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PubMed1984

Analysis of the Complexity and Diversity of mRNAs from Pollen and Shoots of Tradescantia.

The mRNAs of the mature pollen grain of Tradescantia paludosa at anesthesia and of vegetative shoots have been compared by analyzing the kinetics of hybridization between homologous and heterologous reactions of cDNA to poly(A)RNA in excess. The mRNAs in pollen can be divided into three abundance classes with complexities of 5.2 x 10(4), 1.6 x 10(6), and 2.1 x 10(7) nucleotides. The three classes are made up of sequences that constitute 15, 60, and 24% of the mRNAs and each sequence is present on an average at 26,000, 3,400, and 100 copies, respectively, per pollen grain. About 20,000 different genes are expressed in pollen as compared to about 30,000 in vegetative shoots. Estimates have been made of pollen mRNA sequences shared with those of shoot tissue and of shoot sequences common to those in pollen.

Journal Article↗
PubMed2003

Identification of p53 target genes by fluorescent differential display.

Differential display (DD) is a method used worldwide for identifying differentially expressed genes in eukaryotic cells. The mRNA DD technology works by systematic amplification of the 3' terminal regions of mRNAs. Using anchored primers designed to bind 5' boundary of the polyA tails for reverse transcription, followed by polymerase chain reaction (PCR) amplification with additional upstream primers of arbitrary sequences, mRNA subpopulations are separated by denaturing polyacrylamide electrophoresis. This allows direct side-by-side comparison of most of the mRNAs between or among related cells. Because of its simplicity, sensitivity, and reproducibility, the mRNA DD method is finding wide-ranging and rapid applications in developmental biology, cancer research, neuroscience, pathology, endocrinology, plant physiology, and many other areas. Since the recent development of the fluorescent differential display (FDD), the first nonradioactive DD system with equivalent sensitivity to the original 33P isotopic labeling method, it is now possible with this technology to automate, which can greatly increase the throughput and accuracy of mRNA DD.

Cells, Cultured↗
PubMed1982

Isolation of multiple genomic sequences coding for chicken myosin heavy chain protein.

A genomic bank has been constructed using DNA isolated from a dystrophic strain of chickens. The library was screened for myosin heavy chain (MHC) sequences using a cDNA probe and 11 positive recombinants isolated. Identity of the clones was confirmed by positive RNA selection via hybridization of the clones with muscle RNA and subsequent translation of the hybridizable RNA in vitro. Restriction maps indicate that the clones can be divided into 7 distinct groups; some of these groups do, however, share similar or homologous sequences. Hydridization of the clones to RNA derived from leg, breast, heart, and brain reveals that all of the clones code for the muscle-specific MHC isoforms. These experiments also show that there are at least 3 size classes of MHC mRNA sequences, whose relative amounts appear to be modulated in a tissue- and developmental stage-specific manner.

Animals↗
PubMedDate not supplied

A class of edit kernels for SVMs to predict translation initiation sites in eukaryotic mRNAs.

The prediction of translation initiation sites (TISs) in eukaryotic mRNAs has been a challenging problem in computational molecular biology. In this paper, we present a new algorithm to recognize TISs with a very high accuracy. Our algorithm includes two novel ideas. First, we introduce a class of new sequence-similarity kernels based on string editing, called edit kernels, for use with support vector machines (SVMs) in a discriminative approach to predict TISs. The edit kernels are simple and have significant biological and probabilistic interpretations. Although the edit kernels are not positive definite, it is easy to make the kernel matrix positive definite by adjusting the parameters. Second, we convert the region of an input mRNA sequence downstream to a putative TIS into an amino acid sequence before applying SVMs to avoid the high redundancy in the genetic code. The algorithm has been implemented and tested on previously published data. Our experimental results on real mRNA data show that both ideas improve the prediction accuracy greatly and that our method performs significantly better than those based on neural networks and SVMs with polynomial kernels or Salzberg kernels.

Algorithms↗
PubMed1986

Nucleotide sequence of the mRNA encoding the cytosolic form of phosphoenolpyruvate carboxykinase (GTP) from the chicken.

