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At least 1,279 records · Page 71Linked to original sources
PubMed1984

Cell type-specific expression of bovine keratin genes as demonstrated by the use of complementary DNA clones.

Cytokeratins are a family of polypeptides that form the intermediate-sized filament characteristic of epithelial cells. The cytoskeletons of different types of epithelial cells have been reported to possess specific combinations of the members of this protein family. Therefore, we have sought to examine the correspondence between such differential protein expression and the expression of cytokeratin genes at the nucleic acid level. A library of recombinant plasmids carrying cDNA sequences synthesized from bovine epidermal mRNAs was constructed. Clones of about 10(3) base-pairs coding for all the major epidermal keratins of molecular weights of 50,000, 54,000, 59,000, 60,000 and 68,000 were identified by means of hybridization-selection, followed by one and two-dimensional gel electrophoresis of products of translation in vitro. Under stringent conditions, each of these clones hybridizes specifically with its corresponding mRNA and does not show significant cross-hybridization with mRNAs coding for the other keratins, including those belonging to the same subfamily. Using these clones in RNA blot hybridization analysis, we have studied the expression of keratin genes in diverse bovine epithelial tissues (muzzle epidermis, cornea, esophagus, bladder urothelium, liver) and cultured cell lines from kidney (MDBK) and mammary gland (BMGE + H, BMGE -H). In each case we have found a correlation between the respective keratin polypeptides and the corresponding mRNAs. Whereas mRNA coding for keratins Ia and VIb have been found only in epidermis, genes coding for other epidermal keratins are expressed also in certain non-epidermal epithelia and in cells of the BMGE + H line. In contrast, epidermal keratin mRNA sequences have not been detected in liver or bladder tissue, nor in cultured kidney cells (MDBK) or mammary gland cells of the BMGE - H line, which all express a set of cytokeratin polypeptides entirely different from those of epidermis. In all cases, only one mRNA size species has been found, suggesting that in different cell types the same mRNA species is synthesized from the same keratin gene. We conclude that the mechanisms controlling the cell type-specific synthesis of the diverse keratin genes act at a pre-translational level.

Animals↗
PubMed2005

Molecular cloning of Indian jujube (Zizyphus mauritiana) allergen Ziz m 1 with sequence similarity to plant class III chitinases.

Indian jujube (Zizyphus mauritiana) is a sweet fruit that is abundantly cultivated in Taiwan. We have previously identified 42 and 30 kDa allergens that are cross-reactive with latex allergen from crude Indian jujube extract. This study aimed to clone the 30 kDa Ziz m 1 Z. mauritiana allergen. The Ziz m 1 encoding cDNA was isolated from a ZAPII cDNA library constructed from Z. mauritiana mRNA, sequenced and expressed in Pichia pastoris. The protein predicted from the cDNA sequence has 330 amino acids, the first 25 of which constituted a putative signal peptide. The deduced molecular mass of the mature protein is 33.86 kDa, while its isoelectric point is estimated at 4.36. The recombinant Ziz m 1 showed chitinase activity, possessed IgE binding capacity, and had IgE cross-reactivity with the latex allergen. Moreover, anti-recombinant Ziz m 1 antibody-based ELISA was able to detect commercial skin testing latex reagent, laboratory prepared latex and Indian jujube extracts. Recombinant Ziz m 1 showed 87.5% skin reactivities on eight latex- and Indian jujube-sensitive subjects. Although no sequence similarity was found to other known allergens, Ziz m 1 was found to have amino acid sequence identity (39-45.3%) to many plant chitinases including chitinase (45.2%) of Hevea brasiliensis (hevamine), class III chitinases of Vigna angularis (45.3%), Capsicum annuum (44.7%) and Oryza sativa (41.2%). A conserved domain search revealed that Ziz m 1 belongs to the family 18 glycosyl hydrolases. The recombinant allergen may therefore be of value for diagnosis and therapeutic purposes, and the further characterization of Indian jujube allergen may help to elucidate the mechanism underlying latex-fruit syndrome.

