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At least 73 records · Page 4Linked to original sources
PubMed1987

Expression of active human blood clotting factor IX in transgenic mice: use of a cDNA with complete mRNA sequence.

Haemophilia B is a bleeding disorder caused by a functional deficiency of the clotting factor IX. A full length human factor IX complementary DNA clone containing all the natural mRNA sequences plus some flanking intron sequences was constructed with a metallothionein promoter and introduced into transgenic mice by microinjection into the pronuclei of fertilised eggs. The transgenic mice expressed high levels of messenger RNA, gamma-carboxylated and glycosylated protein, and biological clotting activity that are indistinguishable from normal human plasma factor IX. This study demonstrates the feasibility of expressing highly complex heterologous proteins in transgenic mice. It also provides the groundwork for the production of large amounts of human factor IX in larger transgenic livestock for therapeutic use, and the investigation of alternative genetic therapies for haemophilia B.

Animals↗
PubMed1988

Developmental expression of alpha- and gamma-enolase subunits and mRNA sequences in the mouse brain.

Nonneuronal alpha alpha- and neuron-specific alpha gamma- and gamma gamma-enolase activities were measured in the mouse brain during development. The corresponding mRNA sequences were quantified directly by hybridization with cDNA probes. The variations in alpha- and gamma-monomer levels inferred from the enzymatic activities were very similar to those of their respective mRNAs. We conclude that monomer levels are primarily controlled by the amounts of their mRNAs during mouse brain development.

Animals↗
PubMed1977

Presence of histone mRNA sequences in high molecular weight RNA of HeLa cells.

Pulse-labeled HeLa cell RNA centrifuged under denaturing conditions was hybridized with DNA of recombinant phages containing sea urchin histone genes. This cross-hybridization showed the presence of histone mRNA sequences in high molecular weight RNA molecules. Treatment of the cells with actinomycin to stop RNA synthesis resulted in the rapid decay of this high molecular weight RNA followed by an increase of 9S histone mRNA in the cytoplasm. The results are consistent with the presence in HeLa cells of a high molecular weight precursor to histone messengers.

Coliphages↗
PubMed1985

Evolutionary conservation of ribosomal protein mRNA sequences: application for expansion of corresponding cDNA and gene libraries.

Cloned cDNAs, containing ribosomal protein sequences from mouse (five cDNAs) or Xenopus laevis (six cDNAs), were used to estimate the evolutionary conservation, from insects to mammals, of the corresponding mRNA sequences. Northern blot analysis reveals a variable degree of homology between these sequences in different eukaryotes. Thus, among the ribosomal protein cDNA clones utilized, some exhibit complete, others partial, and a few no interphyla cross-hybridization. Melting profile analysis was employed to quantitate this homology. It is proposed that for expansion of eukaryotic ribosomal cDNA and gene libraries, one can exploit the interspecies homology of the corresponding sequences. However, the diverse evolutionary conservation of individual ribosomal protein gene sequences should be taken into account.

Animals↗
PubMed1990

Evolution of nucleic acids coding for ribonucleases: the mRNA sequence of mouse pancreatic ribonuclease.

The cDNA of mouse pancreatic mRNA has been cloned. After the library was screened with a rat ribonuclease cDNA probe, the positive clones were isolated and sequenced. There were no differences from the previously determined protein sequence. The mRNA codes for a preribonuclease of 149 amino acid residues including a signal peptide of 25 amino acids. The 3' noncoding region has a length of 260 bp, and the total mRNA length is approximately 940 bp. Comparison with the rat pancreatic ribonuclease sequence showed a high rate of nucleotide substitution. Within the coding region, nonsynonymous and synonymous substitution rates are 4.3 X 10(-9) and 15 X 10(-9) nucleotide substitutions/site/year, respectively. The latter value is one of the highest rates observed in the molecular evolution of mammalian nuclear genes. In the signal sequences the synonymous substitution rate is much lower and about the same as the nonsynonymous rate. Signal sequences of other mouse and rat proteins also exhibit little difference between synonymous and nonsynonymous rates. The sequences of rat and mouse pancreatic ribonuclease messengers were compared with those of bovine pancreatic, seminal, and brain ribonuclease. While the 3' noncoding regions of rat and mouse are very similar, as are those of the three bovine messengers, there is no significant similarity between both rodent and the three bovine messengers for the greater part of these regions. There is a duplication of approximately 50 nucleotides in the 3' noncoding region of the bovine messengers, with a region rich in A and C in between. The presence of this structural feature may be correlated with recent gene duplications that have occurred in the bovine genome.