We have determined the sequence of the mRNA encoding cytosolic phosphoenolpyruvate carboxykinase (GTP) [GTP:oxaloacetate carboxy-lyase (transphosphorylating), EC 4.1.1.32] from the chicken and have deduced the primary structure of the protein. The message for the enzyme is 2762 bases long and encodes a protein of 622 amino acids with a molecular mass of 69,522 daltons. The 5' untranslated region is 246 nucleotides long and contains two nonfunctional AUG initiator codons. The 3' untranslated sequence is 649 bases long and contains multiple polyadenylylation signals. There are regions of dyad symmetry and an A + U-rich region within the 3' translated and untranslated sequences of the message. Such regions are also present in the mRNA for the enzyme from the rat and may be of functional significance. Conserved regions of the enzyme, that may interact with substrates, were identified by comparing the amino acid sequence of phosphoenolpyruvate carboxykinase with that of other proteins that use guanine nucleotides and phosphoenolpyruvate as substrates.

Amino Acid Sequence↗
PubMed2001

Relationship between the increase of secretion of sTNF alpha induced by lipopolysaccharides and the enhanced expression of TACE mRNA in HL-60 cells and adhesive cells from human spleen.

In order to find out the potential modulators which influence the secretion of TNF alpha, the relationship between the amount of secreted TNF alpha (sTNF alpha) and the level of TNF alpha converting enzyme (TACE) gene expression was studied before and after the stimulation by lipopolysaccharides (LPS) to HL-60 cells and adhered cells isolated from human spleen using cytological and molecular biology techniques and methods (RT-PCR, Dot-blot hybridization, etc.). The experimental results showed that: (1) LPS could induce the increase of expression of TNF alpha mRNA and TACE mRNA, reaching the peak value at 6 h and 10 h respectively after addition of LPS into cell culture medium; (2) The anti-sense oligodeoxyribonucleotide (A-ODN) complementary to TACE mRNA sequence really inhibited the secretion of TNF alpha as a result of blocked translation of TACE gene; (3) Furthermore, it was also observed that RDQ, a kind of injection derived from Chinese traditional herb, had strongly inhibitory effects on the expression of TACE mRNA and secretion of sTNF alpha stimulated by LPS. The above results suggested that the TACE indeed involved in delivering and processing of pro-TNF alpha during the period of LPS stimulation and the study about the regulator/inhibitors of TACE gene expression would be very important to develop new types of therapy agents against toxic and side effects of sTNF alpha during the period of infection/inflammation to human body.

ADAM Proteins↗
PubMed2001

Target gene identification from expression array data by promoter analysis.

DNA microchips and expression arrays yield enormous amounts of data linking cDNA sequences to gene expression patterns. This now allows the characterization of gene expression in normal and diseased tissues as well as the response of tissues to the application of therapeutic reagents. Software currently exists to analyze DNA array/chip data with respect to corresponding mRNA sequences, which facilitates the precise determination of when and where certain groups of genes are expressed. The information concerning transcriptional regulatory networks responsible for the observed expression patterns is not contained within the cDNA sequences used to generate the arrays, but resides often within the promoter sequences of the individual genes (and/or enhancers). The complete sequence of the human genome will provide the molecular basis for the identification of such regulatory regions. Promoter sequences for specific cDNAs can be obtained reliably from genomic sequences simply by exon mapping. Promoter prediction tools can also be used to locate promoters directly in the genomic sequence in many cases in which cDNAs are 5'-incomplete. Once sufficient numbers of promoter sequences have been obtained, the comparative promoter analysis of the co-regulated genes and groups of genes can be applied in order to generate models describing the higher order levels of the transcription factor binding site organization within these promoter regions. As evident from several examples, this approach can identify promoter modules responsible for the common regulation of promoters solely by the application of bioinformatics methods. Such modules represent the molecular mechanisms through which regulatory networks influence gene expression. Another advantage of this approach is that it also provides a powerful alternative for elucidating functional features of genes with no detectable sequence similarity, by linking them to other genes on the basis of their common promoter structures.

