FRINK Linked Data Fragments server

Matches in SemOpenAlex for { <https://semopenalex.org/work/W2012384644> ?p ?o ?g. }

• W2012384644 endingPage "31970" @default.
• W2012384644 startingPage "31962" @default.
• W2012384644 abstract "The transition metal cadmium is a pervasive and persistent environmental contaminant that has been shown to be both a human toxicant and carcinogen. To inhibit cadmium-induced damage, cells respond by increasing the expression of genes encoding stress-response proteins. In most cases, the mechanism by which cadmium affects the expression of these genes remains unknown. It has been demonstrated in several instances that cadmium activates gene transcription through signal transduction pathways, mediated by protein kinase C, cAMP-dependent protein kinase, or calmodulin. A codicil is that cadmium should influence the expression of numerous genes. To investigate the ability of cadmium to affect gene transcription, the differential display technique was used to analyze gene expression in the nematode Caenorhabditis elegans. Forty-nine cDNAs whose steady-state levels of expression change 2–6-fold in response to cadmium exposure were identified. The nucleotide sequences of the majority of the differentially expressed cDNAs are identical to those of C. elegans cosmids, yeast artificial chromosomes, expressed sequence tags, or predicted genes. The translated amino acid sequences of several clones are identical to C. elegansmetallothionein-1, HSP70, collagens, and rRNAs. In addition, C. elegans homologues of pyruvate carboxylase, DNA gyrase, β-adrenergic receptor kinase, and human hypothetical protein KIAA0174 were identified. The translated amino acid sequences of the remaining differentially expressed cDNAs encode novel proteins. The transition metal cadmium is a pervasive and persistent environmental contaminant that has been shown to be both a human toxicant and carcinogen. To inhibit cadmium-induced damage, cells respond by increasing the expression of genes encoding stress-response proteins. In most cases, the mechanism by which cadmium affects the expression of these genes remains unknown. It has been demonstrated in several instances that cadmium activates gene transcription through signal transduction pathways, mediated by protein kinase C, cAMP-dependent protein kinase, or calmodulin. A codicil is that cadmium should influence the expression of numerous genes. To investigate the ability of cadmium to affect gene transcription, the differential display technique was used to analyze gene expression in the nematode Caenorhabditis elegans. Forty-nine cDNAs whose steady-state levels of expression change 2–6-fold in response to cadmium exposure were identified. The nucleotide sequences of the majority of the differentially expressed cDNAs are identical to those of C. elegans cosmids, yeast artificial chromosomes, expressed sequence tags, or predicted genes. The translated amino acid sequences of several clones are identical to C. elegansmetallothionein-1, HSP70, collagens, and rRNAs. In addition, C. elegans homologues of pyruvate carboxylase, DNA gyrase, β-adrenergic receptor kinase, and human hypothetical protein KIAA0174 were identified. The translated amino acid sequences of the remaining differentially expressed cDNAs encode novel proteins. polymerase chain reaction base pair(s) expressed sequence tag heat shock protein. The transition metal cadmium is considered to be a serious occupational and environmental toxin. Cadmium was ranked number 7 on the Agency for Toxic Substances and Disease Registry/Environmental Protection Agency “Top 20 Hazardous Substances Priority List” in 1997 (1Fay R.M. Mumtaz M.M. Food Chem. Toxicol. 1997; 34: 1163-1165Crossref Scopus (127) Google Scholar). In addition, it is a frequently found contaminant at Superfund sites (1Fay R.M. Mumtaz M.M. Food Chem. Toxicol. 