Amino Acid Sequence↗
PubMed1977

Authentic beta-globin mRNA sequences in homozygous betaO-thalassemia.

In a patient with homozygous betaO-thalassemia in whom studies of reticulocyte hemoglobin synthesis showed no beta-globin chain synthesis in vivo and in vitro, molecular hybridization studies revealed RNA sequences complementary to beta-globin cDNA. The fact that these sequences were authentic beta-globin mRNA was shown by fingerprint analysis of T1 ribonuclease-digested mRNA and by sequencing of oligonucleotides unique to beta-globin mRNA. The beta-mRNA that failed to direct beta-globin chain synthesis was not detectably shortened or degraded and contained poly(A) sequences.

Adult↗
PubMed1991

Site-specific cleavage of natural mRNA sequences by newly designed hairpin catalytic RNAs.

The negative strand of tobacco ringspot virus satellite RNA is a self-cleaving RNA. Its catalytic domain and substrate domain have been identified, and the catalytic domain has been named hairpin catalytic RNA. Here we report the construction of a plasmid containing a modified hairpin catalytic RNA sequence that can be transcribed in vitro. Because this plasmid has two specific restriction enzyme recognition sites at both ends of the substrate binding site in the catalytic RNA sequence, it is possible to construct new plasmids by substituting different sequences in the substrate binding site. Using this plasmid, synthetic DNA, and in vitro transcription, we obtained three ribozymes designed to cleave Escherichia coli prolipoprotein signal peptidase (lsp) mRNA at specific sites. All three ribozymes cleaved the lsp mRNA sequence in vitro at the specific sites, and two of them cleaved it efficiently. Kinetic analyses showed that one had a higher kcat/Km value than that of the well-known hammerhead ribozyme. Problems associated with attaining the goal of expressing these ribozymes in vivo also are discussed.

Bacterial Outer Membrane Proteins↗
PubMed2004

Adaptive role of increased frequency of polypurine tracts in mRNA sequences of thermophilic prokaryotes.

The mechanism of an organism's adaptation to high temperatures has been investigated intensively in recent years. It was suggested that the macromolecules of thermophilic microorganisms (especially proteins) have structural features that enhance their thermostability. We compared mRNA sequences of 72 fully sequenced prokaryotic proteomes (14 thermophilic and 58 mesophilic species). Although the differences between the percentage of adenine plus guanine content of whole mRNAs of different prokaryotic species are much lower than those of guanine plus cytosine content, the thermophile purine-pyrimidine (R/Y) ratio within their mRNAs is significantly higher than that of the mesophiles. The first and third codon positions of both thermophiles and mesophiles are purine-biased, with the bias more pronounced by the thermophiles. Thermophile mRNAs that display the highest R/Y ratio (1.43-1.69) are those of the ribosomal proteins, histone-like proteins, DNA-dependent RNA polymerase subunits, and heat-shock proteins. Within mesophilic prokaryotes and five eukaryotic species, the R/Y ratio of the mRNAs of heat-shock proteins is higher than their average over coding part of the genome. Polypurine tracts (R)(n) (with n > or = 5) are much more abundant within the thermophile mRNAs compared with mesophiles. Between two sequential pure-purinic codons of thermophile mRNAs, there is a rather strong tendency for the occurrence of adenine but not guanine tracts. The data suggest that mixed adenine.guanine and polyadenine tracts in mRNAs increase the thermostability beyond the contribution of amino acids encoded by purine tracts, which highlights the importance of ecological stress in the evolution of genome architecture.

Animals↗
PubMed1988

Repetitive extragenic palindromic sequences, mRNA stability and gene expression: evolution by gene conversion? A review.

