bluuebunny/crossref_metadata_2025 huggingface dataset:
Genetic selection for genes encoding sequence-specific DNA-binding proteins.
Authors: Elledge S J , Sugiono P , Guarente L , Davis R W
Published: May 2006

We describe a genetic selection method designed to facilitate the cloning of genes encoding sequence-specific DNA-binding proteins. The strategy selects for clones expressing particular sequence-specific DNA-binding activities from a library of clones encoding other, nonspecific proteins. Specific DNA-binding sites have been placed near the start of transcription of the strong synthetic conII promoter to create promoters that can be repressed by the corresponding sequence-specific DNA-binding proteins. Transcription from the conII derivatives in the absence of repression interferes with the phenotypic expression of an adjacent drug-resistance gene, aadA. Sequence-specific DNA-binding proteins are shown to repress these promoters and alleviate transcriptional interference of aadA, resulting in drug resistance in cells expressing the appropriate DNA-binding protein.
Molecular cloning of a cDNA that encodes the peptide core of a mouse mast cell secretory granule proteoglycan and comparison with the analogous rat and human cDNA.
Authors: Avraham S , Stevens R L , Nicodemus C F , Gartner M C , Austen K F , Weis J H
Published: May 2006

A cDNA that encodes a mouse secretory granule proteoglycan peptide core was isolated from a cDNA library prepared from nontransformed mouse bone marrow-derived mast cells (BMMC) using as a probe a 280-base-pair fragment of a rat cDNA that encodes the proteoglycan peptide core of rat basophilic leukemia (RBL)-1 cells. Based on the consensus nucleotide sequence and deduced amino acid sequence of the cDNA, the mouse BMMC proteoglycan peptide core is 16.7 kDa and contains a 21-amino acid glycosaminoglycan attachment region consisting of alternating serine and glycine residues. When the predicted amino acid sequence of the mouse BMMC proteoglycan peptide core was compared with the predicted amino acid sequences of the homologous molecules expressed in RBL-1 cells and in human promyelocytic leukemia HL-60 cells, the mouse-derived sequence was more closely homologous to the rat sequence than the human sequence except for the length of the serine-glycine repeat region. The N terminus was found to be a highly conserved region of the molecule in the three species, suggesting that this region is important for the structure, function, and/or metabolism of this family of proteoglycans. Nucleotide sequences within the 5' and 3' untranslated regions of the mouse, rat, and human proteoglycan cDNA were conserved. That similar sequences were also present in the corresponding regions of a cDNA that encodes a rat mast cell protease suggests that particular nucleotide sequences may be important for regulation of expression of those proteins that are destined to reside in secretory granules.
Guanine nucleotide-binding protein regulation of melatonin receptors in lizard brain.
Authors: Rivkees S A , Carlson L L , Reppert S M
Published: May 2006

Melatonin receptors were identified and characterized in crude membrane preparations from lizard brain by using 125I-labeled melatonin (125I-Mel), a potent melatonin agonist. 125I-Mel binding sites were saturable; Scatchard analysis revealed high-affinity and lower affinity binding sites, with apparent Kd of 2.3 +/- 1.0 x 10(-11) M and 2.06 +/- 0.43 x 10(-10) M, respectively. Binding was reversible and inhibited by melatonin and closely related analogs but not by serotonin or norepinephrine. Treatment of crude membranes with the nonhydrolyzable GTP analog guanosine 5'-[gamma-thio]triphosphate (GTP[gamma S]), significantly reduced the number of high-affinity receptors and increased the dissociation rate of 125I-Mel from its receptor. Furthermore, GTP[gamma S] treatment of ligand-receptor complexes solubilized by Triton X-100 also led to a rapid dissociation of 125I-Mel from solubilized ligand-receptor complexes. Gel filtration chromatography of solubilized ligand-receptor complexes revealed two major peaks of radio-activity corresponding to Mr greater than 400,000 and Mr ca. 110,000. This elution profile was markedly altered by pretreatment with GTP[gamma S] before solubilization; only the Mr 110,000 peak was present in GTP[gamma S]-pretreated membranes. The results strongly suggest that 125I-Mel binding sites in lizard brain are melatonin receptors, with agonist-promoted guanine nucleotide-binding protein (G protein) coupling and that the apparent molecular size of receptors uncoupled from G proteins is about 110,000.
Molecular cloning and amino acid sequence of human plakoglobin, the common junctional plaque protein.
Authors: Franke W W , Goldschmidt M D , Zimbelmann R , Mueller H M , Schiller D L , Cowin P
Published: May 2006

Plakoglobin is a major cytoplasmic protein that occurs in a soluble and a membrane-associated form and is the only known constituent common to the submembranous plaques of both kinds of adhering junctions, the desmosomes and the intermediate junctions. Using a partial cDNA clone for bovine plakoglobin, we isolated cDNAs encoding human plakoglobin, determined its nucleotide sequence, and deduced the complete amino acid sequence. The polypeptide encoded by the cDNA was synthesized by in vitro transcription and translation and identified by its comigration with authentic plakoglobin in two-dimensional gel electrophoresis. The identity was further confirmed by comparison of the deduced sequence with the directly determined amino acid sequence of two fragments from bovine plakoglobin. Analysis of the plakoglobin sequence showed the protein (744 amino acids; 81,750 Da) to be unrelated to any other known proteins, highly conserved between human and bovine tissues, and characterized by numerous changes between hydrophilic and hydrophobic sections. Only one kind of plakoglobin mRNA (3.4 kilobases) was found in most tissues, but an additional mRNA (3.7 kilobases) was detected in certain human tumor cell lines. This longer mRNA may be represented by a second type of plakoglobin cDNA, which contains an insertion of 297 nucleotides in the 3' non-coding region.
Localization of preferential sites of rearrangement within the BCR gene in Philadelphia chromosome-positive acute lymphoblastic leukemia.
Authors: Denny C T , Shah N P , Ogden S , Willman C , McConnell T , Crist W , Carroll A , Witte O N
Published: May 2006

