Human growth hormone: complementary DNA cloning and expression in bacteria. 1979.
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Amino Acid Sequence↗
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Results for “DNA, Complementary”
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[Evaluation of p63 expression in lung cancer by use of complementary DNA and tissue microarray].
OBJECTIVE: To study p63 expression at mRNA transcript and protein levels in human lung cancers, including squamous cell lung carcinoma (SCC), adenocarcinoma, large cell lung carcinoma (LCLC) and small cell lung carcinoma (SCLC), or the corresponding metastatic foci. The relationship of p63 expression and alterations in p63 locus at chromosomal 3q27-29 was also determined. METHODS: p63 gene expression in 72 cases of SCC, adenocarcinoma, LCLC and SCLC was analyzed by cDNA microarray technology. Tissue microarray of specimens from 150 cases of primary lung cancer was prepared for immunohistochemical study for p63 protein. Possible chromosomal alterations at the p63 locus in 70 cases of primary lung cancer were studied by comparative genomic hybridization (CGH) technology. RESULTS: p63 mRNA transcript expression was significantly increased by more than 10-fold in SCC, as compared with that in other histologic subtypes including adenocarcinoma, LCLC and SCLC. p63 mRNA expression in metastatic foci was also remarkably higher than that in their primary tumors (P < 0.001). Immunostaining showed that p63 protein expression was observed in 94.64% of SCC, whereas only one lung adenocarcinoma (1.79%) was positive. Immunopositivity was also demonstrated in 2 of the 4 LCLC cases studied. None of the SCLC cases was positive. There was a statistically significant difference in p63 expression between pT1 and pT2 tumors (P < 0.05). The CGH results showed that overrepresentation of p63 locus at chromosomal 3q27-29 was a typical finding in SCC. p63 immunopositivity also correlated significantly with pronounced gains of p63 locus at chromosomal 3q27-q29 (P < 0.000 1), suggesting that strong expression of p63 in lung SCC was associated with increased gene amplification. CONCLUSION: p63 may play a role in oncogenesis of human lung squamous cell carcinoma and development of metastasis.
Adenocarcinoma↗
Complete complementary DNA sequence of the third component of complement of lamprey. Implication for the evolution of thioester containing proteins.
Lamprey liver mRNA sequences were amplified by reverse transcriptase-polymerase chain reaction using primers synthesized according to the amino acid sequences at the thioester region common to the mammalian C3, C4, and alpha 2-macroglobulin (alpha 2M). Two different cDNA species were identified that showed a close similarity to the mammalian C3 or alpha 2M sequences, respectively. Using the C3-like sequence as a probe, two overlapping cDNA clones were isolated from the lambda ZAP library, which together covered the entire region encoding the putative lamprey pro-C3. The deduced amino acid sequence of the putative lamprey pro-C3 contained 1660 amino acids and showed 31%, 22%, 23%, and 16% amino acid sequence identity with mouse C3, C4, C5, and human alpha 2M, respectively. The distributions of cysteine residues were completely identical between the mouse C3 and the putative lamprey C3 except that the lamprey sequence had two additional cysteine residues in the alpha-chain. The possible beta-alpha and alpha-gamma processing sites were found at exactly the same positions as in mammalian C4. These results suggest that the putative lamprey C3 retains a close similarity to the common ancestor of the mammalian C3 and C4, which appeared to have had a three-subunit chain structure.
Amino Acid Sequence↗
[Hepatitis E: isolation of a complementary DNA of the enterally transmitted form of non-A, non-B hepatitis virus].
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Base Sequence↗
Characterization of complementary DNA clones encoding the rabbit IL-8 receptor.
IL-8 is a proinflammatory cytokine that functions as a chemoattractant for neutrophils. Recently, cDNA clones encoding the human neutrophil IL-8R were isolated by an expression cloning strategy. The amino acid sequence of the human IL-8R was sufficiently similar to a published sequence for an isoform of the rabbit FMLP receptor that we considered the possibility that the rabbit sequence might bind IL-8 as well. In order to establish its ligand specificity, we have isolated and characterized cDNA clones encoding the rabbit receptor. These cDNA clones, when expressed in mammalian cells, confer high affinity IL-8 binding (Kd = 3.6 nM), lack detectable binding of FMLP, and produce a transient increase in the intracellular Ca2+ concentration in response to IL-8 but not to FMLP. These data demonstrate that the reported rabbit FMLP receptor is the rabbit IL-8R, not an isoform of the FMLP receptor. In addition, the amino acid sequence of the rabbit IL-8R encoded by these cDNA clones differs at 23 amino acids (of 355) from that previously published.
