SEARCH · PubMed Health

Results for “DNA, Complementary”

Explore indexed PubMed citations for clinical trials, systematic reviews and public health research. Read source abstracts and follow each citation to its original PubMed record.

Quote a phrase for an exact phrase match. Source license links do not imply unrestricted reuse.

At least 253 records · Page 14Linked to original sources

PubMedDate not supplied

Amino-acid sequence of a Ca2+ + Mg2+-dependent ATPase from rabbit muscle sarcoplasmic reticulum, deduced from its complementary DNA sequence.

We have cloned and sequenced complementary DNA encoding a Ca2+-ATPase of rabbit muscle sarcoplasmic reticulum. We propose a model of the protein which has 3 cytoplasmic domains joined to a set of 10 transmembrane helices by a narrow, penta-helical stalk. In this model, ATP bound to one cytoplasmic domain would phosphorylate an aspartate in an adjoining cytoplasmic domain, inducing translocation of Ca2+ from binding sites on the stalk.

Amino Acid Sequence↗

PubMed1987

Isolation and sequence of complementary DNA encoding human extracellular superoxide dismutase.

A complementary DNA (cDNA) clone from a human placenta cDNA library encoding extracellular superoxide dismutase (EC-SOD; superoxide:superoxide oxidoreductase, EC 1.15.1.1) has been isolated and the nucleotide sequence determined. The cDNA has a very high G+C content. EC-SOD is synthesized with a putative 18-amino acid signal peptide, preceding the 222 amino acids in the mature enzyme, indicating that the enzyme is a secretory protein. The first 95 amino acids of the mature enzyme show no sequence homology with other sequenced proteins and there is one possible N-glycosylation site (Asn-89). The amino acid sequence from residues 96-193 shows strong homology (approximately 50%) with the final two-thirds of the sequences of all known eukaryotic CuZn SODs, whereas the homology with the P. leiognathi CuZn SOD is clearly lower. The ligands to Cu and Zn, the cysteines forming the intrasubunit disulfide bridge in the CuZn SODs, and the arginine found in all CuZn SODs in the entrance to the active site can all be identified in EC-SOD. A comparison with bovine CuZn SOD, the three-dimensional structure of which is known, reveals that the homologies occur in the active site and the divergences are in the part constituting the subunit contact area in CuZn SOD. Amino acid sequence 194-222 in the carboxyl-terminal end of EC-SOD is strongly hydrophilic and contains nine amino acids with a positive charge. This sequence probably confers the affinity of EC-SOD for heparin and heparan sulfate. An analysis of the amino acid sequence homologies with CuZn SODs from various species indicates that the EC-SODs may have evolved from the CuZn SODs before the evolution of fungi and plants.

Amino Acid Sequence↗

PubMed2001

Differential transcription factor expression in human mononuclear cells in response to amphotericin B: identification with complementary DNA microarray technology.

STUDY OBJECTIVE: To identify genes differentially expressed in human monocytic cells exposed to amphotericin B in vitro. DESIGN: In vitro experiment. SETTING: Hospital laboratory. MATERIAL: Human mononuclear cell line, THP-1. INTERVENTION: Human mononuclear cells were exposed to amphotericin B or media alone for 6 hours. After exposure, total RNA was isolated and reverse transcribed to complementary DNA. Differences in probe hybridization observed during blotting were measured, and genes with altered regulation were described by using human complementary DNA microarray technology. MEASUREMENTS AND MAIN RESULTS: Of 588 genes represented on the array, 16 transcripts were found to be upregulated and 4 transcripts were downregulated in response to amphotericin B. These findings suggest that amphotericin B alters the expression of genes in human monocytic cells that play a role in many cellular functions, including immune response, signal transduction, and cell differentiation. CONCLUSION: Amphotericin B induces alterations in human cell gene transcription. These changes could be used to evaluate differences in toxicity or efficacy observed in patients receiving this agent.

Amphotericin B↗

PubMed2000

Normalizing complementary DNA by quantitative reverse transcriptase-polymerase chain reaction of beta2-microglobulin: molecular monitoring of minimal residual disease in acute promyelocytic leukemia.