Allergens↗
PubMed1979

Sequence complexity of bovine pituitary poly(A)RNA.

Poly(A)RNA extracted from the anterior lobe of bovine-pituitary tissue was transcribed into its complementary DNA with reverse transcriptase. This 3H-labeled cDNA was hybridized with its template RNA. Hybridization kinetics revealed at least 3 abundance classes with the highest abundance class consisting of only a few different sequences. Bovine-liver poly(A)RNA did not contain this highest abundance class when hybridized to the cDNA probe complementary to pituitary poly(A)RNA. This result suggested that the highest abundance class found in bovine-pituitary poly(A)RNA was specific for that tissue and most likely contained the mRNA sequences for the major pituitary hormones.

Animals↗
PubMed2003

Discovery of novel splice forms and functional analysis of cancer-specific alternative splicing in human expressed sequences.

We report here a genome-wide analysis of alternative splicing in 2 million human expressed sequence tags (ESTs), to identify splice forms that are up-regulated in tumors relative to normal tissues. We found strong evidence (P < 0.01) of cancer-specific splice variants in 316 human genes. In total, 78% of the cancer-specific splice forms we detected are confirmed by human-curated mRNA sequences, indicating that our results are not due to random mis-splicing in tumors; 73% of the genes showed the same cancer-specific splicing changes in tissue-matched tumor versus normal datasets, indicating that the vast majority of these changes are associated with tumorigenesis, not tissue specificity. We have confirmed our EST results in an independent set of experimental data provided by human-curated mRNAs (P-value 10(-5.7)). Moreover, the majority of the genes we detected have functions associated with cancer (P-value 0.0007), suggesting that their altered splicing may play a functional role in cancer. Analysis of the types of cancer-specific splicing shifts suggests that many of these shifts act by disrupting a tumor suppressor function. Sur prisingly, our data show that for a large number (190 in this study) of cancer-associated genes cloned originally from tumors, there exists a previously uncharacterized splice form of the gene that appears to be predominant in normal tissue.

Alternative Splicing↗
PubMed2003

Widespread selection for local RNA secondary structure in coding regions of bacterial genes.

Redundancy of the genetic code dictates that a given protein can be encoded by a large collection of distinct mRNA species, potentially allowing mRNAs to simultaneously optimize desirable RNA structural features in addition to their protein-coding function. To determine whether natural mRNAs exhibit biases related to local RNA secondary structure, a new randomization procedure was developed, DicodonShuffle, which randomizes mRNA sequences while preserving the same encoded protein sequence, the same codon usage, and the same dinucleotide composition as the native message. Genes from 10 of 14 eubacterial species studied and one eukaryote, the yeast Saccharomyces cerevisiae, exhibited statistically significant biases in favor of local RNA structure as measured by folding free energy. Several significant associations suggest functional roles for mRNA structure, including stronger secondary structure bias in the coding regions of intron-containing yeast genes than in intronless genes, and significantly higher folding potential in polycistronic messages than in monocistronic messages in Escherichia coli. Potential secondary structure generally increased in genes from the 5' to the 3' end of E. coli operons, and secondary structure potential was conserved in homologous Salmonella typhi operons. These results are interpreted in terms of possible roles of RNA structures in RNA processing, regulation of mRNA stability, and translational control.

Computational Biology↗
PubMed1990

Combined actions of multiple hairpin loop structures and sites of rate-limiting endonucleolytic cleavage determine differential degradation rates of individual segments within polycistronic puf operon mRNA.

Differential expression of the genes within the puf operon of Rhodobacter capsulatus is accomplished in part by differences in the rate of degradation of different segments of the puf transcript. We report here that decay of puf mRNA sequences specifying the light-harvesting I (LHI) and reaction center (RC) photosynthetic membrane peptides is initiated endoribonucleolytically within a discrete 1.4-kilobase segment of the RC-coding region. Deletion of this segment increased the half-life of the RC-coding region from 8 to 20 min while not affecting decay of LHI-coding sequences upstream from an intercistronic hairpin loop structure shown previously to impede 3'-to-5' degradation. Prolongation of RC segment half-life was dependent on the presence of other hairpin structures 3' to the RC region. Inserting the endonuclease-sensitive sites into the LHI-coding segment markedly accelerated its degradation. Our results suggest that differential degradation of the RC- and LHI-coding segments of puf mRNA is accomplished at least in part by the combined actions of RC region-specific endonuclease(s), one or more exonucleases, and several strategically located exonuclease-impeding hairpins.

