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At least 1,315 records · Page 73Linked to original sources
PubMed1992

Identification of skeletal muscle precursor cells in vivo by use of MyoD1 and myogenin probes.

The activation of mononuclear muscle precursor cells after crush injury to mouse tibialis anterior muscles was monitored in vivo by in situ hybridization with MyoD1 and myogenin probes. These genes are early markers of skeletal muscle differentiation and have been extensively studied in vitro. The role in vivo of these regulatory proteins during myogenesis of mature muscle has not been studied previously. MyoD1 and myogenin mRNA were present in occasional mononuclear cells of uninjured muscle. Increased MyoD1 and myogenin mRNA sequences in mononuclear cells were detected as early as 6 h after injury, peaked between 24 and 48 h, and thereafter declined to pre-injury levels at about 8 days. The mRNAs were detected in mononuclear cells throughout the muscle, with the majority of cells located some distance from the site of crush injury. The presence of MyoD1 and myogenin mRNA at 6 to 48 h indicates that transcription of these genes is occurring at the same time as replication of muscle precursor cells in vivo. At no time were significant levels of mRNA for these genes detected in myotubes. MyoD1 and myogenin provide precise markers for the very early identification and study of mononuclear skeletal muscle precursor cells in muscle regenerating in vivo.

Animals↗
PubMed1983

A Markov analysis of DNA sequences.

We present a model by which we look at the DNA sequence as a Markov process. It has been suggested by several workers that some basic biological or chemical features of nucleic acids stand behind the frequencies of dinucleotides (doublets) in these chains. Comparing patterns of doublet frequencies in DNA of different organisms was shown to be a fruitful approach to some phylogenetic questions (Russel & Subak-Sharpe, 1977). Grantham (1978) formulated mRNA sequence indices, some of which involve certain doublet frequencies. He suggested that using these indices may provide indications of the molecular constraints existing during gene evolution. Nussinov (1981) has shown that a set of dinucleotide preference rules holds consistently for eukaryotes, and suggested a strong correlation between these rules and degenerate codon usage. Gruenbaum, Cedar & Razin (1982) found that methylation in eukaryotic DNA occurs exclusively at C-G sites. Important biological information thus seems to be contained in the doublet frequencies. One of the basic questions to be asked (the "correlation question") is to what extent are the 64 trinucleotide (triplet) frequencies measured in a sequence determined by the 16 doublet frequencies in the same sequence. The DNA is described here as a Markov process, with the nucleotides being outcomes of a sequence generator. Answering the correlation question mentioned above means finding the order of the Markov process. The difficulty is that natural sequences are of finite length, and statistical noise is quite strong. We show that even for a 16000 nucleotide long sequence (like that of the human mitochondrial genome) the finite length effect cannot be neglected. Using the Markov chain model, the correlation between doublet and triplet frequencies can, however, be determined even for finite sequences, taking proper account of the finite length. Two natural DNA sequences, the human mitochondrial genome and the SV40 DNA, are analysed as examples of the method.

Base Sequence↗
PubMed2005

Serum amyloid A in geese; cloning and expression of recombinant protein.

We defined the nucleotide-sequence of the full-length goose serum amyloid A and compared it to SAA sequences of the duck. The aim of this work was to clone and express recombinant goose SAA and to produce antibody against this protein: Total RNA was isolated from goose liver and used to synthesise first strand cDNA. The coding region of the goose SAA cDNA was amplified by PCR using primers corresponding to the appropriate conservative regions of duck SAA mRNA. The product was subcloned into pET-15b expression vector to result in a His*Tag fusion protein expression. The protein was purified by affinity chromatography. Rabbits were then immunized against the recombinant purified goose SAA protein. The anti-SAA serum was tested by Western blotting. Full-length goose SAA mRNA sequence has been obtained and sequenced.

Amino Acid Sequence↗
PubMed1995

AGL15, a MADS domain protein expressed in developing embryos.