Amino Acid Sequence↗
Molecular cloning and structural analysis of complementary DNA of human and rat androgen receptors.
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Amino Acid Sequence↗
Transforming growth factor-alpha expression is enhanced in human mammary epithelial cells transformed by an activated c-Ha-ras protooncogene but not by the c-neu protooncogene, and overexpression of the transforming growth factor-alpha complementary DNA leads to transformation.
MCF-10A cells are a spontaneously immortalized normal human mammary epithelial cell line. MCF-10A cells were transfected with two expression vector plasmids containing either a human point-mutated c-Ha-ras protooncogene or the rat c-neu protooncogene. c-Ha-ras-transfected MCF-10A cells grow as colonies in soft agar, exhibit a 3- to 4-fold increase in their growth rate in serum-free medium, and show a reduced mitogenic response to exogenous epidermal growth factor (EGF) or transforming growth factor-alpha (TGF alpha) as compared to MCF-10A cells. c-Ha-ras-transfected MCF-10A cells express a 4- to 8-fold increase in TGF alpha mRNA levels and secrete 4- to 6-fold more TGF alpha protein as compared to MCF-10A cells. Addition of either an anti-TGF alpha neutralizing monoclonal antibody or an anti-EGF receptor blocking monoclonal antibody to the Ha-ras-transformed MCF-10A cells produces a 50 to 80% inhibition of colony formation of these cells in soft agar. c-neu-transfected MCF-10A cells grown in soft agar and exhibit an increase in their growth rate in serum-free medium at a level comparable to that observed in Ha-ras-transformed MCF-10A cells. Addition of an anti-c-erbB-2 monoclonal antibody inhibits the anchorage-independent growth of these cells in soft agar. However, c-neu-transformed MCF-10A cells show no increase in TGF alpha secretion and no change in their responsiveness to exogenous EGF or TGF alpha. A recombinant retroviral vector containing the human TGF alpha gene was also introduced into MCF-10A cells. TGF alpha-infected MCF-10A cells secrete 15- to 20-fold more TGF alpha protein than MCF-10A cells, form colonies in soft agar, exhibit an enhanced growth rate in serum-free medium, and show a decreased mitogenic response to exogenous EGF or TGF alpha at a level equivalent to Ha-ras-transformed MCF-10A cells. Growth of TGF alpha-infected MCF-10A cells in soft agar is completely inhibited by anti-TGF alpha neutralizing or anti-EGF receptor blocking monoclonal antibodies. These results suggest that TGF alpha is an intermediary in the transformation of human mammary epithelial cells by an activated c-Ha-ras gene, but not by the c-neu gene, and demonstrate that overexpression of this growth factor is able to transform immortalized human mammary epithelial cells which also express a sufficient complement of functional EGF receptors.
Blotting, Northern↗
Complementary DNA cloning establishes microfibril-associated glycoprotein (MAGP) to be a discrete component of the elastin-associated microfibrils.
Affinity-purified antibodies to microfibril-associated glycoprotein (MAGP) were used to screen a random-primed, bovine nuchal ligament cDNA library in lambda gt11. A 303-base pair clone, cM5, was isolated which encoded an amino acid sequence homologous with that determined directly from a Lys-C peptide of MAGP. A 936-base pair cDNA clone, cM32, was identified in an oligo(dT)-primed cDNA library using plaque hybridization with clone cM5. Clone cM32 encoded amino acid sequences corresponding to sequences obtained from three Lys-C peptides of MAGP, indicating that the clone was an authentic cDNA for the glycoprotein. The cDNA coded for the entire MAGP polypeptide (21 kDa) of 183 amino acids including a putative signal peptide of 17-19 amino acids. This was confirmed by in vitro translation of synthetic mRNAs transcribed from cM32. The amino acid composition of the encoded protein was virtually identical to that previously published for MAGP. DNA sequence analysis of cM32 indicated that MAGP contains two structurally dissimilar regions, an amino-terminal domain containing high levels of glutamine, proline, and acidic amino acids and a carboxyl-terminal domain containing all 13 of the cysteine residues and most of the basic amino acids. Northern blot hybridization of poly(A+) RNA from fetal nuchal ligament with clone cM32 identified a single mRNA species for MAGP of approximately 1.1 kilobases. The evidence indicates that MAGP is a distinct component of 12-nm microfibrils and that it is not derived from a larger microfibrillar glycopolypeptide.
