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At least 289 records · Page 16Linked to original sources
PubMed1999

Rubisco activase transcript (rca) abundance increases when the marine unicellular green alga Chlorococcum littorale is grown under high-CO2 stress.

cDNA and the corresponding genomic DNA region encoding Rubisco activase were isolated from the unicellular green alga Chlorococcum littorale. The deduced amino acid sequence encoded by the cDNA was 403 amino acids long and exhibited important homology with those of other known Rubisco activases. Its N-terminal sequence was similar to the chloroplastic transit peptides in Chlamydomonas reinhardtii. The mature protein had a predicted molecular mass of 42 kDa. Five introns were located inside the genomic gene encoding Rubisco activase (rca). Genomic Southern blots indicated that two copies of the rca gene were present in the genome of C. littorale. The level of rca messenger RNA increased when cells of C. littorale were subjected to high-CO2 stress (i.e. grown under at least 20% CO2). Hsp70 heat-shock protein was also induced under high-CO2 conditions and, as expected, was also induced at 35 degrees C. The rca gene, in contrast, was not induced at 35 degrees C, indicating that this gene was induced in response to the high CO2 concentration and not to general stress. A search of the promoter-binding proteins by a gel retardation assay showed that, under the high-CO2 conditions, a protein(s) which was probably an activator of the rca transcription was synthesized.

Amino Acid Sequence↗
PubMed2006

Design of microarray experiments for genetical genomics studies.

Microarray experiments have been used recently in genetical genomics studies, as an additional tool to understand the genetic mechanisms governing variation in complex traits, such as for estimating heritabilities of mRNA transcript abundances, for mapping expression quantitative trait loci, and for inferring regulatory networks controlling gene expression. Several articles on the design of microarray experiments discuss situations in which treatment effects are assumed fixed and without any structure. In the case of two-color microarray platforms, several authors have studied reference and circular designs. Here, we discuss the optimal design of microarray experiments whose goals refer to specific genetic questions. Some examples are used to illustrate the choice of a design for comparing fixed, structured treatments, such as genotypic groups. Experiments targeting single genes or chromosomic regions (such as with transgene research) or multiple epistatic loci (such as within a selective phenotyping context) are discussed. In addition, microarray experiments in which treatments refer to families or to subjects (within family structures or complex pedigrees) are presented. In these cases treatments are more appropriately considered to be random effects, with specific covariance structures, in which the genetic goals relate to the estimation of genetic variances and the heritability of transcriptional abundances.

Animals↗
PubMed1998

CYP21 pseudogene transcripts are much less abundant than those from the active gene in normal human adrenocortical cells under various conditions in culture.

The human steroid 21-hydroxylase pseudogene (CYP21P, also termed CYP21A) is transcribed in the adrenal cortex, but the relative abundance of transcripts from CYP21P and from the active CYP21 gene (also termed CYP21B) is not well established. In the present experiments we cultured primary human adrenocortical cells in defined medium and used RNase protection assays to examine whether there might be a selective increase in the relative abundance of CYP21P transcripts under any of the various regulatory factors known to affect expression of 21-hydroxylase. Differences between the sequences of intron 2 in CYP21P and CYP21 allowed the synthesis of gene-specific probes spanning exon 3 and parts of the adjacent introns. CYP21- and CYP21P-specific probes spanning the site of the start of transcription were also synthesized. CYP21 transcripts were readily detectable. In agreement with previous observations on 21-hydroxylase mRNA and enzyme activity in primary cultures of human adrenocortical cells, the abundance of CYP21 transcripts was increased by cyclic AMP analogues (N6-monobutyryl cyclic AMP and 8-bromo cyclic AMP), insulin, IGF-I and tetradecanoyl phorbol acetate (TPA). However, CYP21P transcripts were not detected in the presence of any of the various regulatory factors known to affect expression of 21-hydroxylase.

