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PubMed1993

Molecular cloning and characterization of the complementary DNA for the M(r) 85,000 protein overexpressed in adriamycin-resistant human tumor cells.

An M(r) 85,000 membrane protein was identified by a monoclonal antibody MRK20 raised against an Adriamycin-resistant subline of human myelogenous leukemia K562 (K562/ADM) cells. The M(r) 85,000 protein was found to be overexpressed in both innate and acquired Adriamycin-resistant tumor lines. A complementary DNA (cDNA) clone coding for the M(r) 85,000 protein was isolated by mixed oligonucleotide-primed polymerase chain reaction and further screening of a cDNA library from K562/ADM. Amino acid and nucleotide sequence analysis of the M(r) 85,000 protein revealed that this protein is identical with CD36, a cell surface adhesion molecule of endothelium, platelets, and monocytes. We constructed an expression vector utilizing two different promoters, SV40 and MMTV, and two cDNAs for the M(r) 85,000 protein that have different 3'-ends. DNA transfection experiments were carried out by the calcium phosphate method with a selectable marker using drug-sensitive human tumor lines KB3-1 and A2780 as recipient cells. We obtained transfectant clones expressing the M(r) 85,000 protein stably or inducibly but found no resistance against Adriamycin or vincristine. Direct selection with Adriamycin or vincristine or tumor cells transfected with the SV40 promoter-regulated expression constructs also failed to yield drug-resistant clones. These results indicate that the M(r) 85,000 protein/CD36 cannot confer drug resistance by itself, even though the protein can be an effective marker for Adriamycin resistance.

Amino Acid Sequence↗

PubMed1990

Expression of a human complementary DNA for the multidrug resistance gene in murine hematopoietic precursor cells with the use of retroviral gene transfer.

In multidrug resistance, cells become simultaneously resistant to anthracyclines, vinca alkaloids, epipodophyllotoxins, and certain other natural product cytotoxic drugs. Resistance results from synthesis of a multidrug transporter (P-glycoprotein) encoded by the MDR1 gene (also known as the PGY1 gene). In the present study, a retrovirus vector containing a complementary DNA for the human multidrug resistance gene HaMDR1/A was used to transfer the multidrug resistance phenotype to bone marrow cells of the DBA/2J mouse. A high proportion of transduced bone marrow cells showed resistance to both colchicine and vinblastine, as determined by in vitro colony formation of hematopoietic precursor cells. In addition, brief culturing of the cells in a cytotoxic drug following exposure to the retrovirus vector could be used to increase the proportion of bone marrow cell colonies that were resistant. These results may serve as a model for the generation and selection of bone marrow cells resistant to the toxic effects of chemotherapeutic agents in vivo.

Animals↗

PubMed1988

Complementary DNA clones of chicken proto-oncogene c-ets: sequence divergence from the viral oncogene v-ets.

The avian acute leukemia virus E26 induces erythroblastosis and myeloblastosis in chickens. The oncogene of this virus includes sequences derived from the cellular gene designated c-ets, which is normally expressed in lymphoid cells and whose product is a protein of apparent molecular weight ca. 54,000 daltons. Complementary DNA clones representing the major transcript of the chicken c-ets proto-oncogene were isolated from a spleen cell library. Sequence analysis of the cDNA revealed that it contains an open reading frame encoding a polypeptide of 441 amino acids with a molecular weight of 49,932 daltons. This open reading frame can be transcribed and translated in vitro into a 50 kd protein that is specifically immunoprecipitated with antiserum to the v-ets oncogene product. Within the central region of homology between c-ets and v-ets, there are only 5 nucleotide substitutions resulting in 4 amino acid changes. However, coding sequences at the 5' and 3' ends of the v-ets oncogene and the chicken c-ets cDNA differ from one another. These changes may be responsible for the differential functions of c-ets and v-ets in cells of different hematopoietic lineages and may account for the pathogenic properties of the v-ets oncogene.

Amino Acid Sequence↗

PubMed1981

The construction and partial characterization of plasmids containing complementary DNA sequences to human calcitonin precursor polyprotein.