Reverse transcription (RT)-polymerase chain reaction (PCR) raises unique methodological matters that may hamper the reliability of the procedure, especially when results should direct therapeutic decisions. One of these matters is represented by the RT step. The present study shows that differences in complementary DNA (cDNA) preparations purposely containing increasing amounts of retrotranscribed RNA were not disclosed by nonquantitative RT-PCR by two different housekeeping genes, leading to fictitious results when the expression of a given gene was quantitatively assessed. To overcome this problem, the following are proposed: 1) to evaluate the efficiency of RT step through the quantification, by competitive RT-PCR, of the expression levels of the housekeeping gene beta2-microglobulin (beta2M); 2) to normalize each cDNA preparation to be comprised within 1 standard deviation of the mean value of beta2M absolute level (3.14 +/- 1.14 attomoles/microg RNA) found by analyzing 33 cell lines of hematopoietic origin. To validate this strategy in a clinical setting, serial cDNA samples from patients were checked by conventional and quantitative RT-PCR for beta2M. Again, only a quantitative evaluation of beta2M levels was allowed to unveil significant differences, otherwise undetected, in the efficiency of RT reactions among these cDNA samples. Normalization of samples to obtain cDNA preparations containing comparable beta2M levels, eventually led to an increased sensitivity in the detection of PML-RARalpha fusion transcripts. This approach seems of great value for the monitoring of minimal residual disease in serial patient samples when a tumor-specific marker is available.

Actins↗

PubMed1992

Increased growth of NIH/3T3 cells by transfection with human p120 complementary DNA and inhibition by a p120 antisense construct.

The human nucleolar antigen p120 was detected with an anti-p120 monoclonal antibody in most human malignant tumors but not in most resting human tissues (J. W. Freeman et al., Cancer Res., 48: 1244-1251, 1988) and has been used as a prognostic tumor marker in breast cancer patients (J. W. Freeman et al., Cancer Res., 51: 1973-1978, 1991). After the complementary DNA and gene for the human p120 protein were isolated and sequenced (review: H. Busch, Cancer Res., 50: 4830-4838, 1990), constructs were prepared to study the expression of the sense p120 and its antisense, p021 message. NIH/3T3 cells were transfected by electroporation with pSVX plasmids containing either the p120 complementary DNA (pSVX120) or the antisense, p021 DNA (pSVX021), and clones containing these constructs were selected. The expression of p120 or p021 in these constructs was regulated by Moloney murine leukemia virus long terminal repeats. In pSVX120-transfected NIH/3T3 cells, the expressed human p120 protein was localized to the nucleoli as shown by anti-p120 monoclonal antibody immunofluorescence. Expression of the p120 message and protein was confirmed by Northern (mRNA) and Western (protein) blots. Transfection of the p120 complementary DNA in sense orientation caused malignant transformation of NIH/3T3 cells in vitro and produced rapidly growing tumors in nude mice. Transfection of the antisense p120 constructs markedly delayed the growth of these tumors in vitro and in vivo (L. Perlaky et al., Proc. Am. Assoc. Cancer Res., 32: 1682, 1991). When transformed 3T3/pSVX120 cells were transfected with an inducible antisense p120 construct (pMSG021), dexamethasone induction decreased the growth rate by 62%, and the cell line returned to its normal phenotype. Northern blot analysis showed a decreased level of p120 mRNA, and the immunofluorescence was also markedly reduced.

3T3 Cells↗

PubMed1989

ATP synthase from bovine mitochondria: complementary DNA sequence of the import precursor of a heart isoform of the alpha subunit.

The alpha-subunit of ATP synthase from mitochondria is a major component of the extrinsic membrane sector of the enzyme. It is encoded in nuclear DNA. A family of overlapping complementary DNA clones encoding its precursor has been isolated from a bovine library by using in the first instance a mixture of 128 synthetic oligonucleotides designed on the basis of the known protein sequence, and the sequence of the full-length cDNA has been determined. The deduced protein sequence shows that the alpha-subunit of ATP synthase has a presequence of 43 amino acids that is not present in the mature protein. Presumably it directs the protein into the mitochondrial matrix and is removed during the import process. The encoded protein sequence is also longer by one amino acid at its C-terminal end than the protein isolated from F1-ATPase, but this alanine residue may have been removed artifactually during release of the F1-ATPase particle from the inner mitochondrial membrane. With the exception of one uncertainty caused by an ambiguity at one position in the nucleotide sequence, the mature protein sequence encoded in the cDNA is exactly the same as the sequence determined previously by direct analysis of the protein isolated from bovine heart mitochondria [Walker et al. (1985) J. Mol. Biol. 184, 677-701]. The cDNA sequence differs in 158 nucleotides over a region of alignment of 1097 nucleotides from a partial cDNA for the alpha-subunit that has been isolated from a bovine cDNA derived from liver RNA [Breen (1988) Biochem. Biophys. Res. Commun. 152, 264-269].(ABSTRACT TRUNCATED AT 250 WORDS)