Actin Cytoskeleton↗
Isolation, characterization and expression of the complementary DNA for human tumor necrosis factor (TNF-alpha).
A cDNA for human TNF-alpha (615bp) was isolated by means of polymerase chain reaction (PCR) using first strand cDNA from PMA-induced HL-60 cells as template. The result from sequencing the 615 bp cDNA fragment indicated that it corresponded to the entire sequence of mature human TNF coding region. Direct expression of mature human TNF was achieved using a plasmid pHT-1 constructed by ligation of the cDNA and a synthetic DNA. The IPTG-induced bacterial product (hTNF) showed cytotoxicity to mouse L-929 cells. The TNF activity was further identified by neutralization of a specific monoclonal antibody against human TNF-alpha. Approximately 80,000 units of activity were detected per ml of culture at A600 = 2.
Animals↗
Isolation and characterization of a complementary DNA clone for an algal pre-apoplastocyanin.
We have isolated a cDNA clone for the Chlamydomonas reinhardtii pre-apoplastocyanin. The sequence contains codons for the complete pre-protein including a two-domain, lumen-targeting transit sequence and the mature apoprotein. The transit sequence (47 amino acids) is the shortest one described for chloroplast lumenal proteins, and like other C. reinhardtii lumen-targeting transit sequences appears to lack an uncharged amino-terminal domain usually present in plant lumen-directing sequences. The mature protein is deduced to be 98 amino acids in length and shows highest primary sequence similarity (74-76% identity) to other unicellular algal plastocyanins. Southern hybridization analysis of C. reinhardtii genomic DNA indicates the presence of a single nuclear gene, as is the case for all other plastocyanin genes characterized to date, although the algal gene might be interrupted. Codon usage in this gene reflects the high GC content of C. reinhardtii nuclear DNA, but is more highly biased than that found in the C. reinhardtii copper-repressible gene for the functionally equivalent pre-apocytochrome c552 (perhaps contributing to the more efficient synthesis in vivo of plastocyanin over cytochrome c552). The deduced physical properties of this plastocyanin are compared to those of the C. reinhardtii plastidic cytochrome c552.
Amino Acid Sequence↗
High-level expression of complementary DNA encoding rat calmodulin in Escherichia coli.
We report the production and characterization of a rat calmodulin made in Escherichia coli. To express the rat calmodulin cDNA in E. coli, we have employed an expression vector containing the E. coli trp promoter and trpA terminator. The cDNA was modified so as to delete the 5' nontranslated sequence and to incorporate a consensus sequence for the E. coli ribosome-binding site. Several codons for the N-terminal amino acids were selected to fit the E. coli consensus nucleotide sequence around the translational initiation codon. After induction of expression in E. coli, rat calmodulin accounted for over 30% of total cellular proteins. About 100 mg of recombinant rat calmodulin, purified to over 90% homogeneity by extraction from bacterial lysate followed by phenyl-Sepharose column chromatography, was obtained from 1 liter of E. coli culture. This recombinant calmodulin activated rat brain cyclic AMP phosphodiesterase to the same extent as the native calmodulin purified from rat brain. These results indicate that the overproduction system of the recombinant calmodulin in E. coli facilitates the study of the structure-function relationship by site-specific mutagenesis.
Amino Acid Sequence↗
[Expression of ceruloplasmin gene in mammalian organs from the data of hybridization analysis with complementary DNA probes].
Expression of ceruloplasmin (Cp)-coding gene in rat and human liver and brain tissues was studied by Northern blot hybridization and by in situ hybridization with cloned species-specific cDNA probes. In rat brain structures, different levels of Cp mRNA were detected, the maximal one was found in cerebellum. The steady-state level of Cp mRNA in rat and human brain was several times lower than in parenchymatous liver cells. The size heterogeneity of Cp mRNA was found. Polyadenylated RNA prepared from human liver contains two equally abundant Cp mRNAs differing in their chain length (3.6 and 4.5 kb) while brain polyadenylated RNA contains a single Cp mRNA (4.5 kb).
