, as has been suggested. Thus genesis of extant rain forests in northern Central America and southern Mexico remains unexplained.
Genes for murine fourth complement component (C4) and sex-limited protein (Slp) identified by hybridization to C4- and Slp-specific cDNA.
Authors: Ogata R T , Sepich D S
Published: May 2006

Murine fourth component of complement (C4) and sex-limited protein (Slp) are two closely related serum proteins whose structural genes lie in the S region of the murine H-2 complex. We have cloned two very similar (95% nucleotide sequence identity) cDNAs; they encode amino acid sequences that are distinct but that correspond equally well with the limited amino acid sequence available for murine and human C4. We have identified one of these as C4 cDNA and the other as Slp cDNA by comparing, in a RNA blot hybridization experiment, the extent of hybridization of the two cDNAs to liver mRNAs from inbred mouse strains expressing varying amounts of C4 and Slp proteins in their plasma. The C4 and Slp cDNAs were used in a Southern blot experiment to identify the C4 and Slp genes in the molecular map of the S region.
No evidence for differences in the Epstein-Barr virus genome carried in Burkitt lymphoma cells and nonmalignant lymphoblastoid cells from the same patients.
Authors: Bornkamm G W , von Knebel-Doeberitz M , Lenoir G M
Published: May 2006

Epstein-Barr virus (EBV), although not an indispensable factor for the development of Burkitt lymphoma, is apparently associated with the 20-fold higher incidence of the disease in Equatorial Africa compared to the incidence in other parts of the world. To determine whether different EBV subtypes are associated with the appearance of the malignant phenotype, we have compared the EBV genomes carried in the Burkitt tumor cells with those carried in the nonmalignant lymphoblastoid cells from the same individuals. From three patients with EBV -associated Burkitt lymphoma, tumor cell lines as well as spontaneously established lymphoblastoid cell lines representing the nonmalignant counterparts were obtained. The viral DNA in these cell lines was analyzed by Southern blot hybridization, using a set of cloned EBV DNA fragments as probes that recognize polymorphic regions in the viral genome. Using a number of different polymorphic markers to distinguish one isolate from another, the virus genome found in the tumor cells could also be identified in the nonmalignant cells of the same patient. In one case, in which two independent lymphoblastoid cell lines were established, evidence was obtained that this patient was infected by at least two distinct EBV subtypes. These results strongly suggest that in Burkitt lymphoma, the risk associated with EBV is related to cofactors such as chronic malaria and the mode of infection rather than to peculiar viral subtypes. The situation seems to be totally different from papillomavirus-associated diseases, in which the risk of progression to malignancy appears to be associated with particular viral strains.
Enhanced expression of the human gene N-myc consequent to amplification of DNA may contribute to malignant progression of neuroblastoma.
Authors: Schwab M , Ellison J , Busch M , Rosenau W , Varmus H E , Bishop J M
Published: May 2006

Previous studies had revealed that DNA with partial similarity to the myc oncogene (N-myc) is frequently amplified in human neuroblastoma cell lines and neuroblastoma tumors. We show here for one patient that N-myc amplification is confined to the neuroblastoma tumor and is not present in normal tissue. N-myc mRNA approximately equal to 4.0 kilobases in size is detectable in neuroblastoma cell lines and tumors and in a retinoblastoma cell line. By contrast, appreciable amounts of this RNA were not present in a number of cell lines derived from other human tumors and in fibroblasts from a normal individual and from a neuroblastoma patient. Low levels of N-myc RNA were found in human and murine neuroblastoma cell lines lacking amplification of this gene, up to 80-fold greater levels in all cell lines carrying amplified N-myc. In situ hybridization to sections of neuroblastoma tumors revealed high expression of N-myc predominantly in undifferentiated neuroblasts. We hypothesize that amplification and consequent elevated expression of N-myc may be related to malignant progression.
Immunodeficiency with defective T-cell response to interleukin 1.
Authors: Chu E T , Rosenwasser L J , Dinarello C A , Rosen F S , Geha R S
Published: May 2006