(1) Total poly(A)-containing RNA isolated from human thyroid medullary carcinoma tissue was shown to direct the synthesis in the wheat germ cell-free system of a major (Mr 21000) and several minor forms of human calcitonin precursor polyproteins. Evidence for processing of these precursor(s) by the wheat germ cell-free system is also presented. (2) A small complementary DNA (cDNA) plasmid library has been constructed in the PstI site of the plasmid pAT153, using total human thyroid medullary carcinoma poly(A)-containing RNA as the starting material. (3) Plasmids containing abundant cDNA sequences were selected by hybridization in situ, and two of these (ph T-B3 and phT-B6) were characterized by hybridization--translation and restriction analysis. Each was shown to contain human calcitonin precursor polyprotein cDNA sequences. (4) RNA blotting techniques demonstrate that the human calcitonin precursor polyprotein is encoded within a mRNA containing 1000 bases. (5) The results demonstrate that human calcitonin is synthesized as a precursor polyprotein.

Calcitonin↗

PubMed1985

Human dioxin-inducible cytochrome P1-450: complementary DNA and amino acid sequence.

Induction of cytochrome P1-450 has been linked to susceptibility to certain chemically induced cancers in mouse and man. Treatment of the human cell line MCF-7 with 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) results in high levels of aryl hydrocarbon (benzo[a]pyrene) hydroxylase (P1-450) activity. This cell line was used to isolate a human P1-450 full-length complementary DNA (cDNA) clone. The cDNA is 2566 nucleotides in length, encodes a polyadenylated messenger RNA (2.8 kilobases in length), and has a continuous reading frame producing a protein with 512 residues (molecular weight, 58,151). The human P1-450 cDNA and protein are 63 percent and 80 percent similar to mouse P1-450 cDNA and protein, respectively. Whereas the mouse TCDD-inducible P-450 gene subfamily has two members (P1-450 and P3-450), the human TCDD-inducible gene subfamily appears to have only one gene (P1-450).

Amino Acid Sequence↗

PubMed1987

Isolation of a complementary DNA clone for thyroid microsomal antigen. Homology with the gene for thyroid peroxidase.

The thyroid microsomal antigen (MSA) in autoimmune thyroid disease is a protein of approximately 107 kD. We screened a human thyroid cDNA library constructed in the expression vector lambda gt11 with anti-107-kD monoclonal antibodies. Of five clones obtained, the recombinant beta-galactosidase fusion protein from one clone (PM-5) was confirmed to react with the monoclonal antiserum. The complementary DNA (cDNA) insert from PM-5 (0.8 kb) was used as a probe on Northern blot analysis to estimate the size of the mRNA coding for the MSA. The 2.9-kb messenger RNA (mRNA) species observed was the same size as that coding for human thyroid peroxidase (TPO). The probe did not bind to human liver mRNA, indicating the thyroid-specific nature of the PM-5-related mRNA. The nucleotide sequence of PM-5 (842 bp) was determined and consisted of a single open reading frame. Comparison of the nucleotide sequence of PM-5 with that presently available for pig TPO indicates 84% homology. In conclusion, a cDNA clone representing part of the microsomal antigen has been isolated. Sequence homology with porcine TPO, as well as identity in the size of the mRNA species for both the microsomal antigen and TPO, indicate that the microsomal antigen is, at least in part, TPO.

Amino Acid Sequence↗

PubMed1991

Isolation of partial complementary DNA encoding human thromboxane synthase.

Thromboxane synthase catalyzes the biosynthesis of thromboxane A2 which plays a key role in the proaggregatory and vasoconstrictive processes. In this communication, we reported the successful cloning of thromboxane synthase cDNA from a human lung cDNA library. Oligonucleotides were synthesized according to the direct amino acid sequence of 2 peptides derived from purified human thromboxane synthase. Polymerase chain reaction was carried out using these oligonucleotides as primers to isolate a complementary DNA from human lung cDNA library. The longest cDNA thus obtained was 687 base pairs in length. Amino acid sequences deduced from the cDNA contained all three peptide sequences reported, confirming the authenticity of the cDNA clone.