The Philadelphia chromosome associated with acute lymphoblastic leukemia (ALL) has been linked to a hybrid BCR/ABL protein product that differs from that found in chronic myelogenous leukemia. This implies that the molecular structures of the two chromosomal translocations also differ. Localization of translocation breakpoints in Philadelphia chromosome-positive ALL has been impeded due to the only partial characterization of the BCR locus. We have isolated the entire 130-kilobase BCR genomic locus from a human cosmid library. A series of five single-copy genomic probes from the 70-kilobase first intron of BCR were used to localize rearrangements in 8 of 10 Philadelphia chromosome-positive ALLs. We have demonstrated that these breakpoints are all located at the 3' end of the intron around an unusual restriction fragment length polymorphism caused by deletion of a 1-kilobase fragment containing Alu family reiterated sequences. This clustering is unexpected in light of previous theories of rearrangement in Philadelphia chromosome-positive chronic myelogenous leukemia that would have predicted a random dispersion of breakpoints in the first intron in Philadelphia chromosome-positive ALL. The proximity of the translocation breakpoints to this constitutive deletion may indicate shared mechanisms of rearrangement or that such polymorphisms mark areas of the genome prone to recombination.
Evidence that the familial adenomatous polyposis gene is involved in a subset of colon cancers with a complementable defect in c-myc regulation.
Authors: Erisman M D , Scott J K , Astrin S M
Published: May 2006

Human colorectal carcinomas frequently express elevated levels of c-myc mRNA in the absence of a gross genetic change at the c-myc locus. To test the hypothesis that these tumors are defective in a gene function necessary for the regulation of c-myc expression, we fused an osteosarcoma cell line that exhibits normal c-myc regulation with two colon carcinoma cell lines that express deregulated levels of c-myc mRNA. The levels of c-myc transcripts in all of the hybrid clones examined were normal and were induced normally by a mitogenic stimulus. Since rates of c-myc mRNA turnover in the colon carcinoma cells were found to be comparable to those in normal cells, increased message stability cannot account for the increased steady-state levels of transcripts. Our findings suggest that loss of function of a trans-acting regulator is responsible for the deregulation of c-myc expression in a major fraction of colorectal carcinomas. Analysis of restriction fragment length polymorphisms in tumor/normal tissue pairs from patients with primary colorectal lesions indicated that deregulation of c-myc expression in the tumors is correlated with frequent loss of alleles of syntenic markers on chromosome 5q; allele loss on 5q could be detected in 9 of 19 tumors expressing deregulated levels of c-myc mRNA, but not in any of 8 tumors expressing normal levels of c-myc RNA. Chromosome 5q is the region known to contain the gene for familial adenomatous polyposis, an inherited predisposition to colon cancer. These findings, together with the earlier finding that the colonic distribution of tumors exhibiting deregulated c-myc expression is similar to that reported for familial polyposis, provide evidence that loss of function of the familial adenomatous polyposis gene is involved in a subset of colorectal cancers in which c-myc expression is deregulated.
Induction of multiple independent T-cell lymphomas in rats inoculated with MOloney murine leukemia virus.
Authors: Bellacosa A , Lazo P A , Bear S E , Shinton S , Tsichlis P N
Published: May 2006

Tumor cell DNA derived from different lymphoid organs of 30 rats serially inoculated at birth with Moloney murine leukemia virus (MoMuLV) was examined by Southern blot analysis and hybridization to the following DNA probes: MoMuLV long terminal repeat (LTR), Moloney leukemia virus integration regions 1, 2, 3, and 4 (Mlvi-1, Mlvi-2, Mlvi-3, and Mlvi-4), T-cell receptor beta locus, and immunoglobulin heavy chain locus. This analysis revealed that the tumors segregating in different lymphoid organs in 10% of the animals were clonally unrelated. These findings are consistent with the hypothesis that the MoMuLV-induced rat thymic lymphomas are polyclonal in origin. At least two factors may be responsible for this phenomenon: (i) increase in the number of the available target cells in virus-infected animals, and (ii) genetic instability associated with provirus integration in the developing premalignant clones.
Molecular cloning and sequencing of chicken liver fatty acid synthase cDNA.
Authors: Holzer K P , Liu W , Hammes G G
Published: May 2006

The complete amino acid sequence of chicken liver fatty acid synthase [acyl-CoA:malonyl-CoA C-acyltransferase (decarboxylating, oxoacyl- and enoyl-reducing, and thioester-hydrolyzing), EC 2.3.1.85] has been determined from the corresponding cDNA sequence. A 5.3-kilobase-pair (kbp) region of cDNA coding for chicken fatty acid synthase has been cloned and sequenced that is contiguous to the 2.3-kbp region previously sequenced [Yuan, Z., Liu, W. & Hammes, G.G. (1988) Proc. Natl. Acad. Sci. USA 85, 6328-6331]. The cDNA codes for the remaining 1677 amino acids of the previously unsequenced region of the protein. The amino acid sequence contains peptides known to be associated with the NADPH binding site of the enoylreductase active center, the acetyl/malonyltransacylase active site, the "waiting" site containing cysteine, and a pyridoxal 5'-phosphate binding site. Locations of the NADPH binding site of the beta-ketoacylreductase active site and of the dehydratase active site are proposed on the basis of protein sequence homologies to catalytic sites in other enzymes. The molecular weight of the complete polypeptide chain is 267,288. A linear functional map of the chicken fatty acid synthase derived from its primary sequence is presented.
Mutations in the maize mitochondrial T-urf13 gene eliminate sensitivity to a fungal pathotoxin.
Authors: Braun C J , Siedow J N , Williams M E , Levings C S
Published: May 2006