Normal proliferation of T cells in vitro requires production of and response to the lymphokine interleukin 2 (IL-2). Optimal IL-2 production by T cells is dependent on the monokine interleukin 1 (IL-1). A 10-year-old male with recurrent infections and failure to thrive was evaluated for possible defects in the production and response to IL-1 and IL-2. The patient had normal levels of serum immunoglobulins and a normal distribution of circulating T-cell subsets. However, the in vitro proliferative response of his peripheral blood mononuclear cells (PBMC) to phytohemagglutinin was depressed (40% of normal) and the response of his PBMC to antigens was absent. Delayed hypersensitivity skin tests and in vitro response to tetanus toxoid remained absent despite repeated immunizations. Monocyte function in this patient was normal as judged by the following criteria: normal expression of Ia antigens (77% +), normal IL-1 production, and normal capacity to present tetanus toxoid to a maternal T-cell line specific for tetanus toxoid antigen. The abnormal phytohemagglutinin response of the patient's PBMC was corrected by the addition of exogenous IL-2. IL-2 production by the patient's phytohemagglutin-stimulated PBMC was severely deficient but was corrected by the addition of phorbol 12-myristate 13-acetate, suggesting a defective response to IL-1. T-cell blasts derived from a normal subject but not T-cell blasts derived from the patient absorbed out IL-1 activity from a preparation of purified human IL-1. These results indicate that the patient's T-cell deficiency was due to a defective T-cell response to IL-1 and suggest that IL-1 plays an important role in the in vivo immune response.
Sensory transduction in Escherichia coli: regulation of the demethylation rate by the CheA protein.
Authors: Springer M S , Zanolari B
Published: May 2006

The reversible methylation of three membrane proteins plays an essential role in bacterial chemotaxis. Chemotactic stimuli bring about changes in the levels of methylation of these proteins, at least in part, by regulation of the demethylation reaction. Addition of attractants causes an increase in the methylation level and a transient, but essentially complete, inhibition in the rate of the demethylation reaction, while addition of repellents results in a decrease in level and a transient increase (of at least 25- to 30-fold) in rate. We have now found that the increase, but not the decrease, in rate requires the presence of the cheA gene product, a protein that is distinct from the demethylase. The demethylation reaction is therefore regulated by two distinct mechanisms--one, which involves the CheA protein, that mediates the increase in rate and a second, which does not involve the CheA protein, that mediates the decrease in rate. Several pieces of evidence already in the literature imply that the CheA protein functions downstream of the methylation system at the flagellar end of the chemotactic machinery. These data, in conjunction with the newer results, suggest that the CheA protein helps to regulate the demethylation reaction through a feedback mechanism.
In vivo effects of microinjected alkaline phosphatase and its low molecular weight substrates on the first meiotic cell division in Xenopus laevis oocytes.
Authors: Hermann J , Mulner O , Bellé R , Marot J , Tso J , Ozon R
Published: May 2006

Xenopus laevis oocytes were microinjected with low molecular weight phosphoesters such as 2-glycerophosphate, phosphotyrosine, phosphoserine, phosphothreonine, 4-nitrophenyl phosphate, and orthophosphate. These compounds were able to induce a considerable reduction in the time course of progesterone-induced maturation, with 2-glycerophosphate being the most effective. The basal level of cAMP and its drop during maturation were not affected by the microinjection of 2-glycerophosphate. The injection of alkaline phosphatase (EC 3.1.3.1.) from calf intestine at a low concentration (10 ng per oocyte) was able to decrease or abolish the effect of 2-glycerophosphate. At higher concentration (25 ng per oocyte) this enzyme totally blocked progesterone- or maturation-promoting factor-induced maturation. Alkaline phosphatase might behave in vivo as a phosphoprotein phosphatase active towards phosphotyrosine-containing proteins. In addition, our results indicate that phosphate or phosphoester-containing buffers should be avoided in the course of maturation-promoting factor purification.
Monoclonal antibodies of predefined specificity detect activated ras gene expression in human mammary and colon carcinomas.
Authors: Hand P H , Thor A , Wunderlich D , Muraro R , Caruso A , Schlom J
Published: May 2006

Monoclonal antibodies (MAbs) of predefined specificity have been generated by utilizing a synthetic peptide reflecting amino acid positions 10-17 of the Hu-rasT24 gene product as immunogen. These MAbs, designated RAP-1 through RAP-5 (RA, ras; P, peptide), have been shown to react with the ras gene product p21. Since the Hu-ras reactive determinants (positions 10-17) have been predicted to be within the tertiary structure of the p21 molecule, it was not unexpected that denaturation of cell extracts or tissue sections with Formalin or glutaraldehyde enhanced binding of the RAP MAbs. When paraffin-embedded Formalin-fixed tissue sections and the avidin-biotin complex immunoperoxidase method were used, the RAP MAbs clearly defined enhanced ras p21 expression in the majority of human colon and mammary carcinomas. The majority of all abnormal ducts and lobules from fibroadenoma and fibrocystic disease patients were negative, as were all normal mammary and colonic epithelia examined. The findings reported here form the basis for quantitative radioimmunoassays for a ras translational product and provide a means to evaluate ras p21 expression within individual cells of normal tissues and benign, "premalignant," and malignant lesions.
Acetylcholine receptor (from Electrophorus electricus): a comparison of single-channel current recordings and chemical kinetic measurements.
Authors: Hess G P , Kolb H A , Läuger P , Schoffeniels E , Schwarze W
Published: May 2006