Repetitive extragenic palindromic (REP) sequences are highly conserved inverted repeats present in up to 1000 copies on the Escherichia coli chromosome. We have shown both in vivo and in vitro that REP sequences can stabilize upstream mRNA by blocking the processive action of 3'----5' exonucleases. In a number of operons, mRNA stabilization by REP sequences plays an important role in the control of gene expression. Furthermore, differential mRNA stability mediated by the REP sequences can be responsible for differential gene expression within polycistronic operons. Despite the key role of REP sequences in mRNA stability and gene expression in a number of operons, several lines of evidence suggest that this is unlikely to be the primary reason for the exceptionally high degree of sequence conservation between REP sequences. Other possible functions for REP sequences are discussed. We propose that REP sequences may be a prokaryotic equivalent of 'selfish DNA' and that gene conversion may play a role in the evolution and maintenance of REP sequences.

Biological Evolution↗
PubMed2000

cDNA sequence, mRNA expression and genomic DNA of trypsinogen from the indianmeal moth, Plodia interpunctella.

Trypsin-like enzymes are major insect gut enzymes that digest dietary proteins and proteolytically activate insecticidal proteins produced by the bacterium Bacillus thuringiensis (Bt). Resistance to Bt in a strain of the Indianmeal moth, Plodia interpunctella, was linked to the absence of a major trypsin-like proteinase (Oppert et al., 1997). In this study, trypsin-like proteinases, cDNA sequences, mRNA expression levels and genomic DNAs from Bt-susceptible and -resistant strains of the Indianmeal moth were compared. Proteinase activity blots of gut extracts indicated that the susceptible strain had two major trypsin-like proteinases, whereas the resistant strain had only one. Several trypsinogen-like cDNA clones were isolated and sequenced from cDNA libraries of both strains using a probe deduced from a conserved sequence for a serine proteinase active site. cDNAs of 852 nucleotides from the susceptible strain and 848 nucleotides from the resistant strain contained an open reading frame of 783 nucleotides which encoded a 261-amino acid trypsinogen-like protein. There was a single silent nucleotide difference between the two cDNAs in the open reading frame and the predicted amino acid sequence from the cDNA clones was most similar to sequences of trypsin-like proteinases from the spruce budworm, Choristoneura fumiferana, and the tobacco hornworm, Manduca sexta. The encoded protein included amino acid sequence motifs of serine proteinase active sites, conserved cysteine residues, and both zymogen activation and signal peptides. Northern blotting analysis showed no major difference between the two strains in mRNA expression in fourth-instar larvae, indicating that transcription was similar in the strains. Southern blotting analysis revealed that the restriction sites for the trypsinogen genes from the susceptible and resistant strains were different. Based on an enzyme size comparison, the cDNA isolated in this study corresponded to the gene for the smaller of two trypsin-like proteinases, which is found in both the Bt-susceptible and -resistant strains of the Indianmeal moth. The sequences reported in this paper have been deposited in the GenBank database (accession numbers AF064525 for the RC688 strain and AF064526 for HD198).

Amino Acid Sequence↗
PubMed2006

RNA reprogramming of alpha-mannosidase mRNA sequences in vitro by myxomycete group IC1 and IE ribozymes.

Trans-splicing group I ribozymes have been introduced in order to mediate RNA reprogramming (including RNA repair) of therapeutically relevant RNA transcripts. Efficient RNA reprogramming depends on the appropriate efficiency of the reaction, and several attempts, including optimization of target recognition and ribozyme catalysis, have been performed. In most studies, the Tetrahymena group IC1 ribozyme has been applied. Here we investigate the potential of group IC1 and group IE intron ribozymes, derived from the myxomycetes Didymium and Fuligo, in addition to the Tetrahymena ribozyme, for RNA reprogramming of a mutated alpha-mannosidase mRNA sequence. Randomized internal guide sequences were introduced for all four ribozymes and used to select accessible sites within isolated mutant alpha-mannosidase mRNA from mammalian COS-7 cells. Two accessible sites common to all the group I ribozymes were identified and further investigated in RNA reprogramming by trans-splicing analyses. All the myxomycete ribozymes performed the trans-splicing reaction with high fidelity, resulting in the conversion of mutated alpha-mannosidase RNA into wild-type sequence. RNA protection analysis revealed that the myxomycete ribozymes perform trans-splicing at approximately similar efficiencies as the Tetrahymena ribozyme. Interestingly, the relative efficiency among the ribozymes tested correlates with structural features of the P4-P6-folding domain, consistent with the fact that efficient folding is essential for group I intron trans-splicing.