URF13, the product of the mitochondrial T-urf13 gene, confers on Texas cytoplasmic male-steril maize (Zea mays L.) a unique susceptibility to a fungal pathogen (Bipolaris maydis race T) and sensitivity to its pathotoxin. Expression of URF13 in Escherichia coli imparts pathotoxin sensitivity to the bacterium. We show by ion uptake studies in E. coli that a pathotoxin-URF13 interaction causes membrane permeability. Similarly, mitochondrial dysfunction caused by membrane permeabilization probably accounts for increased colonization of maize carrying the Texas cytoplasm by toxin-producing pathogens. Site-directed mutagenesis studies show that approximately one-quarter of the amino acids at the carboxyl end of URF13 can be eliminated without affecting toxin sensitivity. We have identified two dicyclohexylcarbodiimide (DCCD) binding sites in the URF13 protein and show that one of the sites is involved in conferring DCCD protection against the pathotoxin. Substitutional mutations at this DCCD binding site also eliminate toxin sensitivity.
Tobacco mosaic virus RNA enters chloroplasts
in vivo
Authors: Schoelz James E. , Zaitlin Milton
Published: May 2006

Several lines of evidence are presented to allow us to conclude that tobacco mosaic virus (TMV) RNA enters the chloroplast
in vivo
. Chloroplasts were prepared from either directly inoculated or systemically infected leaves of tobacco plants inoculated with one of several strains of the virus and from uninfected control plants. Intact chloroplasts were isolated on Percoll gradients and treated with pancreatic RNase and thermolysin to destroy potential TMV virions and RNA on the outside or bound to their surfaces. Northern blot analysis of RNA extracted from these chloroplasts demonstrated that full-length TMV RNA was present within the chloroplasts prepared from both directly inoculated and systemically invaded leaves. Only genomic length, but not subgenomic length, RNA was found in the chloroplast extracts, indicating a selectivity of the transport of the viral RNA into the chloroplast. A temperature-sensitive TMV mutant (Ts 38), in which no virions are formed at 35°C, was used to demonstrate that at that restrictive temperature viral RNA is detected in the chloroplast, indicating that free viral RNA can enter the chloroplast rather than intact virions. To our knowledge, the transport of a foreign RNA species into chloroplasts has not been reported previously.
Theileria annulata sporozoite surface antigen expressed in Escherichia coli elicits neutralizing antibody.
Authors: Williamson S , Tait A , Brown D , Walker A , Beck P , Shiels B , Fletcher J , Hall R
Published: May 2006

Theileria annulata is an economically important protozoan parasite that threatens an estimated 250 million cattle with the disease tropical theileriosis. Development of a defined subunit vaccine is one means of trying to develop control measures against the disease. To this end we have characterized a surface antigen complex of the infective stage (sporozoite), by using a monoclonal antibody that neutralizes sporozoite infectivity in vitro. We have cloned the gene coding for this complex and have demonstrated that a fusion protein expressed from a fragment of this gene elicits strong neutralizing antibodies. Furthermore we provide data on the structure and expression of this gene. In particular we show that the region of the gene, expressed in one clone, codes for a protein segment relatively rich in proline residues. Also we demonstrate that expression of this gene appears to be stage specific, transcripts being present only in the sporoblast and sporozoite stages. The relevance of these findings to the production of a defined subunit vaccine is discussed.
Evolutionary expression of the neuronal form of the src protein in the brain.
Authors: Yang X M , Martinez R , Le Beau J , Wiestler O , Walter G
Published: May 2006

The protooncogene src encodes two proteins, designated pp60c-src+ and pp60c-src.pp60c-src+ is expressed only in neurons, whereas pp60c-src is expressed in neuronal and nonneuronal cells. pp60c-src+ differs from pp60c-src in that it contains an insert of 6 amino acids. To study the evolutionary conservation of the 6-amino acid insert, the expression of pp60c-src+ in the brain of animals from different classes was assayed by using pp60c-src+-specific antibodies raised against a synthetic peptide corresponding to the insert. pp60c-src+ was detected only in the brain of mammals, birds, and reptiles, but not amphibians and fish, whereas pp60c-src was present in the brain of all animals tested, including lobster (invertebrate). These findings indicate that pp60c-src+ may play a role in events associated with higher brain function, such as neuronal plasticity.
Role of neuromedin B in the control of the release of thyrotropin in the rat.
Authors: Rettori V , Milenkovic L , Fahim A M , Polak J , Bloom S R , McCann S M
Published: May 2006