We report a direct comparison between two types of measurements of the dynamic properties of the acetylcholine receptor: single-channel currents recorded using the patch-clamp technique and chemical kinetic measurements. Electrophorus electricus electroplax cells, and membrane vesicles prepared from these cells, were used. Such a comparison, and single-channel currents recorded from these cells, have not previously been reported. We first give the theoretical basis for the comparison and define the conditions under which the comparisons are elegantly simple. We relate (i) measurements of currents through receptor channels in the cell membranes to measurements of the rates of ion translocation through the receptor channels in vesicles and (ii) measurements of the lifetimes of receptor states (for instance, the lifetime of the active state of the receptor--i.e., the state in which it can form open channels) to rate coefficients obtained in chemical kinetic measurements (for instance, those for the interconversions between different states of the receptor). In eel Ringer's solution we have found the single-channel conductance (gamma) of the receptor in E. electricus electroplax cells to be 53 pS. From this value, a specific reaction rate for ion translocation, J, of 5 X 10(7) M-1 X sec-1 was calculated. When membrane vesicles prepared from the electroplax cells and the same solution compositions were used, chemical kinetic measurements gave a J value of 3 X 10(7) M-1 X sec-1. The agreement between the two measurements is important because (i) they reflect different experimental conditions, which require different assumptions in interpreting the results, and (ii) it indicates that the two techniques can be used to obtain complementary information: the methods have different time resolutions and can be used in different ranges of acetylcholine concentrations.
Secretion of foreign proteins from Saccharomyces cerevisiae directed by alpha-factor gene fusions.
Authors: Bitter G A , Chen K K , Banks A R , Lai P H
Published: May 2006

Fusions between the cloned yeast alpha-factor structural gene and chemically synthesized DNA segments encoding human protein analogs have been constructed. The gene fusions encode hybrid proteins that include the first 89 amino acids of the native alpha-factor precursor fused to either a small (beta-endorphin, 31 amino acids) or large (alpha-interferon, 166 amino acids) foreign protein. Proteolytic cleavage sites involved in alpha-factor maturation from the native precursor immediately precede the foreign peptide in the hybrid protein. The alpha-factor promoter was utilized to express the gene fusions in Saccharomyces cerevisiae and resulted in the efficient secretion of the foreign proteins into the culture medium. The processing of the hybrid proteins has been characterized by amino acid sequence analysis of the secreted proteins. The proteolytic cleavages involved in the maturation of alpha-factor peptides from the native precursor also occur accurately in the hybrid protein. In addition, cleavages occurred on the carboxyl side of two lysines within the beta-endorphin peptide. Internal cleavages in the interferon protein were also detected. However, in this case, the cleavages occurred at a very low frequency such that greater than 95% of the secreted interferon remained intact.
Immunocytochemical localization of calcium/calmodulin-dependent protein kinase II in rat brain.
Authors: Ouimet C C , McGuinness T L , Greengard P
Published: May 2006

Calcium/calmodulin-dependent protein kinase II (CaM kinase II) is a prominent enzyme in mammalian brain capable of phosphorylating a variety of substrate proteins. In the present investigation, the subcellular and regional distribution of CaM kinase II has been studied by light and electron microscopic immunocytochemistry using an antibody that recognizes the Mr 50,000 and 60,000/58,000 subunits of the enzyme. Light microscopy demonstrates strong immunoreactivity in neuronal somata and dendrites and weak immunoreactivity in axons. Electron microscopy, in addition to confirming light microscopic observations, reveals moderate immunoreactivity in spines and weak immunoreactivity in nerve terminals. An accumulation of immunoreaction product is also present on postsynaptic densities. The presence of CaM kinase II in diverse structures throughout the neuron supports the view that this enzyme may be involved in mediating a variety of calcium-dependent physiological processes. CaM kinase II immunoreactivity is present in neurons throughout the brain, but a marked regional variation in the strength of the immunoreactivity exists. Overall, there is a gradient of staining intensity with the strongest immunoreactivity in the telencephalon and the weakest in the myelencephalon. The most heavily labeled regions of the telencephalon are the hippocampal formation, lateral septum, cortical regions, neostriatum, and amygdaloid complex.
Sigma opiates and certain antipsychotic drugs mutually inhibit (+)-[3H] SKF 10,047 and [3H]haloperidol binding in guinea pig brain membranes.
Authors: Tam S W , Cook L
Published: May 2006