Animals↗
Cloning of a full-length complementary DNA for an Artemia salina glycine-rich protein. Structural relationship with RNA binding proteins.
Overlapping cDNAs have been isolated containing all the coding sequences for Artemia salina protein GRP33, a glycine-rich protein (16.6 mol % glycine), with a molecular weight of 32,992. GRP33 is closely related to HD40, the major protein component of Artemia heterogeneous nuclear ribonucleoprotein particles, and shares certain characteristics with other RNA binding proteins. The C-terminal region (123 amino acids) contains 39 glycine residues. This region has multiple arginine residues flanked by glycines, resembling the glycine-dimethylarginine clusters present in other RNA binding proteins. Secondary structure predictions for the protein reveal two distinct domains: a hydrophilic C-terminal domain with an extended conformation and a larger N-terminal domain with a number of alpha-helices and beta-sheets.
Amino Acid Sequence↗
Molecular analysis of human and rat calmodulin complementary DNA clones. Evidence for additional active genes in these species.
A cDNA clone, lambda rCB1, encoding calmodulin was isolated from a rat brain expression library. The sequence was determined and compared to the structures of the previously described rat genes, lambda SC4 and lambda SC8 (Nojima, H., and Sokabe, H. (1986) J. Mol. Biol. 190, 391-400). Faithful sequence conservation is observed in the coding regions of lambda rCB1 and lambda SC4, the bona fide gene. Both cDNAs encode identical amino acid sequence. Very limited sequence homology, however, is noted in the 3'-untranslated segments of these clones. Surprisingly, when the lambda rCB1 nucleotide structure is compared to the processed intronless gene, lambda SC8, extensive sequence homology is found both in the coding and noncoding regions. The inferred protein sequences of lambda SC8 and lambda rCB1, however, are divergent. Using a fragment of lambda rCB1 to screen an expression library derived from a human embryonic cell line, a calmodulin cDNA clone, lambda hCE1, was isolated and characterized. Comparison of the sequence of lambda hCE1 to the cDNA from human liver, hCWP (Wawrzynczak, E. J., and Perham, R. N. (1984) Biochem. Int. 9, 177-185), reveals substantial structural divergence. Strikingly poor homology is seen in the 5'- and 3'-noncoding segments, but the coding regions were 85% homologous. Both lambda hCE1 and hCWP encode proteins of identical primary structure which is equivalent to the protein sequence deduced from lambda rCB1 and lambda SC4. Taken together these results suggest the existence of an additional actively transcribed calmodulin gene, not previously identified, in each of the human and rat genomes. Transcripts of lambda rCB1 and lambda hCE1 were observed in all tissues examined indicating the absence of tissue-specific expression. Calmodulin gene polymorphisms were detected using TaqI, HindIII, and MspI.
Amino Acid Sequence↗
Isolation and characterization of a complementary DNA (galanin) clone from estrogen-induced pituitary tumor messenger RNA.
The administration of high levels of estrogen is a well established method for producing prolactin-secreting pituitary tumors in rodents but the mechanism of tumor induction is not clear. In this paper we describe a cDNA clone (pEIC) which has been isolated from an estrogen-induced pituitary tumor cDNA library. The mRNA transcript corresponding to the pEIC clone is 0.9 kilobase in length and is not detectable in normal pituitaries but is expressed as early as 3 h after estrogen stimulation. Nucleotide sequence analysis of two 700-base pair recombinant clones shows that they encode a 124-amino acid protein which is 70% identical to the porcine galanin precursor. The sequence of 29 amino acid residues coded for by the pEIC cDNA clone is 88% identical with porcine galanin with only three amino acid substitutions near the C terminus. This extensive homology suggests that the pEIC cDNA clone codes for rat galanin or a protein belonging to the galanin gene family. These results provide the first evidence of a physiological regulator (estrogen) of the expression of the galanin gene. They also imply that galanin is secreted by prolactin-secreting tumors. Because intracerebroventricular injection of galanin can stimulate prolactin secretion and galanin inhibits hypothalamic dopamine release, it is conceivable that galanin may play a role in the induction of prolactin-secreting tumors.