Animals↗
PubMed1989

mRNA sequence and deduced amino acid sequence of the mumps virus small hydrophobic protein gene.

The mRNA of a putative small hydrophobic protein (SH) of mumps virus was identified in mumps virus-infected Vero cells, and its complete nucleotide sequence was determined by sequencing the genomic RNA and cDNA clones and partial sequencing of mRNA. The SH mRNA is 310 nucleotides long excluding the poly(A) and contains a single open reading frame encoding a protein of 57 amino acids with a calculated molecular weight of 6,719. The predicted protein is highly hydrophobic and contains a stretch of 25 hydrophobic amino acids near the amino terminus which could act as a membrane anchor region. There is no homology between the putative SH protein of mumps virus and the SH protein of simian virus 5, even though the SH genes are located in the same locus in the corresponding genome. One interesting observation is that the hydrophobic domain of simian virus 5 SH protein is at the carboxyl terminus, whereas that of mumps virus putative SH protein is near the amino terminus.

Amino Acid Sequence↗
PubMed1993

Bovine mammary lactoferrin: implications from messenger ribonucleic acid (mRNA) sequence and regulation contrary to other milk proteins.

The regulation of bovine mammary lactoferrin, an important component of the antimicrobial defenses of the mammary gland, is poorly understood compared with the other milk proteins. The complete sequence for bovine lactoferrin mRNA shows it to be highly homologous to other lactoferrins and transferrins. However, regional differences in the deduced AA sequence of bovine lactoferrin compared with human lactoferrin and transferrin imply functional differences between them. Steady-state levels of bovine lactoferrin mRNA (by Northern blot) in the bovine mammary gland indicate that bovine lactoferrin expression is minimal in the developing and lactating gland but is strongly induced by mammary involution. The overall regulation of bovine lactoferrin in the mammary gland appears to be contrary to that of the other milk proteins. Features identified in the mRNA of bovine mammary lactoferrin may contribute to the differences in regulation between lactoferrin and other bovine milk proteins and to differences in concentrations of lactoferrin in milk across species. Lactoferrin secretion by bovine mammary cells grown in vitro does not appear to be dependent on prolactin and shows regulation by substratum, serum, and cell population to be different from that for casein. In contrast to casein, efficient secretion of lactoferrin from the cell does not require detachment of collagen substratum.

Amino Acid Sequence↗
PubMed1988

Diagnosis of chronic myeloid and acute lymphocytic leukemias by detection of leukemia-specific mRNA sequences amplified in vitro.

The Philadelphia chromosome is present in more than 95% of chronic myeloid leukemia patients and 13% of acute lymphocytic leukemia patients. The Philadelphia translocation, t(9;22), fuses the BCR and ABL genes resulting in the expression of leukemia-specific, chimeric BCR-ABL messenger RNAs. To facilitate diagnosis of these leukemias, we have developed a method of amplifying and detecting only the unique mRNA sequences, using an extension of the polymerase chain reaction technique. Diagnosis of chronic myeloid and acute lymphocytic leukemias by this procedure is rapid, much more sensitive than existing protocols, and independent of the presence or absence of an identifiable Philadelphia chromosome.

Cell Line↗
PubMed1996

Cloning of mRNA sequences for two antibacterial peptides in a hemipteran insect, Riptortus clavatus.

Escherichia coli injection rapidly induced bactericidal activity in the hemolymph of a hemipteran insect, Riptortus clavatus. This activity reached its maximum at 9 hr after injection and thereafter declined slowly. Two types of cDNA clones involved in this response were isolated by differential screening. The predominant type encoded for an open reading frame of 678 amino acids, which consisted of fourteen tandem repeats. Each repeat was rich in charged residues and had a proline-rich region which had striking sequence similarities to proline-rich antibacterial peptides from other insect species, indicating these clones encode a multipeptide precursor of antibacterial peptides. The other type encoded for a glycine-rich peptide similar to a known antibacterial peptide as well. Northern blot analyses revealed rapid induction of mRNAs corresponding to these clones after the injection. To our knowledge, this is the first report on the mRNA sequences of antibacterial peptides of hemimetabolous insects, and the second report on the occurrence of multipeptide precursor structure in insect antibacterial peptides.