The relationship between binding of antipsychotic drugs and sigma psychotomimetic opiates to binding sites for the sigma agonist (+)-[3H]SKF 10,047 (N-allylnormetazocine) and to dopamine D2 sites was investigated. In guinea pig brain membranes, (+)-[3H]SKF 10,047 bound to a single class of sites with a Kd of 4 X 10(-8) M and a Bmax of 333 fmol/mg of protein. This binding was different from mu, kappa, or delta opiate receptor binding. It was inhibited by opiates that produce psychotomimetic activities but not by opiates that lack such activities. Some antipsychotic drugs inhibited (+)-[3H]SKF 10,047 binding with high to moderate affinities in the following order of potency: haloperidol greater than perphenazine greater than fluphenazine greater than acetophenazine greater than trifluoperazine greater than molindone greater than or equal to pimozide greater than or equal to thioridazine greater than or equal to chlorpromazine greater than or equal to triflupromazine. However, there were other antipsychotic drugs such as spiperone and clozapine that showed low affinity for the (+)-[3H]SKF 10,047 binding sites. Affinities of antipsychotic drugs for (+)-[3H]SKF 10,047 binding sites did not correlate with those for [3H]spiperone (dopamine D2) sites. [3H]-Haloperidol binding in whole brain membranes was also inhibited by the sigma opiates pentazocine, cyclazocine, and (+)-SKF 10,047. In the striatum, about half of the saturable [3H]haloperidol binding was to [3H]spiperone (D2) sites and the other half was to sites similar to (+)-[3H]SKF 10,047 binding sites.
Evidence for the presence of makisterone A in
Drosophila
larvae and the secretion of 20-deoxymakisterone A by the ring gland
Authors: Redfern Christopher P. F.
Published: May 2006

Ring glands or brain—ring gland complexes from third-instar
Drosophila melanogaster
larvae secreted ecdysone [(22
R
)-2β,3β,14α,22,25-pentahydroxy-5β-cholest-7-en-6-one] and two less polar ecdysteroids (LP1 and LP2)
in vitro
. Radioimmunoassay with different antisera indicated that LP1 and LP2 are side-chain-modified analogues of ecdysone. In high-performance liquid chromatography, the retention time of LP2 was equivalent to that of a precursor of makisterone A [(20
R
,22
R
)-2β,3β,14α,20,22,25-hexahydroxy-24-methyl-5β-cholest-7-en-6-one] secreted by
Dysdercus fasciatus
prothoracic glands
in vitro
. LP2 was metabolized
in vitro
by the fat body of
Drosophila
larvae to a product with the characteristics of makisterone A when analyzed by gas chromatography/mass spectrometry (selected ion monitoring). Evidence was obtained for the presence of makisterone A in
Drosophila
larvae. These data suggest that LP2 is 20-deoxymakisterone A (24-methylecdysone) and that makisterone A could function as an additional moulting hormone in
Drosophila
, although 20-deoxymakisterone A production is apparently dependent on the sterol composition of the diet. LP1 has not been identified.
Site-specific mutagenesis of the human fibroblast interferon gene.
Authors: Mark D F , Lu S D , Creasey A A , Yamamoto R , Lin L S
Published: May 2006

Human fibroblast interferon has three cysteine residues, located at amino acid positions 17, 31, and 141. Using the technique of site-specific mutagenesis with a synthetic oligonucleotide primer, we changed the codon for cysteine-17 to a codon for serine. The resulting interferon, IFN-beta Ser-17, retains the antiviral, natural killer cell activation, and antiproliferative activities of native fibroblast interferon. The purified IFN-beta Ser-17 protein has an antiviral specific activity of 2 X 10(8) units/mg, similar to that of purified native fibroblast interferon. In addition, the purified protein is stable to long-term storage at -70 degrees C.
Intrinsic GTPase activity distinguishes normal and oncogenic ras p21 molecules.
Authors: Gibbs J B , Sigal I S , Poe M , Scolnick E M
Published: May 2006

The 21-kilodalton protein (p21) encoded by normal cellular Harvey-ras has been expressed in Escherichia coli as a fusion protein by using the pUC8 vector and has been purified to greater than 95% homogeneity by ion-exchange chromatography and gel filtration. The purified protein molecules possess intrinsic GTPase activity on the basis of the following criteria: (i) elution of the GTPase activity with p21 GDP-binding activity in two different chromatography systems, (ii) parallel thermal inactivation of GTPase activity and p21 GTP-binding activity, and (iii) immunoprecipitation of the GTPase activity with monoclonal antibodies to p21. At 37 degrees C, the rate of GTP hydrolysis by the purified normal p21 assayed in solution was 5.3-6.6 mmol/min per mol of p21. The rate of GTP hydrolysis by a form of p21 [Val12] encoded by a human oncogene was significantly lower (1.4-1.9 mmol/min per mol of p21). The presence of a threonine phosphate acceptor site at residue 59 also decreased p21 GTPase activity. For regulatory proteins that use GTP as part of their biochemical mechanism, the hydrolysis of GTP to GDP reverses the biological activity of the respective proteins. The observation that oncogenic forms of p21 lose GTPase activity suggests that GTP hydrolysis may be a biochemical event that inactivates the growth-promoting effects of a p21 X GTP complex.
On the redox conformational change in cytochrome c.
Authors: Rackovsky S , Goldstein D A
Published: May 2006

