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PubMed2004

Complete cDNA sequence and mRNA expression of dog preproendothelin-3.

The full-length cDNA of dog preproendothelin-3 (PPET3) was cloned from lung tissue using RT-PCR and rapid amplification of cDNA ends. Aside from the poly (A) tail, the full-length cDNA was 1976 bp. A polyadenylation signal sequence and one copy of a consensus sequence, ATTTA, which is related to mRNA turnover, was found in the 3' noncoding region. The cDNA had a 594-bp open reading frame encoding a 198-amino acid polypeptide. Regions corresponding to a bioactive mature ET3 peptide, an intermediate form known as big-ET3, and an ET3-like peptide were observed in dog PPET3. Expression of PPET3 mRNA was detected throughout the organs examined, which included heart, lung, liver, kidney, spleen, stomach, pancreas, duodenum, colon, uterus, ovary and testis.

Amino Acid Sequence↗
PubMed2006

Evolution of +1 programmed frameshifting signals and frameshift-regulating tRNAs in the order Saccharomycetales.

Programmed translational frameshifting is a ubiquitous but rare mechanism of gene expression in which mRNA sequences cause the translational machinery to shift reading frames with extreme efficiency, up to at least 50%. The mRNA sequences responsible are deceptively simple; the sequence CUU-AGG-C causes about 40% frameshifting when inserted into an mRNA in the yeast Saccharomyces cerevisiae. The high efficiency of this site depends on a set of S. cerevisiae tRNA isoacceptors that perturb the mechanism of translation to cause the programmed translational error. The simplicity of the system might suggest that it could evolve frequently and perhaps be lost as easily. We have investigated the history of programmed +1 frameshifting in fungi. We find that frameshifting has persisted in two structural genes in budding yeasts, ABP140 and EST3 for about 150 million years. Further, the tRNAs that stimulate the event are equally old. Species that diverged from the lineage earlier both do not employ frameshifting and have a different complement of tRNAs predicted to be inimical to frameshifting. The stability of the coevolution of protein coding genes and tRNAs suggests that frameshifting has been selected for during the divergence of these species.

Amino Acid Sequence↗
PubMed1975

Changes in globin messenger RNA content during erythroid cell differentiation.

Previous studies have shown that mouse fetal erythroid precursor cells isolated by an immunological technique synthesize little or no globin and contain little, if any, globin mRNA, as assayed in a cell-free system (translatable mRNA). After culture for 10 hours in the presence of erythropoietin, there is a marked increase in globin synthesis and in translatable globin mRNA. The present studies were designed to measure directly the content of globin mRNA sequences during erythroid cell differentiation, by molecular hybridization with 3H-labeled DNA complementary to globin mRNA. The results indicate that few, if any, globin mRNA sequences are present in the total RNA of erythroid precursor cells. There is little or no pool of untranslated globin mRNA in these cells. After 10 hours of culture with erythropoietin, there is an increase in globin mRNA content, as ;easured by a change in the Cot1/2 values obtained by cDNA: mRNA hybridization with (Co) representing the concentration of RNA. Between 0 and 22 hours of culture, there is a 250-fold rise, and between 22 and 44 hours, a further 2-fold increase in globin mRNA content. During the 44 hours in culture, the number of cells in culture increases 2- to 3-fold. The number of globin mRNA molecules rises in erythroid precursor cells to an average value of 1800 molecules/cell during 22 hours of culture. In cultures without added erythropoietin, the absolute number of cells decreases, however, cells presumably induced to differentiate by exposure to erythropoietin in vivo continue to differentiate in vitro, accumulating globin mRNA and initiating globin synthesis.

Animals↗
PubMed1982

Cytoplasmic dot hybridization. Simple analysis of relative mRNA levels in multiple small cell or tissue samples.