Computational Biology↗
PubMed1995

Exon and intron sequences, respectively, repress and activate splicing of a fibroblast growth factor receptor 2 alternative exon.

Two alternative exons, BEK and K-SAM, code for part of the ligand binding site of fibroblast growth factor receptor 2. Splicing of these exons is mutually exclusive, and the choice between them is made in a tissue-specific manner. We identify here pre-mRNA sequences involved in controlling splicing of the K-SAM exon. The short K-SAM exon sequence 5'-TAGGGCAGGC-3' inhibits splicing of the exon. This inhibition can be overcome by mutating either the exon's 5' or 3' splice site to make it correspond more closely to the relevant consensus sequence. Two separate sequence elements in the intron immediately downstream of the K-SAM exon, one of which is a sequence rich in pyrimidines, are both needed for efficient K-SAM exon splicing. This is no longer the case if either the exon's 5' or 3' splice site is reinforced. Furthermore, if the exon inhibitory sequence is removed, the intron sequences are not required for splicing of the K-SAM exon in a cell line which normally splices this exon. At least three elements are thus involved in controlling splicing of the K-SAM exon: suboptimal 5' and 3' splice sites, an exon inhibitory sequence, and intron activating sequences.

Alternative Splicing↗
PubMed1997

cDNAs for a chymotrypsinogen-like protein from two strains of Plodia interpunctella.

Gut proteinases are involved in the solubilization and activation of insecticidal toxins produced by Bacillus thuringiensis and may also be involved in resistance development. Approximately threefold lower chymotrypsin-like enzyme activity was observed in a Bt(entomocidus)-resistant strain of the Indianmeal moth, Plodia interpunctella, than that in the Bt-susceptible strain. Because chymotrypsin-like proteinases are involved in Bt protoxin activation in P. interpunctella, we compared cDNA sequences, mRNA expression levels, and genomic DNA for chymotrypsin-like enzymes in Bt-susceptible and Bt-resistant strains of P. interpunctella. To isolate cDNA coding for chymotrypsinogen-like proteinases, a probe was developed using polymerase chain reaction (PCR) amplification of a cDNA library from the Bt-susceptible strain using a vector primer and a degenerate primer corresponding to a conserved sequence in the active site of serine proteinases. This probe was used to screen cDNA libraries from resistant and susceptible strains. Predicted amino acid sequences from cDNA clones of each strain share similarity with sequences of chymotrypsin-like proteinases and are most similar to a chymotrypsin-like proteinase from the tobacco hornworm, Manduca sexta. cDNAs for putative chymotrypsinogen-like proteins, from both Bt-susceptible and Bt-resistant strains of P. interpunctella share an identical open reading frame of 846 nucleotides. The encoded proteins contain amino acid sequence motifs of serine proteinase active sites, disulfide-bridge cysteine residues, and both zymogen activation and signal peptides. A difference between these cDNAs was observed only in the untranslated region where a substitution of guanine for adenine occurred in the Bt-resistant strain. Southern and Northern blotting analyses indicated that there are no major differences in chymotrypsinogen-like genomic organization and mRNA expression in the two strains. These data suggest that chymotrypsinogen-like proteinase genes and their transcription are similar in the Bt-susceptible and Bt-resistant strains of P. interpunctella.

Amino Acid Sequence↗
PubMed1992

Sequence of the S-layer gene of Thermus thermophilus HB8 and functionality of its promoter in Escherichia coli.

The nucleotide sequence of the slpA gene, which is responsible for the synthesis of the S-layer protein of Thermus thermophilus HB8, is described. This gene is transcribed as a unit in which the coding region is preceded by a 127-base-long leader mRNA sequence. The promoter region is also recognized by the RNA polymerase of Escherichia coli because of the presence of homologous -35 and -10 boxes. Homologies with other promoters from Thermus spp. are also presented.

Amino Acid Sequence↗
PubMed2004

Gene expression profile of the rat eye iridocorneal angle: NEIBank expressed sequence tag analysis.

