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At least 883 records · Page 49Linked to original sources
PubMed1998

Identification of cytochrome P450 1A1 in human brain.

We have isolated cDNA fragments that were originated from P450 1A1 in human brain by polymerase chain reaction (PCR) analysis. Their size matched up to what we expected based on the reported P450 1A1 mRNA sequence of human breast carcinoma cell line MCF-7. To confirm proper PCR products, they were subcloned and their nucleotide sequences were determined. This result showed that PCR products were actually derived from P450 1A1 mRNA in human brain. We demonstrated for the first time that human brain contains a P450 1A1-dependent metabolism system which seems to be important for understanding the metabolism of endogenous and exogenous compounds.

Base Sequence↗
PubMed1998

Loops and bulge/loops in iron-responsive element isoforms influence iron regulatory protein binding. Fine-tuning of mRNA regulation?

A family of noncoding mRNA sequences, iron-responsive elements (IREs), coordinately regulate several mRNAs through binding a family of mRNA-specific proteins, iron regulatory proteins (IRPs). IREs are hairpins with a constant terminal loop and base-paired stems interrupted by an internal loop/bulge (in ferritin mRNA) or a C-bulge (in m-aconitase, erythroid aminolevulinate synthase, and transferrin receptor mRNAs). IRP2 binding requires the conserved C-G base pair in the terminal loop, whereas IRP1 binding occurs with the C-G or engineered U-A. Here we show the contribution of the IRE internal loop/bulge to IRP2 binding by comparing natural and engineered IRE variants. Conversion of the internal loop/bulge in the ferritin-IRE to a C-bulge, by deletion of U, decreased IRP2 binding by >95%, whereas IRP1 binding changed only 13%. Moreover, IRP2 binding to natural IREs with the C-bulge was similar to the DeltaU6 ferritin-IRE: >90% lower than the ferritin-IRE. The results predict mRNA-specific variation in IRE-dependent regulation in vivo and may relate to previously observed differences in iron-induced ferritin and m-aconitase synthesis in liver and cultured cells. Variations in IRE structure and cellular IRP1/IRP2 ratios can provide a range of finely tuned, mRNA-specific responses to the same (iron) signal.

5-Aminolevulinate Synthetase↗
PubMed1985

Nucleotide sequence of cDNA clones of the murine myb proto-oncogene.

We have isolated cDNA clones of murine c-myb mRNA which contain approximately 2.8 kb of the 3.9-kb mRNA sequence. Nucleotide sequencing has shown that these clones extend both 5' and 3' to sequences homologous to the v-myb oncogenes of avian myeloblastosis virus and avian leukemia virus E26. The sequence contains an open reading frame of 1944 nucleotides, and could encode a protein which is both highly homologous, and of similar size (71 kd), to the chicken c-myb protein. Examination of the deduced amino acid sequence of the murine c-myb protein revealed the presence of a 3-fold tandem repeat of 52 residues near the N terminus of the protein, and has enabled prediction of some of the likely structural features of the protein. These include a high alpha-helix content, a basic region toward the N terminus of the protein and an overall globular configuration. The arrangement of genomic c-myb sequences, detected using the cDNA clones as probes, was compared with the reported structure of rearranged c-myb in certain tumour cells. This comparison suggested that the rearranged c-myb gene may encode a protein which, like the v-myb protein, lacks the N-terminal region of c-myb.

Amino Acid Sequence↗
PubMed1986

Measles virus haemagglutinin gene: cloning, complete nucleotide sequence analysis and expression in COS cells.

A measles virus (Hallé strain) cDNA library was prepared by cloning virus-induced mRNA directly into the expression vector PCD. Clones corresponding to the measles virus haemagglutinin (HA) gene were isolated and one, PCD-HA-15, which corresponded to the complete mRNA sequence, was further characterized. After transfection into COS-7 cells, measles virus HA antigen was detected by immunofluorescence. The [35S]methionine-labelled HA protein from transfected cells was immunoprecipitated by both polyclonal and monoclonal measles virus antibodies. Analysis by SDS-polyacrylamide gel electrophoresis revealed that the PCD-HA-15 protein migrated in a manner identical to the virus-induced HA. Nucleotide sequence analysis established that the gene contained 1949 nucleotides [exclusive of poly(A)] and coded for a protein containing 617 amino acids. A single hydrophobic domain likely to represent the transmembrane region was identified at the N-terminus. A second overlapping reading frame coded for a protein containing 70 amino acids. This contained a short hydrophobic region (16 amino acids) and had two potential N-glycosylation sites. Comparison of the HA gene of the Hallé strain with the published sequence of the Edmonston strain showed that there was a high degree of conservation (99.3%).