Amino Acid Sequence↗

PubMed1992

Complementary DNA cloning, messenger RNA expression, and induction of alpha-class glutathione S-transferases in mouse tissues.

Glutathione S-transferases (EC 2.5.1.18) are a multigene family of related proteins divided into four classes. Each class has multiple isoforms that exhibit tissue-specific expression, which may be an important determinant of susceptibility of that tissue to toxic injury or cancer. Recent studies have suggested that alpha-class glutathione S-transferase isoforms may play an important role in the development of cancers. Several alpha-class glutathione S-transferase isozymes have been characterized, purified, and cloned from a number of species, including rats, mice, and humans. Here we report on the cloning, sequencing, and mRNA expression of two alpha-class glutathione S-transferases from mouse liver, termed mYa and mYc. While mYa was shown to be identical to the known alpha-class glutathione S-transferase complementary DNA clone pGT41 (W. R. Pearson et al., J. Biol. Chem., 263: 13324-13332, 1988), the other clone, mYc, was demonstrated to be a novel complementary DNA clone encoding a glutathione S-transferase homologous to rat Yc (subunit 2). The mRNA for this novel complementary DNA is expressed constitutively in mouse liver. It also is the major alpha-class glutathione S-transferase isoform expressed in lung. The levels of expression of the butylated hydroxyanisole-inducible form (mYa) are highest in kidney and intestine. Treatment of mice with butylated hydroxyanisole had little effect on the expression levels of mYc but strongly induced mYa expression in liver. Butylated hydroxyanisole treatment increased expression levels for both mYa and mYc to varying degrees in kidney, lung, and intestine. The importance of the novel mouse liver alpha-class glutathione S-transferase isoform (mYc) in the metabolism of aflatoxin B1 and other carcinogens is discussed.

Amino Acid Sequence↗

PubMed1989

Viral infectious complementary-DNA studies may identify nonviral genes critical to central nervous system disease.

A major interest of modern science and medicine is the delineation of genes that cause disease. In the case of cancer, the study of viral oncogenic genes led to the recognition of similar human genes that play an important role in this disease. In an analogous fashion, the identification of viral genes important in central nervous system disease may lead to the recognition of related cellular genes that are important in nonviral central nervous system disease. New molecular techniques now provide tools for identification of pathogenic viral genes and elucidation of mechanisms of disease production. Positive-strand RNA viruses such as picornaviruses provide an especially attractive model system for studies of central nervous system disease-producing genes. A limitation in molecular studies of these viruses has resulted from an inability to use restriction enzymes, since these enzymes are active against DNA and not RNA. This limitation has recently been overcome with the preparation of infectious picornavirus complementary-DNA. This review highlights the importance of infectious complementary-DNA in pathogenesis studies and provides a glimpse of the impact of such studies on neurology.

DNA↗

PubMed1999

Effect of multiple gene transfers of insulinlike growth factor I complementary DNA gene constructs in rats after thermal injury.

