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At least 1,261 records · Page 70Linked to original sources
PubMed1993

Site-directed cross-linking of mRNA analogues to 16S ribosomal RNA; a complete scan of cross-links from all positions between '+1' and '+16' on the mRNA, downstream from the decoding site.

mRNA analogues containing 4-thiouridine residues at selected sites were used to extend our analysis of photo-induced cross-links between mRNA and 16S RNA to cover the entire downstream range between positions +1 and +16 on the mRNA (position +1 is the 5'-base of the P-site codon). No tRNA-dependent cross-links were observed from positions +1, +2, +3 or +5. Position +4 on the mRNA was cross-linked in a tRNA-dependent manner to 16S RNA at a site between nucleotides ca 1402-1415 (most probably to the modified residue 1402), and this was absolutely specific for the +4 position. Similarly, the previously observed cross-link to nucleotide 1052 was absolutely specific for the +6 position. The previously observed cross-links from +7 to nucleotide 1395 and from +11 to 532 were however seen to a lesser extent with certain types of mRNA sequence from neighbouring positions (+6 to +10, and +10 to +13, respectively); no tRNA-dependent cross-links to other sites on 16S RNA were found from these positions, and no cross-linking was seen from positions +14 to +16. In each case the effect of a second cognate tRNA (at the ribosomal A-site) on the level of cross-linking was studied, and the specificity of each cross-link was confirmed by translocation experiments with elongation factor G, using appropriate mRNA analogues.

Base Sequence↗
PubMed1987

Molecular cloning and serological characterization of an altered c-abl gene product produced in Ph1 CML patients.

The reciprocal translocation between human chromosomes 9 and 22, termed the Philadelphia chromosome (Ph1), is observed in more than 90% of patients with chronic myelogenous leukemia. This translocation fuses sequences from a variable distance 5' to the c-abl locus on chromosome 9 to sequences in a breakpoint cluster region (bcr) on chromosome 22. The appearance of the Ph1 chromosome is correlated with the production of a novel 8.7-kb RNA transcript containing both bcr and c-abl sequences as well as with a 210-kd phosphoprotein (p210c-abl) representing non-abl polypeptide sequences fused to c-abl-derived sequences. Antibodies prepared to a number of different c-abl domains and to bcr determinants were employed to characterize the normal and altered c-abl gene products. By combining a variety of cDNA cloning techniques, we have isolated bcr/abl clones representing 8.7 kb of contiguous mRNA sequence.

Cloning, Molecular↗
PubMed1980

Accumulation of individual pA+ RNAs during oogenesis of Xenopus laevis.

RNA metabolism during amphibian oogenesis has been investigated by the analysis of individual cDNA cloned sequences. Two cDNA clone libraries were constructed from the pA+ RNA of Xenopus ovary and of tadpole. The accumulation of RNAs complementary to individual clones was examined during oogenesis by isolating RNA from oocytes of different stages. There are only two patterns of accumulation during oogenesis. Mitochondrial mRNA sequences accumulate throughout all the stages of oogenesis, whereas all other sequences detected cease accumulation early in oogenesis and remain at a constant steady state level for the remainder of oocyte development. In the earliest stages examined, the individual RNAs are already present at approximately 1/4 their final level, and the subsequent increase of all sequences appears to be coordinant. These basic observations were confirmed by colony screening approximately 500 clones. The pattern of RNA accumulation observed is significant, because it had been determined that the total steady state pA+ RNA levels in the oocyte also ceased to increase at the same stage in oogenesis in which each individual sequenced reached a plateau level. In addition, lampbrush chromosomes, which have traditionally been thought to be the site of oocyte pA+ RNA synthesis, are maximally active after the stage in which all pA+ RNAs seem to have reached their final level of accumulation.

Animals↗
PubMed2004

Crassostrea gigas ferritin: cDNA sequence analysis for two heavy chain type subunits and protein purification.

