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At least 775 records · Page 43Linked to original sources
PubMed2000

Identification of genes differentially expressed in association with acquired cisplatin resistance.

The goal of this study was to identify genes whose mRNA levels are differentially expressed in human cells with acquired cisplatin (cDDP) resistance. Using the parental UMSCC10b head and neck carcinoma cell line and the 5.9-fold cDDP-resistant subline, UMSCC10b/Pt-S15, two suppressive subtraction hybridization (SSH) cDNA libraries were prepared. One library represented mRNAs whose levels were increased in the cDDP resistant variant (the UP library), the other one represented mRNAs whose levels were decreased in the resistant cells (the DOWN library). Arrays constructed with inserts recovered from these libraries were hybridized with SSH products to identify truly differentially expressed elements. A total of 51 cDNA fragments present in the UP library and 16 in the DOWN library met the criteria established for differential expression. The sequences of 87% of these cDNA fragments were identified in Genbank. Among the mRNAs in the UP library that were frequently isolated and that showed high levels of differential expression were cytochrome oxidase I, ribosomal protein 28S, elongation factor 1alpha, alpha-enolase, stathmin, and HSP70. The approach taken in this study permitted identification of many genes never before linked to the cDDP-resistant phenotype.

Cisplatin↗
PubMed1993

Characterization of the structural and functional properties of cloned calcitonin receptor cDNAs.

We have recently cloned CTRs from cDNA libraries prepared from porcine renal and human ovarian cell lines. In situ hybridization and Northern analysis confirm the widespread distribution of CTR mRNA in numerous tissues. Hydropathy plots of the predicted amino acid sequence of the receptors demonstrate multiple hydrophobic regions that could generate 7 transmembrane spanning domains, similar to other G protein-coupled receptors. Searches of databanks for proteins with related amino acid sequences reveals that the CTRs are closely related to the receptors for parathyroid hormone/parathyroid hormone related peptide, secretin, vasoactive intestinal peptide, growth hormone releasing hormone, glucagon-like peptide-1 and glucagon. These receptors have no significant sequence homology to other G protein-coupled receptors, and therefore, appear to comprise a distinct receptor family. Expression of the hCTR or pCTR in COS cells results in expression of high affinity CTRs which are coupled to adenylate cyclase (AC). The hCTR, however, demonstrates higher affinity for human and salmon CT compared to the pCTR. Both CTRs demonstrate low affinity binding and AC activation in response to calcitonin gene related peptide, amylin or secretin, providing a possible explanation for the cross-reactivity among these peptides in vivo. Stable transfectants expressing the pCTR increase cAMP levels and increases in cytosolic free Ca2+ concentration consistent with dual coupling to AC and phospholipase C. Additional studies will help to establish the structural basis for this functional property as well as the evolutionary relationship of the members of this newly identified family of receptors.

Animals↗
PubMed2004

Selenophosphate synthetase genes from lung adenocarcinoma cells: Sps1 for recycling L-selenocysteine and Sps2 for selenite assimilation.

A labile selenium donor compound monoselenophosphate is synthesized from selenide and ATP by selenophosphate synthetase (SPS). In the present study, Sps1 and Sps2 were cloned from a cDNA library prepared from human lung adenocarcinoma cells (NCIH441). The human lung Sps1 has been cloned as an ORF of 1,179 bp, identical in sequence to that of the recently revised human liver Sps1. The in-frame TGA codon of the lung Sps2 was genetically altered to TGT (Cys) to obtain the Sps2Cys gene. Expression of the recombinant plasmids containing Sps1 or Sps2Cys was highly toxic to Escherichia coli host cells grown aerobically. Accordingly, the human lung Sps homologs were characterized by an in vivo complementation assay using a selD mutant strain. An added selenium source and a low salt concentration (0.1-0.25% NaCl) in the medium were required for reproducible and sensitive in vivo complementation. Sps2Cys effectively complemented the selD mutant, and the resulting formate dehydrogenase H activity was as high as that of WT E. coli MC4100. In contrast, only a weak complementation of the selD mutant by the Sps1 gene was observed when cells were grown in selenite media. Better complementation with added l-selenocysteine suggested involvement of a selenocysteine lyase for mobilization of selenium. Based on this apparent substrate specificity of the Sps1 and Sps2 gene products we suggest that the Sps1-encoded enzyme depends on a selenium salvage system that recycles l-selenocysteine, whereas the Sps2 enzyme can function with a selenite assimilation system.

