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At least 649 records · Page 36Linked to original sources
PubMed1981

Mouse complement components C3 and C4. Characterization of their messenger RNA and molecular cloning of complementary DNA for C3.

Mouse liver mRNA species which direct the cell-free synthesis of pro-C3 and pro-C4 polypeptides with an apparent molecular weight of 175,000 and 190,000, respectively, were shown to sediment faster than 28 S. By electrophoresis in a denaturing gel the mRNA for C3 was determined to contain approximately 7,500 ribonucleotides. cDNA was synthesized from size-fractionated mouse liver mRNA and cloned in the plasmid pBR 322. Among the cDNA clones recovered three were identified as being complementary to portions of the mRNA for C3.

Animals↗
PubMed1989

Complementary DNA cloning and hybridization analysis of maize stripe virus RNAs.

Cloned cDNAs were used to assess the relationships of the RNAs isolated from maize stripe virus (MStV) virion-like nucleoproteins. Northern blot hybridization analysis showed no detectable sequence homology between the five different sized RNAs; however, cDNAs which hybridized with a specific ssRNA also hybridized with a specific dsRNA. Hybridization analysis of denatured MStV RNAs using ssRNA riboprobes of opposite polarities showed that for each of the five MStV RNAs, opposite, complementary polarity RNAs were present. However, unequal amounts of complementary polarities for each size RNA were detected, and the polarity of the RNA in excess was primarily detectable as ssRNA.

Blotting, Northern↗
PubMed1981

Cloning of epoxide hydratase complementary DNA.

Tightly membrane-bound polysomes were isolated from livers of rats administered trans-stilbene oxide. Epoxide hydratase mRNA was enriched from these polysomes using immunochemical techniques and oligo(dT)-cellulose chromatography. This resulted in an increase in message concentration over that found in noninduced membrane-bound cDNA, synthesized from enriched mRNA, was inserted into the ampicillin resistance gene of pBR322 using oligo(dG)-oligo(dC) tailing. Clones containing sequences complementary to epoxide hydratase mRNA were selected by differential colony hybridization using [32P]cDNA synthesized from immunoenriched mRNA and [32P]cDNA synthesized from nonenriched mRNA. Plasmids from four clones, which only annealed with the enriched probe, were isolated and shown to specifically hybridize with epoxide hydratase mRNA by hybrid selection-translation. A composite restriction endonuclease map of the plasmid inserts was constructed which spanned 1310 base pairs and represented approximately 80% of the message sequence. The 3'-5' orientation of this map relative to the epoxide hydratase mRNA was also determined.

Cloning, Molecular↗
PubMed1988

Molecular cloning and characterization of human cardiac alpha- and beta-form myosin heavy chain complementary DNA clones. Regulation of expression during development and pressure overload in human atrium.

We have constructed and characterized two types of myosin heavy chain (MHC) cDNA clones (pHMHC2, pHMHC5) from a fetal human heart cDNA library. Comparison of the nucleotide and deduced amino acid sequences between pHMHC2 and pHMHC5 shows 95.1 and 96.2% homology, respectively. The carboxyl-terminal peptide and 3'-untranslated (3'-UT) regions are highly divergent and specific for these cDNA clones. By using the synthetic oligonucleotide probes that are complementary to the unique 3'-UT regions of these cDNA clones, we demonstrate that pHMHC2 is exclusively transcribed in the atrium, whereas the mRNA for pHMHC5 is predominantly expressed in the ventricle. This result indicates that pHMHC2 and pHMHC5 code for alpha- and beta-form MHCs, respectively. Furthermore, we show that beta-form MHC mRNA is expressed in adult atrium at a low level but scarcely expressed in fetal atrium. Finally, we demonstrate that MHC isozymic transition in pressure-overloaded atrium is, at least in part, regulated at a pretranslational level.

Adult↗
PubMed1980

The synthesis and isolation of DNA complementary to histone mRNA from mouse embryos.