1997; 34: 1163-1165Crossref Scopus (127) Google Scholar). Cadmium is used primarily in metal coatings, nickel-cadmium batteries, and pigments (2Friberg L. Kjellstrom T. Nordberg G.F. Friberg L Nordberg G.F. Vouk V. Handbook of the Toxicology of Metals. Elsevier/North-Holland, Amsterdam1986: 130-237Google Scholar, 3Aylett B.J. Webb M. The Chemistry, Biochemistry and Biology of Cadmium. Elsevier/North-Holland, New York1979: 1-43Google Scholar). It is also continuously introduced into the atmosphere through the smelting of ores and the burning of fossil fuels (2Friberg L. Kjellstrom T. Nordberg G.F. Friberg L Nordberg G.F. Vouk V. Handbook of the Toxicology of Metals. Elsevier/North-Holland, Amsterdam1986: 130-237Google Scholar, 3Aylett B.J. Webb M. The Chemistry, Biochemistry and Biology of Cadmium. Elsevier/North-Holland, New York1979: 1-43Google Scholar). It has been suggested that increased industrialization has resulted in higher levels of accumulated cadmium in humans (4Fortoul T.I. Osorio L.S. Tovar A.T. Salazar D. Castilla M.E. Olaiz-Fernandez G. Environ. Health Perspect. 1996; 104: 630-632Crossref PubMed Scopus (50) Google Scholar). The primary routes of nonoccupational exposure in humans are via inhalation and via ingestion of cadmium-containing food (5Waalkes M.P. Coogan T.P. Barter R.A. Crit. Rev. Toxicol. 1992; 22: 175-201Crossref PubMed Scopus (273) Google Scholar). Humans are continuously exposed to cadmium and accumulate the metal throughout their lives in liver, lung, and kidney tissue (3Aylett B.J. Webb M. The Chemistry, Biochemistry and Biology of Cadmium. Elsevier/North-Holland, New York1979: 1-43Google Scholar, 6Bernard A. Lauwerys R. Experientia Suppl. 1986; 50: 114-123Crossref PubMed Scopus (9) Google Scholar). Toxicological responses of cadmium exposure include kidney damage, respiratory diseases such as emphysema, and neurologic disorders (5Waalkes M.P. Coogan T.P. Barter R.A. Crit. Rev. Toxicol. 1992; 22: 175-201Crossref PubMed Scopus (273) Google Scholar,7Chmielnicka J. Cherian M.G. Biol. Trace Elements Res. 1986; 10: 243-256Crossref PubMed Scopus (69) Google Scholar). Cadmium has been classified as a group 1 human carcinogen (8International Agency for Research on Cancer Beryllium, Cadmium, Mercury and Exposures in the Glass Manufacturing Industry. 58. International Agency for Research on Cancer, Lyon, France1993: 377Google Scholar). It induces lung, kidney, prostate, and testicular cancers in rats and mice (5Waalkes M.P. Coogan T.P. Barter R.A. Crit. Rev. Toxicol. 1992; 22: 175-201Crossref PubMed Scopus (273) Google Scholar). Human epidemiological data suggest that it causes tumors of the male reproductive system and induces respiratory tumors (5Waalkes M.P. Coogan T.P. Barter R.A. Crit. Rev. Toxicol. 1992; 22: 175-201Crossref PubMed Scopus (273) Google Scholar, 9Oberdorster G. Scand. J. Work Environ. Health. 1986; 12: 523-537Crossref PubMed Scopus (39) Google Scholar). Intracellular damage associated with cadmium exposure includes protein denaturation, lipid peroxidation, and DNA strand breaks. Proposed mechanisms by which cadmium induces this damage involve (a) metal binding to reduced cysteine residues and (b) the generation of reactive oxygen species, possibly by lowering reduced glutathione levels (10Abe T. Konishi T. Katoh T. Hirano H. Matsukuma K. Kashimura M. Higashi K. Biochim. Biophys. Acta. 1994; 1201: 29-36Crossref PubMed Scopus (35) Google Scholar, 11Manca D. Ricard A.C. Trottier B. Chevalier G. Toxicology. 1991; 67: 303-323Crossref PubMed Scopus (293) Google Scholar, 12Chin T.A. Templeton D.M. Toxicology. 1993; 77: 145-156Crossref PubMed Scopus (92) Google Scholar). To prevent cadmium-induced intracellular damage, cells respond to metal exposure by inducing the transcription of genes that encode defense and repair proteins. These proteins (a) chelate the metal to prevent further damage, (b) remove reactive oxygen species, (c) repair membrane and DNA damage, and (d) renature or degrade unfolded proteins. Cadmium has been shown to affect the steady-state levels of the mRNAs encoding metallothionein (13Hamer D.H. Annu. Rev. Biochem. 1986; 55: 913-951Crossref PubMed Google Scholar), heme oxygenase (14Adam J. Shibahara S. Smith A J. Biol. Chem. 1989; 264: 6371-6375Abstract Full Text PDF PubMed Google Scholar), γ-glutamylcysteine synthetase (15Hatcher E.L. Chen Y. Kang Y.J. Free Radical Biol. Med. 1995; 19: 805-812Crossref PubMed Scopus (89) Google Scholar), low and high molecular weight heat shock proteins (16Wiegant F.A. Souren J.E. van Rijn J. van Wijk R. Toxicology. 