A simple technique for the simultaneous measurement of relative levels of a specific mRNA in numerous small samples of animal cells or tissue is described. The technique involves denaturation of cytoplasmic preparations, followed by dotting of up to 96 samples onto a single sheet of nitrocellulose, hybridization with a 32P-labeled cDNA plasmid, autoradiography, and scanning. By analyzing cytoplasmic preparations instead of purified RNA, manipulations of multiple samples prior to analysis is minimized. Experiments with a clonal line of rat pituitary tumor (GH3) cells showed that this technique can be employed to follow the induction by Ca2+ of prolactin mRNA sequences, employing cytoplasm prepared from as little as 2.5 x 10(4) cells. The specificity of the technique for prolactin mRNA was shown by employing GC cells, a GH3 cell variant lacking detectable prolactin mRNA sequences. Experiments with cultured rat hemipituitaries showed that the prolactin mRNA present in cytoplasm corresponding to as little as 1/100 of a pituitary could be readily detected. This technique is quite simple, requires very small amounts of cells or tissue, and permits the simultaneous analysis of multiple samples. Hence, it should be quite useful for studies with various experimental systems of the regulation of specific mRNA levels.

Animals↗
PubMed1986

[Cloning of mRNA nucleotide sequences amplified during DNA replication in the regenerating liver].

A library of double-stranded cDNA has been constructed using the mRNA of regenerating rat liver 20 hr after partial hepatectomy. The differential screening of the library with the regenerating liver specific and the resting-liver-specific single-stranded cDNA probes has identified 11 cDNA clones which sequences are preferentially expressed in regenerating rat liver. The RNA dot blot hybridization has shown that levels of RNA complementary to these clones are 3 to 8-fold higher in dividing cells as compared with resting cells.

Animals↗
PubMed1992

Molecular modeling to predict the structural and biological effects of mutations in a highly conserved histone mRNA loop sequence.

The 3'-end of histone mRNAs contains a highly conserved sequence motif which is believed to form a 6 base pair stem and a 4 base loop. These sequences are involved in both the efficiency of 3'-end formation and stability of the mature histone mRNA. We have modeled four stem basepairs and the loop portion of this structure using the wildtype sequences and several mutant sequences. A structure for the wildtype stem-loop is proposed that is based on energy minimization using a representative wildtype sequence and comparison with structures obtained using naturally occurring mutations which do not alter loop function. A wildtype structure is proposed in which the top basepair of the stem is broken, forming a six base loop. Mutant sequences with altered bases in the loop and in the stem were also modeled. The effect of these mutations on the proposed wildtype structure is discussed and possible biological consequences considered.

Base Sequence↗
PubMed1993

Molecular cloning of a cDNA showing alternative splicing of the 5'-untranslated sequence of mRNA for human aromatase P-450.

A new type of full-length cDNA clone encoding human aromatase P-450 was isolated from a human placental cDNA library. The clone, designated as pES-4, has a 3130-bp insert. The nucleotide sequences of the translated region and the 3'-untranslated region of the insert of pES-4 are exactly identical with those of the cDNA clone characterized previously. However, the sequence of the 5'-untranslated region of the insert has characteristic feature, i.e. an extra sequence of 109 bp is present at a junction between exon 1 and exon 2 on the processed human aromatase mRNA. Analysis of the genomic clones containing the region between exon 1 and exon 2 of the human aromatase P-450 gene reveals that the 109-bp genomic segment, encoding the same sequence as the extra sequence observed in pES-4, is located approximately 10-kbp downstream of exon 1 and that the nucleotide sequences of the 5'-flanking and the 3'-flanking regions of the segment conform to the GT-AG rule for RNA splicing. By means of reverse transcription and polymerase chain reaction, relative amounts of the pES-4-type mRNA are estimated to be approximately 4.8% and 2.3% of the processed aromatase P-450 mRNA in human placenta and human BeWo choriocarcinoma cells, respectively. These results indicate that the segment of 109 bp between exon 1 and exon 2 is a new exon hitherto unidentified and that heterogeneity observed in the 5'-untranslated sequence of human aromatase P-450 mRNA is, at least in part, caused by alternative splicing of this new exon.

