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At least 19 recordsLinked to original sources
PubMed1979

The activity of two heat shock loci of Drosophila hydei in tissue culture cells and salivary gland cells as analyzed by in situ hybridization of complementary DNA.

Complementary DNA was made to poly A+ nuclear or polysomal RNA isolated from heat shock tissue culture cells of Drosophila hydei. A number of loci other than the four major heat shock loci are labelled after in situ hybridization of these cDNA preparations, while solution hybridization indicated that only about 10% of the cDNA was specific for heat shocked cells. Removal of the fraction of cDNA which could react with 25 degrees C RNA and subsequent in situ hybridization of heat shock specific cDNA indicated that locus 4-81 B, a major salivary gland heat shock locus, is also active at 25 degrees C in tissue culture cells, while locus 4-85 B is specifically activated by heat shock in tissue culture cells. This latter locus is not seen to be clearly puffed in salivary glands, but was shown to be active in that tissue both by direct autoradiography of salivary gland chromosomes after 3H-uridine labeling and by hybridization of cDNA to chromosomal RNA.

Animals↗
PubMed1976

Quantitation of human gamma globin genes and gamma globin mRNA with purified gamma globin complementary DNA.

Complementary DNA (cDNA) specific for gamma-globin nucleotide sequences has been prepared by hybridizing total cDNA made from cord blood messenger RNA (mRNA) as template to an excess of normal adult human globin mRNA and recovering the single-stranded cDNA from hydroxylapatite. The specificity of the gamma cDNA for gamma mRNA sequences is strongly supported by the hybridization of this cDNA at low Cot values (Co, concentration of RNA and t, time in seconds) to RNA samples containing large amounts of functional gamma globin mRNA and the lack of hybridization to RNA samples containing little, if any, gamma-globin mRNA. The absence of cross-hybridization of gamma cDNA with alpha, beta, and delta mRNAs is demonstrated by the complete hybridization of the gamma cDNA to mRNA samples completely lacking either alpha or beta and delta mRNA. An estimate of the number of gamma-globin genes in human cellular DNA was obtained by hybridization of purified gamma cDNA to DNA from spleen and white blood cells of normal and beta-thalassemia subjects and measurement of the percent of gamma cDNA hybridized at saturation. The results indicate that there are between one and two gamma-globin genes per total haploid gene DNA equivalent obtained from both normal and beta-thalassemia subjects. These values are consistent with genetic evidence for the presence of multiple gamma gene loci in human cells. The finding that the number of gamma-globin genes in beta-thalassemia DNA is similar to that in nonthalassemia DNA indicates that a deletion of gamma-globin genes cannot account for either the inadequate gamma-globin synthesis or indirectly for the decreased or absent beta-globin synthesis in beta-thalassemia cells.

Amino Acid Sequence↗
PubMed1988

Human microsomal xenobiotic epoxide hydrolase. Complementary DNA sequence, complementary DNA-directed expression in COS-1 cells, and chromosomal localization.

A lambda gt11 expression library constructed from human liver mRNA was screened with an antibody against human microsomal xenobiotic epoxide hydrolase. The clone pheh32 contains an insert of 1742 base pairs with an open reading frame coding for a protein of 455 amino acids with a calculated Mr of 52,956. The nucleotide sequence is 77% similar to the previously reported rat xenobiotic epoxide hydrolase cDNA sequence. The deduced amino acid sequence of the human epoxide hydrolase is 80% similar to the previously reported rabbit and 84% similar to the deduced rat protein sequence. The NH2-terminal amino acids deduced from the human xenobiotic epoxide hydrolase cDNA are identical to the published 19 NH2-terminal amino acids of the purified human xenobiotic epoxide hydrolase protein. Northern blot analysis revealed a single mRNA band of 1.8 kilobases. Southern blot analysis indicated that there is only one gene copy/haploid genome. The human xenobiotic epoxide hydrolase gene was assigned to the long arm of human chromosome 1. Several restriction fragment length polymorphisms were observed with the human epoxide hydrolase cDNA. pheh32 was expressed as enzymatically active protein in cultured monkey kidney cells (COS-1).

Amino Acid Sequence↗
PubMed1973

Enzymatic synthesis of DNA complementary to purified 14S messenger RNA of immunoglobulin light chain.