HYPOTHESIS: Multiple subcutaneous injections of cholesterol-containing cationic liposomes encapsulating the complementary DNA (cDNA) gene for insulinlike growth factor I (IGF-I) increase the rate of transfected skin cells and result in increased IGF-I protein levels in the skin with subsequent improvement in wound healing when compared with a single injection. SETTING: Laboratory. INTERVENTION: Twenty-four adult male Sprague-Dawley rats (350-375 g) received a full-thickness scald burn on 60% of their body surface. These rats were randomly divided to receive either 1 injection of liposomes containing 2.2 microg-cytomegalovirus-driven cDNA coding for IGF-I and 0.2 microg of the Lac Z gene cDNA construct, or 2 injections of liposomes containing 2.2 microg cytomegalovirus-driven cDNA coding for IGF-I and 0.2 microg of the Lac Z gene cDNA construct. MAIN OUTCOME MEASURES: Transfection rates and IGF-I protein levels in the skin and physiological responses to the IGF-I gene therapy, evaluated from changes in body weight, protein content in serum and liver, and the rate of burn wound healing. RESULTS: There was a significant decrease in transfection rate and IGF-I protein expression distal from the injection site in animals receiving 1 injection, as compared with a consistent increase in rats receiving multiple injections. Multiple injections improved the response to thermal trauma by increasing the extent of the healed burn wound 33 days after thermal injury (single injection, 31% +/- 1% vs multiple injections, 38% +/- 2%), total serum protein (single injection, 52 +/- 0.5 g/L vs multiple injections, 55 +/- 0.6 g/L), and total liver protein (single injection, 82.0 +/- 0.3 mg/mL vs multiple injections, 91.0 +/- 3.8 mg/mL), P<.05. CONCLUSIONS: Gene transfer rates can be increased by multiple injections of liposomes encapsulating IGF-I cDNA constructs. Increased transfer results in greater IGF-I protein skin concentrations, accelerated wound healing, and increased serum and liver protein concentrations. The clinical relevance of these findings is that liposomal gene constructs should be applied in well-defined distances to improve gene transfer in the skin, and thus clinical outcome after thermal injury.

Animals↗

PubMed2002

Oxidative stress is involved in the development of experimental abdominal aortic aneurysm: a study of the transcription profile with complementary DNA microarray.

BACKGROUND: The role of oxidative stress in the formation of aneurysms is not fully understood. We used the complementary DNA (cDNA) microarray technique to determine the transcription profile in the development of elastase-induced abdominal aortic aneurysm in rat models, with an emphasis on the oxidative stress-related genes. MATERIALS AND METHODS: In the experimental group, rat abdominal aortas were perfused with elastase to induce AAA. In the control group, a sham operation was performed with perfusion of the aortas with saline solution. Four or five animals were used for each time point for each of the elastase-treated or saline-treated groups. At day 2, day 7, and day 10 after surgery, the external aortic diameter was measured and AAA formation was estimated. Total RNA was isolated from aortas and subjected to cDNA microarray analysis with the use of the rat genome U34A high-density oligonucleotide DNA chip (Affymetrix, Santa Clara, Calif), which contains a total number of 8799 genes of which 2017 are expressed sequence tag (EST) genes. The data were analyzed with the GENECHIP Data Mining Tool software (Affymetrix). For genes of interest, reverse-transcription polymerase chain reaction was performed to confirm their expression level. RESULTS: Comparison ranking analysis revealed that during AAA development, the expression of 212 genes, including 46 of EST genes, increased by more than two-fold and 229 genes, including 95 of EST genes, decreased by more than two-fold in at least one of the three time points. The regulated genes included those encoding heme oxygenase, inducible nitric oxide synthase, some extracellular matrix proteins, members of the matrix metalloproteinase family, and those associated with prooxidant/antioxidant and inflammatory responses. Reverse-transcription polymerase chain reaction analysis confirmed the upregulation of genes involved in oxidative stress, such as heme oxygenase, inducible nitric oxide synthase, 12-lipoxygenase, and heart cytochrome c oxydase subunit VIa, and the downregulation of antioxidant genes, such as superoxide dismutase, reduced nicotinamide adenine dinucleotide-cytochrome b-5 reductase, and glutathion S-transferase. CONCLUSION: The cDNA microarray technique was useful for investigation of the transcription profiles during the development of AAA. Our results indicate that oxidative stress may play a pivotal role in the pathologic progression of AAA.

Animals↗

PubMed1990

Cloning and sequencing of a complementary DNA encoding a Mr 70,000 human breast epithelial mucin-associated antigen.