To extend our knowledge of genes expressed during early embryogenesis, the differential display technique was used to identify and isolate mRNA sequences that accumulate preferentially in young Brassica napus embryos. One of these genes encodes a new member of the MADS domain family of regulatory proteins; it has been designated AGL15 (for AGAMOUS-like). AGL15 shows a novel pattern of expression that is distinct from those of previously characterized family members. RNA gel blot analyses and in situ hybridization techniques were used to demonstrate that AGL15 mRNA accumulated primarily in the embryo and was present in all embryonic tissues, beginning at least as early as late globular stage in B. napus. Genomic and cDNA clones corresponding to two AGL15 genes from B. napus and the homologous single-copy gene from Arabidopsis, which is located on chromosome 5, were isolated and analyzed. Antibodies prepared against overexpressed Brassica AGL15 lacking the conserved MADS domain were used to probe immunoblots, and AGL15-related proteins were found in embryos of a variety of angiosperms, including plants as distantly related as maize. Based on these data, we suggest that AGL15 is likely to be an important component of the regulatory circuitry directing seed-specific processes in the developing embryo.

Amino Acid Sequence↗
PubMed1998

Isolation and characterization of the Xanthomonas campestris rpoH gene coding for a 32-kDa heat shock sigma factor.

Degenerate oligonucleotide primers corresponding to the conserved regions of bacterial heat shock sigma factor RpoH (sigma 32) were used to amplify a 190-bp fragment by PCR on the X. campestris pv. campestris strain 11 chromosome. Using this fragment as a probe, plasmid pXC57 carrying a 4.7-kb insert was isolated from a genomic library of Xc11. Sequence analysis of a stretch of 2,053 bp from the pXC57 insert revealed an ORF encoding a polypeptide of 291 aa (32,854 dal) which displays 59.6% and 57.3% identity to the rpoH gene products of E. coli and P. aeruginosa, respectively. The Xc11 rpoH gene was able to complement the RpoH deficient E. coli strain A7448. Both amino acid and mRNA sequences deduced from the Xc11 rpoH gene show structural features characteristics of the corresponding sequences from those of the gamma subgroup proteobacteria. The RpoH levels in Xc11 were demonstrated to increase transiently in response to heat shock treatment by immunoblot analysis using the polyclonal antibody raised against the purified Xc11 RpoH.

Amino Acid Sequence↗
PubMed2005

Novel targeting of cyclooxygenase-2 (COX-2) pre-mRNA using antisense morpholino oligonucleotides directed to the 3' acceptor and 5' donor splice sites of exon 4: suppression of COX-2 activity in human amnion-derived WISH and myometrial cells.

Increased expression of cyclooxygenase-2 (COX-2) has been implicated in the onset of both term and preterm labor. In this context, both selective and nonselective COX-2 inhibitors have been used in clinical trials to determine their efficacy in delaying preterm labor. However, recent evidence indicates that these tocolytics may have potentially adverse fetal and maternal side effects. Therefore, the development of more specific and nontoxic agents to inhibit COX-2 needs to be considered. We have evaluated whether antisense morpholino oligonucleotides have therapeutic potential in inhibiting COX-2 by specifically targeting both the 3' and 5' acceptor and donor sites of exon 4 of COX-2's pre-mRNA sequence. Confocal microscopy on "live" cells illustrated high levels of penetrance of antisense morpholino oligonucleotides using the Endo-Porter formula (Gene-Tools, LLC, Philomath, OR), with delivery efficiencies of 82 and 78%, respectively, in amnion-derived WISH and myometrial cells. Substantial inhibition by the morpholino oligonucleotides of COX-2 expression, induced by lipopolysaccharide administration, was observed at both the mRNA and protein levels. Loss of enzymic activity of COX-2 was confirmed using a sensitive COX enzyme activity assay, which reflects the rate of conversion of arachidonic acid to prostaglandin H2. Our results indicate that antisense morpholino oligonucleotides significantly inhibit expression and activity of this enzyme in in vitro cultures of amnion-WISH and myometrial cells. The potential thus exists that a similar approach can be mimicked in vivo to produce a highly specific and nontoxic strategy to inhibit COX-2 activity with its subsequent effects on the better management of preterm labor and other inflammatory conditions.

Amnion↗
PubMed1992

Coordinated regulation of two indole alkaloid biosynthetic genes from Catharanthus roseus by auxin and elicitors.