Animals↗
PubMed1984

Increased abundance of a normal cell mRNA sequence accompanies the conversion of rat mammary cuboidal epithelial cells to elongated myoepithelial-like cells in culture.

Rat mammary cuboidal epithelial cell lines in culture convert to elongated myoepithelial-like cells. This conversion is accompanied by the appearance of a 9,000 molecular weight acidic polypeptide (p9ka), abundant in the elongated convertants, but which is hardly detectable in the cuboidal epithelial cells. A cDNA library corresponding to a low-molecular-weight fraction of poly(A)- containing RNA from a myoepithelial-like cell line, has been constructed. Recombinant plasmids containing cDNA complementary to p9ka mRNA have been identified by hybrid-selected translation. The mRNA for p9ka has been identified by Northern blotting and is found to be at least five-times more abundant in cultured myoepithelial-like rat mammary cells when compared to the cuboidal epithelial cells. This cytoplasmic mRNA sequence, which is present in increased abundance in cultured mammary myoepithelial-like cells, is also present, at lower levels, in normal rat tissues, including the mammary glands.

Animals↗
PubMed2003

Interferon-tau in bovine blastocysts following parthenogenetic activation of oocytes: pattern of secretion and polymorphism in expressed mRNA sequences.

A series of experiments were conducted to examine the pattern of production and secretion of interferon-tau (IFN-tau) by blastocysts following parthenogenetic activation of bovine oocytes. In the first experiment, 36.8, 24.1, and 33.2% of IVF-derived and parthenogenetically activated oocytes cultured in the presence or absence of a monolayer of buffalo rat liver cells, respectively, reached the blastocyst stage. Following individual culture of blastocysts, IFN-tau concentration in medium droplets was similar among the three groups, although IVF-derived blastocysts contained significantly more cells. In the second experiment, 156 IVF-derived blastocysts were sexed by PCR with 75 and 81, respectively, being male and female. IFN-tau secretion of these was compared to that of 70 parthenogenetic blastocysts. Female and parthenogenetic blastocysts produced significantly more IFN-tau than their male counterparts. In the third experiment, the ability of hatched blastocysts to form outgrowths and the pattern of their IFN-tau secretion were examined. Of the 48 IVF-derived blastocysts, 44 formed outgrowths compared to 41 of the 42 hatched parthenotes. Parthenogenetic outgrowths were significantly larger after 7 days, but this difference had disappeared after 14 days. IFN-tau secretion did not differ between the two groups. Lastly, sequence analyses of expressed mRNA from individual parthenogenetic blastocyst outgrowths showed four different transcript types which, based on their predicted amino acid sequence, belong to two subgroups, IFN-tau1 and IFN-tau3. In addition, one new transcript sequence was identified, encoding a new protein isoform.

Animals↗
PubMed1977

Size distribution of rat liver nuclear RNA containing mRNA sequences.

Total rat liver poly(A)-containing polysomal mRNA was size-fractionated on polyacrylamide gels in 98% formamide. Complementary DNA (cDNA) was prepared from the 8--14-S mRNA fraction and separated into sequences representing abundant and non-abundant mRNAs. The cDNA complementary to the abundant small mRNA of the rat liver cell (approximately 20 species) was hybridized to nuclear RNA of different lengths to determine the size distribution of nuclear RNA molecules which contain these messenger sequences. It was found that: 1. All abundant 8--14-S poly(A)-containing mRNAs have larger nuclear precursor molecules; 20% of the different messenger sequences are found in nuclear RNA of several times their cytoplasmic length. 2. 70% of the mass of the examined nuclear messenger sequences is in RNA molecules of a size similar to their polysomal mRNA; 30% are in larger than 18-S RNA and 2% are between 37 S and 44 S. 3. The majority of small messenger-containing RNA molecules in the RNA prepared from isolated nuclei are of true nuclear origin, since their frequency distribution differs significantly from that of the polysomal 8--14-S mRNA.

Animals↗