Neuromedin B (NB), a bombesin-like peptide, was first isolated from porcine spinal cord and subsequently found in the central nervous systems of rat, cat, and human. Immunocytochemical studies have shown that NB is present in hypothalamus and various other regions of the brain and in thyrotrophs of the anterior pituitary of the rat. The possible physiological role of NB in the hypothalamic-pituitary axis is not known. Therefore, we studied the in vivo effect of this peptide on the plasma levels of thyrotropin (TSH) after administering NB to conscious freely moving adult male rats. When injected i.v., only the highest dose of NB (50 micrograms, 44.2 nmol) significantly lowered plasma TSH levels relative to levels in saline controls. When injected intraventricularly, NB doses of 0.5 and 5 micrograms (0.44 and 4.42 nmol, respectively) lowered plasma TSH levels with a 15-min latency period. Responsiveness of the pituitary to TSH-releasing hormone (TRH) was tested after the effective dose of NB was administered i.v. The increase in plasma TSH levels after the i.v. injection of 1 microgram of TRH was not altered by NB. NB at 10(-9) to 10(-7) M also reduced TSH release from hemipituitaries incubated in vitro without decreasing the response to TRH. Consequently, NB appears to have a direct inhibitory effect on TSH release from thyrotrophs. Since the response to TRH was not depressed, NB appears to act on a separate NB thyrotroph receptor that suppresses TSH release by a TRH-independent mechanism. After antiserum directed against NB was injected into the third ventricle, there was a highly significant elevation of the plasma level of TSH, beginning at 4 hr and increasing at 6 hr, relative to initial levels and levels in normal rabbit serum-injected controls. These results indicate that NB has a tonic suppressive effect on TSH release and is a physiologically significant TSH-release-inhibitory factor.
Phorbol ester binding to protein kinase C requires a cysteine-rich zinc-finger-like sequence.
Authors: Ono Y , Fujii T , Igarashi K , Kuno T , Tanaka C , Kikkawa U , Nishizuka Y
Published: May 2006

Protein kinase C normally has a tandem repeat of a characteristic cysteine-rich sequence in C1, the conserved region of the regulatory domain. These sequences resemble the DNA-binding zinc finger domain. For the gamma subspecies of rat brain protein kinase C, various deletion and point mutants in this domain were constructed, and the mutated proteins were expressed in Escherichia coli by using the T7 expression system. Radioactive phorbol 12,13-dibutyrate binding analysis indicated that a cysteine-rich zinc-finger-like sequence was essential for protein kinase C to bind phorbol ester and that one of two sequences was sufficient for the phorbol ester binding. Conserved region C2, another region in the regulatory domain, was apparently needed for the enzyme to require Ca2+ for phorbol ester binding activity.
Linkage mapping of a mouse gene, iv, that controls left-right asymmetry of the heart and viscera.
Authors: Brueckner M , D'Eustachio P , Horwich A L
Published: May 2006

Inherited single gene defects have been identified in both humans and mice that lead to loss of developmental control over the left-right asymmetry of the heart and viscera. In mice the recessively inherited mutation iv leads to such apparent loss of control over situs: 50% of iv/iv mice exhibit situs inversus and 50% exhibit normal situs. The affected gene product has not been identified in these animals. To study the normal function of iv, we have taken an approach directed to the gene itself. As a first step, we have mapped iv genetically, by examining its segregation in backcrosses with respect to markers defined by restriction fragment length polymorphisms. The iv locus lies 3 centimorgans (cM) from the immunoglobulin heavy-chain constant-region gene complex (Igh-C) on chromosome 12. A multilocus map of the region suggests the gene order centromere-Aat (alpha 1-antitrypsin gene complex)-(11 cM)-iv-(3 cM)-Igh-C-(1 cM)-Igh-V (immunoglobulin heavy-chain variable-region gene complex).
Distribution of neurotensin/neuromedin N mRNA in rat forebrain: unexpected abundance in hippocampus and subiculum.
Authors: Alexander M J , Miller M A , Dorsa D M , Bullock B P , Melloni R H , Dobner P R , Leeman S E
Published: May 2006

We have used in situ hybridization to determine the regional distribution of mRNA encoding the neurotensin/neuromedin N (NT/N) precursor in the forebrain of the adult male rat. Cells containing NT/N mRNA are widely distributed in the forebrain. These areas include the septum, bed nucleus of the stria terminalis, preoptic area, hypothalamus, amygdala, accumbens nucleus, caudate-putamen, and piriform and retrosplenial cortex. In general, the regional distribution of NT/N mRNA corresponds to the previously determined distribution of neurotensin-immunoreactive cell bodies; however, several notable exceptions were observed. The most striking difference occurs specifically in the CA1 region of the hippocampus, where intense labeling is associated with the pyramidal cell layer despite the reported absence of neurotensin-immunoreactive cells in this region. Analysis of microdissected tissue by S1 nuclease protection assay confirmed the abundance of authentic NT/N mRNA in CA1. A second major discrepancy between NT/N mRNA abundance and neurotensin-immunoreactivity occurs in the intensely labeled subiculum, a region that contains only scattered neurotensin-immunoreactive cells in the adult. These results suggest that, in specific regions of the forebrain, NT/N precursor is processed to yield products other than neurotensin. In addition, these results provide an anatomical basis for studying the physiological regulation of NT/N mRNA levels in the forebrain.
The v-sea oncogene of avian erythroblastosis retrovirus S13: another member of the protein-tyrosine kinase gene family.
Authors: Smith D R , Vogt P K , Hayman M J
Published: May 2006

The cloning and sequencing of the oncogene of the avian erythroblastosis virus S13 is described. The oncogene, termed v-sea, was found to be another member of the protein-tyrosine kinase gene family. The oncogene was fused in frame with the retrovirus S13 envelope gene, thus generating a fusion protein with a structure resembling that of a growth factor receptor. Sequence comparisons revealed that the v-sea gene was most closely related to the insulin receptor family of protein-tyrosine kinases, the greatest similarity being with the human MET oncogene.
An alternative cytoplasmic domain of the integrin beta 3 subunit.
Authors: van Kuppevelt T H , Languino L R , Gailit J O , Suzuki S , Ruoslahti E
Published: May 2006