Catharanthus roseus (periwinkle) produces a wide range of terpenoid indole alkaloids, including several pharmaceutically important compounds, from the intermediate strictosidine. The complete mRNA sequence for the enzyme strictosidine synthase (SSS) was determined. Comparison of the primary structure of the encoded protein with the amino-terminal sequence of purified SSS indicated the presence of a signal peptide of 31 amino acids in the putative primary translation product. SSS is encoded by a single-copy gene indicating that isoenzymes reported by others are formed post-translationally from a single precursor. The sss gene and the tryptophan decarboxylase gene (tdc), encoding another enzyme essential for indole alkaloid biosynthesis, are coordinately regulated. In plants steady-state mRNA levels are highest in roots. In cell suspension cultures the genes are rapidly down-regulated by auxin. In contrast, both genes are strongly induced by fungal elicitors such as Pythium aphanidermatum culture filtrate or yeast extract. Induction is a rapid, transcriptional event occurring independent of de novo protein synthesis. These results show that a first important regulatory step in the complex process leading to indole alkaloid accumulation in C. roseus suspension cells is transcription of the biosynthetic genes.

Alkaloids↗
PubMed1988

The human thyrotropin beta-subunit gene differs in 5' structure from murine TSH-beta genes.

The gene encoding the beta-subunit of human thyrotropin (hTSH-beta) was isolated, and its nucleotide sequence was determined. The gene is 4.3 kb in length, consists of three exons and two introns, and is present as a single copy as determined by Southern blot analysis of total genomic DNA. The protein coding portion of the gene, which includes exons 2 and 3, was isolated from a human genomic phage library, while exon 1, which encodes only 5' untranslated mRNA sequence, was isolated from a plasmid library of size-selected genomic DNA fragments. Here we describe the isolation of the 5' untranslated exon of the hTSH-beta subunit and 5'-flanking region. The structure of the hTSH-beta gene is very similar to the previously characterized TSH-beta genes from mouse and rat. The genes from all three species have two distinct promoter regions, but while both promoters are utilized by the murine TSH-beta genes, the human TSH-beta gene apparently utilizes only the proximal promoter for transcription initiation. A striking difference in hTSH-beta gene structure compared to the murine genes is that exon 1 of the human gene is 36 nucleotides. An analysis of the mouse, rat, and human exon 1 and 5'-flanking region shows a high percentage of sequence homology, with the exception of a 9-nucleotide insertion 13 bases 3' from the proximal TATA box found in the human gene but not found in the other two species. We propose that this insertion results in the additional length of human exon 1 compared to the mouse and rat genes. By isolating the promoter region of the hTSH-beta gene, we can begin to identify specific sequences involved in the regulation of hTSH gene expression.

Animals↗
PubMed1991

The bovine chromogranin A gene: structural basis for hormone regulation and generation of biologically active peptides.

The structure of the gene encoding bovine chromogranin-A has been determined by characterization of two isolated genomic clones. Chromogranin-A is encoded by eight exons, which organize the coding region into several distinct structural and functional domains. Exons 1-5 represent the highly conserved signal peptide and N-terminal domain, which are separated into regions corresponding to the signal peptide, N-terminal sequence, disulfide-bonded loop, and remainder of the conserved N-terminal domain. Exon 6 represents the variable domain and encodes a region that is identical to the novel chromogranin-A-derived peptide chromostatin. Exon 7 encodes the biologically active peptide pancreastatin as well as most of the conserved C-terminal domain, with the remainder found on exon 8. The mRNA sequence obtained from the gene contains five nucleotide differences from the consensus sequence of four reported bovine chromogranin-A cDNA clones. Two of the differences in the gene result in two amino acid changes in the region encoded by exon 6. The structural organization of the chromogranin-A gene resembles that of the chromogranin-B gene in the exons corresponding to the signal peptide, N-terminal sequence, disulfide loop, and C-terminal sequence.(ABSTRACT TRUNCATED AT 250 WORDS)

Adrenal Medulla↗
PubMed2000

Target site search and effective inhibition of leukaemic cell growth by a covalently closed multiple anti-sense oligonucleotide to c-myb.