PURPOSE: To characterize gene expression pattern in the combined tissues of the rat iridocorneal angle by expressed sequence tag (EST) analysis, as part of the NEIBank project. METHODS: RNA was extracted from dissected tissues of the rat iridocorneal angle (iris, ciliary body, trabecular meshwork, and Schlemm's canal) and used to construct unamplified, non-normalized cDNA libraries in the pSPORT1 vector. Approximately 5000 clones were sequenced from the 5'-end. Clones were clustered and identified using the GRIST software, a procedure based on BLAST comparisons. Complete sequences of several novel cDNAs showing eye-preferred expression patterns were obtained. The expression patterns of several genes have been investigated by Northern blot and in situ hybridization, as well as by RT-PCR. RESULTS: After analysis and removal of non-mRNA sequences, 2195 independent clusters, potentially representing individual eye angle-expressed clones were obtained. The expression profile of the combined rat eye angle tissues was more similar to that of the human iris than to human trabecular meshwork. Several cDNAs encoding transcription factors essential for normal eye development and function including Pax-6, Six3, c-Maf, Maf1, Sox-4, Foxc1, Rx, and Ldb2 were present among sequenced clones. A number of tested cDNAs showed eye-preferred expression patterns. Myocilin, which is abundant in human eye angle tissues, was not observed in the rat collection; however, transcripts for three other olfactomedin-domain proteins were seen. Latrotoxin receptor (CL1AA) and optimedin were shown to be expressed in the iris and ciliary body, as well as in the ganglion and inner nuclear cell layers of the retina, whereas the rat orthologue of the human HNOEL-iso gene was expressed in the iris and sclera and less actively in the trabecular meshwork, retina, and optic nerve. CONCLUSIONS: The iridocorneal libraries are a good source of novel uncharacterized genes and molecular markers for the tissues of the eye angle. Although myocilin is not abundantly expressed in rat eye angle, other olfactomedin-containing genes are expressed there and may play important roles in normal eye function and disease.

Amino Acid Sequence↗
PubMed2003

The cone visual pigments of an Australian marsupial, the tammar wallaby (Macropus eugenii): sequence, spectral tuning, and evolution.

Studies on marsupial color vision have been limited to very few species. There is evidence from behavioral, electroretinographic (ERG), and microspectrophotometric (MSP) measurements for the existence of both dichromatic and trichromatic color vision. No studies have yet investigated the molecular mechanisms of spectral tuning in the visual pigments of marsupials. Our study is the first to determine the mRNA sequence, infer the amino acid sequence, and determine, by in vitro expression, the spectra of the cone opsins of a marsupial, the tammar wallaby (Macropus eugenii). This yielded some information on mechanisms and evolution of spectral tuning of these pigments. The tammar wallaby retina contains only short-wavelength sensitive (SWS) and middle-wavelength sensitive (MWS) pigment mRNAs. This predicts dichromatic color vision, which is consistent with conclusions from previous behavioral studies ( Hemmi 1999). We found that the wallaby has a SWS1 class pigment of 346 amino acids. Sequence comparison with eutherian SWS pigments predicts that this SWS1 pigment absorbs maximally (lambdamax) at 424 nm and, therefore, is a blue rather than a UV pigment. This (lambdamax) is close to that of the in vitro-expressed wallaby SWS pigment (lambdamax of 420 +/- 2 nm) and to that determined behaviorally (420 nm). The difference from the mouse UV pigment (lambdamax of 359 nm) is largely accounted for by the F86Y substitution, in agreement with in vitro results comparing a variety of other SWS pigments. This suggests that spectral tuning employing F86Y substitution most likely arose independently in the marsupials and ungulates as a result of convergent evolution. An apparently different mechanism of spectral tuning of the SWS1 pigments, involving five amino acid positions, evolved in primates. The wallaby MWS pigment has 363 amino acids. Species comparisons at positions critical to spectral tuning predict a lambdamax near 530 nm, which is close to that of the in vitro-expressed pigment (529 +/- 1 nm), but quite different from the value of 539 nm determined by microspectrophotometry. Introns interrupt the coding sequences of the wallaby, mouse, and human MWS pigment sequences at the same corresponding nucleotide positions. However, the length of introns varies widely among these species.