The relationship between the crystal structures of oxidized and reduced tuna cytochrome c has been reexamined by a superposition method motivated by recent studies of the cytochrome c-cytochrome c peroxidase complex. It is shown that the observed structural changes precisely reflect the binding face suggested by chemical modification studies. It is further suggested that the large observed motion of lysine-27 and a smaller overall motion of the two binding edges constitute a redox binding-affinity switch and that the driving force for the conformational change of the protein is provided by the internal conformational change and charge redistribution of the heme, which cause it to tilt, under the influence of covalent and nonbonded interactions, within its protein envelope. A picture is presented of the molecule as an electron storage/transfer machine with three elements--a binding module, an electron storage module, and a conformational energy-storage module.
Thyrotropin controls transcription of the thyroglobulin gene.
Authors: Van Heuverswyn B , Streydio C , Brocas H , Refetoff S , Dumont J , Vassart G
Published: May 2006

The availability of rat thyroglobulin cDNA clones was exploited to study the regulation of thyroglobulin gene transcription by thyrotropin (TSH). Groups of rats were subjected to treatments leading to reduction or increase in the rat serum TSH (rTSH) levels. Thyroid gland nuclei were isolated, incubated in vitro in the presence of 32P-labeled uridine triphosphate, and thyroglobulin transcripts were quantitated by hybridization to immobilized rat thyroglobulin cDNA clones. Transcription of the thyroglobulin gene was found to be very active in thyroid nuclei from control animals. It represented about 10% of total RNA polymerase II activity. Chronic hyperstimulation of the thyroid glands with endogenous rTSH was achieved in rats treated with the goitrogen propylthiouracil. No significant increase of thyroglobulin gene transcription could be measured in thyroid nuclei from these animals. On the contrary, a dramatic decrease in thyroglobulin gene transcription was observed in those animals in which endogenous rTSH levels had been suppressed by hypophysectomy or by the administration of triiodothyronine. Injection of exogenous bovine TSH in such animals readily restored transcriptional activity of the gene. Our results identify transcription as an important regulatory step involved in TSH action. They suggest that normal TSH levels induce close to maximal expression of the thyroglobulin gene but that continuous presence of TSH is required in order to maintain the gene in an activated state.
Synthesis of a gene for human growth hormone and its expression in Escherichia coli.
Authors: Ikehara M , Ohtsuka E , Tokunaga T , Taniyama Y , Iwai S , Kitano K , Miyamoto S , Ohgi T , Sakuragawa Y , Fujiyama K
Published: May 2006

A gene coding for human growth hormone, which consists of 192 amino acids, was chemically synthesized. The synthesis entailed ligating 78 deoxyribooligonucleotides, which had been synthesized on polymer supports by the phosphotriester method with frequently occurring amino acid codons of Escherichia coli. The chemically synthesized gene was inserted into an E. coli plasmid downstream from the E. coli trp promoter, with a modified ribosome-binding region carried on pBR322. E. coli cells transformed with this recombinant plasmid synthesized 2.9 X 10(6) molecules per cell of human growth hormone upon induction. The induced polypeptide was identical with natural human growth hormone in size and in immunological properties, as well as in biological activity as examined by the tibial test with hypophysectomized rats.
Cotranscription of genes encoding the small and large subunits of ribulose-1,5-bisphosphate carboxylase in the cyanobacterium Anabaena 7120.
Authors: Nierzwicki-Bauer S A , Curtis S E , Haselkorn R
Published: May 2006

In the filamentous cyanobacterium Anabaena, the gene for the small subunit (rbcS) of ribulose-1,5-bisphosphate carboxylase is linked to and transcribed together with the gene encoding the large subunit (rbcL) of the same enzyme. The two genes are separated by a spacer of 545 base pairs (bp) that does not contain an open reading frame. Both genes hybridize with a predominant 3.1-kilobase transcript that initiates 414 bp upstream from the rbcL coding region. The nucleotide sequence 14-8 bp preceding the transcription start site resembles a good Escherichia coli promoter, but the sequence in the -35 region does not. There is no obvious relationship between the sequence flanking the amino terminus of the cyanobacterial small subunit gene and the transit peptide of eukaryotic small subunit genes. The Anabaena rbcS gene is 61% homologous at the amino acid level with the gene from the unicellular cyanobacterium Anacystis and 37-38% homologous with the corresponding nuclear genes from eukaryotes. In contrast, the Anabaena rbcL gene is approximately equal to 86% homologous at the amino acid level with the rbcL genes from plant chloroplasts. Cotranscription satisfies one of the requirements for coordinate expression of the two genes whose products are needed in equimolar amounts in the mature enzyme. The rbcL-rbcS transcript is equally abundant in Anabaena azollae grown in the light or on fructose in the dark.
Involvement of the carboxyl-terminal domain of tubulin in the regulation of its assembly.
Authors: Serrano L , de la Torre J , Maccioni R B , Avila J
Published: May 2006