Amino Acid Sequence↗
In vitro synthesis of the active tissue inhibitor of metalloproteinases encoded by a complementary DNA from virus-infected murine fibroblasts.
We have recently described the characterization and expression of a murine gene highly homologous to the human tissue inhibitor of metalloproteinases/erythroid potentiating activity (TIMP/EPA) gene. We have also reported that expression of this gene is regulated in response to virus infection. In the present report we describe the use of a cDNA clone derived from mRNA isolated from Newcastle disease virus-induced murine cells to direct in vitro synthesis of proteins encoded by this murine TIMP/EPA gene. This approach was used to analyze structural and functional parameters of the TIMP/EPA protein. Translation experiments using microsomes revealed a murine protein similar in size to that of human TIMP: Mr of approximately 22,000 for the core protein and 28,000 for the processed protein. Processing in microsomes involved N-glycosylation and cleavage of the signal peptide. Both the processed and unprocessed proteins were able to inhibit degradation of collagen by collagenase but unable to inhibit virus replication. Synthesis of truncated TIMP proteins showed that the collagenase-inhibiting activity was not encoded within a delimited portion of the molecule. This result suggests that conformation is probably essential for TIMP activity.
Animals↗
A melanocyte-specific complementary DNA clone whose expression is inducible by melanotropin and isobutylmethyl xanthine.
Two groups of cDNA clones were isolated by screening a lambda gt11 cDNA library of normal human melanocytes with antityrosinase antibodies: one group of 13 was related to the human tyrosinase gene. The properties of the other group of three cDNA clones was investigated by the use of a representative clone, Pmel 17-1. The cDNA hybridized to an mRNA species of approximately 2600 bases from human and murine melanocytes. The transcript of Pmel 17-1 (17-1 mRNA) was expressed preferentially in melanocytes and its abundance paralleled the melanin content. The expression of Pmel 17-1 mRNA increased after stimulation of human and murine melanoma cells with agents that increase the levels of melanization. Immunocompetition assays with monoclonal antibodies to gp75, a known pigmentation-associated antigen of melanocytes, suggested that Pmel 17-1 encodes a 75,000 Mr glycoprotein that is highly abundant in melanotic cells and shares some immunological homology with tyrosinase. The gene for Pmel 17-1 did not map at or near the c-albino locus in mice. The cDNA of Pmel 17-1 detected a single hybridizing restriction fragment in both human and murine DNA, indicating that the gene has been conserved between these two species and exists as a single gene in each.
1-Methyl-3-isobutylxanthine↗
Cloning of a complementary DNA encoding a new mouse B lymphocyte differentiation antigen, homologous to the human B1 (CD20) antigen, and localization of the gene to chromosome 19.
The human B1 (CD20) molecule is a differentiation Ag found only on the surface of B lymphocytes. This structurally unique phosphoprotein plays a role in the regulation of human B cell proliferation and differentiation. In order to determine whether this structure is also expressed by murine B cells, cDNA clones that encode the mouse equivalent of the B1 molecule were isolated. The longest murine cDNA clone isolated, pmB1-1, contained a 1.4-kb insert with an 873 base pair open reading frame that encodes a protein of 32 kDa. The predicted mouse B1 protein contains three hydrophobic domains that may span the membrane four times and shares a 73% amino acid sequence homology with the human B1 protein. The pmB1-1 cDNA probe was used to examine mB1 mRNA expression. Northern blot analysis indicated that pmB1-1 hybridized with two mRNA species of 2.3 and 3.0 kb that were expressed only in murine spleen lymphocytes, in B lineage cell lines representing mature B cells, and were weakly expressed in one of two plasmacytoma cell lines. pmB1-1 failed to hybridize with RNA isolated from murine T cell lines, thymus, and nonlymphoid tissues. Southern blot analysis indicated that mB1 was encoded by a single copy gene. In situ hybridization localized the mB1 gene to chromosome 19 band B, a region that also contains the genes that encode the Ly-1, Ly-10, and Ly-12 Ag. These results suggest that only B cells express this heretofore undescribed murine cell-surface protein that is structurally homologous with the membrane-embedded human B1 Ag.
Amino Acid Sequence↗