Amino Acid Sequence↗
PubMed2001

Differential global gene expression in red and white skeletal muscle.

The differences in gene expression among the fiber types of skeletal muscle have long fascinated scientists, but for the most part, previous experiments have only reported differences of one or two genes at a time. The evolving technology of global mRNA expression analysis was employed to determine the potential differential expression of approximately 3,000 mRNAs between the white quad (white muscle) and the red soleus muscle (mixed red muscle) of female ICR mice (30-35 g). Microarray analysis identified 49 mRNA sequences that were differentially expressed between white and mixed red skeletal muscle, including newly identified differential expressions between muscle types. For example, the current findings increase the number of known, differentially expressed mRNAs for transcription factors/coregulators by nine and signaling proteins by three. The expanding knowledge of the diversity of mRNA expression between white and mixed red muscle suggests that there could be quite a complex regulation of phenotype between muscles of different fiber types.

Animals↗
PubMed2004

Isolation of novel mouse genes that were differentially expressed in W/W(V) mouse fundus.

BACKGROUND: Using cDNA microarray analysis that displays a total of 8734 mouse genes, we have identified 21 known and novel transcripts, including mouse expressed sequence tags (ESTs) that were consistently up- or downregulated in the fundus of wild-type mice and W/W(V) mice, where intramuscular interstitial cells of Cajal (ICC; IC-IM) are lost. METHODS: By using these tags as part of the full-length mRNA sequence of the murine genes, we screened a mouse-brain cDNA library and the FASTA database (http://www.ebi.ac.uk/fasta33/). RESULTS: Three of the queries were identified as novel mouse genes, whereas six transcripts had their human counterparts that were known to encode functional proteins. Four transcripts, DRIM (downregulated in metastasis), SLP8 (tumor antigen), PTK7/CCK4 (receptor protein tyrosine kinase-like molecule-7/colon carcinoma kinase-4), and a novel gene AWMS2 were increased in W/W(V) mice. Expression of another five genes was decreased in W/W(V) mice: BB1 (overexpressed in bladder and breast carcinoma), HTATIP/CPLA2 (HIV-1 TAT interactive protein/cytosolic phospholipase A2), Tenascin-receptor like (Hexabrachion, Cytotactin, Neuronectin, Myotendinous antigen), and two novel transcripts: DRWMS1 and DRWMS2. Molecular profiling generated by cDNA microarray analysis from the fundus of W/W(V) mice and its complete list of full-length cDNAs will be shown on our web site (http://www.yamanashi.ac.jp/). CONCLUSIONS: Differential gene comparisons between control and mutant animals with losses in specific populations of ICCs will contribute significantly to our understanding of motility disorders associated with the loss of these cells and electrical slow waves in the gastrointestinal tract.

Animals↗
PubMed1992

The nucleotide sequence of the variable region in Trypanosoma brucei completes the sequence analysis of the maxicircle component of mitochondrial kinetoplast DNA.

The nucleotide sequence of two non-contiguous DNA fragments of 4.0 and 2.2 kb, respectively, of the kinetoplast maxicircle of Trypanosoma brucei brucei EATRO strain 427 has been determined, completing the sequence analysis of the so-called variable region (see also de Vries et al., 1988, Mol. Biochem. Parasitol. 27, 71-82). Analysis of the entire 8-kb variable region sequence revealed the presence of a 5.2-kb cluster of imperfect, tandemly repeated sequences, flanked by DNA of unique sequence. Both repetitive and unique DNA evolve rapidly, but comparison to the closely related strain EATRO 164 indicated that the repetitive cluster is more prone to sequence and size divergence. The variable region is transcribed into RNAs of varying lengths but appears to be devoid of genes encoding mitochondrial proteins or tRNAs, as judged from computer analysis. Moreover, genes that could encode guide RNAs involved in producing the known edited mitochondrial mRNA sequences are also absent. The repetitive DNA cluster within this region consists of 14 blocks each containing one 130 bp repeat and a variable number of 19 bp repeats. A duplicated sequence was identified (5'-GGGGTTGGTGT) which proved to be identical to the eleven 5'-terminal residues of the universal minicircle dodecamer involved in initiation of leading strand synthesis. This suggests a role for these sequences in the initiation of maxicircle DNA replication. With the data presented in this report, the nucleotide sequence analysis of the 23016 bp maxicircle of T. brucei brucei EATRO strain 427 has been completed.