Limited proteolysis of phosphocellulose-purified tubulin with subtilisin resulted in cleavage of both alpha and beta tubulin subunits, with the formation of two major fragments (S alpha, and S beta, 48 kDa) and a small peptide (4 kDa) containing the carboxyl-terminal region of tubulin. Interestingly, tubulin cleaved under the present conditions showed an increased ability to assemble into large polymers in the absence of MAPs and under conditions that do not promote assembly of undigested tubulin--i.e., low magnesium concentrations and the absence of taxol and polyalcohols. The critical concentrations for the subtilisin-cleaved tubulin assembly was similar to that of MAPs-promoted tubulin assembly. Assembly product from subtilisin-cleaved tubulin consisted mainly of protofilament bundles, hooked polymer, and open tubules, structures showing equatorial and longitudinal spacings of 50 and 40 A, respectively. The existence of junctions between polymer walls indicates that the carboxyl-terminal removal facilitates polymer-polymer interactions. These results, together with previous studies on the involvement of the carboxyl-terminal domain of tubulin in its interaction with MAP-2, suggest a regulatory role for this domain in tubulin assembly. Thus, in general terms the tubulin molecule can be analyzed as a protein containing two essential domains with functional significance, one domain playing a major role in self-association and the other (the carboxyl-terminal moiety) playing a regulatory role in modulating the interactions responsible for self-association.
Cell-cycle-specific cDNAs from mammalian cells temperature sensitive for growth.
Authors: Hirschhorn R R , Aller P , Yuan Z A , Gibson C W , Baserga R
Published: May 2006

A library of double-stranded cDNA was constructed from ts13 cells, a G1-specific temperature-sensitive hamster cell line. The cDNAs, cloned into pBR322, were prepared from poly(A)+ mRNA isolated from ts13 cells 6 hr after serum stimulation at the permissive temperature of 34 degrees C. Differential screening of the library with G1-specific and G0-specific single-stranded cDNA probes prepared from the same cells identified five cDNA clones whose sequences were preferentially expressed in G1. Levels of RNA complementary to these clones were 3- to 6-fold higher in G1 than in other phases of the cell cycle. When ts13 cells were arrested in G1 at the restrictive temperature of 39.6 degrees C, the levels of RNA complementary to p13-2A9 and p13-4F1 were as high as 10 times that found in a resting population, while the expression of sequences complementary to p13-2A8 did not significantly change from levels found in G0. RNA and Southern gel blot analysis suggest that these cell-cycle-specific clones represent either low copy or moderately repetitive gene sequences. Results with another ts mutant of the cell cycle, tsAF8, which is a ts mutant of RNA polymerase II, showed that these cell-cycle-specific sequences have a rapid turnover. The use of G1-specific ts mutants of the cell cycle provides an approach to determine which cell-cycle-dependent genes are most relevant to cell cycle progression.
Cloned avirulence gene of
Pseudomonas syringae
pv.
glycinea
determines race-specific incompatibility on
Glycine max
(L.) Merr
Authors: Staskawicz Brian J. , Dahlbeck Douglas , Keen Noel T.
Published: May 2006

A genomic library of
Pseudomonas syringae
pv.
glycinea
race 6 DNA was constructed in the mobilizable cosmid vector pLAFR1 and maintained in
Escherichia coli
HB101. Completeness of the library was estimated by assaying clones for the expression of ice-nucleating activity in
E. coli
. Ice-nucleation activity was represented approximately once in every 600 clones. Six hundred eighty random race 6 cosmid clones were mobilized from
E. coli
by plasmid pRK2013 in individual conjugations to a race 5 strain of
P. s. glycinea
. A single clone (pPg6L3) was detected that changed the race specificity of race 5 from virulent (compatible) to avirulent (incompatible) on the appropriate soybean cultivars. The clone was also mobilized from
E. coli
into race 1 and race 4 strains of
P. s. glycinea
, and it conferred on these transconjugants the same host range incompatibility as the wild-type race 6 strain. The cosmid clone was mapped by restriction endonucleases, and two adjacent
Eco
RI fragments were identified by transposon Tn5 mutagenesis to be important in determining race specificity. Southern blot analysis showed that the two
Eco
RI fragments are unique to race 6 and are not present in the other races tested. The cosmid clone pPg6L3 was also mobilized to
Pseudomonas fluorescens
and
Rhizobium japonicum
. However, neither these isolates nor
E. coli
harboring pPg6L3 elicited a hypersensitive reaction in soybean leaves.
Cytomegalovirus infects human lymphocytes and monocytes: virus expression is restricted to immediate-early gene products.
Authors: Rice G P , Schrier R D , Oldstone M B
Published: May 2006