1994; 94: 143-159Crossref PubMed Scopus (48) Google Scholar), and ubiquitin (17Muller-Taubenberger A. Hagmann J. Noegel A. Gerisch G. J. Cell Sci. 1988; 90: 51-58PubMed Google Scholar). In addition, increases in superoxide dismutase, catalase, glutathione peroxidase, and glucose-6-phosphate dehydrogenase activities are observed following cadmium exposure in cultured cells and whole animals (18Kostic M.M. Ognjanovic B. Dimitrijevic S. Zikic R.V. Stajn A. Rosic G.L. Zivkovic R.V. Eur. J. Haematol. 1993; 51: 86-92Crossref PubMed Scopus (89) Google Scholar, 19Salovsky P. Shopova V. Dancheva V. Marev R. Hum. Exp. Toxicol. 1992; 11: 217-222Crossref PubMed Scopus (16) Google Scholar). The mechanism(s) by which this metal modulates the levels of expression of most of these genes remains unknown. Cadmium-activated transcription may occur through specific metal-responsive upstream regulatory elements found in the promoters of cadmium-responsive genes. These may include MRE sequences, found in most metallothionein genes (20Stuart G.W. Searle P.F. Chen H.Y. Brinster R.L. Palmiter R.D. Proc. Natl. Acad. Sci. U. S. A. 1984; 81: 7322-7381Crossref Scopus (247) Google Scholar, 21Searle P.F. Nucleic Acids Res. 1990; 18: 4690-4863Google Scholar, 22Culotta V.C. Hamer D.H. Mol. Cell. Biol. 1989; 9: 1376-1380Crossref PubMed Scopus (161) Google Scholar), or cadmium-responsive elements, as found in the human heme oxygenase gene (23Takeda K. Ishizawa S. Sato M. Yoshida T. Shibahara S. J. Biol. Chem. 1994; 265: 14061-14064Google Scholar). Cadmium may also affect gene expression by influencing signal transduction pathways. Cadmium affects the activities of protein kinase C, cAMP-dependent protein kinase, and calmodulin (24Wang Z. Templeton D.M. J. Biol. Chem. 1998; 273: 73-79Abstract Full Text Full Text PDF PubMed Scopus (98) Google Scholar, 25Beyersmann D. Hechtenberg S. Toxicol. Appl. Pharmacol. 1997; 144: 247-261Crossref PubMed Scopus (498) Google Scholar). It has been suggested that cadmium-induced transcription of the proto-oncogenes jun and fos is mediated via protein kinase C and calmodulin (25Beyersmann D. Hechtenberg S. Toxicol. Appl. Pharmacol. 1997; 144: 247-261Crossref PubMed Scopus (498) Google Scholar). Thus, cadmium can modulate the activities of complex signal transduction pathways that in turn can influence the expression of a myriad of genes. However, relatively few cadmium-responsive genes have been identified. In addition, there is a paucity of information on the influence of cell-specific and developmental factors on metal-inducible gene expression. We have used the reverse transcriptase-PCR1 protocol of differential display to identify new cadmium-responsive genes from the nematode Caenorhabditis elegans. The nonparasitic nematode C. elegans provides an excellent model system for obtaining an integrated picture of cellular, developmental, and molecular aspects of the regulation of cadmium-responsive gene expression. The adult hermaphrodite is composed of 959 somatic cells but contains highly differentiated muscle, nervous, digestive, and reproductive systems (26Sulston J. Wood W.B. The Nematode Caenorhabditis elegans. Cold Spring Harbor Laboratory, Cold Spring Harbor, NY1988: 123-155Google Scholar, 27Kenyon C. Science. 1988; 240: 1448-1453Crossref PubMed Scopus (62) Google Scholar). The developmental and cellular biology of C. elegans is thoroughly understood in exceptional detail (26Sulston J. Wood W.B. The Nematode Caenorhabditis elegans. Cold Spring Harbor Laboratory, Cold Spring Harbor, NY1988: 123-155Google Scholar, 27Kenyon C. Science. 1988; 240: 1448-1453Crossref PubMed Scopus (62) Google Scholar). High levels of evolutionary conservation between C. elegans and higher organisms are observed in many signal transduction, gene regulatory, and developmental pathways (28McGhee J.D. Krause M.W. Riddle D.L. Blumenthal T. Meyer B.J. Priess J.R. C. elegans II. Cold Spring Harbor Laboratory, Cold Spring Harbor, NY1997: 147-184Google Scholar, 29Han M. Sternberg P. Cell. 1990; 65: 921-931Abstract Full Text PDF Scopus (316) Google Scholar, 30Clark S.G. Stern M.J. Horvitz H.R. Nature. 