Alternative Splicing↗
PubMed1989

A splicing defect in the mouse transferrin gene leads to congenital atransferrinemia.

We have analyzed the biochemical defect in a mutant line of mice that produces less than 1% of the normal level of serum transferrin. This mouse line (Hp) transcribes the transferrin gene in liver at the same rate observed in normal mice, but the steady state levels of transferrin mRNA sequences are less than 20% of normal. Further hybridization studies reveal that most of the transferrin mRNA sequences present in homozygous Hp mouse liver are in the form of a 5 kb nuclear precursor instead of the mature 2.5 kb transferrin mRNA seen in normal mice. Using several different exon and intron probes from the mouse transferrin gene, we have shown that the 5 kb RNA precursor retains the last two introns of the transferrin gene but that the 5' and middle introns have been removed by processing. The defect in transferrin mRNA processing also extends to nonhepatic tissues and we find the same lack of mature mRNA and increased precursor accumulation in brain RNA. Since Southern blot analysis does not reveal gross changes in the structure of the transferrin gene in Hp mice, we suggest that the Hp defect is due to a small deletion or point mutation that either disrupts splicing signals or uncovers cryptic splice signals that interfere with processing of the last two introns in the transferrin gene. This Hp mouse line provides an opportunity to study the effects of transferrin deficiency on development and iron homeostasis.

Anemia↗
PubMed1997

Cloning and sequencing of a new 15-hydroxyprostaglandin dehydrogenase related mRNA.

NAD+-dependent 15-hydroxyprostaglandin dehydrogenase (type-I 15-PGDH) inactivates prostaglandins. We recently reported an mRNA sequence coding for a predicted isomer (PGDH(rI)) of this enzyme. The TT cell line, derived from medullary thyroid carcinoma (MTC), expresses mRNAs for both isomers. We report here the expression by TT cells and MTC of a third 15-PGDH related mRNA (PGDH(rII)), 241 nt shorter than type-I 15-PGDH. RNase protection assays confirmed that TT cells expressed this mRNA (PGDH(rII)). Thus different splicing patterns could be involved in the post-transcriptional regulation of type-I 15-PGDH gene in MTC.

Amino Acid Sequence↗
PubMed1976

The effect of 5-bromodeoxyuridine on messenger RNA production in cultured cells.

Cells of clone B(5)59, a derivative of the murine B16 melanoma, are highly tumorigenic, and produce both melanin and plasminogen activator. Cells of clone C(3)471, a line obtained by continued growth and maintenance of B(5)59 cells in medium containing 5-bromodeoxyuridine are nontumorigenic, amelanotic, and have no plasminogen activator. Differences in the mRNA complement of these two syngenic mouse melanoma clones have been determined by hybridization kinetics between complementary DNA transcribed from mRNA of either B(5)59 OR C(3)471 cells, and mRNA isolated from both sources. To detect the presence of unique mRNA sequences produced in B(5)59 cells, complementary DNA produced using B(5)59 mRNA as a template was exhaustively hybridized to C(3)471 mRNA. The results indicate that (i) polyadenylated mRNA produced by either clone can be divided into three main groups based upon the relative complexity of mRNA species within each group, (ii) more than 98% of the mRNA species produced by the B(5)59 cells are also produced by the cells of the C(3)471 clone, (iii) approximately 25% of the mRNA species produced by the cells of the C(3)471 clone in moderate abundance (500 molecules per cell) are not produced by the B(5)59 clone. Despite the fact that at least two of the proteins synthesized by B(5)59 cells are not detectable in C(3)471 cells, our results support the hypothesis that the major effect of BrdUrd incorporation into DNA is the induction rather than the repression of transcription of polyadenylated mRNA sequences.

Animals↗
PubMed1998

Epidermal cell kinetics by combining in situ hybridization and immunohistochemistry.