A cDNA encoding a new form of the shared beta subunit (beta 3) of the platelet integrin gpIIb/IIIa and the vitronectin receptor was isolated from a placental cDNA library by screening with a beta 3 (gpIIIa) DNA probe. This beta 3 variant differs from the previously reported beta 3 in that the cytoplasmic domain is 8 amino acids shorter and has an alternative, 13-amino acid COOH-terminal peptide. The 3' untranslated region of the cDNA also differs from the previously reported sequence, while the region coding for the transmembrane domain and extracellular domain is identical to it. Reverse transcription combined with polymerase chain reaction was used to show that human placental tissue and two human cell lines contain the variant mRNA. The sequences of the cDNAs for the previously known beta 3 and the variant beta 3 described here suggest that the difference between the cytoplasmic domains of these subunits arises as a result of an alternative mRNA splicing. These cytoplasmic domains may provide alternative means for the beta 3 integrins to interact with cytoskeletal components.
A general method for detecting rearrangements in a bacterial genome.
Authors: Au L C , Ts'o P O
Published: May 2006

An effective method was developed to monitor genome rearrangement in bacteria. The whole procedure consists of five steps. (i) Genomic DNAs of reference cells and test cells are digested with the same restriction enzyme. (ii) The DNA restriction fragments from the test cells are radioactively labeled. (iii) The labeled DNA fragments of test cells are mixed with unlabeled DNA fragments from reference cells that are 100- to 1000-fold in excess and the mixture is electrophoresed in an agarose gel. (iv) After electrophoresis, DNA fragments are alkali-denatured; this is followed by renaturation in situ in the gel. The labeled rearranged DNA fragments from the test cells will renature much slower, as compared with the nonrearranged fragments, since in this location of the gel these rearranged fragments do not have a counterpart in the driver DNA, which is in excess. (v) The DNA gel is electrophoresed in a second dimension perpendicular to the first dimension after renaturation. The denatured rearranged DNAs are revealed after autoradiography, since single-stranded DNA fragments have higher electrophoretic mobility than double-stranded fragments of the same sizes. This process of detection has been demonstrated in this report by using Escherichia coli HB101 as the reference strain and E. coli HB101 carrying lambda phage DNA (1:1 genomic ratio) as the test strain.
Early estrogen-induced metabolic changes and their inhibition by actinomycin D and cycloheximide in human breast cancer cells: 31P and 13C NMR studies.
Authors: Neeman M , Degani H
Published: May 2006

Metabolic changes following estrogen stimulation and the inhibition of these changes in the presence of actinomycin D and cycloheximide were monitored continuously in perfused human breast cancer T47D clone 11 cells with 31P and 13C NMR techniques. The experiments were performed by estrogen rescue of tamoxifen-treated cells. Immediately after perfusion with estrogen-containing medium, a continuous enhancement in the rates of glucose consumption, lactate production by glycolysis, and glutamate synthesis by the Krebs cycle occurred with a persistent 2-fold increase at 4 hr. The content of phosphocholine had increased by 10% to 30% within the first hour of estrogen stimulation, but the content of the other observed phosphate metabolites as well as the pH remained unchanged. Pretreatment with either actinomycin D or cycloheximide, at concentrations known to inhibit mRNA and protein synthesis, respectively, and simultaneous treatment with estrogen and each inhibitor prevented the estrogen-induced changes in glucose metabolism. This suggested that the observed estrogen stimulation required synthesis of mRNA and protein. These inhibitors also modulated several metabolic activities that were not related to estrogen stimulation. The observed changes in the in vivo kinetics of glucose metabolism may provide a means for the early detection of the response of human breast cancer cells to estrogen versus tamoxifen treatment.
Yeast TATA-binding protein TFIID binds to TATA elements with both consensus and nonconsensus DNA sequences.
Authors: Hahn S , Buratowski S , Sharp P A , Guarente L
Published: May 2006

The DNA binding properties of the yeast TATA element-binding protein TFIID were investigated. The affinity (apparent equilibrium dissociation constant) of TFIID for the adenovirus major late promoter consensus TATA element is 2 x 10(-9) M, a value similar to the affinity of gene-specific regulatory proteins for their binding sites. TFIID binding is highly specific and recognizes nonspecific sites with approximately 10(5)-fold lower affinity. Despite this specificity, TFIID also binds with high affinity to several TATA elements that do not match the consensus TATA sequences (TATAAA and TATATA): the yeast LEU2 TATA (TATTATTTA), the simian virus 40 TATA (CTTATTTAT), and the yeast CYC1 -10 TATA (TTATACATT) all bound TFIID. Furthermore, TFIID was active in promoting transcription in vitro from the nonconsensus TATA elements. Thus, contrary to previous suggestions, the existence of nonconsensus TATA elements does not itself indicate the existence of multiple TATA-binding factors.
Isolation and characterization of cDNA encoding the alpha subunit of Cap Z(36/32), an actin-capping protein from the Z line of skeletal muscle.
Authors: Casella J F , Casella S J , Hollands J A , Caldwell J E , Cooper J A
Published: May 2006