Adrenal Cortex↗
PubMed2005

Sugarbeet sucrose synthase genes differ in organ-specific and developmental expression.

A full-length sucrose synthase (SBSS2) cDNA clone was isolated from sugarbeet. Comparison of its composition and organ-specific and developmental expression with a previously isolated sugarbeet sucrose synthase gene (SBSS1) revealed distinct differences between the two genes. The two genes share 80% similarity in deduced amino acid sequence but belong to different sucrose synthase subclasses based on phylogenic analysis. Both sucrose synthases were highly expressed in roots, and had low levels of expression in leaf tissue. Transcript abundance of SBSS2, relative to SBSS1, was greater in young vegetative and floral tissues, and reduced in mature vegetative tissues. The organ-specific and developmental expression of SBSS1 and SBSS2 proteins was similar to SBSS1 and SBSS2 transcript levels, although developmental changes in protein abundance lagged transcriptional changes by many weeks. The similarities and differences in transcript and protein abundance suggest that both transcriptional and post-transcriptional regulatory mechanisms are likely to contribute to sucrose synthase expression in sugarbeet.

Base Sequence↗
PubMed2005

EST sequencing for gene discovery in Chinese hamster ovary cells.

Chinese hamster ovary (CHO) cells are one of the most important cell lines in biological research, and are the most widely used host for industrial production of recombinant therapeutic proteins. Despite their extensive applications, little sequence information is available for molecular based research. To facilitate gene discovery and genetic engineering, two cDNA libraries were constructed from three CHO cell lines grown under various conditions. The average insert size for both libraries is approximately 800-850 bp, and each library has comparable redundancy levels of 36%-38% for the sequences isolated. Random sequencing of 4,608 ESTs yielded 2,602 unique assemblies, 76% of which were annotated as orthologs of sequences in the GenBank database. A high abundance of mitochondrial genome transcripts facilitated the assembly of the complete mitochondrial genome by PCR walking. Comparative analysis of sequences from both mitochondrial and nuclear genomes with orthologous genes from other species shows that CHO sequences are generally most similar to mouse; however, examples with highest similarity to rat or human are common. A cDNA microarray, including all 4,608 ESTs, was constructed. The microarray results reveal a high level of consistency between transcript abundance in the libraries and fluorescence intensities. Inclusion of redundant clones in the microarray, additionally, allows small changes in abundant mRNAs to be discerned with a high degree of confidence. The information and tools generated provide access to genomic technology for this important cell line.

Amino Acid Sequence↗
PubMed1995

Molecular cloning and heterologous expression of acridone synthase from elicited Ruta graveolens L. cell suspension cultures.

Cell suspension cultures of Ruta graveolens L. produce a variety of acridone alkaloids, and the accumulation can be stimulated by the addition of fungal elicitors. Acridone synthase, the enzyme catalyzing the synthesis of 1,3-dihydroxy-N-methylacridone from N-methylanthraniloyl-CoA and malonyl-CoA, had been isolated from these cells, and the partial enzyme polypeptide sequence, elucidated from six tryptic fragments, revealed homology to heterologous chalcone synthases. Poly(A)+ RNA was isolated from Ruta cells that had been treated for 6 h with a crude cell wall elicitor from Phytophthora megasperma f. sp. glycinea, and a cDNA library was constructed in lambda 2AP. Clones harboring acridone synthase cDNA were isolated from the library by screening with a synthetic oligonucleotide probe complementary to a short stretch of sequence of the enzyme peptide with negligible homology to chalcone synthases. The identity of the clones was substantiated by DNA sequencing and by recognition of five additional peptides, determined previously from tryptic acridone synthase digests, in the translated sequence. An insert of roughly 1.4 kb encoded the complete acridone synthase, and alignments at both DNA and protein levels corroborated the high degree of homology to chalcone synthases. Expression of the enzyme in vector pET-11c in the Escherichia coli pLysS host strain proved the identity of the cloned cDNA. The heterologous enzyme in the crude E. coli extract exhibit high acridone but no chalcone synthase activity. The results were fully supported by northern blot hybridizations which revealed that the specific transcript abundance did not increase but rather decreased upon white light irradiation of cultured Ruta graveolens L. cells, a condition that commonly induces the abundance of chalcone synthase transcripts.