The 14S messenger RNA, which contains poly(adenylic acid), of MOPC 41 (mouse plasmocytoma) immunoglobulin light chain, purified to a single peak as shown by polyacrylamide gel electrophoresis, was used to synthesize complementary DNA with the RNA-dependent DNA polymerase of avian myeloblastosis virus. DNA synthesis is entirely dependent on added RNA template and oligo(dT) primer. Both the size and the concentration of the primer affect the reaction. The product behaves similarly to DNA during centrifugation in cesium sulfate density gradients. It is shown by hybridization that the DNA made is complementary to the purified template, light-chain mRNA. The high specific activity of the complementary DNA should make it suitable for gene-dosage experiments. According to alkaline sucrose gradient analyses, some complete complementary DNA transcripts of the 14S mRNA seem to be made. Oligo(dG) can also function as a primer for DNA synthesis, possibly by annealing to an internal cluster of cytidines in the mRNA, that correspond to the bases coding for amino-acids 119 and 120 of the MOPC 41 light chain.

Animals↗
PubMed1987

A novel progesterone-induced messenger RNA in rabbit and human endometria. Cloning and sequence analysis of the complementary DNA.

Complementary DNAs (cDNAs) prepared from messenger RNAs (mRNAs) isolated from endometria of 5 day pregnant rabbits were inserted into the plasmid pBR322. A library of 2400 recombinant plasmid clones was prepared and screened by differential in situ hybridization with cDNAs prepared from mRNAs of rabbits either injected with progesterone or untreated by the hormone. Clones encoding uteroglobin were identified and discarded. Several progesterone-induced and progesterone-repressed clones were identified. One of them corresponded to a relatively frequent mRNA (0.2% of clones in the library) of 2300 nucleotides. The induction of this messenger RNA by progesterone was totally suppressed by the antagonist RU486. This compound displayed a limited agonistic activity when administered alone. A very small increase in mRNA concentration was observed after estradiol administration. The messenger RNA was also found in the liver (where it was constitutively expressed), the ovaries, and the Fallopian tubes of rabbits. A cross-hybridizing messenger RNA was detected in human endometrium during the luteal phase. Sequence analysis showed that the messenger RNA encoded a protein of 370 amino acids with a calculated molecular weight of 40,800. A search in Genbank and National Biomedical Research Foundation data banks showed no identity or marked similarity with previously published DNA or protein sequences.

Animals↗
PubMed1980

Primary sequence of ovomucoid messenger RNA as determined from cloned complementary DNA.

Ovomucoid messenger RNA (mRNAom) comprises approximately 8% of the total mRNA in the estrogen-stimulated oviduct. The recombinant plasmid pOM100 contained DNA complementary to the 3' end of mRNAom. DNA complementary to the 5' end of mRNAom was obtained from a partially purified preparation of mRNAom by polymerization by reverse transcriptase in the presence of a restriction fragment primer from pOM100. The complementary DNA mixture was amplified by molecular cloning using poly dG/dC tailing to form recombinant bacterial plasmids. Recombinant plasmids containing ovomucoid DNA sequences were selected by in situ hybridization to 32P-labeled pOM100 fragments. The longest plasmid containing ovomucoid DNA sequences was designated pOM502. The complete DNA sequence of both pOM100 and pOM502 was determined. The two plasmids appear to contain sequences complementary to the entire length of mRNAom. The nucleic acid sequence agrees with the known amino acid sequences for both ovomucoid and its N-terminal signal peptide. Highly homologous sequences occur in two regions that coincide with structural domains of the protein. Comparison of the sequence of mRNAom with that for other eucaryotic mRNAs allowed identification of possible functional regions in the mRNA molecule.

Animals↗
PubMedDate not supplied

Site-specific cleavage of RNA and DNA by complementary DNA--bleomycin A5 conjugates.

Bleomycin displays clinical chemotherapeutic activity, but is so nonspecifically toxic that it is rarely administered. It was therefore of interest to determine whether bleomycin could be directed to cleave RNA or DNA at a specific site by conjugation to a complementary oligonucleotide. A 15 nt MYC complementary oligodeoxynucleotide (HMYC55) bearing a 5' bleomycin A5 (Blm) residue was designed to base-pair with nt 7047-7061 of human MYC mRNA. Reactivity of the Blm-HMYC55 conjugate (and mismatch controls) with a MYC mRNA 30-mer, a MYC DNA 30-mer, and a MYC 2'-O-methyl RNA 30-mer, nt 7041-7070, was analyzed in 100 microM FeNH(4)SO(4), 50 mM beta-mercaptoethanol, 200 mM LiCl, 10 mM Tris-HCl, pH 7.5, at 37 degrees C. Cleavage of the substrate RNA or DNA occurred primarily at the junction of the complementary DNA-target RNA duplex, 18-22 nt from the 5' end of the RNA. Reaction products with lower mobility than the target RNA or DNA also formed. Little or no reaction was observed with more than three mismatches in a Blm-oligodeoxynucleotide conjugate. Neither the short RNA or DNA cleavage fragments nor the low mobility products were observed in the absence of Fe(II), or the presence of excess EDTA. The target RNA was also cleaved efficiently by bleomycin within a hybrid duplex with a preformed single-nucleotide bulge in the RNA strand. New Blm-oligodeoxynucleotide conjugates containing long hexaethylene glycol phosphate based linkers between oligodeoxynucleotide and bleomycin were designed to target this bulge region. These conjugates achieved 8-18% cleavage of the target RNA, depending on the length of the linker. Blm-oligodeoxynucleotide conjugates thus demonstrated sequence specificity and site specificity against RNA and DNA targets.