In this investigation, we studied the ability of human cytomegalovirus to infect peripheral blood mononuclear cells. With monoclonal antibody technology, we demonstrated that cytomegalovirus could infect human lymphocytes of T- and B-cell lineage, natural killer cells, and monocytes. Furthermore, virus expression was limited to the synthesis of immediate-early cytomegalovirus polypeptides. These peripheral blood mononuclear cells did not produce infectious virus, nor were mature virions visualized by electron microscopy. This abortive infection of mononuclear cells was most convincingly shown with stocks of cytomegalovirus that had been recently isolated from infected patients and passaged minimally in fibroblasts. This argues for an increased lymphotropic effect of some isolates of cytomegalovirus, compared to strains of virus that are extensively adapted to growth in fibroblasts. Furthermore, immunocompetent cells that were shown to be abortively infected with cytomegalovirus lost selected differentiated functions.
Human T-cell leukemia virus (HTLV-I) transcripts in fresh and cultured cells of patients with adult T-cell leukemia.
Authors: Franchini G , Wong-Staal F , Gallo R C
Published: May 2006

We examined the DNA and RNA of fresh leukemic cells (five patients) and long-term cultured T lymphocytes (four patients) from patients with adult T-cell leukemia for the presence of human T-cell leukemia virus (HTLV-I) sequences. In all cases HTLV-I provirus was found and several species of viral mRNAs were observed in all cell lines. We used HTLV DNA probes representing the gag, pol, and env genes and pX regions and the U3- and U5-specific sequences to characterize the genetic content of the viral transcripts. Although similar RNA sequences were expressed in one HTLV-I provirus-positive specimen of fresh primary leukemic cells, no viral transcripts were found in four other similar specimens. These findings are consistent with the idea that the expression of one or more HTLV-I genes may be involved in initiation of transformation, but consistent expression is not needed for maintaining the neoplastic state.
Ionizing radiation and tritium transmutation both cause formation of 5-hydroxymethyl-2'-deoxyuridine in cellular DNA.
Authors: Teebor G W , Frenkel K , Goldstein M S
Published: May 2006

HeLa cells grown in the presence of [methyl-3H]thymidine contained large amounts of 5-hydroxymethyl-2'-deoxyuridine (HMdU) in their DNA. When the cells were grown in [6-3H]thymidine and their DNA was labeled to the same specific activity, no HMdU was present. When such [6-3H]thymidine-labeled cells were exposed to increasing amounts of gamma-radiation, small but increasing amounts of HMdU were formed in their DNA. This indicates that HMdU can be formed in DNA by two distinct mechanisms. The first is the result of the transmutation of 3H to 3He (beta decay) in the methyl group of thymidine, leading to formation of a carbocation. This short-lived ion reacts with hydroxide ions of water, yielding the hydroxymethyl group. HMdU that is formed by this mechanism is formed at the rate of beta decay of 3H. It appears only in [methyl-3H]thymidine residues and is present in the DNA of both nonirradiated and gamma-irradiated cells. The second mechanism is the result of the radiolysis of water caused by ionizing radiation. The resultant radical species, particularly hydroxyl radicals, may react with many sites on DNA. When the methyl group of thymine is attacked by hydroxyl radicals, the hydroxymethyl group is formed. The formation of HMdU by this mechanism was detected only when [6-3H]thymidine-labeled cells were used, since transmutation of 3H in position 6 of thymine cannot yield HMdU.
Chloroplast tRNA
Gly
gene contains a long intron in the D stem: Nucleotide sequences of tobacco chloroplast genes for tRNA
Gly
(UCC) and tRNA
Arg
(UCU)
Authors: Deno Hiroshi , Sugiura Masahiro
Published: May 2006

The nucleotide sequences of tobacco chloroplast genes for tRNA
Gly
(UCC) and tRNA
Arg
(UCU) have been determined. The tRNA
Gly
gene has a 691-base-pair intron located in the D stem while the tRNA
Arg
gene does not have any intron. The tRNA
Gly
and tRNA
Arg
genes are encoded on the same strand and separated by a 169-base-pair spacer. The tRNA
Gly
gene is transcribed as a 900-base precursor RNA molecule. The tRNA
Gly
and tRNA
Arg
deduced from the DNA sequences show 84% and 55% sequence homologies with
Escherichia coli
tRNA
Gly
(UCC) and phage T4 tRNA
Arg
(UCU), respectively.
Preferential binding of estrogen-receptor complex to a region containing the estrogen-dependent hypomethylation site preceding the chicken vitellogenin II gene.
Authors: Jost J P , Seldran M , Geiser M
Published: May 2006