Amino Acid Sequence↗

PubMed2002

Identification of differentially expressed genes in rheumatoid arthritis by a combination of complementary DNA array and RNA arbitrarily primed-polymerase chain reaction.

OBJECTIVE: There is increasing evidence that T cell-independent pathways, such as the up-regulation of protooncogenes and the production of growth factors and matrix-degrading enzymes, lead to progressive destruction of affected joints. Therefore, identification of differentially regulated genes restricted to rheumatoid arthritis (RA) synovial fibroblasts is essential. A combination of RNA arbitrarily primed-polymerase chain reaction (RAP-PCR) and complementary DNA (cDNA) array with defined genes was used for a highly sensitive differential screening using small amounts of RNA. METHODS: RNA was extracted from cultured synovial fibroblasts obtained from 6 patients with RA and 6 patients with osteoarthritis (OA). RAP-PCR was performed using different arbitrary primers for first- and second-strand synthesis. PCRs were hybridized to cDNA array membranes. RA samples were compared with OA samples for differentially expressed genes. RESULTS: In contrast to standard cDNA array, the identification of 12 differentially expressed genes in RA compared with OA (approximately 6%) was possible. Differentially expressed genes of interest were confirmed using semiquantitative RT-PCR and in situ hybridization. CONCLUSION: Numerous variants of the differential display method and continuous improvements, including RAP-PCR, have proven to be both efficient and reliable for examining differentially regulated genes. Our results show that RAP-PCR combined with cDNA arrays is a suitable method for identifying differentially expressed genes in rheumatoid synovial fibroblasts, using very small amounts of RNA.

Arthritis, Rheumatoid↗

PubMedDate not supplied

Isolation of complementary DNA clones encoding the human lymphocyte glycoprotein T1/Leu-1.

The T1/Leu-1/CD5 molecule, a human T-cell surface glycoprotein of relative molecular mass (Mr) 67,000, has been implicated in the proliferative response of activated T cells and in T-cell helper function. A similar involvement in T-cell proliferation has been reported for Ly-1, the murine homologue of T1. Here we report the complete amino-acid sequence of the T1 precursor molecule deduced from complementary DNA clones. The protein contains a classical signal peptide; a 347-amino-acid extracellular segment; a transmembrane region; and a 93-amino-acid intracellular segment. The extracellular segment contains many cysteine residues and is composed of two related structural domains separated by a proline/threonine-rich region. The T1 molecule has structural features characteristic of other receptor molecules.

Amino Acid Sequence↗

PubMed1984

Statistical evaluation of the coding capacity of complementary DNA strands.

Two independent methods are used to evaluate the protein-coding information content in different classes of DNA sequences. The first method allows to evaluate the statistical relevance of finding unidentified reading frames, longer than 100 codons, on both DNA strands of: a) 117 DNA sequences that code for 142 nuclear proteins; b) 39 stable RNA coding sequences and c) 36 other DNA sequences which include regulatory and as yet unknown function sequences. The finding of 50 reading frames longer than 100 codons (complementary inverted proteins or c.i.p. genes) located on the DNA strand complementary to the protein-coding one is drastically in excess of the number predicted by chance alone. An independent method (testcode) applied to c.i.p. gene sequences, which assigns the probability of coding to a given sequence, predicts that more than 50% of these genes are translated in a functional product. These analyses indicate the existence of a new class of protein-coding genes, located on the DNA sequences complementary to the protein-coding DNA strand.

Animals↗

PubMed1982

Cloning of DNA complementary to ornithine aminotransferase mRNA.