Binding Sites↗
PubMed1997

Reversal of 1,3-bis(2-chloroethyl)-1-nitrosourea-induced severe immunodeficiency by transduction of murine long-lived hemopoietic progenitor cells using O6-methylguanine DNA methyltransferase complementary DNA.

Bone marrow toxicity is a dose-limiting side effect of chloroethylnitrosourea (CNU) chemotherapeutic alkylating agents. A major determinant of CNU cytotoxicity is the methylation of guanine at the O6-position and the subsequent formation of interstrand DNA cross-links. O6-Methylguanine DNA methyltransferase (MGMT) removes alkyl groups from the O6 position of guanine and has been shown to repair CNU-induced DNA damage. We have previously demonstrated that transplantation of murine bone marrow cells transduced with a recombinant retroviral vector expressing MGMT via the human phosphoglycerate kinase promoter (PGK-MGMT) protects animals in vivo from acute myelotoxicity associated with CNU treatment. In the present study, we examined the effects of 1,3-bis(2-chloroethyl)-1-nitrosourea (BCNU), a commonly used CNU, on long term recovery of the lymphoid compartment, including thymus reconstitution, peripheral T and B cell populations, and lymphocyte mitogen responses in mice reconstituted with PGK-MGMT-transduced hemopoietic cells. Mice transplanted with either mock-infected control or PGK-MGMT-transduced stem cells were treated with five weekly doses of BCNU. Analysis of the lymphoid compartment demonstrated significant damage 3 mo after the last BCNU dose in control animals. In contrast, the profound deficiency in CD4+CD8+ double-positive thymocytes and mature lymphocytes observed in control mice surviving BCNU treatment was completely reversed in mice transplanted with PGK-MGMT-transduced bone marrow and was associated with molecular evidence of in vivo selection of transduced cells in the lymphoid compartment. Thus, long term immunodeficiency following CNU therapy may be prevented by genetic modification of murine hemopoietic stem cells with MGMT, leading to significant improvement in post-transplant immune function.

Animals↗
PubMed1982

Alphafetoprotein messenger RNA in human embryonal carcinoma grown in nude mice, and cloning of its complementary DNA.

Messenger RNA for human apha-fetoprotein (AFP) was partially purified from human testicular embryonal carcinoma grown in nude mice. DNA complementary to the mRNA was synthesized, tailed with oligo(dC) and inserted at the PstI site of the plasmid pBR322 tailed with oligo(dG). Escherichia coli strain LE392 was transformed with the chimeric plasmid and colonies were screened for human AFP mRNA sequences by hybridization with DNA complementary to mouse AFP mRNA (greater than 95% pure). Five colonies showed positive hybridization. The plasmid pHAF2, containing the largest insert, 900 nucleotides, was further characterized by hybrid-arrest of translation of AFP mRNA, restriction endonuclease mapping and partial nucleotide sequence analysis. It was found that the AFP complementary DNA (cDNA) sequence in pHAF2 corresponded to the 3'-end of AFP messenger RNA (mRNA), and showed a restriction map entirely different from that of mouse AFP cDNA clones previously reported.

Animals↗
PubMed1984

Characterization of complementary DNA clones coding for two forms of 3-methylcholanthrene-inducible rat liver cytochrome P-450.