The human milk fat globule (HMFG) membrane contains several glycoproteins that have been referred to as breast differentiation antigens and that are expressed in normal breast, breast tumors, breast tumor-derived cell lines, and are found in breast cancer patient serum. These antigens include a high molecular weight mucin and several smaller components including Mr 150,000; 70,000; and 46,000 glycoproteins. We have used 2 monoclonal antibodies (McR2 and Mc13) that bind the Mr 70,000 component of HMFG to immunoscreen a lambda gt11 expression library prepared from human lactating breast tissue. We report here the sequence of a complementary DNA clone (BA70-1) that codes for a peptide that binds both McR2 and Mc13 but not monoclonal antibodies to the breast mucin or other components of HMFG.A 1.8-kilobase RNA was detected in 9 of 9 breast tumor cell lines using 32P-labeled BA70-1 as probe. The BA70-1 RNA was highly expressed in 6 of 9 cells lines of breast and several other carcinomas lines compared with a lymphoblastoid cell line (Raji). The BA70-1 complementary DNA sequence has no extensive homology with previously reported sequences including the high-molecular weight mucin complementary DNA. Since the Mr 70,000 molecule appears to be associated with the breast mucin by disulfide bonds, its study could help elucidate the structure of this latter complex and how it is organized in the cell membrane, and prove useful in diagnosis and therapy of breast cancer.

Antibodies, Monoclonal↗

PubMed1995

Expression cloning of gastric mucin complementary DNA and localization of mucin gene expression.

BACKGROUND & AIMS: Secretory mucins play an important role in gastric cytoprotection and are derived from a heterogeneous family of genes. The aim of this study was to determine the specific type and location of mucin gene expression in the human stomach. METHODS: Expression cloning was performed by screening a human gastric complementary DNA expression library with antisera against deglycosylated gastric mucin. RNA analysis and immunohistochemistry were used to quantify and localize mucin gene expression. RESULTS: Sequencing of positive clones revealed two clones containing tandem repeats. The first contained a 169-amino acid repeat and was named MUC6 (as previously described). The second contained the same 8-amino acid repeat consensus sequence (APTTSTTS) as complementary DNAs previously isolated from a tracheobronchial complementary DNA library and was labeled MUC5 (or MUC5AC). RNA analysis indicated that the gastric epithelium contains high levels of MUC5 and MUC6 messenger RNA with little or no MUC2, MUC3, and MUC4 messenger RNA. Immunohistochemical analysis showed that surface mucous cells of the cardia, fundus, and antrum expressed MUC5 peptide. In contrast, MUC6 peptide expression was limited to mucous neck cells of the fundus, antral-type glands of the antrum and cardia, and Brunner's glands of the duodenum. CONCLUSIONS: MUC5 and MUC6 represent major secretory mucins in the stomach and are localized to distinct cell types.

Amino Acid Sequence↗

PubMed1976

Importance of full size complementary DNA in nucleic acid hybridization.

The size of the DNA product synthesized by RNA-directed DNA polymerase (isolated from avian myeloblastosis virus) was found to be important for complementary DNA (cDNA)-mRNA hybridization reactions. Incomplete cDNA to rabbit reticulocyte globin mRNA formed poor hybrids and presumably lacked sequences needed for hybridization. The size of the cDNA synthesized was influenced by the reaction conditions used. The complementary DNA product contained 10 S material when synthesis was done at high deoxynucleoside triphosphate concentrations (greater than 50 muM) while the product was smaller than the template when synthesis was at lower concentrations. The concentration and size (oligo(dT)6 to (dT)10) of primer had little or no effect on the product size. Increasing the concentration of 10 S globin mRNA caused the cDNA product to contain more small material. The cDNA synthesized at high deoxynucleoside triphosphate concentrations was fractionated into heavy, medium, and light fractions by alkaline sucrose density centrifugation. All hybridized to globin mRNA. The larger cDNAs had a higher TM when hybridized to globin mRNA, a lower dTMP/dCMP ratio (indicating that the poly(dT) region constituted a smaller fraction of the molecule), and gave increased protection of 125I-labeled mRNA from nuclease digestion. The full size cDNA was especially useful for studying the RNA transcribed from chromatin by RNA polymerase. The complement of the 5' end of the mRNA is contained only in full size cDNA; the 5' end is the part of the mRNA first transcribed by the RNA polymerase assuming correct transcription. Thus, full size cDNA can hybridize more effectively to the short RNA transcripts that are obtained than partial cDNA. RNA transcribed from rabbit bone marrow chromatin by Escherichia coli RNA polymerase hybridized twice as efficiently to complete cDNA as it did to partial cDNA demonstrating the usefulness of full size cDNA.