Double labelling can serve as a useful tool for providing information about cell kinetics in normal and hyperproliferative tissues in general, and skin in particular. We have developed a double-labelling method that combines immunohistochemistry using the monoclonal antibody MIB1 and non-isotopic in situ hybridization using either a digoxigenin-labelled RNA probe specific for histone 3 mRNA sequences or a Fluorescein-labelled oligonucleotide probe specific for histone 2b, 3, 4 mRNA sequences. Double labelling was performed on normal, tape-stripped normal skin and psoriatic skin. The three proliferation markers were also examined by single labelling. The ratio of cells in the S-phase (Ns) and the growth fraction (Ncy) was determined. In normal skin, psoriatic skin and tape-stripped normal skin after 24 h and after 48 h, we calculated that 15%, 16%, 3% and 12% of growth fraction consisted of cells in the S-phase respectively. The S-phase lasts approximately 10 h, so the cell cycle time in normal and psoriatic skin is approximately 62.5 h. At present, the MIB1/H3 digoxigenin or MIB1/H2b-H3-H4 Fluorescein double-labelling technique cannot be used routinely. Therefore, in order to understand the cell kinetic processes better, experiments are recommended to optimize these methods. From a practical point of view and for reasons of specificity and sensitivity, we prefer the Fluorescein-labelled oligonucleotide probe method.

Cell Division↗
PubMed1979

Effect of hypophysectomy and growth hormone treatment on albumin mRNA levels in the rat liver.

A complementary DNA copy of purified rat albumin mRNA was employed in RNA-excess hybridization reactions to examine albumin mRNA levels in the liver of the hypophysectomized rat. In normal rat liver, albumin mRNA sequences were found to be about 10% of the total poly(A)-containing RNA population. Hypophysectomy reduced albumin mRNA levels by approximately 50%. Growth hormone treatment of hypophysectomized animals returned albumin mRNA to nearly normal levels. These effects on albumin mRNA sequences agreed closely with the changes in the synthesis of albumin that are assoicated with these conditions. The results suggest that hypophysectomy and growth hormone influence albumin synthesis by affecting the level of albumin mRNA.

Albumins↗
PubMed1996

Manduca sexta hemolymph ferritin: cDNA sequence and mRNA expression.

A cDNA clone encoding a subunit of the tobacco hornworm Manduca sexta (Ms) hemolymph (serum) ferritin (Fer) has been identified and sequenced. The deduced amino acid (aa) sequence shows approx. 50% similarity to vertebrate Fer subunit sequences, and the nucleotide sequence contains a stem-loop structure in the 5' untranslated region that could serve as an iron-responsive element (IRE). The stem-loop of this putative IRE exhibits high identity to vertebrate IRE that play an essential role in the control of Fer synthesis. The Ms Fer subunit lacks one of the three Tyr residues required for the rapid biomineralization of iron shown in vertebrate heavy-chain Fer. In addition, aa residues that comprise the putative ferroxidase centers generally are not conserved, suggesting that the Ms Fer subunit more closely resembles the vertebrate light-chain subunit. Northern blot analyses indicate that the fer mRNA is expressed in the midgut, fat body and hemocytes, with the greatest expression in the midgut.

Amino Acid Sequence↗
PubMed1996

Evolution of the nad3-rps12 gene cluster in angiosperm mitochondria: comparison of edited and unedited sequences.

We have analyzed the nad3-rps12 locus for eight angiosperms in order to compare the utility of mitochondrial DNA and edited mRNA sequences in phylogenetic reconstruction. The two coding regions, containing from 25 to 35 editing sites in the various plants, have been concatenated in order to increase the significance of the analysis. Differing from the corresponding chloroplast sequences, unedited mitochondrial DNA sequences seem to evolve under a quasi-neutral substitution process which undifferentiates the nucleotide substitution rates for the three codon positions. By using complete gene sequences (all codon positions) we found that genomic sequences provide a classical angiosperm phylogenetic tree with a clear-cut grouping of monocotyledons and dicotyledons with Magnoliidae at the basal branch of the tree. Conversely, owing to their low nucleotide substitution rates, edited mRNA sequences were found not to be suitable for studying phylogenetic relationships among angiosperms.