Double-stranded DNA complementary to the partially purified mRNA prepared from 3-methylcholanthrene (MC)-treated rat liver was constructed and cloned in Escherichia coli. Twenty clones were verified to carry a complementary DNA (cDNA) insert coding for MC-inducible cytochrome P-450 by positive hybridization translation assay and immunochemical assay with anti-cytochrome P-450 antibody. The identified cDNA clones were divided into at least two groups on the basis of comparison of restriction maps of the cDNA inserts. A clone pAU157 whose cDNA insert was approximately 2.7 kb in length contained nearly full-length mRNA information for cytochrome P-450MC or P-450c, which is the major form of MC-inducible cytochrome P-450. Other cDNA clones pTZ286-pTZ330 contained the 1.2 kb sequence complementary to cytochrome P-450d mRNA. RNA blot analysis revealed that pAU157 and pTZ286-pTZ330 cDNA clones were derived from 22S and 18S mRNAs, respectively, both of which were induced in rat liver by MC treatment. Sequence analysis revealed that there were closely homologous sequence regions in pAU157 and pTZ286-pTZ330 cDNA inserts and most of the homologous sequences were localized in two limited coding regions of the two cytochrome P-450 species. pAU157 encoded the total amino acid sequence of cytochrome P-450MC or P-450c and pTZ286-pTZ330 coded for the C-terminal 368 amino acid residues of cytochrome P-450d. Two highly homologous regions were found in the amino acid sequences of these cytochrome P-450 species.

Amino Acid Sequence↗
PubMed1981

Isolation of globin pre-messenger RNA on thiol-agarose by terminally mercurated complementary DNA.

A mercurated DNA complementary to globin mRNA was prepared by the addition of mercurated poly(dC) tails to the 3'-end of the molecule using the enzyme terminal deoxynucleotidyl transferase. The mercurated complementary DNA was retained efficiently on thiol-agarose from which it was eluted by 2-mercaptoethanol. Hybridization of the mercurated probe to globin mRNA led to a specific selection of the latter from a mixed population of RNA through hybrid retention on thiol-agarose. In some pilot experiments this technique was applied for the isolation of globin gene-specific pre-mRNA. Pulse-labeled RNA up to 3 X 10(6) MW was thus isolated with prominent peaks of 1.5 kb ('15 S'), 4.0 kb ('28 S') and up to about 10 kb. Electron microscopical analysis revealed pre-mRNA molecules of up to 1.2 mu (about 4.5 kb) in length isolated by hybridization to (Hg)cDNA; in control experiments, hybridization of this high MW RNA was competed out by highly purified globin mRNA. These data provide another indication for the existence of globin gene transcripts in the 10 kb range, i.e. transcripts larger than the about 1 500 nucleotides long '15 S' pre-mRNA, the substrate to final splicing. Such 'giant' transcripts can be interpreted as either the obligatory primary pre-mRNA or only facultative transcripts of the globin genes.

Animals↗
PubMedDate not supplied

Cloning, sequencing and expression of complementary DNA encoding the muscarinic acetylcholine receptor.

Cloning and sequence analysis of DNA complementary to porcine cerebral messenger RNA encoding the muscarinic acetylcholine receptor predict the complete amino-acid sequence of this protein. Expression of the complementary DNA produced functional muscarinic receptor in Xenopus oocytes. The muscarinic receptor is homologous with the beta-adrenergic receptor and rhodopsin in both amino-acid sequence and suggested transmembrane topography.

Amino Acid Sequence↗
PubMed2002

Development of a murine hematopoietic progenitor complementary DNA microarray using a subtracted complementary DNA library.

With the goal of creating a resource for in-depth study of myelopoiesis, we have executed a 2-pronged strategy to obtain a complementary DNA (cDNA) clone set enriched in hematopoietic genes. One aspect is a library subtraction to enrich for underrepresented transcripts present at early stages of hematopoiesis. For this, a hematopoietic cDNA library from primary murine bone marrow cells enriched for primitive progenitors was used as tester. The subtraction used 10 000 known genes and expressed sequence tags (ESTs) as driver. The 2304 randomly picked clones from the subtracted cDNA libraries represent 1255 distinct genes, of which 622 (50%) are named genes, 386 (30%) match uncharacterized ESTs, and 247 (20%) are novel. The second aspect of our strategy was to complement this subtracted library with genes known to be involved in myeloid cell differentiation and function. The resulting cDNAs were arrayed on polylysine-coated glass slides. The microarrays were used to analyze gene expression in primary and cultured murine bone marrow-derived progenitors. We found expression of various types of genes, including regulatory cytokines and their receptors, signal transduction genes, and transcription factors. To assess gene expression during myeloid differentiation, we examined patterns of change during induced differentiation of EML cells. Several hundred of the genes underwent fluctuations in expression level during myeloid cell differentiation. The complete database, accessible on the World Wide Web at http://yale130132115135.med.yale.edu/, allows for retrieval of information regarding these genes. Our microarray allows for genomewide expression analysis of myeloid stem cells, which will help in defining the regulatory mechanisms of stem cell differentiation.