Acyltransferases↗
PubMed1991

Corticotropin-releasing factor upregulates expression of two truncated pro-opiomelanocortin transcripts in murine lymphocytes.

Lymphocytes harbor a pro-opiomelanocortin (POMC) mRNA. In this report, a novel procedure was used to study the exonic arrangement of this transcript in lymphocytes. Poly(A)+ mRNA, purified from both corticotropin-releasing factor (CRF)-treated and nontreated lymphocytes, was selectively reverse-transcribed using an antisense oligonucleotide primer complementary to the 3' junction of the translated/nontranslated region of exon 3 of POMC. Alkaline agarose gel analysis of first-strand cDNA synthesis showed an upregulation of POMC transcripts in CRF-treated cells. This first-strand cDNA was amplified in a polymerase chain reaction (PCR) using the complementary antisense primer and selective sense primers homologous to the 5' ends of exons 1, 2, and 3, as well as a region immediately 5' to the ACTH/beta-lipotropin coding region of exon 3 of pituitary POMC. Primers directed at exons 1 and 2 did not amplify a POMC product in nontreated control or CRF-treated cells. However, with both treated and nontreated cells, the internal exon 3 primer amplified the expected size exon 3 DNA fragment (approximately 549 bp). Interestingly, a primer directed at the 5' end of exon 3 apparently did not amplify a POMC product in nontreated cells but did amplify a full-size POMC exon 3 from CRF-treated cells (approximately 615 bp). However, upon reamplification of the original PCR products from nontreated cells, full-length exon 3 product was also observed. Southern gel analysis using a pituitary POMC cDNA probe showed that all of the above PCR products were POMC-related. The results of this study show that lymphocytes basally transcribe at least two POMC transcripts that are upregulated by CRF. These two transcripts lack exons 1 and 2 but contain either part or all of exon 3. The smaller exon 3 transcript was the most abundant transcript under all conditions examined.

Adrenocorticotropic Hormone↗
PubMed2002

Interferon-gamma reduces melanosomal antigen expression and recognition of melanoma cells by cytotoxic T cells.

In malignant melanoma, tumor-infiltrating lymphocytes are frequently reactive with melanosomal antigens. Achieving complete remissions by peptide therapy is frequently hampered by metastases evading immune recognition. The tumor microenvironment seems to favor reduced expression of target antigens by melanoma cells. Among candidate factors, interferon-gamma (IFN-gamma) (10(2) to 10(3) U/ml) suppressed expression of antigens MART-1, TRP-1, and gp100 by M14 melanoma cells as shown by immunohistology and fluorescence-activated cell sorting analysis, reducing MART-1 expression by >65%. Northern blot analysis revealed that reduced expression was regulated at the transcriptional level, demonstrating a 79% reduction in MART-1 transcript abundance after 32 hours of IFN-gamma treatment. To evaluate consequences of IFN-gamma exposure for immune recognition, MART-1-responsive T cells were reacted with pretreated HLA-matched melanoma cells. Cytotoxicity was reduced up to 78% by IFN-gamma pretreatment, and was restored by addition of MART-1 peptide AAGIGILTV for 2 hours. Examination of melanoma lesions by quantitative reverse transcriptase-polymerase chain reaction revealed up to 188-fold more abundant IFN-gamma transcripts when compared to control skin. Laser capture microdissection and immunohistology localized most IFN-gamma-producing T cells to the tumor stroma. Reduced MART-1 expression was frequently observed in adjacent tumor cells. Consequently, IFN-gamma may enhance inflammatory responses yet hamper effective recognition of melanoma cells.