Amino Acid Sequence↗
PubMed1997

Latent periodicity of DNA sequences of many genes.

A method of latent periodicity search being developed. Mutual information is used to reveal of DNA or mRNA sequence latent periodicity. The latent periodicity of DNA sequence is a periodicity with low level of homology between any two periods inside DNA sequence. The mutual information between artificial numerical sequence and DNA sequence is calculated. The length of the artificial sequence period is varied from 2 to 150. The high level of the mutual information between artificial and DNA sequences allows to find any type of latent periodicity of DNA sequence. The latent periodicity of many DNA coding regions has been found. Potential significance of latent periodicity is discussed.

Base Sequence↗
PubMed1993

Methodologies for specific intron and exon RNA localization in cultured cells by haptenized and fluorochromized probes.

We have determined optimal conditions for the detection of mRNA sequences in cultured cells by nonradioactive in situ hybridization. For this purpose a number of different cell lines have been used: rat 9G cells for the detection of human cytomegalovirus immediate early mRNA, and HeLa as well as 5637 carcinoma cells for the detection of housekeeping gene mRNAs. Extensive optimization of fixation and pretreatment conditions revealed that most intense hybridization signals are obtained when cells are grown on glass microscope slides, fixed with a mixture of formaldehyde and acetic acid, pretreated with pepsin and denatured prior to hybridization. In addition, we also studied the potential of fluorochromized probes for the direct detection of multiple RNA sequences. The optimized in situ hybridization procedure revealed that immediate early mRNA transcripts are, in addition to a cytoplasmic localization, localized within nuclei of rat 9G cells. Double hybridization experiments showed that intron and exon sequences colocalize within the main nuclear signal. In addition, the presence of small, intron-specific, fluorescent spots scattered around the main nuclear signals indicates that intron sequences which are spliced out can be visualized. Additional information about the functioning of cells could be obtained by the detection of mRNA simultaneously with bromodeoxyuridine, incorporated during S-phase, or its cognate protein. The sensitivity of these methods is such that mRNAs of abundantly expressed housekeeping genes can be detected in a variety of cell lines with high signal to noise ratios.

Animals↗
PubMed1992

Sequence analysis of M2 mRNA of bovine respiratory syncytial virus obtained from an F-M2 dicistronic mRNA suggests structural homology with that of human respiratory syncytial virus.

The nucleotide sequences of the F and M2 mRNAs of strain A51908 of bovine respiratory syncytial virus (BRSV) were determined by sequencing cDNA of an intracellular dicistronic mRNA. Comparison of the F mRNA sequence with those of other BRSV strains showed that there was extensive sequence identity at both the nucleotide (95% identity) and amino acid (94% identity) levels. Alignment of the nucleotide and encoded amino acid sequences of M2 mRNA of BRSV with those of human respiratory syncytial virus (HRSV) M2 mRNA showed 69% identity at the nucleotide level and 80% identity at the amino acid level. The general features of BRSV F and M2 proteins are similar to those described previously for the HRSV proteins. The M2 mRNA of BRSV also contained a second internal, overlapping open reading frame (ORF) similar to one reported for HRSV. The predicted products of the second ORFs of BRSV and HRSV shared 43% amino acid identity. As described for HRSV, the 3'-terminal end of the M2 mRNA overlaps with the 5' end of the L gene by 68 nucleotides. The identity between the N-terminal regions of the L proteins of BRSV and HRSV is 75%. In addition, the intergenic sequence of the F-M2 gene junction of BRSV was determined.

Amino Acid Sequence↗
PubMed1987

Hepatoma variants (C2) are defective for transcriptional and post-transcriptional actions from both endogenous and viral genomes.