Ferritin has been shown as being the principal iron storage in the majority of living organisms. In marine species, ferritin is also involved in high-level accumulation of (210)Po. As part of our work on the investigation of these radionuclides' concentration in natural environment, ferritin was searched at the gene and protein level. Ferritin was purified from the visceral mass of the oyster Crassostrea gigas by ion-exchange chromatography and HPLC. SDS-PAGE revealed one band of 20 kDa. An Expressed Sequence Tag (EST) library was screened and led to the identification of two complementary DNA (cDNA) involved in ferritin subunit expression. The complete coding sequences and the untranslated regions (UTRs) of the two genes were obtained and a 5' Rapid Amplification of cDNA Ends (RACE) was used to obtain the two iron-responsive elements (IREs) with the predicted stem-loop structures usually present in the 5'-UTR of ferritin mRNA. Sequence alignment in amino acid of the two new cDNA showed an identity with Pinctada fucata (85.4-88.3%), Lymnaea stagnalis (79.3-82.2%) and Helix pomatia (79.1-79.1%). The residues responsible for the ferroxidase center, conserved in all vertebrate H-ferritins, are present in the two oyster ferritin subunits. Oyster ferritins do not present the special characteristics of other invertebrate ferritins like insect ferritins but have some functional similarities with the vertebrate H chains ferritin.

Amino Acid Sequence↗
PubMed2001

Expression of serotonin receptors in bone.

The 5-hydroxytryptamine (5-HT) receptors 5-HT(2A), 5-HT(2B), and 5-HT(2C) belong to a subfamily of serotonin receptors. Amino acid and mRNA sequences of these receptors have been published for several species including man. The 5-HT(2) receptors have been reported to act on nervous, muscle, and endothelial tissues. Here we report the presence of 5-HT(2B) receptor in fetal chicken bone cells. 5-HT(2B) receptor mRNA expression was demonstrated in osteocytes, osteoblasts, and periosteal fibroblasts, a population containing osteoblast precursor cells. Pharmacological studies using several agonists and antagonists showed that occupancy of the 5-HT(2B) receptor stimulates the proliferation of periosteal fibroblasts. Activity of the 5-HT(2A) receptor could however not be excluded. mRNA for both receptors was shown to be equally present in adult mouse osteoblasts. Osteocytes, which showed the highest expression of 5-HT(2B) receptor mRNA in chicken, and to a lesser extent osteoblasts, are considered to be mechanosensor cells involved in the adaptation of bone to its mechanical usage. Nitric oxide is one of the signaling molecules that is released upon mechanical stimulation of osteocytes and osteoblasts. The serotonin analog alpha-methyl-5-HT, which preferentially binds to 5-HT(2) receptors, decreased nitric oxide release by mechanically stimulated mouse osteoblasts. These results demonstrate that serotonin is involved in bone metabolism and its mechanoregulation.

Alkaline Phosphatase↗
PubMed1999

Differential gene expression in CD3epsilon- and RAG1-deficient thymuses: definition of a set of genes potentially involved in thymocyte maturation.

A set of 3000 mouse thymus cDNAs was analyzed by extensive measurement of expression using complex-probe hybridization of DNA arrays ("quantitative differential screening"). The complex probes were initially prepared using total thymus RNA isolated from C57BL/6 wild-type (WT), CD3epsilon- and RAG1-deficient mice. Over 100 clones displaying over- or under-expression by at least a factor of two between WT and knockout (KO) thymuses were further analyzed by measuring hybridization signatures with probes from a wide range of KO thymuses, cell types, organs, and embryonic thymuses. A restricted set of clones was selected by virtue of their expression spectra (modulation in KO thymuses and thymocytes, lymphoid cell specificity, and differential expression during embryonic thymus development), sequenced at one extremity, and compared to sequences in databases. Clones corresponding to previously identified genes (e.g., Tcrbeta, Tcf1 or CD25) showed expression patterns that were consistent with existing data. Ten distinct clones corresponding to new genes were subjected to further study: Northern blot hybridization, in situ hybridization on thymus sections, and partial or complete mRNA sequence determination. Among these genes, we report a new serine peptidase highly expressed in cortical epithelial cells that we have named thymus-specific serine peptidase (TSSP), and an acidic protein expressed in thymocytes and of unknown function that we have named thymus-expressed acidic protein (TEAP). This approach identifies new molecules likely to be involved in thymocyte differentiation and function.