Animals↗

PubMed1992

Fluorescent-labeled oligonucleotides that exhibit a measurable signal in the presence of complementary DNA.

Oligonucleotide derivatives with a fluorescent dye were designed for exhibiting a measurable signal only when they bind to complementary DNA in aqueous solution. The oligonucleotide with a dansyl group at the specific 2'-sugar residue was synthesized by using the protected 2'-dansylaminouridine phosphorobisamidite. The dansyl-oligonucleotide conjugate binds to its complementary DNA to form duplex with a normal stability and exhibits enhanced fluorescence together with a blue-shift in emission maxima after the hybridization. Another possible candidate involved the use of pyrene-excimer emission upon forming ternary complex between two pyrene-labeled oligonucleotide probes with target DNA. A new and general method for introduction of a pyrene fluorophore into the 3'- or 5'-terminal hydroxyl group of oligonucleotides via different linkers was developed.

DNA↗

PubMed1997

p53 abnormalities in primary prostate cancer: single-strand conformation polymorphism analysis of complementary DNA in comparison with genomic DNA. The Cooperative Prostate Network.

BACKGROUND: The reported frequency of mutation of the p53 tumor suppressor gene (also known as TP53) in human carcinomas of the prostate has varied widely, ranging from 3% to 42%. This variability may be a consequence of tumor heterogeneity and/or the use of different methods of analysis. Since p53 mutation has been associated with clinical outcome for a number of cancer types, determination of its true frequency in primary carcinomas of the prostate is important. PURPOSE: The principal aims of this study were as follows: 1) to validate the utility of detecting p53 gene mutations by means of polymerase chain reaction-single-strand conformation polymorphism (PCR-SSCP) analysis of complementary DNA (cDNA) (synthesized from prostate tissue RNA and 2) to study the concordance of RNA- and DNA-based PCR-SSCP assays in detecting p53 mutations in individual tumor fragments. METHODS: RNA and genomic DNA were isolated by means of standard techniques from specimens of 19 carcinomas of the prostate, selected on the basis of p53 data obtained in a previous analysis of cDNA (indicating that 14 were mutant and five were wild-type). RNA was converted into cDNA by means of reverse transcription (RT); the cDNA was then amplified by means of nonisotopic (i.e., nonradioactive) PCR, and the PCR products were subjected to SSCP analysis in polyacrylamide gels (RT-PCR-SSCP analysis). Genomic DNA was examined by means of SSCP analysis of isotopically labeled (32PO4) PCR products (DNA-PCR-SSCP analysis). In both approaches, the protein coding region of the p53 gene was divided into multiple, smaller fragments for study. PCR products exhibiting abnormal migration in SSCP gels were subjected to direct nucleotide sequencing or to cloning and sequencing of multiple clones. RESULTS: RT-PCR-SSCP and DNA-PCR-SSCP identified p53 gene abnormalities in 15 of the 19 selected carcinomas, including one previously reported to be wild-type for p53. Overall, PCR-SSCP analysis identified 18 p53 fragments with abnormalities; three carcinomas showed two abnormalities each. Six (33%) of the 18 abnormalities were detected by both RT-PCR-SSCP and DNA-PCR-SSCP, 10 (56%) were detected by RT-PCR-SSCP alone, and two (11%) were detected by DNA-PCR-SSCP alone. The 18 abnormalities were caused by 20 changes in the sequence of the p53 gene; in one carcinoma, double mutations in two individual p53 exons were identified. CONCLUSIONS AND IMPLICATIONS: PCR-SSCP analysis of both RNA and DNA allows the detection of more mutations than the analysis of either alone. Some primary carcinomas of the prostate contain more than one altered p53 gene, consistent with the possibility of intratumoral heterogeneity of mutation of this gene. For comprehensive analysis of p53 mutations in carcinomas of the prostate, and perhaps in other tumor tissues, SSCP analysis of cDNA should be used in combination with SSCP analysis of genomic DNA.

DNA, Complementary↗

PubMed1997

Nucleotide sequence of rat steroidogenic acute regulatory protein complementary DNA.