Free polysomes were isolated from the livers of rats maintained on a 60% casein diet to induce ornithine aminotransferase mRNA. Ornithine aminotransferase-synthesizing polysomes were immunoadsorbed using monospecific, affinity-purified antibody and Staphylococcus aureus cells. Poly(A+)RNA prepared from these polysomes by oligo(dT)-cellulose chromatography was used as a template for the synthesis of double-stranded cDNA by reverse transcriptase. Using the deoxyguanosine-5'-triphosphate-deoxycytidine-5'-triphosphate tailing method and pBR322 as a vector, recombinant molecules were produced and used to transform Escherichia coli. Two clones containing DNA complementary to ornithine aminotransferase mRNA were identified by colony hybridization and hybrid-arrest translation. A partial restriction endonuclease map of the ornithine aminotransferase cDNA inserts was constructed which spanned 1066 base pairs.

Animals↗

PubMedDate not supplied

Probing the initiation complex formation on E coli ribosomes using short complementary DNA oligomers.

Interactions between Escherichia coli 16S rRNA sequences (as components of 30S ribosomal subunits or tight-couple 70S ribosomes) with the ligands poly(U), poly(AGU), tRNAPhe, tRNAfMet, and the initiation factors have been studied. The ligands were employed as competitors for selected sites on 16S rRNA known to be accessible for hybridization to cDNA oligomers, regions 517-528, 1397-1404, and 1534-1542. The binding of cDNAs 1534-1541 and 1398-1403 decreased in the presence of the ligand pair poly(U)/tRNAPhe. Only the binding of cDNA 1534-1541 was affected by poly(AGU), while none of the complementary DNA oligomer binding was affected by tRNAPhe or tRNAfMet alone. The poly(AGU)/tRNAfMet ligand pair caused an additional decline in the binding of cDNA 1534-1541, relative to that caused by poly(AGU) alone, but the ligand pair did not affect the binding of the cDNA oligomers 517-528 or 1398-1403. The inclusion of the initiation factors did not significantly alter the binding level decreases observed for cDNA 1534-1541 in the presence of mRNAs or tRNA. At the 517-528 and 1398-1403 regions, the inclusion of the initiation factors, in either the presence or absence of the other ligands, caused a large decrease in the binding of the cDNA oligomers. The oligomers complementary to 16S bases 517-528 and 1398-1403 did not bind to tight-couple or reassociated 70S ribosomes. The data are discussed in terms of the decoding site hypothesis, and in terms of the mRNA alignment mechanism proposed by Trifonov [1].

Base Sequence↗

PubMed1985

Molecular cloning of the complementary DNA for human tumor necrosis factor.

Tumor necrosis factor (TNF) is a soluble protein that causes damage to tumor cells but has no effect on normal cells. Human TNF was purified to apparent homogeneity as a 17.3-kilodalton protein from HL-60 leukemia cells and showed cytotoxic and cytostatic activities against various human tumor cell lines. The amino acid sequence was determined for the amino terminal end of the purified protein, and oligodeoxyribonucleotide probes were synthesized on the basis of this sequence. Complementary DNA (cDNA) encoding human TNF was cloned from induced HL-60 messenger RNA and was confirmed by hybrid-selection assay, direct expression in COS-7 cells, and nucleotide sequence analysis. The human TNF cDNA is 1585 base pairs in length and encodes a protein of 233 amino acids. The mature protein begins at residue 77, leaving a long leader sequence of 76 amino acids. Expression of high levels of human TNF in Escherichia coli was accomplished under control of the bacteriophage lambda PL promoter and gene N ribosome binding site.

Amino Acid Sequence↗

PubMedDate not supplied

Identification of genes responsive to UV-A radiation in human lens epithelial cells using complementary DNA microarrays.