Adenocarcinoma↗
PubMed2002

ATP6S1 elicits potent humoral responses associated with immune-mediated tumor destruction.

An important goal of cancer immunology is the identification of antigens associated with tumor destruction. Vaccination with irradiated tumor cells engineered to secrete granulocyte/macrophage colony-stimulating factor (GM-CSF) generates potent, specific, and long-lasting antitumor immunity in multiple murine tumor models. A phase I clinical trial of this vaccination strategy in patients with advanced melanoma demonstrated the consistent induction of dense CD4(+) and CD8(+) T lymphocyte and plasma cell infiltrates in distant metastases, resulting in extensive tumor destruction, fibrosis, and edema. Antimelanoma antibody and cytotoxic T cell responses were associated with tumor cell death. To characterize the targets of these responses, we screened an autologous cDNA expression library prepared from a densely infiltrated metastasis with postvaccination sera from a long-term responding patient. High-titer IgG antibodies detected ATP6S1, a putative accessory unit of the vacuolar H(+)-ATPase complex. A longitudinal analysis of this patient revealed an association between the vaccine-induced increase in antibodies to ATP6S1 and tumor destruction. Three additional vaccinated melanoma patients and three metastatic non-small cell lung carcinoma patients vaccinated with autologous GM-CSF-secreting tumor cells similarly showed a correlation between humoral responses to ATP6S1 and tumor destruction. Moreover, a chronic myelogenous leukemia patient who experienced a complete remission after CD4(+) donor lymphocyte infusions also developed high-titer antibodies to ATP6S1. Lastly, vaccination with GM-CSF-secreting B16 melanoma cells stimulated high-titer antibodies to ATPS1 in a murine model. Taken together, these findings demonstrate that potent humoral responses to ATP6S1 are associated with immune-mediated destruction of diverse tumors.

Animals↗
PubMed2000

An ethylene-induced cDNA encoding a lipase expressed at the onset of senescence.

A cDNA clone encoding a lipase (lipolytic acyl hydrolase) expressed at the onset of petal senescence has been isolated by screening a cDNA expression library prepared from carnation flowers (Dianthus caryophyllus). The cDNA contains the lipase consensus sequence, ITFAGHSLGA, and encodes a 447-amino acid polypeptide with a calculated molecular mass of 50.2 kDa that appears to be a cytosolic protein. Over-expression of the clone in Escherichia coli yielded a protein of the expected molecular weight that proved capable of deesterifying fatty acids from p-nitrophenylpalmitate, tri-linolein, soybean phospholipid, and Tween in both in vitro and in situ assays of enzyme activity. The abundance of the lipase mRNA increases just as carnation flowers begin to senesce, and expression of the gene is also induced by treatment with ethylene. Southern blot analyses of carnation genomic DNA have indicated that the lipase is a single copy gene. The lipase gene is also expressed in carnation leaves and is up-regulated when the leaves are treated with ethylene. Deesterification of membrane lipids and ensuing loss of membrane structural integrity are well established early events of plant senescence, and the expression pattern of this lipase gene together with the lipolytic activity of its cognate protein indicate that it plays a fundamentally central role in mediating the onset of senescence.

Amino Acid Sequence↗
PubMed2002

Identification and cloning of human polynucleotide phosphorylase, hPNPase old-35, in the context of terminal differentiation and cellular senescence.

Terminal differentiation and cellular senescence display common properties including irreversible growth arrest. To define the molecular and ultimately the biochemical basis of the complex physiological changes associated with terminal differentiation and senescence, an overlapping-pathway screen was used to identify genes displaying coordinated expression as a consequence of both processes. This approach involved screening of a subtracted cDNA library prepared from human melanoma cells induced to terminally differentiate by treatment with fibroblast IFN and mezerein with mRNA derived from senescent human progeria cells. This strategy identified old-35, which encodes an evolutionary conserved gene, human polynucleotide phosphorylase (hPNPase(old-35)), that is regulated predominantly by type I IFNs. The hPNPase(OLD-35) protein localizes in the cytoplasm of human cells and induces RNA degradation in vitro, as does its purified bacterial protein homologue. Ectopic expression of hPNPase(old-35) in human melanoma cells reduces colony formation, confirming inhibitory activity of this RNA-degradation enzyme. Identification of hPNPase(old-35), an IFN-inducible 3'-5' RNA exonuclease, provides additional support for a relationship between IFN action and RNA processing and suggests an important role for this gene in growth control associated with terminal differentiation and cellular senescence.