DNA, Mitochondrial↗
PubMed1998

Prediction of splice sites in plant pre-mRNA from sequence properties.

Heterologous introns are often inaccurately or inefficiently processed in higher plants. The precise features that distinguish the process of pre-mRNA splicing in plants from splicing in yeast and mammals are unclear. One contributing factor is the prominent base compositional contrast between U-rich plant introns and flanking G + C-rich exons. Inclusion of this contrast factor in recently developed statistical methods for splice site prediction from sequence inspection significantly improved prediction accuracy. We applied the prediction tools to re-analyze experimental data on splice site selection and splicing efficiency for native and more than 170 mutated plant introns. In almost all cases, the experimentally determined preferred sites correspond to the highest scoring sites predicted by the model. In native genes, about 90% of splice sites are the locally highest scoring sites within the bounds of the flanking exon and intron. We propose that, in most cases, local context (about 50 bases upstream and downstream from a potential intron end) is sufficient to account for intrinsic splice site strength, and that competition for transacting factors determines splice site selection in vivo. We suggest that computer-aided splice site prediction can be a powerful tool for experimental design and interpretation.

Animals↗
PubMed2001

Effective inhibition of herpes simplex virus 1 gene expression and growth by engineered RNase P ribozyme.

Using an in vitro selection procedure, we have previously isolated ribonuclease P (RNase P) ribozyme variants that efficiently cleave an mRNA sequence in vitro. In this study, an M1GS RNA variant was used to target the mRNA encoding human herpes simplex virus 1 (HSV-1) major transcription activator ICP4. The variant is about 15 times more efficient in cleaving the ICP4 mRNA sequence in vitro than the ribozyme derived from the wild type RNase P ribozyme. Moreover, the variant is also more effective in inhibiting viral ICP4 expression and growth in HSV-1-infected cells than the wild type ribozyme. A reduction of approximately 90% in the expression level of ICP4 and a reduction of 4000-fold in viral growth were observed in cells that expressed the variant. In contrast, a reduction of <10% in the ICP4 expression and viral growth was observed in cells that either did not express the ribozyme or produced a catalytically inactive ribozyme mutant. These results provide direct evidence that RNase P ribozyme variants can be highly effective in inhibiting HSV-1 gene expression and growth and furthermore, demonstrate the feasibility of developing highly effective RNase P ribozyme variants for anti-HSV applications by using in vitro selection procedures.

Animals↗
PubMed1990

Different Temporal and Spatial Gene Expression Patterns Occur during Anther Development.

We studied the temporal and spatial regulation of three mRNA sequence sets that are present exclusively, or at elevated levels, in the tobacco anther. One mRNA set accumulates in the tapetum and decays as the tapetum degenerates later in anther development. The second mRNA set accumulates after the tapetal-specific mRNAs, is localized within the stomium and connective, and also decays as these cell types degenerate during anther maturation. The third mRNA sequence set persists throughout anther development and is localized within most anther tissues. A tapetal-specific gene, designated as TA29, was isolated from a tobacco genome library. Runoff transcription studies and experiments with chimeric [beta]-glucuronidase and diphtheria toxin A-chain genes showed that the TA29 gene is regulated primarily at the transcriptional level and that a 122-base pair 5[prime] region can program the tapetal-specific expression pattern. Destruction of the tapetum by the cytotoxic gene had no effect on the differentiation and/or function of surrounding sporophytic tissues but led to the production of male-sterile plants. Together, our studies show that several independent gene expression programs occur during anther development and that these programs correlate with the differentiated state of specific anther cell types.

Journal Article↗