Antigens, Neoplasm↗
PubMed2005

Gene expression profiling in the brains of human cocaine abusers.

Chronic cocaine abuse induces long-term neurochemical, structural and behavioural changes thought to result from altered gene expression within the nucleus accumbens and other brain regions playing a critical role in addiction. Recent methodological advances now allow the profiling of gene expression in human postmortem brain. In this article, we review studies in which we have used Affymetrix oligonucleotide microarrays to identify transcripts that are differentially expressed in the nucleus accumbens of cocaine abusers in comparison to well-matched control subjects. Of the approximately 39,000 gene transcripts interrogated, the expression of only a fraction of 1% is significantly modified in cocaine abusers. Found within this list are equivalent incidences of increased and decreased transcript abundance, including known gene transcripts clustered into several functional categories. A striking exception is a group of myelin-related genes, consisting of multiple transcripts representing myelin basic protein (MBP), proteolipid protein (PLP) and myelin-associated oligodendrocyte basic protein (MOBP), which as a group are substantially decreased in cocaine abusers compared to controls. These data, suggesting a possible dysregulation of myelin in cocaine abusers, are discussed in the context of myelin-related changes in other human brain disorders. Finally, the effects of cocaine abuse on the profile of gene expression in some other brain regions critical for addiction (the prefrontal cortex and ventral midbrain) are briefly reviewed.

Brain↗
PubMed2003

Determination of the transcript profile of human endometrium.

The response of the human endometrium to the ovarian hormones, estrogen and progesterone, has been the focus of decades of research. In order to understand this critical aspect of endometrial physiology, we undertook a genome-wide analysis of transcript abundance and changes in transcript level between normal endometrium in the proliferative and secretory phases of the menstrual cycle. A high-density, oligonucleotide gene array, comprising 60 000 gene targets, was used to define the gene expression profile of proliferative and secretory phase endometrium. Results from the arrays were verified using real-time PCR. The expression levels of 149 transcripts differed significantly between the two phases of the cycle determined by stringent range limits (99.99%), calculated using local variance values. These transcripts include previously documented steroidally responsive genes (such as placental protein 14 and stromelysin-3) and novel transcripts not previously linked to either endometrial physiology or steroid regulation (such as intestinal trefoil factor and a number of expressed sequence tags). Examination of the 5' promoter regions of these genes identified many putative estrogen and progesterone receptor DNA binding domains, suggesting a direct response of these genes to the ovarian hormones.

Base Sequence↗
PubMed1995

Minimal requirements of the Streptomyces lividans 66 oriC region and its transcriptional and translational activities.

Deletion analysis of a previously constructed minichromosome revealed that a stretch of DNA which is longer than 623 bp but shorter than 837 bp is required for autonomous replication of the Streptomyces lividans chromosome. Each of the dnaA and dnaN genes flanking the oriC region is individually transcribed from two promoters. Within the intergenic, nontranslatable region between the dnaA and dnaN genes, five main transcripts and several less abundant transcripts of various lengths as well as one of the promoters were identified. The introduction of additional DnaA boxes in S. lividans led to a significant increase in dnaA gene transcripts and to an enhanced level of the DnaA (73-kDa) protein. In summary, the data suggest that dnaA gene transcription is autoregulated and that initiation of the S. lividans chromosome is tightly controlled.

Amino Acid Sequence↗
PubMed1975

RNA synthesis in cells infected with herpes simplex virus. IX. Evidence for accumulation of abundant symmetric transcripts in nuclei.