Systematic secondary structure simulation of a target mRNA sequence is shown to be effective for locating a good anti-sense target site. Multiple selected anti-sense sequences were placed in a single molecule. The anti-sense oligonucleotide (oligo) was covalently closed to avoid exonuclease activities and was designated CMAS (covalently closed multiple anti-sense)-oligo. CMAS-oligo was found to be stable, largely preserving its structural integrity after 24 h of incubation in the presence of either exonuclease III or serum. When human c-myb mRNA was targeted by the c-myb CMAS-oligo, expression of the gene was completely abolished. Further, tumour cell growth was inhibited by 82+/-3% as determined by an MTT [3-(4, 5-dimethylthiazol-2-yl)-2,5-diphenyl-2H-tetrazolium bromide] assay and by 90+/-1% by [(3)H]thymidine incorporation. When a leukaemic cell line K562 was treated with CMAS-oligo, colony formation on soft agarose was also decreased by 93%. In contrast, treatment with a scrambled control oligo did not significantly inhibit leukaemic cell growth. These results suggest that a rational target site search is possible for an anti-sense oligo and that CMAS-oligo can be employed as an effective anti-sense agent with enhanced stability.

Cell Division↗
PubMed1992

Three widely separated positions in the 16S RNA lie in or close to the ribosomal decoding region; a site-directed cross-linking study with mRNA analogues.

Synthetic mRNA analogues were prepared by T7 transcription, each containing several thio-uridine residues at selected positions. After binding to the ribosome in the presence of cognate tRNA, the thio-U residues were activated by UV irradiation and the resulting sites of cross-linking to 16S RNA analysed. Three distinct cross-links were consistently observed: (i) from position '+6' of the mRNA (the 3'-base of the A-site codon) to base 1052 of 16S RNA; (ii) from position '+7' of the mRNA to base 1395; and (iii) from '+11' to base 532. Individual yields of the cross-links were strongly dependent on the particular mRNA sequence in each case. The '+11/532' and '+6/1052' cross-links were always entirely tRNA-dependent, whereas the '+7/1395' cross-link was observed at lower intensity in the absence of tRNA. In the presence of a second (A-site bound) tRNA the +6/1052 cross-link was markedly reduced. A cross-link to the 1050 region was again observed when a message carrying a thio-U at position '+9' was translocated on the ribosome so as to bring the thio-U to position +6. Taken together, the data are incompatible with some current models both for the three-dimensional arrangement of 16S RNA and for the orientation of the tRNA-mRNA complex in the ribosome.

Base Sequence↗
PubMed1987

Molecular cloning of the cDNA for rat spleen thymosin beta 10 and the deduced amino acid sequence.

A rat spleen cDNA library was prepared and employed for the molecular cloning of the cDNA for thymosin beta 10, a peptide that previously had been found to accompany the closely related peptide, thymosin beta 4, in several species of mammals (S. Erickson-Viitanen, S. Ruggieri, P. Natalini, and B. L. Horecker (1983) Arch. Biochem. Biophys. 225, 407-413). First-round screening with a synthetic oligodeoxynucleotide probe yielded 55 positive clones, and sequence analysis of 11 of these clones revealed that they all coded for a peptide containing the thymosin beta 10 sequence, except for an additional arginyl residue at position 39. This peptide, designated thymosin beta 10arg, had been identified previously in rabbit tissues and reported as a variant of thymosin beta 10 (S. Ruggieri, S. Erickson-Viitanen, and B.L. Horecker (1983) Arch. Biochem. Biophys. 226, 388-392). Analysis of the 55 positive clones using a specific oligodeoxynucleotide probe constructed to correspond to the mRNA sequence, including the codon for Arg39, confirmed that they all coded for the amino acid sequence including Arg39. Based on these results, the existence of a molecular species lacking Arg39 is considered unlikely, and we conclude that thymosin beta 10 contains 43, rather than 42, amino acid residues, with identity to thymosin beta 4 in 32 of the 43 residues. We propose that the name thymosin beta 10 be used to refer to the peptide containing Arg39 and that the designation thymosin beta 10arg be dropped. In the cDNA sequence the codons for Ala1 and Ser43 of thymosin beta 10 are flanked by initiator and terminator codons, respectively; thus, both the thymosin beta 4 and thymosin beta 10, which coexist in mammalian cells and tissues, are synthesized without the formation of larger polypeptide precursors.

Amino Acid Sequence↗
PubMed2006

QGRS Mapper: a web-based server for predicting G-quadruplexes in nucleotide sequences.