Base Sequence↗
PubMed1992

Evolutionarily conserved structure of the 3' non-translated region of a Chinese hamster polyubiquitin gene.

From a V79 Chinese hamster genomic library, we isolated a clone containing a polyubiquitin gene (designated as CHUB1), and determined its nucleotide sequence. The coding region of the CHUB1 gene consisted of five direct repeats of the ubiquitin unit with no spacer, followed by a single tyrosine residue. Northern hybridization analysis with a synthesized probe specific to the 3' non-translated region of the CHUB1 gene revealed that it codes for a 1.8 kb mRNA. An evident homology to the human polyubiquitin gene UbB and the chicken UbI gene was observed in the region corresponding to the full extent of the mature mRNA sequence, suggesting that these three genes belong to a common polyubiquitin gene subfamily, and that the sequence in the 3' non-translated region of the CHUB1 gene is unique to this subfamily.

Amino Acid Sequence↗
PubMed2000

EID: the Exon-Intron Database-an exhaustive database of protein-coding intron-containing genes.

To aid studies of molecular evolution and to assist in gene prediction research, we have constructed an Exon-Intron Database (EID) in FASTA format. Currently, the database is derived from GenBank release 112, and it contains 51 289 protein-coding genes (287 209 exons) that harbor introns, along with extensive descriptions of each gene and its DNA and protein sequences, as well as splice motif information. There is 17% redundancy inherited from GenBank-a purge at the 99% identity level reduced the database to 42 460 genes (243 589 exons). We have created subdatabases of genes whose intron positions have been experimentally determined. One such database, constructed by comparing genomic and mRNA sequences, contains 11 242 genes (62 474 exons). A larger database of 22 196 genes (105 595 exons) was constructed by selecting on keywords to eliminate computer-predicted genes. By examining the two nucleotides adjacent to the intron boundary, we infer that there is a 2% rate of errors or other deviations from the standard GTellipsisAG motif in nuclear genes. This criterion can be used to eliminate 4921 genes from the overall database. Various tools are provided to enable generation of user-specific subsets of the EID. The EID distribution can be obtained from http://mcb.harvard.edu/gilbert/EID

Base Sequence↗
PubMed1993

Detection of growth hormone, prolactin and human beta-chorionic gonadotropin mRNA in growth hormone-secreting pituitary adenomas and in prolactin-secreting pituitary adenomas by in situ hybridization using a non-isotopic detection method.

A non-isotopic in situ hybridization method with digoxigenin-labelled probes was used to examine growth hormone (GH), prolactin (PRL) and human beta-chorionic gonadotropin (beta-hCG(LH)) gene expression in 63 pituitary tumours in acromegaly and 20 adenomas in hyperprolactinaemia. hCG and LH were detected simultaneously because of the extensive homology (more than 90%) of their mRNA sequences (1). A comparison with former results obtained with 35S-labelled probes shows the value of the easier and faster non-isotopic method. Additionally, immunohistochemical data are included to give even more evidence for the synthesis of the respective hormones by the tumour cells. In all 63 adenomas in acromegaly, GH mRNA was revealed in 59 PRL mRNA and in 36 beta-hCG(LH) mRNA. A positive immunostaining for GH was found in all, for PRL in 40, and for beta-hCG(LH) in 34 adenomas. The comparison of the two in situ hybridization methods revealed no differences concerning GH mRNA detection, but not all tumours positive after non-isotopic PRL and beta-hCG(LH) mRNA detection showed signals with the radioactive method. Referring to the 20 PRL-secreting adenomas, PRL gene expression was demonstrable in all, GH mRNA in 12, and beta-hCG(LH) mRNA in 2 cases. Comparing the positive results of immunohistochemistry with those of in situ hybridization, correspondence was found in 19 cases for PRL, in 5 cases for GH and in no case for beta-hCG(LH).