Amino Acid Sequence↗
PubMed1982

Isolation of cloned cDNAs to auxin-responsive poly(A)RNAs of elongating soybean hypocotyl.

Auxin-responsive cDNA clones have been isolated from a cDNA library prepared from elongating soybean hypocotyl poly(A)(+)RNA. The expression of two such sequences has been assessed by RNA blot hybridization analyses during normal developmental transitions in the soybean hypocotyl and during incubation of sections excised from the region of cell elongation. The concentrations of these poly(A)(+)RNAs are higher in the elongating zone than in the apical and mature zones of the hypocotyl. Both poly(A)(+)RNAs are depleted during incubation of the sections in the absence of auxin. The loss of one of these sequences (pJCW1) is prevented by the addition of auxin to the incubation medium while the other sequence (pJCW2) increases above the initial level in the presence of auxin. The addition of auxin to auxin-depleted tissue in which the sequences are depleted results in rapid accumulation of these poly(A)(+)RNAs; pJCW1 accumulates to the control level while pJCW2 increases well above the control level. These data along with others [Baulcombe, D. C. & Key, J. L. (1980) J. Biol. Chem. 255, 8907-8913] demonstrate directly a highly selective effect of auxin on the expression of a small number of mRNAs in tissues undergoing both cell elongation and cell division in response to auxin. Although the data are suggestive of a close association betwen auxin action and altered gene expression, a causal relationship is not established. It seems highly unlikely, however, that such specific effects of auxin on gene expression are unimportant in auxin physiology.

Journal Article↗
PubMed1982

Molecular cloning of 3-hydroxy-3-methylglutaryl coenzyme a reductase and evidence for regulation of its mRNA.

A recombinant plasmid containing a 1.2-kilobase cDNA for 3-hydroxy-3-methylglutaryl coenzyme A reductase was isolated from a cDNA library prepared from UT-1 cells, a clone of Chinese hamster ovary cells that has markedly elevated reductase activity. This plasmid, designated pRed-10, was identified by differential colony hybridization and hybrid-selected mRNA translation. The mRNA that hybridized to pRed-10 directed the synthesis in vitro of a 90,000-dalton protein that was immunoprecipitated by an antireductase antibody. The same 90,000-dalton protein was immunoprecipitated when UT-1 cells were pulse labeled with [35S]methionine in vivo and rapidly solubilized with boiling NaDodSO4. By blot hybridization, pRed-10 hybridized to mRNAs of 4.2 and 4.7 kilobases in UT-1 cells. Both mRNAs were reduced to undetectable levels when low density lipoprotein, a suppressor of the reductase, was present in the culture medium. These data indicate that the primary translation product of reductase mRNA is a 90,000-dalton protein and that LDL suppresses the reductase in UT-1 cells by drastically reducing the level of its mRNA.

Animals↗
PubMed1984

Primary structure of the gene encoding rat preprosomatostatin.

The somatostatins are peptides of 14 and 28 amino acids that are produced in a variety of endocrine and nonendocrine tissues. These peptides inhibit the secretion of many different pituitary, pancreatic, and gastrointestinal hormones. Previously, we have reported the isolation and nucleotide sequence of a cDNA derived from a rat medullary thyroid carcinoma that encoded preprosomatostatin , a 116-amino-acid precursor of somatostatin. We now report the structural characterization of the rat somatostatin gene isolated from recombinant bacteriophage libraries prepared from rat liver DNA. The gene spans 1.2 kilobases and is interrupted within the coding sequence of prosomatostatin by a single intron of 630 bases. A sequence characteristic of a Goldberg- Hogness promoter ("TATA" box), T-T-T-A-A-A-A, is located 31 bases upstream from the transcriptional initiation site. A repetitive DNA sequence, highly reiterated in the rat genome, is located in the 5' flanking region of the gene within 900 bases of the initiation site.

Amino Acid Sequence↗
PubMed1984

Identification and primary sequence of an unspliced human urokinase poly(A)+ RNA.

Human urokinase cDNA clones have been identified from a cDNA library prepared from total RNA of human fibroblasts transformed by simian virus 40 [Okayama, H. & Berg, P. (1983) Mol. Cell. Biol. 3, 280-289]. Synthetic oligonucleotides, corresponding to urokinase protein sequence, were used as probes. The cloned cDNA covers most of the coding sequence and the entire 3' untranslated region. The nucleotide sequence of one of the clones identifies this as a copy of a partially spliced polyadenylylated precursor to urokinase mRNA. The introns separate functionally different domains of the enzyme. Human urokinase mRNA has been identified by RNA blot and its size was estimated at 2500 nucleotides.