A series of rat hepatoma cell lines was infected with a recombinant adenovirus bearing the rat albumin promoter. Transcription from this promoter was scored directly and was highest in FAO, the differentiated parent, undetectable in C2, a cell variant that has lost almost all hepatocytic characteristics, and high again in C2-Rev7, a 'revertant' cell line derived from C2 that has regained the ability to produce many proteins characteristic of hepatocytes. The endogenous albumin gene is not transcribed in C2 cells, and at a very low rate in C2-Rev7 cells, which accumulate endogenous albumin mRNA at close to normal amounts. Thus the C2-Rev7 'recovery' of albumin mRNA concentration for the endogenous gene is based mainly on post-transcriptional events while the ability of C2-Rev7 to transcribe the albumin promoter in the viral genome is based on a transcriptional factor(s). We also showed that the C2 phenotype included post-transcriptional effects for other genes: transcription of phenylalanine hydroxylase and phosphoenolpyruvate carboxykinase mRNA sequences continue in C2 at rates equivalent to FAO but these C2 cells have no mRNA for these proteins while FAO does. In addition, C2 cells transcribed certain early adenovirus transcription units (E2 and 4) as well as FAO cells but accumulated E2 mRNAs poorly if at all. The changes that led to the C2-Rev7 cell line produced a return to normal of the ability to accumulate these viral mRNAs. Thus a major event in the C2 to C2-Rev7 transition involves post-transcriptional processes as well as the ability to transcribe the albumin promoter positioned in the virus genome.

Adenoviridae↗
PubMed2000

Reverse transcription - 3' rapid amplification of cDNA ends-nested PCR of ACT1 and SAP2 mRNA as a means of detecting viable Candida albicans in an in vitro cutaneous candidiasis model.

The presence of viable cells of Candida albicans, in broth or in a reconstructed living skin equivalent, was determined by the detection of amplicons of partial mRNA sequences of the genes encoding fungal actin (ACT1) and secreted aspartyl proteinase 2 (SAP2). The mRNA of both genes were amplified by reverse transcription-3' rapid amplification of cDNA ends-nested polymerase chain reaction. Single bands of ACT1 (315 bp) and SAP2 (162 bp) mRNA were amplified from total RNA extracts of C. albicans grown in yeast carbon base-albumin broth or in living skin equivalent tissue; only the former was amplified from Sabouraud broth-grown organisms. Primer pairs targeted for ACT1 and SAP2 were Candida genus-specific and C. albicans-specific, respectively. The sensitivity limits of the assay were 100 fg of total RNA or 10 cells of C. albicans, by ethidium bromide staining. When C. albicans-infected living skin equivalent was exposed to amorolfine, amplicons of ACT1 and SAP2 mRNA were not detected in total RNA extracts. Non-amplification of the mRNA correlated with the absence of C. albicans growth in Sabouraud agar cultures of living skin equivalent samples. Reverse transcription-3' rapid amplification of cDNA ends-nested polymerase chain reaction of the mRNA encoding specific proteins of an organism has potential application in determining the viability of the organism in tissue, thus monitoring the efficacy of an antimicrobial therapy, and in detecting mRNA expressed in very little amounts in tissue.

Actins↗
PubMed1988

T-cell receptor alpha-chain variable-region haplotypes of normal and autoimmune laboratory mouse strains.

We used Southern blotting and mRNA analysis to characterize allelic polymorphisms among genes of the T-cell antigen receptor (TCR) alpha-chain variable-region (V alpha) locus in a large panel of normal and autoimmune-susceptible or autoimmune-contributing strains of laboratory mice. Four major V alpha haplotypes were defined on the basis of multiple restriction fragment length polymorphisms for each of nine V alpha subfamily probes used. Southern blotting also revealed haplotype-specific loss of bands within some V alpha subfamilies, consistent with the deletion of particular V alpha genes or sets of genes from haplotype to haplotype. In contrast to the situation in the V beta locus, however, deletion of entire V alpha subfamilies was not observed. The nature of V alpha allelic variability was further explored by using an RNase protection assay to analyze expressed V alpha mRNA sequences in thymocyte RNA. Such analysis revealed both shared and unique patterns of V alpha mRNA expression among the different haplotypes and supported the conclusion that haplotype differences sometimes involve V alpha gene deletions. Interestingly, a disproportionate number of, but not all, autoimmune-susceptible strains, including NZB, SJL, SWR, PL/J, and NOD, share a common V alpha haplotype. The identification of murine TCR V alpha haplotypes should provide a basis for understanding the role of TCR diversity in normal immunoregulatory and immune-response phenomena, as well as autoimmune-disease predisposition.