1992; 356: 340-344Crossref PubMed Scopus (461) Google Scholar). In addition, homologues of many of the proteins induced as part of metal-activated stress responses in vertebrates have been identified in C. elegans. These include metallothionein (31Slice L.W. Freedman J.H. Rubin C.S. J. Biol. Chem. 1990; 265: 256-263Abstract Full Text PDF PubMed Google Scholar, 32Freedman J.H. Slice L.W. Dixon D. Fire A. Rubin C.S. J. Biol. Chem. 1993; 268: 2554-2564Abstract Full Text PDF PubMed Google Scholar), superoxide dismutase (33Giglio A.M. Hunter T. Bannister J.V. Bannister W.H. Hunter G.J. Biochem. Mol. Biol. Int. 1994; 33: 41-44PubMed Google Scholar, 34Giglio M.P. Hunter T. Bannister J.V. Bannister W.H. Hunter G.J. Biochem. Mol. Biol. Int. 1994; 33: 37-40PubMed Google Scholar), ubiquitin (35Zhen M. Heinlein R. Jones D. Jentsch S. Candido E.P.M. Mol. Cell. Biol. 1993; 13: 1371-1377Crossref PubMed Scopus (41) Google Scholar, 36Stringham E.G. Jones D. Candido E.P.M. Gene (Amst.). 1992; 113: 165-173Crossref PubMed Scopus (11) Google Scholar), heat shock protein 70 (37Heschl M.F.P. Baillie D.L. DNA. 1989; 8: 233-243Crossref PubMed Scopus (55) Google Scholar), glutathioneS-transferase (38Weston K. Yochem J. Greenwald I. Nucleic Acids Res. 1989; 17: 2138-2139Crossref PubMed Scopus (25) Google Scholar), and catalase (39Ebert R.H. Shammas M.A. Sohal B.H. Sohal R.S. Egilmez N.K. Ruggles S. Shmookler Reis R.J. Dev. Genet. 1996; 18: 131-143Crossref PubMed Scopus (25) Google Scholar). With the exception of metallothionein, the effect of cadmium on the transcription of theseC. elegans genes remains unknown. C. elegans also contains homologues to many of the signal transduction proteins that have been implicated in modulating the cellular/molecular response to metal exposure (40Gross R.E. Bagchi S. Lu X. Rubin C.S. J. Biol. Chem. 1990; 265: 6896-6907Abstract Full Text PDF PubMed Google Scholar, 41Lu X-Y. Gross R.E. Bagchi S. Rubin C.S. J. Biol. Chem. 1990; 265: 3293-3303Abstract Full Text PDF PubMed Google Scholar, 42Land M. Islas-Trejo A. Rubin C.S. J. Biol. Chem. 1994; 269: 14820-14827Abstract Full Text PDF PubMed Google Scholar, 43Land M. Islas-Trejo A. Freedman J.H. Rubin C.S. J. Biol. Chem. 1994; 269: 9234-9244Abstract Full Text PDF PubMed Google Scholar). One of the major advantages in using C. elegans as a model system to identify new metal-responsive genes is the magnitude of cDNA and genomic DNA sequence data currently available. The nematode genome is relatively small (∼108 bp), and an abundance of information is available on the genetic and physical maps of its chromosomes (44Waterston R.H. Sulston J.E. Coulson A.R. Riddle D.L. Blumenthal T. Meyer B.J. Priess J.R. C. elegans II. Cold Spring Harbor Laboratory, Cold Spring Harbor, NY1997: 23-46Google Scholar). Currently, sequencing of the entire C. elegans genome is >80% completed, and >50,000 ESTs have been cloned and sequenced. Megabases of genomic and cDNA sequence data are readily available through GenBankTM, the C. elegans Genome Project (45Coulson A. Biochem. Soc. Trans. 1996; 24: 289-291Crossref PubMed Scopus (16) Google Scholar), and the C. eleganscDNA Sequencing Project. 2Sequence data and information about the C. elegans cDNA Project can be obtained on the World Wide Web athttp://www.ddbj.nig.ac.jp/c-elegans/html/CE_INDEX.html. We have identified 53 differentially expressed DNA fragments from a mixed stage population (i.e. a population at all stages of development) of cadmium-exposed C. elegans. Subsequent analysis confirms that the steady-state level of expression of 48 of these clones increases 2–6-fold following cadmium exposure. In addition, a single clone was isolated whose level of expression decreased ∼2-fold. Sequence analysis has identified C. elegans cosmids, predicted structural genes, and ESTs that are identical to the differentially expressed mRNAs. Furthermore, the cadmium-responsive cDNAs are the products of 32 independent genes. The N2 strain of C. elegans was grown in liquid S medium (0.1 mNaCl, 50 mm potassium phosphate, pH 6.0, 5 μg/ml cholesterol, 10 mm potassium citrate, 3 mmCaCl2, 3 mm MgCl2, 50 μm EDTA, 25 μm FeSO4, 10 μm MnCl2, 10 μmZnSO4, and 1 μm CuSO4) usingEscherichia coli OP50 as a food source (46Brenner S. Genetics. 