Alternative Splicing↗
PubMed2002

An active role for endogenous beta-1,3-glucanase genes in transgene-mediated co-suppression in tobacco.

Post-transcriptional gene silencing (PTGS) is characterized by the accumulation of short interfering RNAs that are proposed to mediate sequence-specific degradation of cognate and secondary target mRNAs. In plants, it is unclear to what extent endogenous genes contribute to this process. Here, we address the role of the endogenous target genes in transgene-mediated PTGS of beta-1,3-glucanases in tobacco. We found that mRNA sequences of the endogenous glucanase glb gene with varying degrees of homology to the Nicotiana plumbaginifolia gn1 transgene are targeted by the silencing machinery, although less efficiently than corresponding transgene regions. Importantly, we show that endogene-specific nucleotides in the glb sequence provide specificity to the silencing process. Consistent with this finding, small sense and antisense 21- to 23-nucleotide RNAs homologous to the endogenous glb gene were detected. Combined, these data demonstrate that a co-suppressed endogenous glucan ase gene is involved in signal amplification and selection of homologous targets, and show that endogenous genes can actively participate in PTGS in plants. The findings are introduced as a further sophistication of the post-transciptional silencing model.

Base Sequence↗
PubMed1991

The complete sequence of perlecan, a basement membrane heparan sulfate proteoglycan, reveals extensive similarity with laminin A chain, low density lipoprotein-receptor, and the neural cell adhesion molecule.

A heparan sulfate proteoglycan is a component of all basement membranes. This molecule consists of three heparan sulfate side chains linked to a large core protein of approximately 400 kDa. We have isolated seven overlapping murine cDNA clones that encode the entire mRNA sequence of 12.685 kilobases of this molecule. This sequence has a single open reading frame of 3,707 amino acids that encodes for a protein of 396 kDa. Identical or near identical matchups with nine peptide sequences derived from the core protein of the molecule isolated from the Engelbreth-Holm-Swarm tumor were found with the deduced sequence. Sequence analysis and data base comparison of the deduced sequence show the protein to consist of five different domains, most of which contain internal repeats. Domain I contains a start methionine followed by a typical signal transfer sequence and a unique segment of 172 amino acids that contains the three probable sites of heparan sulfate attachment, SGD. Domain II contains four cysteine- and acidic amino acid-rich repeats that are very similar to those found in the LDL receptor and proteins such as GP330. Domain III consists of cysteine-rich and globular regions, both of which show similarity to those in the short arm of the laminin A chain. Domain IV contains 14 repeats of the immunoglobulin superfamily that are most highly similar to the immunoglobulin-like repeats in the neural cell adhesion molecule. Domain V contains three repeats with similarity to the laminin A chain G domain that are separated by epidermal growth factor-like regions not found in the laminin A chain. As the primary structural data agree with the appearance of the molecule in the electron microscope as a series of globules separated by rods, or "beads on a string," we have adopted the name perlecan for this molecule. The variety of domains in perlecan suggest multiple interactions with other molecules.

Amino Acid Sequence↗
PubMed1978

Precursors of globin mRNA in erythroid-enriched bone marrow cells of mouse.

RNA from erythroid-enriched bone marrow cells of mouse was analyzed under two kinds of denaturing conditions (in 99% formamide and formaldehyde treatment), and globin mRNA sequence-containing RNAs larger than 18S in addition to 16S and 10S were detected by hybridization at a comparatively high Rot with complementary DNA (cDNA) to globin mRNA (10S). The existence of precursor RNAs larger than 18S was confirmed by two different methods; (i) recentrifugation in 99% formamide of RNA collected from the corresponding regions of RNA fractions and following detection of globin mRNA sequences, (ii) the specific isolation of [3H]labeled precursor RNA with a cDNA-cellulose column and fractionation in a 99% formamide-sucrose gradient. Furthermore, pulse-chase experiments indicated that some large RNAs with globin mRNA sequences could be chased to 10S RNA during a 30 min chase in the presence of a high concentration of actinomycin D (10 microgram/ml). From the results it was assumed that 35-37S RNAs with globin mRNA sequences were possible primary transcripts of globin genes and other RNAs with globin mRNA sequences in 16S, 20S, and 26S regions were intermediate precursors of 10S globin mRNA.