RNA extracted from nuclei of 8-h infected cells drove approximately 50% of herpes virus DNA into DNA-RNA hybrid. The same RNA, preannealed under conditions which allowed base pairing to take place, drove only 35% of the DNA into DNA-RNA hybrid; further annealing of the RNA did not diminish the amount of RNA sequences remaining available for subsequent hybridization with DNA. Upon denaturation of the preannealed RNA, the RNA sequences sequestered during preannealing became available again for hybridization with DNA. The base pairing that occurred during preincubation of the RNA was inter-molecular, since it was RNA concentration dependent and was not affected by limited alkaline hydrolysis. The nuclear viral transcripts that remained available for hybridization, after preannealing of the RNA, were subset of the RNA sequences that accumulated in the cytoplasm of infected cells. In addition, a small amount (derived from 5% or less of the viral DNA) of complementary transcripts was detected in the cytoplasm.

Base Sequence↗
PubMed2006

A statistical method for predicting splice variants between two groups of samples using GeneChip expression array data.

BACKGROUND: Alternative splicing of pre-messenger RNA results in RNA variants with combinations of selected exons. It is one of the essential biological functions and regulatory components in higher eukaryotic cells. Some of these variants are detectable with the Affymetrix GeneChip that uses multiple oligonucleotide probes (i.e. probe set), since the target sequences for the multiple probes are adjacent within each gene. Hybridization intensity from a probe correlates with abundance of the corresponding transcript. Although the multiple-probe feature in the current GeneChip was designed to assess expression values of individual genes, it also measures transcriptional abundance for a sub-region of a gene sequence. This additional capacity motivated us to develop a method to predict alternative splicing, taking advance of extensive repositories of GeneChip gene expression array data. RESULTS: We developed a two-step approach to predict alternative splicing from GeneChip data. First, we clustered the probes from a probe set into pseudo-exons based on similarity of probe intensities and physical adjacency. A pseudo-exon is defined as a sequence in the gene within which multiple probes have comparable probe intensity values. Second, for each pseudo-exon, we assessed the statistical significance of the difference in probe intensity between two groups of samples. Differentially expressed pseudo-exons are predicted to be alternatively spliced. We applied our method to empirical data generated from GeneChip Hu6800 arrays, which include 7129 probe sets and twenty probes per probe set. The dataset consists of sixty-nine medulloblastoma (27 metastatic and 42 non-metastatic) samples and four cerebellum samples as normal controls. We predicted that 577 genes would be alternatively spliced when we compared normal cerebellum samples to medulloblastomas, and predicted that thirteen genes would be alternatively spliced when we compared metastatic medulloblastomas to non-metastatic ones. We checked the consistency of some of our findings with information in UCSC Human Genome Browser. CONCLUSION: The two-step approach described in this paper is capable of predicting some alternative splicing from multiple oligonucleotide-based gene expression array data with GeneChip technology. Our method employs the extensive repositories of gene expression array data available and generates alternative splicing hypotheses, which can be further validated by experimental studies.

Algorithms↗
PubMed2004

[Study of the profile of gene expression in the endometrium].

Implantation in humans is a complex process that is temporally and spatially restricted. Over the past decade, using a one-by-one approach, several genes and gene products that may participate in this process have been identified in secretory phase endometrium. In order to understand this critical aspect of endometrial physiology, Smith group undertook a genome-wide analysis of transcript abundance and changes in transcript level between normal endometrium in the proliferative and secretory phases of the menstrual cycle, between normal and ectopic endometrium (endometriosis) and between normal and RU486-exposed endometrium. A high-density, oligonucleotide gene array was used to define the gene expression profiles of endometrium. Results from the arrays were verified using real-time PCR. Several gene products, known to be differentially expressed in the implantation window or in secretory endometrium, were identified. High density oligonucleotide microarray technology is a valid technique to investigate global changes in gene expression in human endometrium.

Embryo Implantation↗
PubMed1986

Sequences complementary to the brain-specific "identifier" sequences exist in L-type pyruvate kinase mRNA (a liver-specific messenger) and in transcripts especially abundant in muscle.