Acromegaly↗
PubMed1997

Pantropic retroviral vector integration, expression, and germline transmission in medaka (Oryzias latipes).

Pantropic retroviral vectors were used to introduce transgenes into Japanese medaka (Oryzias latipes). These vectors contain the long terminal repeat (LTR) sequence of Moloney murine leukemia virus (Mo-MLV) and a reporter gene (neo or lacZ) regulated by the LTR sequence of rous sarcoma virus (RSV). Because these pseudotyped retroviral vectors contain the vesicular stomatitis virus envelope glycoprotein (VSV-G), they have an extremely broad host cell range and can infect many no mammalian species. Newly fertilized medaka eggs (intact or dechorionated) were electroporated at different voltage settings in the presence of 4 x 10(4) cfu of pantropic retroviral vector. The survival rates of the pantropic retroviral vector-treated embryos ranged from 65% to 20% with increasing amplitude of electroporation. Dechorionation did not substantially affect the survival rate of embryos. PCR amplification demonstrated proviral sequences in up to 60% of the 2-month-old fish. The efficiency of gene transfer was enhanced by dechorionation. Furthermore, overnight incubation of dechorionated embryos with pantropic retroviral vectors without electroporation also resulted in proviral integration in 60% of the embryos without compromising survival rate. Southern blot analysis of DNA samples isolated from polymerase chain reaction (PCR) as positive F1 reaction animals confirmed the integration of a single copy of the provirus into the host genome. Three P1 transgenic females transmitted the proviral sequence to 50% of their F1 progeny in a back cross with wild-type males, suggesting that the entire germline of these P1 fish was transformed by the pantropic retroviral vector. Expression of the neomycin phosphotranferase transgene in F1 transgenic individuals was detected by reverse transcription (RT)-PCR amplification of the neo mRNA sequence. Furthermore, expression of a beta-galactosidase transgene was also observed in 4-day-old F1 transgenic individuals. Thus, pantropic retroviral vectors provide a convenient method to stably introduce and express foreign genes in medaka.

Animals↗
PubMed2005

Impact of formalin-fixation and paraffin-embedding on the ratio between mRNA copy numbers of differently expressed genes.

Several studies have shown that specific mRNA sequences can be successfully detected in formalin-fixed, paraffin-embedded tissues using reverse transcriptase-polymerase chain reaction (RT-PCR). Here, we test the hypothesis that gene expression levels can be accurately quantified in formalin-fixed, paraffin-embedded tissues by determining the ratio between the copy number of the mRNA molecule of interest and the mRNA copy number of a so-called housekeeping gene. The mRNA copy numbers of the variably expressed multiple drug resistance gene (MDR)-1 and four housekeeping genes (hypoxanthine phosphoribosyl-transferase-1, glyceraldehyde-3-phosphate dehydrogenase, beta-actin, and elongation factor-1a) were quantified by real-time-quantitative RT-PCR before and after formalin-fixation and paraffin-embedding of 576 tissue samples (heart, kidney, spleen, liver) from three beagle dogs. The results indicate that fixation and embedding drastically altered the ratios between the different mRNA copy numbers and that the relative expression levels of MDR-1 per any of the housekeeping genes were artificially increased or decreased up to more than tenfold. It would thus appear questionable to normalize quantitative expression data from fixed and embedded tissues by using housekeeping genes as reference. In contrast, tissue autolysis of up to 24 h and long-term storage of embedded tissues of up to 20 years had no additional effects.

Animals↗
PubMed1983

Nucleotide sequence of cloned complementary deoxyribonucleic acid for the alpha subunit of bovine pituitary glycoprotein hormones.