The quadruplex structures formed by guanine-rich nucleic acid sequences have received significant attention recently because of growing evidence for their role in important biological processes and as therapeutic targets. G-quadruplex DNA has been suggested to regulate DNA replication and may control cellular proliferation. Sequences capable of forming G-quadruplexes in the RNA have been shown to play significant roles in regulation of polyadenylation and splicing events in mammalian transcripts. Whether quadruplex structure directly plays a role in regulating RNA processing requires investigation. Computational approaches to study G-quadruplexes allow detailed analysis of mammalian genomes. There are no known easily accessible user-friendly tools that can compute G-quadruplexes in the nucleotide sequences. We have developed a web-based server, QGRS Mapper, that predicts quadruplex forming G-rich sequences (QGRS) in nucleotide sequences. It is a user-friendly application that provides many options for defining and studying G-quadruplexes. It performs analysis of the user provided genomic sequences, e.g. promoter and telomeric regions, as well as RNA sequences. It is also useful for predicting G-quadruplex structures in oligonucleotides. The program provides options to search and retrieve desired gene/nucleotide sequence entries from NCBI databases for mapping G-quadruplexes in the context of RNA processing sites. This feature is very useful for investigating the functional relevance of G-quadruplex structure, in particular its role in regulating the gene expression by alternative processing. In addition to providing data on composition and locations of QGRS relative to the processing sites in the pre-mRNA sequence, QGRS Mapper features interactive graphic representation of the data. The user can also use the graphics module to visualize QGRS distribution patterns among all the alternative RNA products of a gene simultaneously on a single screen. QGRS Mapper can be accessed at http://bioinformatics.ramapo.edu/QGRS/.

Computer Graphics↗
PubMed1999

Mutation in the betaA3/A1-crystallin encoding gene Cryba1 causes a dominant cataract in the mouse.

During the mouse ENU mutagenesis screen, mice were tested for the occurrence of dominant cataracts. One particular mutant was discovered as a progressive opacity (Po). Heterozygotes show opacification of a superficial layer of the fetal nucleus, which progresses and finally forms a nuclear opacity. Since the homozygotes have already developed the total cataract at eye opening, the mode of inheritance is semidominant. Linkage analysis was performed using a set of genome-wide microsatellite markers. The mutation was mapped to chromosome 11 distal of the marker D11Mit242 (9.3 +/- 4.4 cM) and proximal to D11Mit36 (2.3 +/- 2.3 cM). This position makes the betaA3/A1-crystallin encoding gene Cryba1 an excellent candidate gene. Mouse Cryba1 was amplified from lens mRNA. Sequence analysis revealed a mutation of a T to an A at the second base of exon 6, leading to an exchange of Trp by Arg. Computer analysis predicts that the fourth Greek key motif of the affected betaA3/A1-crystallin will not be formed. Moreover, the mutation leads also to an additional splicing signal, to the skipping of the first 3 bp of exon 6, and finally to the deletion of the Trp residue. Both types of mRNA are present in the homozygous mutant lenses. The mutation will be referred to as Cryba1(po1). This particular mouse mutation provides an excellent animal model for a human congenital zonular cataract with suture opacities, which is caused by a mutation in the homologous gene.

Amino Acid Sequence↗
PubMed2006

Effect of shRNA inhibiting HiF1alpha gene on TIMP1 expression in RPE cells.

Small hairpin RNA (shRNA) was used to silence the HIF1alpha gene in human retinal pigment epithelial cells (RPE) under hypoxia in order to observe the effect of gene silencing on the expression of matrix metalloproteinase tissue inhibitor 1 (TIMP1). By using chemical hypoxic inducer CoCl2, to mimic RPE hypoxic environment, shRNA against the targeting region of HIF1alpha mRNA sequence was synthesized by a method of in vitro transcription, and the HIF1alpha was interfered in RPE cultured under hypoxia (induced by 150 micromol/L CoCl2). RT-PCR was employed to detect the expression of HIF1alpha and TIMP1. The expression levels of HIF1alpha and TIMP1 were measured by using Western blotting. The results showed that after the RPE were transfected with specific shRNA against HIF1alpha mRNA, RT-PCR revealed that under hypoxia, the efficacy of HIF1alpha gene silencing in RPE was 83.4%. Western blotting revealed that the expression levels of HIF1alpha protein was dramatically dropped. In addition. RT-PCR results demonstrated that the expression of TIMP1 mRNA was decreased by 28.9%, and the expression levels of TIMP1 protein were also significantly reduced by Western blotting. It was suggested that shRNA targeted against HIF1alpha mRNA could effectively silence the HIF1alpha gene, subsequently effectively inhibit the hypoxia-induced up-regulation of TIMP1.