UV-A radiation produces cataract in animals, enhances photoaging of the lens and skin and increases the phototoxicity of drugs. However, the nature of genes that are activated or repressed after cellular exposure to UV-A radiation remains enigmatic. Because lens epithelial cells exposed to UV-A radiation undergo apoptosis 4 h after exposure to the stress, we sought to establish the change in gene expression in cells by UV-A radiation using gene expression profiling using complementary DNA microarrays containing about 12 000 genes. We identified 78 genes abnormally expressed in UV-A-irradiated cells (showing >2.5-fold change at P < 0.05). These genes are implicated in various biological processes, including signal transduction and nucleic acid binding, and genes encoding enzymes. A majority of the genes were downregulated. Our analysis revealed that the expression of genes for the transcription factors ATF-3 and Pilot increased four-fold, whereas the gene for the apoptosis regulator NAPOR-1 decreased five-fold. These changes were confirmed by real-time quantitative reverse transcriptase-polymerase chain reaction. The calpain large polypeptide 3 (CANP3) gene also increased nine-fold after UV-A radiation. In addition, peroxisomal biogenesis factor 7, glucocorticoid receptor-alpha and tumor-associated calcium signal transducer genes decreased three- to eight-fold. Western blot analysis further confirmed the increase in protein expression of ATF-3 and CANP3 and decreased expression of glucocorticoid receptor-alpha in the irradiated cells. Surprisingly, most of these genes had not been previously shown to be modulated by UV-A radiation. Our results show that human lens epithelial cells respond to a single dose of UV-A radiation by enhancing or suppressing functionally similar sets of genes, some of which have opposing functions, around the time at which apoptosis occurs. These studies support the intriguing concept that activation of competing pathways favoring either cell survival or death is a means to coordinate the response of cells to UV-A stress.

Crystallins↗

PubMed1991

Complementary DNA encoding human colon cancer manganese superoxide dismutase and the expression of its gene in human cells.

Manganese superoxide dismutase (MnSOD) is a member of a family of metalloenzymes that catalyze the dismutation of the superoxide anion to H2O2. It has been shown that MnSOD activity in tumor cells is lower than that in their normal counterparts. To investigate the molecular basis for the reduced level of MnSOD activity in human tumor cells, the primary structure of human MnSOD has been determined from complementary DNA (cDNA) isolated from a human colon carcinoma (HT-29) cDNA library. The sequence of the mature protein is composed of 198 amino acids preceded by a 24-amino acid leader peptide. DNA sequence analysis revealed that the translated region of the human tumor MnSOD is virtually identical to the MnSOD sequence isolated from normal human sources but exhibits differences in both the 5'- and 3'-untranslated regions. DNA blot analysis of genomic DNA isolated from HT-29, simian virus-transformed human lung fibroblast (SV-40/WI-38), and parental human lung fibroblast (WI-38) cells showed an identical pattern of hybridization to that of MnSOD cDNA. RNA blot analysis revealed that tumor cells have lower levels of MnSOD mRNA. However, the half-life of the mRNA was the same (approximately 10 h) in tumor and normal cells. Immunological measurement of the level of MnSOD in both normal and tumor cells also showed a reduced level of MnSOD protein in the tumor cells. These results suggest that the reduced level of MnSOD activity observed in human tumor cells is not due to a defect in the primary structure of the MnSOD protein, a change in the dosage of the MnSOD gene, or a decrease in the stability of MnSOD mRNA in tumor cells but rather is due to a defect or defects in the expression of the gene.

Base Sequence↗

PubMed1990

Renaturation of complementary DNA strands mediated by purified mammalian heterogeneous nuclear ribonucleoprotein A1 protein: implications for a mechanism for rapid molecular assembly.

Purified heterogeneous nuclear ribonucleoprotein (hnRNP) A1 protein, which is found in vivo associated with heterogeneous nuclear RNA (hnRNA), promotes the rapid renaturation of nucleic acid strands. Maximal renaturation activity requires the glycine-rich carboxyl-terminal one-third of the protein, although the amino-terminal two-thirds also has activity. The A1-mediated reaction is second-order with respect to complementary DNA concentration, and the renaturation rate constant at 37 degrees C with A1 is about 3000-fold greater than in the absence of the protein. At 60 degrees C, the A1-mediated renaturation rate is even faster, and is about 300-fold greater than protein-free reactions carried out at 68 degrees C in 1 M NaCl. Provided that sufficient A1 protein is present to coat all strands in solution, the presence of nonhomologous, single-stranded DNA does not significantly inhibit the reaction. Moreover, renaturation of short strands to their complement contained in very long strands is nearly as efficient as between two short strands. These results indicate that A1 may be useful for procedures that rely on nucleic acid renaturation. We propose that A1 promotes rapid renaturation primarily by reducing the entropic barrier of bimolecular strand association through relatively transient interactions between A1-coated strands. Such interactions, mediated by flexible repeating domains, may act generally to increase the association kinetics of highly specific molecular assemblies in processes such as RNA maturation, transcription, translation, and transport.