1974; 77: 71-94Crossref PubMed Google Scholar). In experiments where nematodes were exposed to cadmium, the medium was supplemented with 100 μm CdCl2 (32Freedman J.H. Slice L.W. Dixon D. Fire A. Rubin C.S. J. Biol. Chem. 1993; 268: 2554-2564Abstract Full Text PDF PubMed Google Scholar). C. eleganswere grown in the presence of metal for 8 or 24 h at ∼20 °C. Nematodes were then collected following centrifugation at 800 ×g for 5 min. Pellets were suspended in 50 mmNaCl containing 35% sucrose (final concentration), and viable nematodes were collected from the top of the solution following centrifugation at 1000 × g for 5 min at 4 °C. Nematodes were then washed three times by suspension in M9 buffer (22 mm KH2PO4, 42 mmNa2HPO4, 85 mm NaCl, 1 mm MgSO4) followed by sedimentation at 800 × g. Washed nematode pellets were finally suspended in a small volume of M9 buffer, rapidly frozen in liquid nitrogen, and stored at −80 °C. Total RNA was isolated from mixed stage populations of C. elegans exposed to 100 μmCdCl2 for 8 and 24 h and control, nonexposed nematodes. Frozen worms were first ground into a fine powder using a liquid nitrogen-cooled mortar and pestle. Powdered C. elegans (200 mg) were then homogenized in 2 ml of TRIzol (Life Technologies, Inc.). RNA was then collected from the aqueous phase following the addition of chloroform, precipitated by adding isopropyl alcohol, and then air-dried. The dried RNA pellet was then dissolved in diethyl pyrocarbonate-treated water. For some experiments, poly(A+) RNA was subsequently isolated using the Poly(A)Tract® mRNA isolation system following the manufacturer’s instructions (Promega). Differential display was performed following the protocol of Liang and Pardee (47Liang P. Pardee A.B. Science. 1992; 257: 967-971Crossref PubMed Scopus (4698) Google Scholar). Briefly, 50 μg of total RNA isolated from either of three populations of C. elegans, controls, or those grown in the presence of cadmium for 8 or 24 h, was treated with 10 units of RNase-free DNase I (Boehringer Mannheim) in 10 mm Tris-Cl buffer, pH 8.3, containing 50 mm KCl and 1.5 mmMgCl2. The DNA-free RNA was precipitated with ethanol and dissolved in diethyl pyrocarbonate-treated water. First-strand cDNAs were generated in reverse transcriptase reactions containing 0.2 μg of DNA-free total RNA, reverse transcriptase buffer (25 mm Tris-Cl, pH 8.3, 38 mm KCl, 1.5 mm MgCl2, 5 mm dithiothreitol), a 5 μm concentration of each dNTP, and a 1 μmconcentration of one of four 3′-degenerate anchored oligo(dT) primers. The 3′-degenerate anchored oligo(dT) primers have the following sequences: T12MG, T12MA, T12MT, and T12MC, where M is 3-fold degenerate for G, A, and C. Primers were annealed to the RNA template by incubating the reaction mixture for 5 min at 65 °C and then for 10 min at 37 °C. First strand cDNA synthesis was achieved following the addition of 100 units of Moloney murine leukemia virus reverse transcriptase (Life Technologies) and incubation at 37 °C for 50 min. The reaction was terminated by heating at 95 °C for 5 min, which inactivates the reverse transcriptase. Amplification of cDNA fragments was performed in 20-μl reactions. Each PCR mixture contained 2 μl of the products from one of the four reverse transcriptase reactions above and 18 μl of a solution containing Taq-PCR buffer (10 mm Tris-Cl, pH 8.4, 50 mm KCl, 1.5 mm MgCl2, 0.01% gelatin), a 1 μm concentration of the same 3′-degenerate anchored oligo(dT) primer used in the first-strand synthesis reaction, four dNTPs (2 μm each), 10 μCi of [α-35S]dATP (Amersham Pharmacia Biotech), 1 unit of AmpliTaq DNA polymerase (Perkin-Elmer), and a 0.2 μmconcentration of one of 20 5′-arbitrary decamers. The sequences of the 5′-arbitrary primers used in these reactions are presented in Table I. Reaction mixtures were subjected to 40 cycles of the PCR using the following parameters: denature at 94 °C for 30 s, anneal at 42 °C for 2 min, elongate at 72 °C for 30 s. All PCRs were performed in duplicate. The amplified cDNAs produced from duplicate reactions of RNA isolated from control, 8-h treated and 24-h treated C. elegans were size-fractionated in parallel by polyacrylamide gel electrophoresis in 6% acrylamide, 8 m urea gels.Table ISequences of the 5′-arbitrary decamer primers used in differential displayPrimer designationSequenceAP-3AGGTGACCGTAP-4GGTACTCCACAP-6GCAATCGATCAP-7CCGAAGGAATAP-8GGATTGTGCGAP-9CGTGGCAATAAP-10TAGCAAGTGCAP-13AGTTAGGCACAP-15AGGGCCTGTTAP-18CTGAGCTAGGRT-1TACAACGAGGRT-2TGGATTGGTCRT-3CTTTCTACCCRT-4TTTTGGCTCCRT-5GGAACCAATCRT-6AAACTCCGTCRT-7TCGATACAGGRT-8TGGTAAAGGGRT-9TCGGTCATAGRT-10GGTACTAAGC Open table in a new tab Following electrophoresis, gels were dried onto Whatman 3MM paper and exposed to Kodak X-AR film for 24 h. Differentially expressed cDNAs were visualized by autoradiography. To