A sequence complementary to the brain-specific identifier sequence has been found in the 3' untranslated extension of the heavy 3.2-kilobase (kb) long liver L-type pyruvate kinase mRNA while it is absent in the other two 2- and 2.2-kb long pyruvate kinase mRNA species. A 53-base fragment corresponding to this identifier sequence was subcloned in both orientations in the single-stranded bacteriophage M13, both strands being used as probes to detect homologous sequence in different tissues. Both strands are transcribed in various tissues and are detected in heterogeneous high molecular weight RNA species which are especially abundant in the adult muscle. In addition, the probe identical to the identifier sequence recognized a discrete 0.6-kb RNA species in the muscle and the probe complementary to the identifier-sequence recognized the expected two small brain-specific identifier BC-1 and BC-2 RNAs described by Sutcliffe et al. (Sutcliffe, J. G., Milner, R. J., Bloom, F. E., and Lerner, R. A. (1982) Proc. Natl. Acad. Sci. U. S. A. 79, 4942-4946; Sutcliffe, J. G., Milner, R. J., Gottesfeld, J. M., and Lerner, R. A. (1984) Nature 308, 237-241; Milner, R. J., Bloom, F. E., Lai, C., Lerner, R. A., and Sutcliffe, J. G. (1984) Proc. Natl. Acad. Sci. U. S. A. 81, 713-717; Sutcliffe, J. G., Milner, R. J., Gottesfeld, J. M., and Reynolds, W. (1984) Science 225, 1308-1314).

Animals↗
PubMed2003

Characterization of the gene expression profile of neuroblastoma cell line IMR-5 using serial analysis of gene expression.

The serial analysis of gene expression (SAGE) technique was used to generate a database of the most abundant transcripts of the MYCN-amplified neuroblastoma cell line IMR-5. A total of 8568 tags were sequenced and shown to represent 4034 unique tags, each of which corresponds to an individual transcript. Expression levels of genes are reflected by the frequency of occurrence of the respective tags. To validate fidelity of SAGE data, relative abundances of seven transcripts were evaluated by semiquantitative reverse transcriptase-polymerase chain reaction. Transcripts that were detected nine times or more (>0.1% of the total tag population) accounted for 36% of the total messenger RNA mass but only 3% of the total number of individual transcripts. A strong preponderance of genes involved in protein synthesis, in particular those encoding for ribosomal proteins, were observed among these high-abundance transcripts. Tags corresponding to the amplified gene DDX1 were conspicuously overrepresented in comparison to the other amplified genes MYCN, neuroblastoma amplified gene and MEIS1, which suggests an additional mechanism apart from genomic amplification contributing to the strong upregulation of this gene. This study provides a comprehensive gene expression profile of neuroblastoma cell line IMR-5 and may be used as a reference database for identification of candidate genes that are involved in etiology and pathogenesis of neuroblastoma.

Databases as Topic↗
PubMed1989

Differential expression of gap and pgk genes within the gap operon of Zymomonas mobilis.

In Zymomonas mobilis, the genes encoding glyceraldehyde-3-phosphate dehydrogenase (GAP) and phosphoglycerate kinase (PGK) are encoded in an operon that is transcribed from tandem promoters. The promoter-proximal gap gene is expressed at six- to ninefold higher levels than the pgk gene from chromosomal genes and from multiple copies of plasmid-borne genes. Two dominant transcripts were identified. The smaller, most abundant transcript contained primarily the gap message, whereas the larger, less abundant message contained both genes. The ratio of message levels for gap and pgk was calculated to be 5:1 and is sufficient to account for the observed differences in levels of GAP and PGK. The differences in message abundance are proposed to result from either transcriptional attenuation or preferential degradation of the 3' region encoding pgk. Increases in gene dosage were accompanied by one-third the expected increase in enzymatic activity on the basis of estimates of copy number, consistent with the presence of a limiting, positive regulatory factor. However, GAP and PGK expressions were not reduced from the chromosome in recombinants that contained multiple copies of the gap operon with inactive genes.

Bacteria↗