Recombinant DNA plasmids containing sequences coding for the alpha subunit of the bovine pituitary glycoprotein hormones have been isolated. The nucleotide sequences of three different cDNA clones have been determined. The largest alpha-subunit cDNA clone was found to contain 713 bases including 77 nucleotides from the 5'-untranslated region, 72 nucleotides coding for a precursor segment, 288 nucleotides coding for the mature alpha subunit, and 276 nucleotides from the 3'-untranslated region of the mRNA followed by a poly(A) segment. This cDNA likely represents most of the bovine alpha-subunit mRNA sequence. Nucleotide sequences were obtained from the cDNA inserts of two other alpha-subunit clones, and several differences among the three cDNA sequences have been detected. These differences in nucleotide sequence may represent either individual variation in genomic sequence or cloning artifacts. Comparison of the bovine alpha-subunit cDNA sequence to the sequences of human, rat, and mouse alpha-subunit cDNAs reveals that the bovine sequence has greater than 70% homology with the other cDNAs. The cloned alpha-subunit cDNA should provide a useful probe for further studies of the structure and expression of this interesting gene.

Amino Acid Sequence↗
PubMed1979

Chicken lysozyme gene contains several intervening sequences.

The organization of the chicken lysozyme gene and its neighboring sequences was examined by a comparison of the restriction map of the lysozyme structural gene with the map of the lysozyme gene in genomic DNA. Chicken DNA was cleaved with restriction endonucleases and the DNA fragments were separated by agarose gel electrophoresis. After transfer of the fragments onto nitrocellulose filters, those fragments that contain lysozyme mRNA sequences were detected by hybridization of the filters to labeled probes generated from pls-1, a recombinant plasmid carrying the lysozyme structural gene. This analysis revealed the presence of at least three intervening sequences, two of which interrupt the protein coding region and one of which is located in the 3' untranslated region. When oviduct DNA and sperm DNA were compared, no difference was observed in the size and number of restriction fragments that contain either lysozyme or ovalbumin structural gene sequences.

Animals↗
PubMed1993

A weight array method for splicing signal analysis.

A new method of sequence analysis, using a weight array method (WAM), which generalizes the traditional Staden weight matrix method (WMM), is proposed. With the help of a statistical mechanical model, the discriminant function is identified with the energy function describing macromolecular interactions. The method is applied to the study of 5'-splice signals in Schizosaccharomyces pombe pre-mRNA sequences. The results show that there may exist weak pairwise correlations within the signals and that our method can help to better discriminate these signals. Experiments are proposed to test the predictions of the theory.

Animals↗
PubMed2006

Controlling the rate of organic reactions: rational design of allosteric Diels-Alderase ribozymes.

Allosteric mechanisms are widely used in nature to control the rates of enzymatic reactions, but little is known about RNA catalysts controlled by these principles. The only natural allosteric ribozyme reported to date catalyzes an RNA cleavage reaction, and so do almost all artificial systems. RNA has, however, been shown to accelerate a much wider range of chemical reactions. Here we report that RNA catalysts for organic reactions can be put under the stringent control of effector molecules by straight-forward rational design. This approach uses known RNA sequences with catalytic and ligand-binding properties, and exploits weakly conserved sequence elements and available structural information to induce the formation of alternative, catalytically inactive structures. The potential and general applicability is demonstrated by the design of three different systems in which the rate of a catalytic carbon-carbon bond forming reaction is positively regulated up to 2100-fold by theophylline, tobramycin and a specific mRNA sequence, respectively. Although smaller in size than a tRNA, all three ribozymes show typical features of allosteric metabolic enzymes, namely high rate acceleration and tight allosteric regulation. Not only do these findings demonstrate RNA's power as a catalyst, but also highlight on RNA's capabilities as signaling components in regulatory networks.

Allosteric Regulation↗
PubMed1994

Isolation, characterization and structural organization of the gene and pseudogene for the dihydrolipoamide succinyltransferase component of the human 2-oxoglutarate dehydrogenase complex.