cDNA encoding the alpha chain of Cap Z has been isolated by screening a lambda gt11 library with affinity-purified antibodies. A single cDNA insert (designated CE2) of 2153 base pairs (bp) contains an open reading frame of 836 bp, which is incomplete at its 5' end. The technique of "rapid amplification of cDNA ends" has been used to extend the 5' end of this open reading frame to a potential transcription initiation site that is preceded by 320 bp of an apparently untranslated region. The protein predicted by the resulting nucleotide sequence has a Mr of 32,960 and contains four regions that show close homology with four alpha-chymotryptic digestion fragments of the alpha chain. The amino acid composition of the alpha chain of Cap Z and the predicted protein are also similar. Northern blot analysis of whole chicken embryos shows two mRNA species of 1.9 and 2.4 kilobases, respectively, that hybridize with CE2. Three potential polyadenylylation signals in two regions of CE2 460 bp apart are identified, suggesting that the two messages may result from the use of alternative polyadenylylation sites. Comparison of the sequence data with that of other known actin-capping and severing proteins shows no significant homologies, suggesting that Cap Z may be a member of a unique group of capping, nonsevering proteins.
Mutator phenotype of Werner syndrome is characterized by extensive deletions.
Authors: Fukuchi K , Martin G M , Monnat R J
Published: May 2006

Werner syndrome (WS) is a rare autosomal-recessive disorder characterized by the premature appearance of features of normal aging in young adults. The extensive phenotypic overlap between WS and normal aging suggests they may also share pathogenetic mechanisms. We reported previously that somatic cells from WS patients demonstrate a propensity to develop chromosomal aberrations, including translocations, inversions, and deletions, and that WS cell lines demonstrate a high spontaneous mutation rate to 6-thioguanine resistance. We report here the biochemical and molecular characterization of spontaneous mutations at the X chromosome-linked hypoxanthine phosphoribosyltransferase (HPRT) locus in 6-thioguanine-resistant WS and control cells. Blot hybridization analysis of 89 independent spontaneous HPRT mutations in WS and control mutants lacking HPRT activity revealed an unusually high proportion of HPRT deletions in WS as compared with control cells (76% vs. 39%). Approximately half (58%) of the deletions in WS cells consisted of the loss of greater than 20 kilobases of DNA from the HPRT gene. These results suggest that an elevated somatic mutation rate, and particularly deletions, may play pathogenetically important roles in WS and in several associated age-dependent human disease processes.
Decrease by chronic energy intake restriction of cellular proliferation in the intestinal epithelium and lymphoid organs in autoimmunity-prone mice.
Authors: Ogura M , Ogura H , Ikehara S , Dao M L , Good R A
Published: May 2006

In previous studies we demonstrated that chronic energy intake restriction (CEIR) by a diet relatively low in fat, relatively high in carbohydrate, and reduced 40% in total calories extends life span and delays development of autoimmune disease in autoimmunity-prone mice. To investigate a possible cellular basis for this dramatic action of CEIR, we analyzed the rate of incorporation of [3H]thymidine by cells of the intestinal epithelium, thymus, spleen, and mesenteric lymph nodes in ad libitum-fed mice vs. CEIR mice of three autoimmunity-prone strains. In New Zealand Black (NZB), MRL/MP-lpr/lpr (MRL/lpr), and BXSB mice, CEIR slowed the rate of uptake of [3H]thymidine and, by inference, the rate of cellular proliferation among epithelial cells along the entire length of the gastrointestinal tract. Furthermore, CEIR decreased the apparent proliferative rate of lymphoid cells of the thymus, spleen, and mesenteric lymph nodes. This action by CEIR on the proliferative rate of cells of these rapidly replicating cell populations may point to an important mechanism by which calorie restriction inhibits the development of autoimmune disease and extends longevity in autoimmunity-prone mice.
Human endomembrane H+ pump strongly resembles the ATP-synthetase of Archaebacteria.
Authors: Südhof T C , Fried V A , Stone D K , Johnston P A , Xie X S
Published: May 2006

Preparations of mammalian H+ pumps that acidify intracellular vesicles contain eight or nine polypeptides, ranging in size from 116 to 17 kDa. Biochemical analysis indicates that the 70- and 58-kDa polypeptides are subunits critical for ATP hydrolysis. The amino acid sequences of the major catalytic subunits (58 and 70 kDa) of the endomembrane H+ pump are unknown from animal cells. We report here the complete sequence of the 58-kDa subunit derived from a human kidney cDNA clone and partial sequences of the 70- and 58-kDa subunits purified from clathrin-coated vesicles of bovine brain. The amino acid sequences of both proteins strongly resemble the sequences of the corresponding subunits of the vacuolar H+ pumps of Archaebacteria, plants, and fungi. The archaebacterial enzyme is believed to use a H+ gradient to synthesize ATP. Thus, a common ancestral protein has given rise to a H+ pump that synthesizes ATP in one organism and hydrolyzes it in another and is highly conserved from prokaryotes to humans. The same pump appears to mediate the acidification of intracellular organelles, including coated vesicles, lysosomes, and secretory granules, as well as extracellular fluids such as urine.
Stimulation of phosphorylation of lipocortin at threonine residues by epidermal growth factor (EGF) and the EGF receptor: addition of protein kinase P with polylysine inhibits this effect.
Authors: Abdel-Ghany M , Kole H K , el Saad M A , Racker E
Published: May 2006