DNA-cellulose competition binding assays were used to measure the ability of cloned DNA fragments of the chicken vitellogenin II gene to displace the estrogen-receptor complex from total chicken DNA coupled to cellulose. The DNA fragment that gave the highest competition is situated in the upstream region of the gene between nucleotides -458 and -725. This DNA fragment has four small clusters of A + T-rich sequences and contains the estrogen-dependent hypomethylation site. In vitro methylation of the Msp I site does not change the capacity of the DNA fragment to compete for estrogen-receptor complex, whereas cleavage of the C-C-G-G (Msp I site) results in a complete loss of competition of this fragment for estrogen-receptor complex. These results, combined with deoxyribonuclease I protection experiments, suggest that the most probable binding site for estrogen-receptor complex is . . .G-C-G-T-G-A-C-C-G-G-A-G-C-T-G-A-A-A-G-A-A-C-A-C. . . . This sequence has 73% homology with the core enhancer sequence of simian virus 40, . . .G-G-T-G-T-G-G-A-A-A-G. . . (identical bases italicized).
Cytosolic calcium regulates epidermal growth factor endocytosis in rat pancreas and cultured fibroblasts.
Authors: Korc M , Matrisian L M , Magun B E
Published: May 2006

Cholecystokinin octapeptide (CCK8), the COOH-terminal moiety of cholecystokinin (CCK), exerted a rapid inhibitory effect on total cell-associated 125I-labeled epidermal growth factor (125I-EGF) binding by decreasing the rate of EGF internalization in isolated rat pancreatic acini. Removal of CCK8 from incubation medium followed by extensive washing of acini did not abolish its inhibitory effect, indicating that its action was not readily reversible. Proglumide, a competitive antagonist of CCK8, blocked the inhibitory action of the secretagogue. Addition of CCK8 to cells previously exposed to 125I-EGF did not enhance the release of cell-associated 125I activity. CCK8 did not inhibit the binding of 125I-labeled insulin to pancreatic acini. Other pancreatic secretagogues that enhance digestive-enzyme release through Ca2+, including caerulein, bombesin, carbachol, gastrin, and the Ca2+ ionophore A23187, also inhibited cell-associated 125I-EGF radioactivity. Further, at 37 degrees C the ionophore A23187 inhibited specific 125I-EGF binding in human A-431 carcinoma cells, Swiss 3T3 cells, and Rat-1 fibroblasts, and this effect was abolished when 125I-EGF internalization was reduced by incubating cells at 4 degrees C. It is concluded that alterations in cellular Ca2+ in the pancreas and other cells lead to inhibition of EGF endocytosis.
On the mechanism of nucleolar dominance in mouse-human somatic cell hybrids.
Authors: Onishi T , Berglund C , Reeder R H
Published: May 2006

The mechanism of nucleolar dominance was studied in two lines of mouse-human somatic hybrids. Both lines had preferentially lost human chromosomes but had retained significant amounts of both mouse and human ribosomal genes (genes coding for the 18S, 5.8S, and 28S RNAs of ribosomes). However, the human ribosomal genes were repressed, and only mouse ribosomal genes were expressed. Soluble transcription extracts from the hybrids were able to initiate RNA synthesis accurately on a cloned mouse ribosomal gene but were unable to initiate accurately on a human ribosomal gene. This suggests that nucleolar dominance in these hybrids is due to the loss or inactivation of the gene for a specificity factor required to recognize the human ribosomal gene promoter.
Amounts, synthesis, and some properties of intracisternal A particle-related RNA in early mouse embryos.
Authors: Pikó L , Hammons M D , Taylor K D
Published: May 2006

Early mouse embryos express two morphological subtypes of intracisternal A-type particles, one resembling those occurring in mouse tumors (referred to as IAP) and the other apparently specific for early embryos [referred to as IAP(epsilon)]. Using cloned fragments of IAP genes as labeled probes in dot-hybridization experiments, we detected IAP-related RNA sequences in mouse oocytes and preimplantation embryos. IAP RNA is relatively abundant in ovarian oocytes, is reduced in amount to approximately equal to 1/10th in the ovulated egg, and increases approximately equal to 100 times (from approximately equal to 1.3 X 10(3) to approximately equal to 1.5 X 10(5) molecules per embryo) between the one-cell stage and late blastocyst stage. Most of the IAP RNA consists of a single size class of about 5.4 kilobases, and a major fraction of this RNA is polyadenylylated. Quantitative considerations suggest that only a few percent of the IAP RNA in embryos are associated with particles. In two-cell embryos, the number of IAP RNA molecules is less than 1/10th the number of IAP(epsilon) particles, suggesting that IAP(epsilon) is genetically distinct from IAP and presumably represents a family of as yet unidentified retrovirus-like elements.