Alleles↗
PubMed2004

The Iccare web server: an attempt to merge sequence and mapping information for plant and animal species.

The Iccare web server, http://genopole.toulouse.inra.fr/bioinfo/Iccare, provides a simple yet efficient tool for crude EST (expressed sequence tag) annotation specifically dedicated to comparative mapping approaches. Iccare uses all the EST and mRNA sequences from public databases for an organism of interest (query species) and compares them to all the transcripts of one reference organism (Homo sapiens or Arabidopsis thaliana). The results are displayed according to the location of the genes on the chromosomes of the reference organism. Gene structure information and sequence similarities are combined in a graphical representation in order to pinpoint the nature of the transcript query sequence. The user can subsequently design primers or probes for the purpose of physical or genetic mapping. In addition to the query organisms already available in Iccare, users can perform a tailor-made search with their own sequences against the animal or plant reference organism genes.

Animals↗
PubMed1987

Insulin-like growth factor I induces c-fos messenger ribonucleic acid in L6 rat skeletal muscle cells.

Insulin-like growth factor I (IGF-I) mediates growth promotion of GH on specific target cells. As other growth factor action has been associated with oncogene induction, we examined whether IGF-I activated cellular oncogenes in L6 rat muscle cells, known target cells of IGF-I stimulatory action. Quiescent cells were incubated in serum-free defined medium with IGF-I (Amgen recombinant analog, Thr59) from 5 min to 4 h. 32P-Labeled DNA was first prepared from IGF-I-treated cell poly(A) RNA using avian myeloblastosis virus reverse transcriptase. This labeled probe was hybridized against a blot containing 12 different immobilized oncogene DNAs. The v-fos DNA showed a positive hybridization signal, indicating the presence of c-fos mRNA sequences. Northern analysis with [32P]v-fos DNA revealed a major c-fos mRNA species (2.2 kilobases) induced by treatment of cells with IGF-I (100 ng/ml) or fetal calf serum (15%) for 45 min. We, therefore, measured c-fos induction by cytoplasmic RNA blot hybridization. IGF-I (6.25 ng/ml) stimulated c-fos mRNA 4-fold. Maximum stimulation (18-fold) was seen with 100 ng/ml IGF-I. Peak c-fos induction occurred after 30-min exposure to IGF-I and remained elevated for up to 2 h. Treatment of cells with a high dose of insulin (100 nM) also resulted in a modest (27%) increase in relative levels of c-fos mRNA. The results show that c-fos mRNA is induced by IGF-I in L6 cells. This novel observation suggests that IGF-I action at least in part may be mediated by c-fos, a cellular oncogene thought to play a critical role in cell differentiation and proliferation.

Animals↗
PubMed2001

Sequencing and chromosomal assignment of the rat endothelial-derived lipase gene (Lipg).

Part of the nucleotide sequence of the Lipg gene in the rat was established using primers based on the mRNA sequence described in the mouse. The rat intron sequence served as a template for designing primers for the specific amplification of rat Lipg. A rat-hamster radiation hybrid (RH) panel was used for chromosomal assignment of the rat Lipg gene. The Lipg gene was found to be located on rat chromosome 18 in the vicinity of the marker D18Mit11; a region reported to be homologous with both human and mouse chromosome 18.

Animals↗
PubMed2006

[Knockdown of PLK1 mRNA by special siRNA].

To study knockdown effect of small interfering RNA (siRNA) to PLK1 (Polo-like kinase 1) mRNA in colorectal cancer cell line SW480 and its mitosis and growth was changed. Ten special siRNA molecules were designed targeting different sites of PLK1 mRNA sequence and chemically synthesized. The siRNA molecules were transfected into SW480 by Oligofectamine. The gene mRNA level was assayed by Real-Time PCR. The changes of PLK1 protein, SW480 cell cycle and survival percentage was checked by Western-blot, Flow cytometry and Cell counter assays respectively. All 10 siRNA molecules knocked PLK1 mRNA down in different level. Of them P1, P4 and P9 showed over 80% knockdown efficiency and the others had more than 20% knockdown efficiency to PLK1 mRNA. The best knockdown effect over 95% of all groups was at 25 nmol/L of a mixture with P1, P4 and P9 siRNA equally. In this situation the protein was very less and the cells were blocked at G2 phase of cell cycle. After 72 h cell survival percentages were consistent with PKL1 mRNA level change by siRNA gradient concentration. The results showed that siRNA targeting PLK1 mRNA had effectively knocked PLK1 mRNA down in SW480 cell line. And a blended siRNAs held the best knockdown effect. The cell was blocked on the mitosis and growth.