isolate differentially expressed cDNA fragments, regions of dried gels corresponding to the cDNAs were excised. Gel slices were rehydrated in 100 μl of distilled H2O following a 10-min incubation at room temperature. The cDNA was then extracted from the rehydrated gels by incubating at 100 °C for 15 min in tightly capped microcentrifuge tubes. cDNA was recovered by ethanol precipitation in the presence of 0.3 m sodium acetate and 50 μg of glycogen (Boehringer Mannheim). The eluted cDNA was reamplified in a 40-μl reaction with the identical pair of primers used in the mRNA differential display reaction. PCR reaction conditions were similar to those above, except the concentration of the dNTPs was increased to 20 μm, and the [α-35S]dATP was omitted. Amplified cDNA fragments were resolved by gel electrophoresis using 1.5% agarose gels and then purified using QIAEXII kits (QIAGEN). Gel-purified cDNAs were directly inserted into the T-A cloning vector pGEM-T (Promega). DNA inserts were subsequently sequenced using T7 and SP6 primers by the dideoxynucleotide chain termination procedures of Sanger et. al (48Sanger F. Nicklen S. Coulson A.R. Proc. Natl. Acad. Sci. U. S. A. 1977; 74: 5463-5467Crossref PubMed Scopus (52655) Google Scholar) (U.S. Biochemical Corp. Sequenase Kit, version 2.0). Analysis of cDNA sequence data including sequence comparisons, alignments, and assembly of cDNA sequences were performed using PC/GENE-Intelli-Genetics software. BLAST analysis (49Altschul S.F. Gish W. Miller W. Myers W. Lipman D.J. J. Mol. Biol. 1990; 215: 403-410Crossref PubMed Scopus (70663) Google Scholar) was carried out through the National Center for Biotechnology Information and the C. elegans Genome Project Internet servers using the nonredundant, C. elegans genome and C. elegans EST data bases. For some sequence analysis, the “A C. elegans data base” (ACeDB) software was used (50Eeckman F.H. Durbin R. Methods Cell Biol. 1995; 48: 583-605Crossref PubMed Scopus (74) Google Scholar). Predicted C. elegans genes were identified by theC. elegans Genome Project using the GENEFINDER program (51Favello A. Hillier L. Wilson R.K. Methods Cell Biol. 1995; 48: 551-569Crossref PubMed Scopus (20) Google Scholar). Samples of total RNA (20 μg) or poly(A+) RNA (2 μg) were denatured in a 2.2 mformaldehyde, 50% (v/v) formamide buffer and then subjected to denaturing gel electrophoresis on a 1.5% agarose, 2.2 mformaldehyde gel. Size-fractionated RNAs were then transferred to Nytran membrane (Schleicher and Schuell). Membranes were probed with32P-labeled cDNA fragments of the differentially expressed mRNAs. cDNAs to be used as probes were generated by the PCR from the cloned DNA fragments recovered from differential display gels, as described above. cDNAs were labeled with [α-32P]dCTP (Amersham Pharmacia Biotech) by random-primed labeling. Membranes were hybridized in 6× SSC (1× SSC: 0.15 m sodium chloride, 15 mm sodium citrate, pH 7.0), 1.25× Denhardt’s solution, 0.5% SDS, 300 ng of denatured sonicated salmon sperm DNA, and heat-denatured probe at 42 °C for 16 h. Following hybridization, membranes were washed at a high stringency of 50 °C for 30 min in 0.1× SSC, 0.1% SDS. The amount of probe hybridizing to the RNA was determined by PhosphorImager analysis (Molecular Dynamics, Inc., Sunnyvale, CA). After images were obtained, membranes were incubated at 95 °C for 1 h in 0.1% SDS to remove the bound probe. They were then hybridized with a32P-labeled C. elegans myosin light chain probe, which served as a loading control (32Freedman J.H. Slice L.W. Dixon D. Fire A. Rubin C.S. J. Biol. Chem. 1993; 268: 2554-2564Abstract Full Text PDF PubMed Google Scholar). As a positive control, membranes were also hybridized to a 32P-labeled C. elegans metallothionein-2 (mtl-2) cDNA probe (32Freedman J.H. Slice L.W. Dixon D. Fire A. Rubin C.S. J. Biol. Chem. 1993; 268: 2554-2564Abstract Full Text PDF PubMed Google Scholar). Quantification of radioactivity was performed using the ImageQuant program (Molecular Dynamics). Steady-state levels of mRNA expression were all normalized to that of the constitutively expressed myosin light chain mRNAs (32Freedman J.H. Slice L.W. Dixon D. Fire A. Rubin C.S. J. Biol. Chem. 1993; 268: 2554-2564Abstract Full Text PDF PubMed Google Scholar, 52Cummins C. Anderson P. Mol. Cell. Biol. 