Animals↗
PubMed1991

Molecular cloning and expression of rat placental lactogen-Iv, a variant of rPL-I present in late pregnant rat placenta.

Late pregnant rat placenta was found to contain a messenger RNA (mRNA) that encodes an additional member of the prolactin-growth hormone family. This polypeptide was detected by hybridization of complementary DNA (cDNA) for rPL-I to a 1 kilobase mRNA transcript in late pregnant rat placenta. A cDNA clone for the new polypeptide was isolated from a phage lambda-gt10 library containing cDNA synthesized from day 18 rat placental mRNA. Sequencing of the day 18 cDNA clone revealed that it was most closely related structurally to rPL-I and at the amino acid terminal was identical to the rPL-I variant (rPL-Iv). The sequence of rPL-Iv (223 amino acids) is 85% homologous to rPL-I (230 amino acid). There are 71 base pair changes (85% nucleotide homology) between rPL-Iv and rPL-I which are not confined to any specific region of the molecule and which result in 36 amino acid changes. In vitro translation of rPL-Iv mRNA produced by transcription of the cDNA template yielded a 26 kilodalton polypeptide, the size of the expected precursor protein. In situ hybridization studies indicated that mRNA for rPL-Iv was present primarily in cytotrophoblasts of the basal zone as early as day 15 of gestation with some hybridization in a few giant cells as well.

Amino Acid Sequence↗
PubMed2001

[Cloning and expression of Trichoderma reesei endoglucanase III (EG III) gene in Saccharomyces cerevasiae].

Using Congo red-staining method, one positive clone with CMCase activity was isolated from the Trichoderma ressei cDNA gene bank constructed in Saccharomyces cerevasiae. Sequencing result showed that the 1.5 kb-length DNA fragment inserted in the recombinant plasmid encoded EG III gene from T. reesei. Enzymatic characterization of the EG III produced by recombinant S. cerevasiae was analyzed. The experimental results indicated that the optimum pH and temperature for EG III are 5.0 and 60 degrees C, respectively. The effects of secretory system components SSO 2 and SEB1 of S. cerevisiae on EG III secretion were examined. The results indicated that the amount of EG III secreted by the strain with SSO 2-overexpression was highest among the different recombinant S. cerevisiae strains, showed that SSO 2 is a rate-limiting component of the secretory machinery in the process of EG III secretion. Furthermore, the EG III expression level was increased 5.3 times by deletion. Furthermore, the EG III expression level was increased 5.3 times by deletion of the 98 bp in 5' untranslated region of eg3 mRNA sequence. This result suggested that the regulation region could exist in the 5' untranslated region of EG III mRNA, which is recognized by the gene expression related factors of S. cerevasiae.

Cellulase↗
PubMed1987

Evolution of the primary and secondary structures of the E1a mRNAs of the adenovirus.

In this paper we investigate and compare (evolutionary) patterns in the primary and secondary structure of four homologous E1a mRNAs of the adenovirus. Our main results are as follows: (1) The similarity of the coding regions of the mRNA sequences reflects both similarity in function (i.e., oncogenicity) and evolutionary divergence. (2) The similarity of the leader and the trailer regions reflects host specificity (i.e., human or simian) and must therefore arise from convergence. (3) Minimal energy foldings of the mRNAs show similar secondary structures (in particular around the splice sites). The conservation of pre-mRNA secondary structure shows that mRNAs are subject to selection constraints in addition to those associated with proteins. (4) The conserved secondary (helical) structures consist of nonhomologous subsequences, i.e., shifts have occurred. The observed shifts near the splice sites seem to be the simplest way of dealing with the dual constraints.

Adenoviridae↗
PubMed1982

N6,O2'-dibutyryl cycle AMP and glucose regulate the amount of messenger RNA coding for hepatic phosphoenolpyruvate carboxykinase (GTP).