Animals↗
PubMed1981

Specifically primed synthesis in vitro of full-length DNA complementary to potato-spindle-tuber viroid.

Potato spindle tuber viroid (PSTV) RNA is transcribed in vitro by reverse transcriptase into complementary DNA in the presence of synthetic oligodeoxyribonucleotides as primers. In the case of priming with the pentadecadeoxyribonucleotide d(T-T-C-T-T-T-T-T-T-C-T-T-T-T-C) complementary to PSTV RNA from nucleotides 49 to 63, specificity of transcription initiation allows rapid sequencing of part of the viroid genome using chain-terminating dideoxyribonucleoside triphosphates. The DNA transcripts obtained represent distinct molecular species with the largest product being a full-length copy of the viroid RNA template. Molecular hybridization with 32P-labeled complementary DNA detects sequence homologies among different viroid species.

Base Composition↗
PubMed1976

Gene specific priming of complementary DNA synthesis.

DNA, complementary to chicken globin mRNA was synthesized using either Avian Myeloblastosis virus reverse transcriptase, or E. coli DNA polymerase I. Transcriptase cDNA sediments at 9 S on sucrose gradients, and is 620 nucleotides in length, representing a complete copy of globin mRNA template. In contrast, Polymerase I cDNA sediments at 4 S, is 100 to 200 nucleotides in length, and is a copy of a small region at the 3'(poly A) end of globin mRNA. Similarly, Transcriptase cDNA and Polymerase I cDNA hybridize to globin mRNA template with characteristic, individual Crot1/2 values. The Crot1/2 value for Transcriptase cDNA hybridization is 7 X 10(-4) mol s 1(-1), and that for Polymerase I cDNA is 5 X 10(-3). This work shows that Avian Myeloblastosis virus reverse transcriptase can use Polymerase I cDNA to prime further cDNA synthesis along the mRNA template. The product of extended cDNA synthesis is identical in length and hybridization properties to oligo (dT) primed transcriptase cDNA.

Avian Myeloblastosis Virus↗
PubMed1978

Recombinant plasmids containing Xenopus laevis globin structural genes derived from complementary DNA.

Details are presented of the in vitro synthesis of double-stranded DNA complementary to purified Xenopus globin messenger RNA, using a combination of reverse transcriptase, fragment 'A' of E. coli DNA polymerase 1 and S1 endonuclease. After selection of duplex DNA molecules approaching the length of Xenopus globin messenger RNA by sedimentation of the DNA through neutral sucrose gradients, the 3'-OH termini of the synthetic globin gene sequences were extended with short tracts of oligo dGMP using terminal transferase. This material was integrated into oligo dCMP-extended linear pCR1 plasmid DNA and amplified by transfection of E. coli. Plasmids carrying globin sequences were identified by hybridization of 32P-labelled globin mRNA to total cellular DNA in situ, by hybridization of purified plasmids to globin cDNA in solution, by analysis of recombinant DNA on polyacrylamide and agarose gels, and by heteroduplex mapping. The results show that extensive DNA copies of Xenopus globin mRNA have been integrated into recombinant plasmids.

Animals↗
PubMed1982

The synthesis of DNA complementary to polyadenylate-containing RNA from Bacillus subtilis.

We had shown earlier (Gopalakrishna, Y., Langley, D., Sarkar, N. (1981) Nucleic Acid Res. 9, 3545-3554) that a substantial fraction of mRNA of various bacterial species carries 3'-terminal polyadenylate sequences. In this paper, we show that poly(A)-containing RNA from Bacillus subtilis can serve as template for the synthesis of complementary DNA by avian myeloblastosis virus reverse transcriptase, provided that oligodeoxythymidylate is added as primer. Poly(A)-RNA purified by affinity chromatography on oligo(dT)-cellulose was 20 times more effective as template for cDNA formation than total bacterial RNA, whereas rRNA was inactive. The average chain length of the cDNA was 400 nucleotides (range = 230-800 nucleotides), and 95% of the cDNA could be degraded by the single-strand specific S1 nuclease after denaturation. The small fraction (5%) that was resistant to S1 nuclease may represent duplex hairpin structures. Annealing with poly(A)-RNA protected cDNA from degradation by S1 nuclease, indicating that cDNA indeed contains nucleotide sequences complementary to poly(A)-RNA. These results constitute independent evidence that a large fraction (about 40%) of B. subtilis mRNA is polyadenylated. Moreover, the synthesis of cDNA to bacterial mRNA provides an important new tool for the study of bacterial mRNA structure.

Bacillus subtilis↗