1988; 8: 5334-5349Crossref Scopus (30) Google Scholar). Changes in the steady-state levels of differentially expressed mRNAs in C. elegans following cadmium exposure were also determined by reverse Northern dot-blot analysis by the modified procedure of Zhang et al. (53Zhang H. Zhang R. Liang P. Nucleic Acids Res. 1996; 24: 2454-2455Crossref PubMed Scopus (95) Google Scholar). Briefly, differentially expressed cDNAs that were previously cloned into pGEM-T were amplified using primers that anneal to the T7 and SP6 RNA polymerase binding sites, which flank the cDNA insert. cDNAs were amplified and subsequently purified using PCR spin columns (QIAGEN). Approximately 100 ng of each amplified cDNA were denatured by mixing with 0.1 n NaOH (final concentration) and incubating at 100 °C for 5 min. The solution was neutralized following the addition of 3× SSC (final concentration), and then the volume was adjusted to 700 μl with distilled H2O. 200 μl of each sample was applied to one of three Nytran membranes in a Bio-Dot microfiltration apparatus (Bio-Rad). Membranes were then baked for 30 min at 80 °C under vacuum. As positive and loading controls, 100 ng of mtl-1 cDNA and myosin light chain DNA were also applied to each membrane, respectively. Three pools of single-stranded 32P-labeled cDNA probes were prepared from poly(A+) RNA isolated from control and 8- and 24-h cadmium-treated nematodes. cDNAs were generated from a mixture of mRNAs in 25-μl reverse transcriptase reactions, which contained 2 μg of poly(A+) RNA; 1 μg of oligo(dT)18 primer; reverse transcriptase buffer; 800 μm dATP, dGTP, and dTTP; 4.5 μm dCTP; 100 μCi of [α-32P]dCTP (3000 Ci/mmol); 20 units of RNase inhibitor; and 200 units of Moloney murine leukemia virus reverse transcriptase. The reaction mixture was incubated at 37 °C for 1 h and then at 95 °C for 5 min to terminate the reaction. Unincorporated nucleotides were separated from the labeled cDNAs by using a G-25 spin column (Boehringer Mannheim). Equal amounts (5 × 106 cpm/ml) of each radioactive cDNA mixture were heat-denatured and then hybridized separately to one of the three membranes at 42 °C for 16 h in hybridization buffer (see above). Membranes were washed at a high stringency of 0.1× SSC, 0.1% SDS at 55 °C for 30 min. The amount of 32P-labeled probe bound to each differentially expressed cDNA was quantified by PhosphorImager analysis, and levels of expression of the cognate mRNAs were normalized to that of the myosin light chain mRNA, as described above. The level of theC. elegans mtl-2 mRNA was measured by Northern blot analysis to confirm that the cadmium exposure protocol outlined above affects gene expression (32Freedman J.H. Slice L.W. Dixon D. Fire A. Rubin C.S. J. Biol. Chem. 1993; 268: 2554-2564Abstract Full Text PDF PubMed Google Scholar). A 32P-labeled oligonucleotide probe that is specific for the 3′-end of the mtl-2 mRNA was hybridized to a membrane that contained RNA prepared from controlC. elegans or nematodes exposed to 100 μmCdCl2 for 24 h (Fig. 1). The steady-state level of mtl-2 mRNA increased in response to cadmium exposure to that previously reported (32Freedman J.H. Slice L.W. Dixon D. Fire A. Rubin C.S. J. Biol. Chem. 1993; 268: 2554-2564Abstract Full Text PDF PubMed Google Scholar). This verified that the cadmium treatment protocol alters gene expression in C. elegans and can be used for the differential display analysis. mRNA expression patterns of nontreated C. elegans and those exposed to cadmium for 8 and 24 h were compared by mRNA differential display in order to identify new genes whose transcription is regulated by cadmium. A total of 20 5′-arbitrary decamers, including five that have sequences that are homologous to the mtl-1 cDNA, were used. Each of the 20 decamers was paired with one of four 3′-degenerate anchored oligo(dT) primers and used to amplify cDNAs prepared from control and cadmium-treated C. elegans. All amplification experiments were performed in duplicate using RNA prep" @default.
• W2012384644 created "2016-06-24" @default.
• W2012384644 creator A5052369153 @default.
• W2012384644 creator A5089490515 @default.
• W2012384644 date "1998-11-01" @default.
• W2012384644 modified "2023-09-27" @default.
• W2012384644 title "Cadmium-regulated Genes from the NematodeCaenorhabditis elegans" @default.