Rat liver phosphoenolpyruvate carboxykinase (GTP) (EC 4.1.1.32) mRNA was purified to 25% of total mRNA activity (greater than 50-fold enrichment) by polysome immunoprecipitation. This preparation was used as template for the synthesis of cDNA that was subsequently cloned in Escherichia coli. The resulting clones were screened by in situ hybridization and by hybrid-selected translation of phosphoenolpyruvate carboxykinase mRNA. The cDNA insert of one plasmid, pPC2, was complementary to phosphoenolpyruvate carboxykinase mRNA as determined by these screening procedures. pPC2 cDNA was 760 base pairs in length and a partial restriction enzyme map was constructed. pPC2 was labeled with 32P by nick translation and was used as a hybridization probe to quantitate phosphoenolpyruvate carboxykinase mRNA following N6,O2'-dibutyryl cAMP (Bt2cAMP) injection or glucose feeding. Bt2cAMP increased whereas glucose decreased the level of hybridizable phosphoenolpyruvate carboxykinase mRNA and in all cases the changes were proportional to the in vitro translational activities measured in a reticulocyte lysate system. The half-life of phosphoenolpyruvate carboxykinase mRNA sequences was measured by an indirect procedure involving their quantitation, by hybridization assay, during deinduction and induction. The half-life was approximately 10-40 min during deinduction by glucose or during induction stimulated by Bt2cAMP. Our data indicate that cAMP enhances some step in the generation of phosphoenolpyruvate carboxykinase mRNA.

Animals↗
PubMed1985

Hemagglutinin-neuraminidase protein of the paramyxovirus simian virus 5: nucleotide sequence of the mRNA predicts an N-terminal membrane anchor.

The nucleotide sequence of a cloned cDNA copy of the mRNA coding for the hemagglutinin-neuraminidase of the paramyxovirus SV5 was determined. There was a single large open reading frame on the mRNA which encoded a protein of 565 amino acids with a molecular weight of 62,134. The deduced amino acid sequence indicated that the only major hydrophobic region in the protein sufficiently long to anchor the protein in the membrane is located near the N terminus (amino acids 18 to 36). It is suggested that, like the influenza virus neuraminidase, hemagglutinin-neuraminidase of paramyxoviruses is oriented with its N terminus inserted into the membrane.

Amino Acid Sequence↗
PubMed2002

PALS db: Putative Alternative Splicing database.

PALS db is a collection of Putative Alternative Splicing information from 19 936 human UniGene clusters and 16 615 mouse UniGene clusters. Alternative splicing (AS) sites were predicted by using the longest messenger RNA (mRNA) sequence in each UniGene cluster as the reference sequence. This sequence was aligned with related sequences in UniGene and dbEST to reveal the AS. This information was presented with six features: (i) literature aliases were used to improve the result of a gene name search; (ii) the quality of a prediction can be easily judged from the color-coded similarity and the scaled length of an alignment; (iii) we have clustered those EST sequences that support the same AS site together to enhance the users' confidence on a prediction; (iv) the users can also set up the alignment criteria interactively to recover false negatives; (v) tissue distribution can be displayed by placing the mouse cursor over an alignment; (vi) gene features will be analyzed at foreign sites by submitting the selected mRNA or its encoded protein as a query. Using these features, the users cannot only discover putative AS sites in silico, but also make new observations by combining AS information with tissue distributions or with gene features. PALS db is available at http://palsdb.ym.edu.tw/.

Alternative Splicing↗
PubMed1989

A monoclonal antibody against the platelet fibrinogen receptor contains a sequence that mimics a receptor recognition domain in fibrinogen.