A 9S poly(A)-RNA preparation isolated from mouse embryos was shown to stimulate the synthesis of histones in an ascites cell free extract. This RNA preparation was used for the synthesis of a highly labelled cDNA probe complementary to histone mRNA. Hybridisation of this cDNA probe to rRNA showed that 52% of the cDNA consisted of sequences complementary to rRNA. The histone mRNA specific cDNA was purified by hybridising the impure cDNA to rRNA followed by removal of the single-stranded histone cDNA by hydroxylapatite chromatography.

Animals↗
PubMed1980

Characterization of cloned complementary DNA covering more than 6000 nucleotides (97%) of avian vitellogenin mRNA.

The messenger RNA coding for chicken vitellogenin, a precursor of the egg-yolk proteins lipovitellin and phosvitin, is synthesized in the liver following estrogen injection. This mRNA is 6600 nucleotides long. We have previously reported the cloning and preliminary characterization of some cDNA fragments representing portions of the vitellogenin mRNA [Biochem. Biophys. Acta, 606, 34--46 (1980)]. In this paper we report the full characterization of a larger series of such clones, representing almost the entire length of the mRNA, by restriction endonuclease mapping, R-loop mapping, RNA-DNA hybridization and by translation in vitro of the RNA which hybridizes to the cloned DNA. From the results we conclude that the chicken vitellogenin mRNA, unlike that of Xenopus laevis, does not vary in sequence over most of its length, although some variations in the cDNA sequences were detected particularly in clones derived from the 3' terminus of the RNA. All sequence variants appear to be present in RNA prepared from single animals. The possible origins of these minor species are discussed. Furthermore, we describe a cDNA clone complementary to an mRNA which is about the same size as vitellogenin mRNA and which codes for an egg yolk protein antigenically related to lipovitellin. This mRNA is synthesized constitutively.

Animals↗
PubMed1998

Non-complementary DNA helical structure induced by positive torsional stress.

We have induced a local conformational transition by positive torsional stress in small synthetic circular DNA molecules containing cruciforms with immobile or tetramobile branched junctions. The immobile species correspond to the extruded and intruded extrema of the tetramobile junction. Under normal conditions the sequences of all the branched species prevent them from being re-absorbed into the circle. We have induced positive stress by addition of ethidium to the circle, in a low ionic strength medium. Alterations in gel electrophoretic mobility under increasing concentrations of ethidium suggest that the cruciforms undergo a transition under torsional stress. The product of this transition contains mispaired nucleotides, but interwound backbones. By comparing the electrophoretic mobilities of circles containing these structures with that of a completely complementary circle of the same length, we conclude that the twist in the mispairing region is similiar to that of completely paired species.

DNA, Circular↗
PubMed1974

A limited number of globin genes in human DNA.

The number of globin genes in human cells was determined by hybridizing DNA from human spleens to (3)H-labeled DNA complementary to human globin mRNA. Assuming the rates of reannealing of complementary DNA and cellular DNA are similar, the extent of hybridization of complementary DNA at various ratios of cellular DNA to complementary DNA indicate that there are fewer than 10 globin gene copies per haploid human genome. An alternative analysis of the data, which introduces no assumptions concerning the relative rates of reaction of complementary DNA and cellular DNA, indicates fewer than 20 globin gene copies are present. DNA isolated from the spleen of a patient with beta(+) thalassemia contained a number of globin gene copies similar to that of normal DNA.

Base Sequence↗
PubMed1983

Cloning of DNA complementary to cytochrome P-450 induced by pregnenolone-16 alpha-carbonitrile. Characterization of its mRNA, gene, and induction response.