In the present study, the dihydrolipoamide succinyltransferase gene of the 2-oxoglutarate dehydrogenase complex was isolated from a human genomic DNA library and its entire nucleotide sequence was determined. This gene was approximately 23 kbp in size with 15 exons and 14 introns. All of the donor and acceptor splice sites of this gene conformed to the GT/AG rule. A guanine residue 43 bases upstreams of the ATG initiating translation codon was the transcription initiation site of the human dihydrolipoamide succinyltransferase mRNA. Sequence analysis of the promoter-regulatory region showed the presence of a CAAT-box-like sequence but the presence of a TATA-box-like sequence was not evidenced. Also located in this region were sequences resembling glucocorticoid-responsive and cAMP-responsive elements, and an Sp1 binding site. No nucleotide sequence corresponding to the E3-binding and/or E1-binding domain was found in any region of the gene. Therefore, the exon coding for the E3-binding and/or E1-binding domain may have been lost from the gene during evolution. Moreover, a processed pseudogene of dihydrolipoamide succinyltransferase was isolated and sequenced. The nucleotide sequence of the pseudogene is 93% similar to the sequence of the human dihydrolipoamide succinyltransferase cDNA, but the pseudogene is not functional for base changes, deletions and insertions of the pseudogene. Southern-blot analysis showed the presence of a single copy of this gene and a single copy of a pseudogene in the human genome. In addition, a possible relationship between dihydrolipoamide succinyltransferase and familial Alzheimer's disease is discussed.

Acyltransferases↗
PubMed2017

OCA2 splice site variant in German Spitz dogs with oculocutaneous albinism.

We investigated a German Spitz family where the mating of a black male to a white female had yielded three puppies with an unexpected light brown coat color, lightly pigmented lips and noses, and blue eyes. Combined linkage and homozygosity analysis based on a fully penetrant monogenic autosomal recessive mode of inheritance identified a critical interval of 15 Mb on chromosome 3. We obtained whole genome sequence data from one affected dog, three wolves, and 188 control dogs. Filtering for private variants revealed a single variant with predicted high impact in the critical interval in LOC100855460 (XM_005618224.1:c.377+2T>G LT844587.1:c.-45+2T>G). The variant perfectly co-segregated with the phenotype in the family. We genotyped 181 control dogs with normal pigmentation from diverse breeds including 22 unrelated German Spitz dogs, which were all homozygous wildtype. Comparative sequence analyses revealed that LOC100855460 actually represents the 5'-end of the canine OCA2 gene. The CanFam 3.1 reference genome assembly is incorrect and separates the first two exons from the remaining exons of the OCA2 gene. We amplified a canine OCA2 cDNA fragment by RT-PCR and determined the correct full-length mRNA sequence (LT844587.1). Variants in the OCA2 gene cause oculocutaneous albinism type 2 (OCA2) in humans, pink-eyed dilution in mice, and similar phenotypes in corn snakes, medaka and Mexican cave tetra fish. We therefore conclude that the observed oculocutaneous albinism in German Spitz is most likely caused by the identified variant in the 5'-splice site of the first intron of the canine OCA2 gene.

Albinism, Oculocutaneous↗
PubMed1992

Antiviral effects of herpes simplex virus specific anti-sense nucleic acids.

We have targeted mRNA sequences encompassing the translation initiation codon of the essential herpes simplex virus type 1 (HSV-1) IE3 gene with three kinds of anti-sense molecule. Addition of a 15mer oligodeoxyribonucleoside methylphosphonate to tissue culture cells resulted in suppression of viral replication. HSV-1 replication was also inhibited in cultured cells containing anti-sense vectors expressing transcripts complementary to the IE3 mRNA. We have also constructed a ribozyme which upon base pairing with the target IE3 mRNA induces cleavage at the predicted GUC site. A major obstacle to anti-sense studies in animals is drug delivery of preformed antisense molecules to ganglionic neurons, the site of HSV latency and reactivation. We speculate as to how this may be accomplished through carrier compounds which are taken up by nerve terminals and transported by retrograde axoplasmic flow. By the same route, HSV itself may be used as an anti-sense vector.

Animals↗