The binding of fibrinogen to its platelet receptor, the glycoprotein IIb-IIIa complex, is mediated, in part, by an Arg-Gly-Asp (RGD) sequence within the fibrinogen A alpha chain. PAC1 is an IgM-kappa murine monoclonal antibody that binds to the platelet fibrinogen receptor, and its binding is inhibited by both fibrinogen and RGD-containing peptides. To identify the regions of PAC1 that interact with the fibrinogen receptor, we determined the mRNA sequences of PAC1 immunoglobulin heavy and light chain variable regions. Five out of the six complementarity-determining regions (CDRs) of PAC1 had entirely germline sequences with no regions of similarity to fibrinogen. However, CDR3 of the PAC1 heavy chain (H-CDR3) was very large and unique due to the insertion of a novel D region segment. H-CDR3 contained a sequence, Arg-Tyr-Asp (RYD), that, if present in the proper conformation, might behave like the RGD sequence in fibrinogen. A 21-residue synthetic peptide encompassing the H-CDR3 region inhibited fibrinogen-dependent platelet aggregation as well as the binding of PAC1 (Ki = 10 microM) and fibrinogen (Ki = 5 microM) to activated platelets. The RYD region of H-CDR3 appeared to be central to its function, because substitution of the tyrosine with glycine increased the inhibitory potency of the peptide by 10-fold, while replacing the tyrosine with D-alanine or inverting the RYD sequence sharply reduced the inhibitory potency. Thus, the linear sequence, RYD, within H-CDR3 of PAC1 appears to mimic the RGD receptor recognition sequence in fibrinogen. This type of immunologic approach could be useful in studying the structural basis of other receptor-ligand interactions.

Amino Acid Sequence↗
PubMed1997

In situ assessment of mRNA accessibility in heterogeneous tissue samples using elongation factor-1 alpha (EF-1 alpha).

Elongation factor-1 alpha (EF-1 alpha) is an evolutionarily highly conserved universal cofactor of protein synthesis in all living cells. In this study, its use as a positive control in situ hybridization assays for specific detection of mRNA sequences was evaluated. Northern blot analysis of various non-neoplastic and neoplastic cultured cells of different stages of confluence, cell shape, and cell cycle status revealed that EF-1 alpha had a lower and more homogeneous expression than did beta-actin. In situ hybridization assays using digoxigenin-labeled riboprobes for the detection of EF-1 alpha mRNA in routinely formalin-fixed, paraffin-embedded tissue sections showed that EF-1 alpha is a suitable positive control in all types of cells. However, variation of protease pretreatments demonstrated distinct and sometimes mutually exclusive digestion conditions for different cell types within the same tissue sample. Our results indicate that detection of EF-1 alpha mRNA is an appropriate internal standard for in situ hybridization assays and that it is useful to control artifacts such as false negatives caused by inappropriate protease pretreatments. The observed variability of optimal protease pretreatments for different cell types within the same tissue section strengthens the importance of a positive control in in situ hybridization assays.

3T3 Cells↗
PubMed1984

Effect of adenovirus infection on expression of human histone genes.

The influence of adenovirus type 2 infection of HeLa cells upon expression of human histone genes was examined as a function of the period of infection. Histone RNA synthesis was assayed after run-off transcription in nuclei isolated from mock-infected cells and after various periods of adenovirus infection. Histone protein synthesis was measured by [3H]leucine labeling of intact cells and fluorography of electrophoretically fractionated nuclear and cytoplasmic proteins. The cellular representation of RNA species complementary to more than 13 different human histone genes was determined by RNA blot analysis of total cellular, nuclear or cytoplasmic RNA by using a series of 32P-labeled cloned human histone genes as hybridization probes and also by analysis of 3H-labeled histone mRNA species synthesized in intact cells. By 18 h after infection, HeLa cell DNA synthesis and all parameters of histone gene expression, including transcription and the nuclear and cytoplasmic concentrations of core and H1 mRNA species, were reduced to less than 5 to 10% of the control values. By contrast, transcription and processing of other cellular mRNA sequences have been shown to continue throughout this period of infection. The early period of adenovirus infection was marked by an inhibition of transcription of histone genes that accompanied the reduction in rate of HeLa cell DNA synthesis. These results suggest that the adenovirus-induced inhibition of histone gene expression is mediated in part at the transcriptional level. However, the persistence of histone mRNA species at concentrations comparable to those of mock-infected control cells during the early phase of the infection, despite a reduction in histone gene transcription and histone protein synthesis, implies that histone gene expression is also regulated post-transcriptionally in adenovirus-infected cells. These results suggest that the tight coupling between histone mRNA concentrations and the rate of cellular DNA synthesis, observed when DNA replication is inhibited by a variety of drugs, is not maintained after adenovirus infection.

Adenoviruses, Human↗