The major form of cytochrome P-450 (P-450PCN) was isolated from rats administered pregnenolone-16 alpha-carbonitrile (PCN). Messenger RNA coding for P-450PCN was enriched by polysome immunoadsorption and utilized to construct a library of cDNA clones in pBR322. P-450PCN clones were isolated from this library by differential colony hybridization using [32P]cDNA probes transcribed from PCN-induced and PCN-induced P-450PCN immunoenriched poly(A) RNA. The P-450PCN clone with the largest cDNA insert (pP450PCN-10) was verified to contain sequences complementary to P-450PCN mRNA by hybrid selection-translation. pP450PCN-10 was composed of approximately 1900 base pairs and had a restriction map that overlapped at least 3 other cDNA clones selected by differential colony hybridization. Denaturing-agarose gel electrophoresis and nitrocellulose blot-hybridization using nick-translated 32P-labeled pP450PCN-10 indicated that pP450PCN mRNA is 2500 +/- 150 nucleotides in length; pP450PCN-10, therefore, represents approximately 76% of its corresponding mRNA sequence. Southern blot analysis of rat DNA using pP450PCN revealed that approximately 50 to 60 kilobases of DNA reacted with the PCN probe, suggesting the P-450PCN gene is either a very large gene or other genomic segments exist that react with the probe, such as pseudogenes or related P-450 genes that share homology. The mechanism of P-450PCN induction was examined by isolating poly(A) RNA at various times after steroid administration and quantitating for P-450PCN mRNA using pP450PCN-10 as a hybridization probe. PCN administration produced a rapid elevation of P-450PCN mRNA which reached maximal levels (7-fold above control) 12 h after administration. In contrast, cytochrome P-450b mRNA, which is readily induced by phenobarbital, was only slightly elevated (approximately 2-fold) after PCN administration.

Animals↗
PubMed1990

Sequencing and expression of complementary DNA for the general transcription factor BTF3.

The initiation of transcription of eukaryotic genes involves the ordered assembly of a multiprotein complex on proximal promoter elements such as the TATA box. In addition to RNA polymerase II (otherwise RNA pol II, RNA polymerase B), four general transcription factors are required for initiation of transcription: BTF1 (also referred to as TFIID) which has recently been cloned from yeast, BTF2, BTF3 and STF. The first step in assembly of the initiation complex is the stable binding of BTF1 to the TATA box, which is facilitated by STF. Neither BTF2 nor BTF3 bind directly to the promoter proximal elements, but BTF3 can form a stable complex with RNA pol II. We recently purified BTF3, which is a protein of relative molecular mass 27,000, but further studies have been hampered by its low abundance in cells. On the basis of sequences from peptides of BTF3, we have now cloned two complementary DNAs, one for a protein (BTF3a) with all the characteristics of purified BTF3, and one for a shorter protein (BTF3b) lacking the first 44 residues of BTF3a and which is transcriptionally inactive, despite its ability to bind RNA pol II.

Amino Acid Sequence↗
PubMed1975

Synthesis of DNA complementary to separated human alpha and beta globin messenger RNAs.

Human globulin messenger RNA, purified by oligo(dT)-cellulose column chromatography, is reproducibly separated into two bands by polyacrylamide gel electrophoresis in the presence of 99% formamide. The more rapidly migrating (fast) band is somewhat more abundant than the slow band in normal (nonthalassemic) total reticulocyte globin messenger RNA. In alpha-thalassemic (Hb H disease) messenger RNA, the slow band is 6.5 times more abundant than the fast band, whereas in beta-thalassemic messenger RNA the fast band is three times more abundant than a second band, which has a slightly greater mobility than the slow band of normal and alpha-thalassemic RNA. The RNA bands of nonthalassemic globin messenger RNA were eluted from the gel and efficiently transcribed into DNA copies by use of the RNA-dependent DAN polymerase of avian myeloblastosis virus. Hybridization of these copy DNAs to fast and slow band RANs and to nonfractionated normal, alpha-thalassemic, and geta-thalassemic messenger RNAs revealed that the eluted fast band RNA contains predominantly alpha-chain specific sequences, whereas the eluted slow band RNA contains predominantly beta-chain specific sequences. Nucleotide sequence analysis of 32-P-labeled RNA transcribed from the slow band copy DNA also indicated that the slow band RNA is beta messenger RNA.

Avian Myeloblastosis Virus↗