Base Sequence↗
PubMed1984

Xenopsin: the neurotensin-like octapeptide from Xenopus skin at the carboxyl terminus of its precursor.

We have synthesized two oligodeoxyribonucleotide mixtures that contain sequences complementary to different parts of the hypothetical mRNA sequence of xenopsin, a biologically active octapeptide found in skin extracts from Xenopus laevis. The two primer pools were independently used to initiate reverse transcription on skin poly(A)+ RNA and the resulting cDNAs were then used to screen in parallel a cDNA library prepared from skin poly(A)+ RNA. One of the clones that hybridized with both probes was subjected to sequence analysis. It contains a nearly full-length DNA copy of a mRNA of approximately equal to 490 nucleotides that encodes a xenopsin precursor protein. The deduced precursor is 80 amino acids long, exhibits a putative signal sequence at the NH2 terminus, and contains the biologically active peptide at the COOH terminus. The region corresponding to the NH2-terminal portion of the xenopsin precursor shows a striking nucleotide and amino acid sequence homology with the precursor of PYLa, another recently described peptide from Xenopus skin.

Amino Acid Sequence↗
PubMed1984

Characterization of the genes specifying two metacyclic variable antigen types in Trypanosoma brucei rhodesiense.

Bloodstream trypanosomes evade the immune system of their mammalian host by sequentially expressing a large number of different variable surface glycoproteins (VSGs). In contrast, metacyclic trypanosomes, the final developmental stage in the tsetse fly, express a much more restricted set of VSGs. These metacyclic VSGs are the first to be exposed to the immune system of the mammalian host after infection and may offer the potential for the eventual development of a vaccine. We have identified cDNAs for two VSGs in cDNA libraries prepared from amplified metacyclic populations of Trypanosoma brucei rhodesiense and show that they correspond to two different metacyclic serotypes. Determination of the cDNA sequences shows that metacyclic VSG mRNAs are similar to VSG mRNAs expressed during the bloodstream stage. Southern blots demonstrate that the metacyclic VSG genes are located near chromosomal telomeres. No evidence of gene rearrangement associated with expression of these VSGs was found.

Animals↗
PubMed1984

Isolation and characterization of a mouse cDNA clone that expresses mast-cell growth-factor activity in monkey cells.

A cDNA sequence coding for mouse mast-cell growth-factor (MCGF) has been cloned from a cDNA library prepared from mRNA derived from a concanavalin A-activated mouse T-cell clone. Cloning was achieved using the pcD vector that permits expression of cDNA inserts in mammalian cells. The DNA sequence codes for a polypeptide of 166 amino acid residues including a putative signal peptide. The supernatant fluid obtained after transfection of COS-7 monkey cells with the pcD-MCGF plasmid had mouse MCGF activity. The MCGF amino acid sequence between amino acids 33 and 41, deduced from the nucleotide sequence of its cDNA, is identical to the NH2-terminal sequence of the mouse lymphokine, interleukin 3, suggesting that MCGF is related to interleukin 3.

Amino Acid Sequence↗
PubMed1985

Cloning and expression of cDNA for salmon growth hormone in Escherichia coli.

cDNA clones encoding chum salmon (Oncorhynchus keta) growth hormone (sGH) have been isolated from a cDNA library prepared from chum salmon pituitary gland poly(A)(+) RNA. Synthetic oligodeoxynucleotide mixtures based on amino acid residues 23-28 of sGH were used as hybridization probes to select recombinant plasmids carrying the sGH coding sequence. The complete nucleotide sequence of sGH cDNA has been determined. The cDNA sequence codes for a polypeptide of 210 amino acids, including a putative signal sequence of 22 amino acids. The 5' and 3' untranslated regions of the message were 64 and 426 bases long, respectively. Mature sGH was efficiently expressed in Escherichia coli carrying a plasmid in which the sGH cDNA was under control of the E. coli trp promoter; sGH comprised about 15% of the total cellular protein in such bacteria. The partially purified sGH from E. coli stimulated the growth of rainbow trout and the activity was indistinguishable from that of natural sGH.

Journal Article↗
PubMed1985

Remarkable homology among the internal repeats of erythroid and nonerythroid spectrin.