Adult↗
PubMed1999

The LIM proteins FHL1 and FHL3 are expressed differently in skeletal muscle.

We have determined the complete mRNA sequence of FHL3 (formerly SLIM2). We have confirmed that it is a member of the family of LIM proteins that share a similar secondary protein structure, renamed as Four-and-a-Half-LIM domain (or FHL) proteins in accordance with this structure. The "half-LIM" domain is a single zinc finger domain that may represent a subfamily of LIM domains and defines this particular family of LIM proteins. The distribution of FHL mRNA expression within a variety of murine tissues is complex. Both FHL1 and FHL3 were expressed in a number of skeletal muscles while FHL2 was expressed at high levels in cardiac muscle. Localisation of FHL3 to human chromosome 1 placed this gene in the proximity of, but not overlapping with, alleles associated with muscle diseases. FHL1 and FHL3 mRNAs were reciprocally expressed in the murine C2C12 skeletal muscle cell line and this suggested that the pattern of expression was linked to key events in myogenesis.

Amino Acid Sequence↗
PubMed1984

Changes in gene expression following neoplastic transformation of rat myogenic cells.

Two malignant sublines, M4 and RMS4 , were previously derived from the recloned L6 line of rat myogenic cells. Comparative studies in tissue culture and inoculation into suckling rats indicated that M4 cells and RMS4 cells may be considered as low-malignant and high-malignant cells, respectively, while L6 cells are not malignant. In the present work, we used extracts from L6 cells, M4 cells, and RMS4 cells collected during the period of exponential growth, to compare their polyadenylic acid-containing messenger RNA (mRNA) populations and the corresponding cell-free translation products. Analysis of the hybridization kinetics between radioactive complementary DNA and homologous or heterologous cellular RNAs indicated that L6 cells contained about 28,000 distinct polyadenylic acid-containing mRNA sequences of 1.8 kilobases each, of which 2,000 to 2,500 and 4,000 to 5,000 were missing (or at least were very infrequent) in M4 cells and RMS4 cells, respectively. Using a minor fraction of the RMS4 cell complementary DNAs, partially purified through repeated complementary DNA-RNA hybridization cycles, it was further shown that RMS4 cells contained at least 700 to 800 distinct mRNA species, mainly belonging to the class of low abundance, which appeared to be absent in L6 cells. Most of these mRNA species were also found with a lower frequency in M4 cells. Bidimensional analysis of the cell-free translation products directed by polyadenylic acid-containing mRNA revealed some remarkable differences, in particular the synthesis in a RMS4 cell extract of at least three major polypeptides, possibly related either to the neoplastic process itself or to the stage of malignant transformation.

Animals↗
PubMed2005

Silencing the cardiac potassium channel Kv4.3 by RNA interference in a CHO expression system.

RNA interference (RNAi) is a powerful technique for gene silencing, in which the downregulation of mRNA is triggered by short RNAs complementary to a target mRNA sequence, with consequent reduction of the encoded protein. The aim of this study was to test the effects of silencing the expression of the cardiac potassium channel Kv4.3 in a heterologous expression system, in order to investigate the effect of RNAi on channel properties. A Chinese hamster ovary cell line stably expressing Kv4.3 and the accessory beta-subunit KChIP2 was transfected with small-interfering RNAs (siRNAs) targeting Kv4.3. Effects of RNAi were monitored at the mRNA, protein, and functional levels. Real-time PCR and immunofluorescence staining revealed significant reduction of Kv4.3 mRNA and protein expression. These results were confirmed by functional patch-clamp measurements of the transient outward current (I(to)) which was reduced up to 80% by RNAi. We conclude that the use of siRNAs reagents for post-transcriptional gene silencing is a new effective method for the reduction of the expression and function of different ionic channels which may be adapted for studying their role also in native cells.

Animals↗