• W2012384644 cites W11767146 @default.
• W2012384644 cites W1513576576 @default.
• W2012384644 cites W1535466932 @default.
• W2012384644 cites W1546961183 @default.
• W2012384644 cites W1572224339 @default.
• W2012384644 cites W1588947966 @default.
• W2012384644 cites W1603910537 @default.
• W2012384644 cites W1621199903 @default.
• W2012384644 cites W1703289108 @default.
• W2012384644 cites W194357103 @default.
• W2012384644 cites W1944127002 @default.
• W2012384644 cites W1969931373 @default.
• W2012384644 cites W1971059223 @default.
• W2012384644 cites W1972765329 @default.
• W2012384644 cites W1978368163 @default.
• W2012384644 cites W1979864331 @default.
• W2012384644 cites W1980390665 @default.
• W2012384644 cites W1981686594 @default.
• W2012384644 cites W1986291674 @default.
• W2012384644 cites W1987798850 @default.
• W2012384644 cites W1989420807 @default.
• W2012384644 cites W1992257729 @default.
• W2012384644 cites W1995522877 @default.
• W2012384644 cites W2008690956 @default.
• W2012384644 cites W2009582725 @default.
• W2012384644 cites W2013581305 @default.
• W2012384644 cites W2016157964 @default.
• W2012384644 cites W2019001895 @default.
• W2012384644 cites W2021964203 @default.
• W2012384644 cites W2028634080 @default.
• W2012384644 cites W2031826603 @default.
• W2012384644 cites W2033764083 @default.
• W2012384644 cites W2037067089 @default.
• W2012384644 cites W2038234450 @default.
• W2012384644 cites W2041420418 @default.
• W2012384644 cites W2045625541 @default.
• W2012384644 cites W2050430335 @default.
• W2012384644 cites W2050944318 @default.
• W2012384644 cites W2055043387 @default.
• W2012384644 cites W2062832120 @default.
• W2012384644 cites W2065747563 @default.
• W2012384644 cites W2067084274 @default.
• W2012384644 cites W2068259648 @default.
• W2012384644 cites W2070242176 @default.
• W2012384644 cites W2070501544 @default.
• W2012384644 cites W2072374971 @default.
• W2012384644 cites W2075921988 @default.
• W2012384644 cites W2081736437 @default.
• W2012384644 cites W2081981245 @default.
• W2012384644 cites W2103444229 @default.
• W2012384644 cites W2138270253 @default.
• W2012384644 cites W2151215356 @default.
• W2012384644 cites W2166649538 @default.
• W2012384644 cites W2169161439 @default.
• W2012384644 cites W2253427875 @default.
• W2012384644 cites W2320867237 @default.
• W2012384644 cites W2499754438 @default.
• W2012384644 cites W3023647119 @default.
• W2012384644 cites W4250221563 @default.
• W2012384644 cites W4302785827 @default.
• W2012384644 doi "https://doi.org/10.1074/jbc.273.48.31962" @default.
• W2012384644 hasPubMedId "https://pubmed.ncbi.nlm.nih.gov/9822667" @default.
• W2012384644 hasPublicationYear "1998" @default.
• W2012384644 type Work @default.
• W2012384644 sameAs 2012384644 @default.
• W2012384644 citedByCount "101" @default.
• W2012384644 countsByYear W20123846442012 @default.
• W2012384644 countsByYear W20123846442013 @default.
• W2012384644 countsByYear W20123846442014 @default.
• W2012384644 countsByYear W20123846442015 @default.
• W2012384644 countsByYear W20123846442016 @default.
• W2012384644 countsByYear W20123846442017 @default.
• W2012384644 countsByYear W20123846442018 @default.
• W2012384644 countsByYear W20123846442020 @default.
• W2012384644 countsByYear W20123846442022 @default.
• W2012384644 countsByYear W20123846442023 @default.
• W2012384644 crossrefType "journal-article" @default.
• W2012384644 hasAuthorship W2012384644A5052369153 @default.
• W2012384644 hasAuthorship W2012384644A5089490515 @default.
• W2012384644 hasBestOaLocation W20123846441 @default.
• W2012384644 hasConcept C104317684 @default.
• W2012384644 hasConcept C178790620 @default.
• W2012384644 hasConcept C185592680 @default.
• W2012384644 hasConcept C2778944004 @default.
• W2012384644 hasConcept C54355233 @default.
• W2012384644 hasConcept C544657597 @default.
• W2012384644 hasConcept C86803240 @default.
• W2012384644 hasConcept C95444343 @default.
• W2012384644 hasConceptScore W2012384644C104317684 @default.
• W2012384644 hasConceptScore W2012384644C178790620 @default.
• W2012384644 hasConceptScore W2012384644C185592680 @default.

• next