A cDNA clone for nonerythroid alpha-spectrin was identified by direct immunological screening of a chicken smooth muscle cDNA library. A library prepared in the expression plasmids pUC8 and pUC9 was screened with an antiserum specific for chicken alpha-spectrin. Blots of poly(A)+ RNA from various tissues of chicken and mouse show that the cDNA hybridizes to an 8-kilobase mRNA. The cDNA hybridizes to a single-copy sequence on Southern blots of chicken genomic DNA. The complete nucleic acid sequence of the clone has a single 1419-base open reading frame. The derived amino acid sequence is organized into two partial and three complete 106-amino-acid repeats that show homology to the repeats described for human erythroid alpha- and beta-spectrin. Immunological and biochemical data indicate that chicken nonerythroid and human erythroid alpha-spectrin are two of the more widely diverged members of the spectrin family of proteins. In this respect, the degree of homology found between them was unexpected. Our data suggest a common evolutionary origin for these two alpha-spectrins and allow some predictions concerning spectrin gene structure.

Amino Acid Sequence↗
PubMed1985

Cloning and expression in Escherichia coli of the cDNA for murine tumor necrosis factor.

A murine tumor necrosis factor (MuTNF) cDNA was isolated from a cDNA library prepared by using mRNA from the murine macrophage-like cell line PU5-1.8 induced with 4 beta-phorbol 12 beta-myristate 13 alpha-acetate. The cDNA encodes a polypeptide consisting of a 79 amino acid pre sequence followed by a mature MuTNF sequence of 156 amino acids. The 235 amino acid murine pre-TNF polypeptide is 79% homologous to the human pre-TNF protein. There is one potential N-linked glycosylation site on MuTNF, in contrast to human TNF, which lacks any such site. The MuTNF cDNA, when engineered for expression in Escherichia coli, was found to direct the synthesis of biologically active MuTNF as determined by its cytotoxicity against several transformed cell lines.

Amino Acid Sequence↗
PubMed1985

Molecular approach to thermogenesis in brown adipose tissue: cDNA cloning of the mitochondrial uncoupling protein.

The uncoupling protein (UCP) of mammalian brown fat is a specialized and unique component responsible for energy dissipation as heat. Translation and immunoprecipitation from sucrose-fractionated mRNA indicated that the mRNA of UCP sedimented at 14-16 S. A recombinant cDNA library prepared from mRNA of thermoactive brown fat enriched for UCP mRNA has been constructed and cloned in Escherichia coli. Recombinant plasmids were screened by differential colony hybridization to a cDNA probe complementary to poly(A)+ RNA isolated from thermogenic or from weakly thermogenic brown fat. Several differentially hybridizing plasmids were shown to contain UCP cDNA sequences by their ability to select a mRNA coding for an in vitro translation product that was immunoprecipitable with antibodies against UCP. Blot hybridization of brown fat mRNA to a 32P-labeled UCP cDNA probe revealed two major species of mRNA (15S and 18S). As compared to non-thermogenic tissue, a strikingly increased hybridization to the probe was observed with brown fat mRNA from thermoactive tissue. Moreover, hybridization was observed with RNA of brown adipose tissue from rat, hamster, or mouse but not with RNA from rat or mouse liver.

Adipose Tissue, Brown↗
PubMed1985

Chromosomal location of the gene encoding the neural cell adhesion molecule (N-CAM) in the mouse.

The gene encoding the neural cell adhesion molecule, N-CAM, has been localized on mouse chromosome 9. A BALB/cJ mouse genomic library prepared in lambda bacteriophage EMBL4 was screened by using a cDNA probe, pEC204, that corresponds to the coding region of the chicken N-CAM gene. Four weakly reactive and one strongly reactive recombinant phage were isolated. A region of the latter that was strongly homologous to pEC204 was subcloned to yield a new probe, pEC501. RNA transfer blots and nucleotide sequencing indicated that pEC501 encoded part of the mouse N-CAM gene. This probe defined a unique genetic locus, Ncam, associated with a restriction fragment length polymorphism that allowed the definition of two alleles. The locus could be provisionally assigned either to chromosome 9 or to chromosome 10 by correlating the presence or absence of mouse-specific DNA fragments reactive with the probe in a panel of somatic hybrid cell lines with the presence or absence of the various mouse chromosomes. Analysis of the inheritance of the Ncam-associated DNA polymorphism in recombinant inbred strains of mice revealed close linkage between Ncam and the Lap-1, Sep-1, and Thy-1 loci on chromosome 9. This result suggests an additional linkage between Ncam and the locus for the cerebellar mutation staggerer (sg). The Ncam locus provides an important reference point for mapping the genes for additional cell adhesion molecules as well as genes for other molecules involved in neural development and function.

Animals↗