Blotting, Western↗
PubMed1980

Predicted structure of two adenovirus tumor antigens.

Early adenovirus type 2(Ad2) mRNA sequences have been cloned by using the pBR322 plasmid as a vector. Two clones that include sequences from region E1B were identified and their DNAs were characterized by hybridization, restriction enzyme cleavage, and DNA sequence analysis. The results showed that the clones were derived from two different spliced mRNAs. By combining our results with the established DNA sequence for region E1B of the closely related adenovirus type 5[Maat, J., van Beveren, C.P. & van Ormondt, H. (1980) Gene, in press] it was possible to deduce the structure of a 13S and a 22S mRNA. The two mRNAs differ from each other by the size of their intervening sequences. If translation starts at the first AUG following the cap, the 22S mRNA encodes a Mr 67,000 polypeptide that is terminated by a UGA stop codon located immediately before the splice, whereas the 13S mRNA encodes a Mr 20,000 polypeptide that is translated in different reading frames before and after the splice. The Mr 20,000 and 67,000 polypeptides correspond in molecular weight to two proteins that invariably are precipitated from infected cell extracts by antisera from animals carrying adenovirus-induced tumors.

Adenoviruses, Human↗
PubMed1989

Cloning of cDNAs that encode human mast cell carboxypeptidase A, and comparison of the protein with mouse mast cell carboxypeptidase A and rat pancreatic carboxypeptidases.

Human skin and lung mast cells and rodent peritoneal mast cells contain a carboxypeptidase in their secretory granules. We have screened human lung cDNA libraries with a mouse mast cell carboxypeptidase A (MC-CPA) cDNA probe to isolate a near-full-length cDNA that encodes human MC-CPA. The 5' end of the human MC-CPA transcript was defined by direct mRNA sequencing and by isolation and partial sequencing of the human MC-CPA gene. Human MC-CPA is predicted to be translated as a 417 amino acid preproenzyme which includes a 15 amino acid signal peptide and a 94 amino acid activation peptide. The mature human MC-CPA enzyme has a predicted size of 36.1 kDa, a net positive charge of 16 at neutral pH, and 86% amino acid sequence identity with mouse MC-CPA. DNA blot analyses showed that human MC-CPA mRNA is transcribed from a single locus in the human genome. Comparison of the human MC-CPA with mouse MC-CPA and with three rat pancreatic carboxypeptidases shows that these enzymes are encoded by distinct but homologous genes.

Amino Acid Sequence↗
PubMed1984

Transcriptional and post-transcriptional control of specific messenger RNAs in adult and embryonic liver.

The rate of synthesis and the concentrations of a variety of messenger RNA sequences have been compared between adult mouse liver cells and cells in other adult tissues, and between cells in fetal and neonatal liver. The sequences were distinguished as "liver-specific" or "common" in a previous report, where liver was compared with brain cells and cultured cells. Most of the liver-specific mRNAs are greatly decreased or absent in a large group of other adult mouse tissues. In two cases, kidney shares mRNA sequences with liver. The levels of the common mRNAs varied from two to fivefold in various tissues. For the liver-specific mRNAs, the transcription rates in nuclei from adult tissues and from fetal liver showed a good correspondence to the presence and the level of mRNA. However, most of the common RNA sequences were transcribed at similar rates in all nuclei despite their different cytoplasmic concentrations. In addition, two liver-specific RNAs were transcribed in fetal liver nuclei but were not present as mRNA. Thus, the presence of tissue-specific mRNAs in adult cells is based first (and probably most importantly) on transcriptional control, but several instances were observed where post-transcriptional control also contributes to the level of mRNA.

Aging↗