In this paper we show that epidermal growth factor (EGF) stimulates the phosphorylation of lipocortin 1, at threonine as well as at tyrosine residues, by a highly purified preparation of the EGF receptor. The phosphorylation of threonine residues is catalyzed by an enzyme that contaminates the receptor preparations, since crude extracts of A431 plasma membranes contain larger amounts of the threonine kinase than does the receptor preparation. Protein kinase P (2.5 ng) inhibits both threonine and tyrosine phosphorylation of lipocortin 1 while greatly stimulating the autophosphorylation of the EGF receptor. Acetyllipocortin 1 is poorly phosphorylated at tyrosine residues by the EGF receptor kinase, but it becomes readily phosphorylated in the presence of polylysine. The most likely explanation for this observation is that there is an interaction between polylysine and acetyllipocortin that converts the latter into a suitable substrate for the EGF receptor. These and other experiments described in this paper point to a role of surface charges in the susceptibility of substrates to attach by protein kinases.
Cloning human telomeric DNA fragments into Saccharomyces cerevisiae using a yeast-artificial-chromosome vector.
Authors: Riethman H C , Moyzis R K , Meyne J , Burke D T , Olson M V
Published: May 2006

Telomeric fragments of human DNA ranging in size from 50 to 250 kilobases were cloned into Saccharomyces cerevisiae using a yeast-artificial-chromosome (YAC) vector. Six human-telomeric YAC (HTY) strains were selected by virtue of the specific hybridization of their DNA with the human telomeric terminal-repeat sequence (TTAGGG)n, and the telomeric localization of this sequence within each YAC was demonstrated by its sensitivity to nuclease BAL-31. In situ hybridization of DNA from three of these HTY strains with human metaphase chromosomes yielded discrete patterns of hybridization signals at the telomeres of a limited number of human chromosomes, different for each clone. DNA from selected cosmid subclones of one of the HTY strains was used to localize the origin of the cloned telomeric DNA by in situ hybridization to the tip of the long arm of chromosome 7.
In vitro propagation of human hepatitis B virus in a rat hepatoma cell line.
Authors: Shih C H , Li L S , Roychoudhury S , Ho M H
Published: May 2006

A rat hepatoma cell line (Q7) of Morris hepatoma origin was transfected with a construct containing the tandem dimer genome of human hepatitis B virus (HBV) and the neomycin-resistant selection marker. The culture medium of several neomycin-resistant single-cell clones was found to accumulate high levels of secreted HBV surface antigen and core-related e antigen. HBV-specific replication intermediates, including relaxed circular and single-stranded DNA with a minus-strand polarity, could be found in both the intracellular fraction and the extracellular culture medium by the Southern blot procedure. One of these clones, designated Q7 HBV-21, was characterized in further detail. DNA polymerase activity was present in the virus particles produced by Q7 HBV-21 cells. Characteristic transcripts of HBV, including the 3.5-, 2.5-, and 2.1-kilobase mRNA as well as a core-gene-related transcript of 2.2 kilobases could be detected. Electron microscopic examination of the conditioned medium from Q7 HBV-21 cells identified 42-nm Dane-like particles as well as 22-nm subviral particles with a spherical or filamentous shape. This Q7 HBV-21 cell line has been maintained in the absence of neomycin for 1 year without losing the properties of HBV DNA replication and Dane-like particle production. Our results strongly suggest that the species barrier of HBV infection is at an early step of viral absorption onto or penetration into the target hepatocytes. This nonhuman system for HBV production in culture could be used to complement the human HepG2 system.
Epstein-Barr virus-transformed pro-B cells are prone to illegitimate recombination between the switch region of the mu chain gene and other chromosomes.
Authors: Altiok E , Klein G , Zech L , Uno M , Henriksson B E , Battat S , Ono Y , Ernberg I
Published: May 2006

Six independently maintained sublines of FLEB 14, a fetal-liver-derived Epstein-Barr virus-transformed pro-B cell line that has not yet rearranged its immunoglobulin genes, were examined after in vitro propagation during 19-36 months. Two lines showed no immunoglobulin heavy chain gene rearrangement, whereas one allele was rearranged with breakpoints inside the switch region of the mu chain gene in the remaining four. These rearrangements had been generated by the translocation of different chromosome fragments to the immunoglobulin heavy chain gene cluster-carrying 14q32 band in each of the four lines. Previously, a similar rearrangement was found in a fifth subline concurrently with a reciprocal 6;14 translocation. The transposed pieces have been derived from chromosomes 16 and 18 in two of the more recently rearranged lines. Their origins could not be determined in the remaining two lines, but they were different from each other and the other three 14q+ markers. The 14q+ marker-carrying variant has replaced its diploid progenitor suggesting that the translocation has conveyed some in vitro growth advantage on its carrier. This was also supported by the duplication of the 14q+ marker and the loss of its normal chromosome 14 homologue in one subline during serial culturing. The vulnerability of the switch region of the mu chain gene to illegitimate recombination at the pro-B stage and the possible relevance of this finding for the origin of the Burkitt lymphoma-associated 8;14 (immunoglobulin heavy chain gene cluster/MYC) translocation is discussed.
Mechanism of oligonucleotide uptake by cells: involvement of specific receptors?
Authors: Yakubov L A , Deeva E A , Zarytova V F , Ivanova E M , Ryte A S , Yurchenko L V , Vlassov V V
Published: May 2006