Animals↗
PubMed2001

ARED: human AU-rich element-containing mRNA database reveals an unexpectedly diverse functional repertoire of encoded proteins.

The adenylate uridylate-rich elements (AREs) mediate the rapid turnover of mRNAs encoding proteins that regulate cellular growth and body response to exogenous agents such as microbes, inflammatory and environmental stimuli. However, the full repertoire of ARE-containing mRNAs is unknown. Here, we explore the distribution of AREs in human mRNA sequences. Computational derivation of a 13-bp ARE pattern was performed using multiple expectation maximization for motif elicitations (MEME) and consensus analyses. This pattern was statistically validated for the specificity towards the 3'-untranslated region and not coding region. The computationally derived ARE pattern is the basis of a database which contains non-redundant full-length ARE-mRNAs. The ARE-mRNA database (ARED; http://rc.kfshrc.edu.sa/ared) reveals that ARE-mRNAs encode a wide repertoire of functionally diverse proteins that belong to different biological processes and are important in several disease states. Cluster analysis was performed using the ARE sequences to demonstrate potential relationships between the type and number of ARE motifs, and the functional characteristics of the proteins.

Adenosine↗
PubMedDate not supplied

mRNA expression of components of the insulin-like growth factor system in breast cancer cell lines, tissues, and metastatic breast cancer cells.

IGF-1 and 2 are thought to be important growth factors for breast cancer. However, gene expression of IGFs or IGF receptors in breast cancer tissues, and especially in metastatic breast cancer cells, is not well known. Expression of mRNA encoding for IGF-1, IGF-2, IGF-receptor 1 and 2, IGF binding proteins- 1 to -6, insulin receptor and insulin was determined in the NIH MCF-7 breast cancer cell line, in specimens from breast cancer tissues, and in 6 primary breast cancer cell cultures obtained from metastatic breast cancer, using rT-PCR technique. Specific mRNA sequences encoding for IGF-receptor 1 and 2, IGFBP-2, -4 and insulin receptor were identified in all cell cultures and most of the tissue specimens. Though in most of the tissues additional expression of IGF-1 and IGF-2 was detected, there was no mRNA encoding for these proteins in MCF-7 cell cultures as well as in the primary cell cultures of metastatic breast cancers. In none of our specimens mRNA encoding for IGFBP-1, -3, -5, -6 and insulin was detectable. IGF-receptor expression in cancer tissues and metastatic breast cancer cells supports the hypothesis that IGFs increase tumor cell proliferation in vivo. Expression of IGF-1 and IGF-2 in tumor tissues but not in cancer cell cultures indicates an IGF expression located predominantly in stromal parts of cancer tissues.

Breast Neoplasms↗
PubMed1985

Ubiquitin is a heat shock protein in chicken embryo fibroblasts.

Clones containing heat-inducible mRNA sequences were selected from a cDNA library prepared from polyadenylated RNA isolated from heat-shocked chicken embryo fibroblasts. One recombinant DNA clone, designated clone 7, hybridized to a 1.2-kilobase RNA that was present in normal cells and increased fivefold during heat shock. Clone 7 also hybridized to an RNA species of 1.7 kilobases that was present exclusively in heat-shocked cells. In vitro translation of mRNA hybrid selected from clone 7 produced a protein product with a molecular weight of approximately 8,000. Increased synthesis of a protein of similar size was detected in chicken embryo fibroblasts after heat shock. DNA sequence analysis of clone 7 indicated its protein product has amino acid sequences identical to bovine ubiquitin. In addition, clone 7 contains tandem copies of the ubiquitin sequences contiguous to each other with no untranslated sequences between them. We discuss some possible roles for ubiquitin in the heat shock response.

Amino Acid Sequence↗