Coliphages↗

PubMedDate not supplied

Enhanced immunity to breast cancer in mice immunized with fibroblasts transfected with a complementary DNA expression library from breast cancer cells: Enrichment of the vaccine for immunotherapeutic cells.

Breast cancer cells express an array of weakly immunogenic tumor-associated antigens (TAAs). Under appropriate circumstances, immunity to breast cancer can be induced, with potential benefits for patients with the disease. Here, we report a new cell-based vaccination strategy resulting in enhanced immunity to breast cancer in tumor-bearing mice. The strategy was designed to enrich the vaccine for highly immunogenic cells. The vaccine was prepared by transfer of unfractionated complementary DNA (cDNA) derived from a highly malignant breast neoplasm that arose spontaneously in a C3H/He mouse (SB5b) into an immunogenic fibroblast cell line. As the transferred cDNA spontaneously integrates into the genome of the recipient cells and is replicated as the cells divide, sufficient DNA to prepare the vaccine could be obtained from as few as 10(7) cells (4-mm tumor). Because only a small proportion of the transfected cell population was expected to have incorporated genes responsible for inducing immunity to the breast cancer, we devised a novel approach designed to enrich the transfected cell population for cells that induced immunity to the neoplasm. Aliquots of the transfected population were divided into small pools (initial inoculum = 4 x 10). Afterward, the cell number from each pool was allowed to expand in vitro. Pools containing greater numbers of immunogenic cells (identified by 2 independent assays) were subdivided for additional rounds of immune selection. Enhanced immunity to the neoplasm was detected in tumor-bearing mice treated solely by immunization with the enriched cell population. The immunity, mediated by CD8+ T cells, was sufficient to prolong the survival of mice with established breast cancer.

Animals↗

PubMed1984

Cloning and characterization of DNA complementary to rat liver UDP-glucuronosyltransferase mRNA.

UDP-glucuronosyltransferase (transferase) clones were isolated from a cDNA bank constructed in pBR322 using transferase-enriched mRNA from the livers of phenobarbital-treated rats. The enrichment of mRNA was accomplished by polysome immunoadsorption with antibody to purified mouse liver transferase. This antibody was shown to bind specifically to rat transferase by Ouchterlony double diffusion analysis, immunoadsorption of glucuronidating activities, and selective inhibition of the immunoadsorption of in vitro synthesized enzyme by purified rat liver transferase. The isolated clones were verified to contain DNA complementary to transferase mRNA by hybrid translation-selection. Three classes of transferase cDNAs were characterized by restriction endonuclease mapping, and the largest insert-containing clone of each class was designated pUDPGTr-1, pUDPGTr-2, and pUDPGTr-3. Their insert sizes were approximately 2,400, 2,000, and 2,000 bp, respectively. All three cDNAs hybridized with a 2,300 +/- 150 bp mRNA, and each selected the translation of a 52,000-dalton polypeptide. Immunoadsorption of the 35S-labeled translation product could be competitively inhibited in each case by the addition of purified rat liver transferase. pUDPGTr-1 and pUDPGTr-3 inserts shared extensive sequence homology. This was demonstrated by Southern blot analysis using purified inserts and electron microscopic heteroduplex analysis. Southern blot analysis revealed that these cDNAs hybridized to overlapping genomic fragments. pUDPGTr-2 shared less sequence homology with the other two classes of cDNAs, based on the above criteria. In addition, mRNA corresponding to pUDPGTr-2 was elevated 5-fold by phenobarbital treatment, whereas the other mRNAs levels were unaffected. These studies demonstrate that in rat liver there are a minimum of three distinct transferase mRNAs, two of which may be associated with a common gene or gene family.

Animals↗