We have investigated the interaction of oligonucleotides and their alkylating derivatives with mammalian cells. In experiments with L929 mouse fibroblast and Krebs 2 ascites carcinoma cells, it was found that cellular uptake of oligodeoxynucleotide derivatives is achieved by an endocytosis mechanism. Uptake is considerably more efficient at low oligomer concentration (less than 1 microM), because at this concentration a significant percentage of the total oligomer pool is absorbed on the cell surface and internalized by a more efficient absorptive endocytosis process. Two modified proteins were detected in mouse fibroblasts that were treated with the alkylating oligonucleotide derivatives. The binding of the oligomers to the proteins is inhibited by other oligodeoxynucleotides, single- and double-stranded DNA, and RNA. The polyanions heparin and chondroitin sulfates A and B do not inhibit binding. These observations suggest the involvement of specific receptor proteins in binding of oligomers to mammalian cells.
Bacterial luciferase alpha beta fusion protein is fully active as a monomer and highly sensitive in vivo to elevated temperature.
Authors: Escher A , O'Kane D J , Lee J , Szalay A A
Published: May 2006

A 2.2-kilobase-pair (kbp) DNA fragment from Vibrio harveyi contains the luxA and luxB genes separated by a 26-base-pair (bp) intergenic region. The two genes were converted to a single open reading frame by site-specific mutagenesis. A full-length fusion protein is obtained when the new gene is placed under transcriptional control of a T7 promoter in Escherichia coli. Bioluminescence of colonies containing the gene fusion is 0.002% of the wild-type luciferase [alkanal monooxygenase (FMN-linked); alkanal, reduced-FMN:oxygen oxidoreductase (1-hydroxylating, luminescing), EC 1.14.14.3] at 37 degrees C. Growth at 23 degrees C results in a greater than 50,000-fold increase in light emission in cells containing fusion protein, whereas only a 3-fold increase in observed with cells containing the luxAB dicistron. Purified fusion protein isolated from E. coli grown at 19 degrees C exists in both monomeric and dimeric forms with specific bioluminescence activities comparable to the heterodimeric wild-type enzyme at 23 degrees C and 37 degrees C. These findings show that the alpha beta fusion polypeptide is functional as a monomer and suggest that its folding is drastically affected at elevated temperature. We hypothesize that the two-subunit bacterial luciferase may have evolved from a monomer as a result of a temperature increase in the environment.
Human platelet glycoprotein IX: an adhesive prototype of leucine-rich glycoproteins with flank-center-flank structures.
Authors: Hickey M J , Williams S A , Roth G J
Published: May 2006

The glycoprotein (GP) Ib-IX complex on the surface of human platelets functions as the von Willebrand factor receptor and mediates von Willebrand factor-dependent platelet adhesion to blood vessels. GPIX is a relatively small (Mr, 17,000) protein that may provide for membrane insertion and orientation of the larger component of the complex, GPIb (Mr, 165,000). Using antibody screening, we cloned a cDNA encoding GPIX from a human erythroleukemia cell cDNA library constructed in phage lambda gt11. Lacking a 5' untranslated region and start codon, the cDNA sequence includes 604 nucleotides, beginning with 495 bases at the 5' end coding for 165 amino acids, followed by a stop codon and 106 noncoding bases at the 3' end. By Northern blot analysis, the GPIX cDNA hybridizes with a single 1.0-kilobase species of platelet poly(A)+ RNA. Translation of the cDNA sequence gives a predicted protein sequence beginning with a truncated putative signal sequence of 5 amino acid followed by a sequence of 17 amino acids matching that determined directly by Edman degradation of intact GPIX. The predicted amino acid sequence of mature GPIX includes an NH2-terminal extracytoplasmic domain of 134 residues, a transmembrane domain of 20 residues, 6 intracytoplasmic residues, and 1 N-linked glycosylation site. GPIX contains a leucine-rich glycoprotein (LRG) sequence of 24 amino acids similar to conserved LRG sequences in GPIb and other proteins from humans, Drosophila, and yeast. "Flanking" sequences of approximately 22 amino acids are present at the NH2 and/or COOH sides of the "central" LRG sequence(s) in GPIX, GPIb, and the other human and Drosophila members of the LRG family. The role of the flank-LRG center-flank structure in the evolution and function of the LRG proteins remains to be defined.
Differential regulation of oligodendrocyte markers by glucocorticoids: post-transcriptional regulation of both proteolipid protein and myelin basic protein and transcriptional regulation of glycerol phosphate dehydrogenase.
Authors: Kumar S , Cole R , Chiappelli F , de Vellis J
Published: May 2006

During neonatal development glucocorticoids potentiate oligodendrocyte differentiation and myelinogenesis by regulating the expression of myelin basic protein, proteolipid protein, and glycerol phosphate dehydrogenase (sn-glycerol-3-phosphate: NAD+ 2-oxidoreductase, EC 1.1.1.8). The actual locus at which hydrocortisone exerts its developmental influence on glial physiology is, however, not well understood. Glycerol phosphate dehydrogenase is glucocorticoid-inducible in oligodendrocytes at all stages of development both in vivo and in vitro. In newborn rat cerebral cultures, between 9 and 15 days in vitro, a 2- to 3-fold increase in myelin basic protein and proteolipid protein mRNA levels occurs in oligodendrocytes within 12 hr of hydrocortisone treatment. Immunostaining demonstrates that this increase in mRNAs is followed by a 2- to 3-fold increase in the protein levels within 24 hr. In vitro transcription assays performed with oligodendrocyte nuclei show an 11-fold increase in the transcriptional activity of glycerol phosphate dehydrogenase in response to hydrocortisone but no increase in transcription of myelin basic protein or proteolipid protein. These results indicate that during early myelinogenesis, glucocorticoids influence the expression of key oligodendroglial markers by different processes: The expression of glycerol phosphate dehydrogenase is regulated at the transcriptional level, whereas the expression of myelin basic protein and proteolipid protein is modulated via a different, yet uncharacterized, mechanism involving post-transcriptional regulation.