The steroidogenic acute regulatory (StAR) protein is a key regulator for the steroidogenesis in acute response to trophic hormone. A rat complementary DNA of the StAR protein was cloned and its complete nucleotide sequence was determined. The deduced amino acid sequence of the clone has an additional 86 amino acid stretch at amino terminus when it was compared with those sequences in other species. The other part of the amino acid sequence has 94% identity to mouse StAR protein sequence. Three transcripts (1.3 kb, 1.6 kb, and 3.5 kb) which are hybridizing to the clone were detected in testis, ovary and adrenal gland. When the cDNA was expressed in COS1 cells, 30 kDa and 47 kDa proteins specific to the anti-StAR antibody were detected.

Amino Acid Sequence↗

PubMed1994

Human very-low-density lipoprotein receptor complementary DNA and deduced amino acid sequence and localization of its gene (VLDLR) to chromosome band 9p24 by fluorescence in situ hybridization.

A complementary DNA for the very-low-density lipoprotein receptor (VLDLR) that codes for a protein of 873 amino acids was cloned from a human heart cDNA library. The mature protein of 846 amino acids, preceded by a 27-residue signal peptide, shares 97% amino acid sequence identity with the rabbit VLDLR. Like the low-density lipoprotein receptor, the VLDLR contains five different domains, all of which are highly conserved between human and rabbit. A tetrapeptide NPVY that potentially serves as a signal for clustering of the VLDLR on coated pits is present in the cytoplasmic domain, which is 100% conserved between human and rabbit. We localized the VLDLR gene to chromosome 9p24 by fluorescence in situ hybridization using the cloned cDNA as hybridization probe. The high amino acid sequence homology of the VLDLR between two mammalian species suggests that the receptor plays a fundamental role in lipoprotein metabolism and that energy metabolism mediated by triglyceride utilization may be an evolutionarily highly conserved mechanism.

Amino Acid Sequence↗

PubMed1994

Episomal expression of sense and antisense insulin-like growth factor (IGF)-binding protein-4 complementary DNA alters the mitogenic response of a human colon cancer cell line (HT-29) by mechanisms that are independent of and dependent upon IGF-I.

HT-29 cells express and secrete insulin-like growth factor (IGF)-II and only one of the six IGF-binding proteins, IGFBP-4. In the present study, the physiological role of endogenous IGFBP-4 in regulating the growth response of HT-29 cells to exogenous and endogenous IGFs was examined. Both the basal and the IGF-stimulated growth of HT-29 cells was significantly increased over control values in the presence of IGFBP-4 antibody, suggesting that endogenous IGFBP-4 is a potent inhibitor of the mitogenic effects of endogenous and exogenous IGFs. In order to further confirm the inhibitory role of endogenous IGFBP-4, sense and antisense complementary DNA fragments of human IGFBP-4 were ligated into an episomal mammalian expression vector (pCEP4). Restriction mapping and Southern blot analysis were used to confirm directional cloning of the IGFBP-4 complementary DNA fragments in the sense and antisense directions in the pCEP4 vectors. HT-29 cells were transfected with either the control (no insert, C-P), sense (S-P), or antisense (AS-P) vectors and subjected to hygromycin selection. The functional nature of the transfectants was confirmed by measuring IGFBP-4 concentrations in the conditioned media (CM) of 10(7) cells by ligand and immunoblot analysis. IGFBP-4 concentrations were 7.4 +/- 1.7-fold higher in the CM of S-P cells compared to that in the CM of C-P cells, while IGFBP-4 concentrations in the CM of AS-P cells were significantly lower than those present in the CM of C-P cells. Both the basal and the IGF-I-stimulated growth of the AS-P cells was significantly higher than that of the C-P and S-P cells. The basal (non-stimulated) and the IGF-I-stimulated growth of the S-P cells was not significantly different from that of the C-P cells, suggesting that overexpression of IGFBP-4 was not inhibitory to the growth of the HT-29 cells. The basal growth of the S-P and C-P cells was significantly increased in the presence of IGFBP-4 antibody, once again suggesting that endogenous IGFBP-4 was a potent inhibitor of autocrine effects of endogenous factors (IGF-II). Addition of IGFBP-4 antibody had no significant effect on the basal growth of the AS-P cells, confirming that the difference between the growth response of the AS-P, C-P, and S-P cells was largely contributed by the difference in the endogenous secretion of IGFBP-4 by the cells.(ABSTRACT TRUNCATED AT 400 WORDS)

Antibodies↗