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PubMed2001

Serial analysis of gene expression in differentiated cultures of human epidermal keratinocytes.

Keratinocyte gene expression was surveyed more comprehensively than before, by means of serial analysis of gene expression. A total of 25,694 tags derived from expressed mRNA, were analyzed in a model for normal differentiation and in a model where cultured keratinocytes were stimulated for a prolonged period of time with tumor necrosis factor-alpha, thus mimicking aberrant differentiation in the context of cutaneous inflammation. Serial analysis of gene expression revealed many transcripts derived from unknown genes and a large number of genes that are not known to be expressed in keratinocytes; furthermore, these data provide quantitative information about the relative abundance of transcripts, allowing the identification of differentially expressed genes. A major part of the identified transcripts accounted for genes involved in energy metabolism and protein synthesis. A large proportion of all transcripts (6%) corresponded to genes associated with terminal differentiation and barrier formation. Another highly expressed functional group of genes (2% of all transcripts) corresponded to proteins involved in host protection such as antimicrobial proteins and proteinase inhibitors. Three of these genes were not known to be expressed in keratinocytes, and some were upregulated after prolonged tumor necrosis factor-alpha exposure. Our data on expressed genes in keratinocytes are consistent with the known function of human epidermis, and provide a first step to generate a transcriptome of human keratinocytes.

Apoptosis↗
PubMed1990

Structural analysis of the mouse mdr1a (P-glycoprotein) promoter reveals the basis for differential transcript heterogeneity in multidrug-resistant J774.2 cells.

In multidrug-resistant mouse J774.2 cells, the differential overproduction of functionally distinct phosphoglycoprotein isoforms reflects the amplification or transcriptional activation or both of two mdr gene family members, mdr1a and mdr1b. The mdr1a gene is a complex transcriptional unit whose expression is associated with multiple transcript sizes. Independently selected multidrug-resistant J774.2 cell lines differentially overexpress either 4.6- and 5.0-kilobase (kb) or 4.7- and 5.1-kb mdr1a transcripts. However, abundant overproduction of the mdr1a gene product was observed only in cell lines which overexpressed the 4.6- and 5.0-kb mRNAs. In order to determine the basis for mdr1a transcript heterogeneity and the relationship between transcript size and steady-state mdr1a protein levels, genomic and cDNA sequence analyses of the 5' and 3' ends of the mdr1a gene were carried out. Promoter sequence analysis and primer extension mapping indicated that mdr1a transcripts were differentially initiated from two putative promoters to generate either 5.1- and 4.7-kb or 5.0- and 4.6-kb transcripts in four multidrug-resistant J774.2 cell lines. Sequence analysis of 3' cDNA variants and a 3' genomic fragment revealed that the 5.1- and 5.0-kb mRNAs had identical 3'-untranslated regions which differed from those of the 4.7- and 4.6-kb mRNAs as a result of the utilization of a more downstream alternative poly(A) addition signal. Transcript initiation from the putative upstream promoter correlated with a 70 to 85% decrease in steady-state mdr1a protein levels relative to transcript levels. In addition, the identification of putative AP-1 and AP-2 promoter elements suggests a possible role for protein kinase A and protein kinase C in the regulation of mdr1a. The implications of these findings for mdr gene expression and regulation are discussed.

ATP Binding Cassette Transporter, Subfamily B, Mem↗
PubMed1986

UV crosslinking of RNA to nylon membrane enhances hybridization signals.

An improvement in the detection by nucleic acid hybridization of size-fractionated RNA immobilized to nylon-based membranes is described. Electrophoretic transfer of RNA to nylon membranes permits a quantitative determination of different RNA transcripts on the same membrane after sequential hybridization using different 32P-labeled DNA probes. UV crosslinking of the RNA to the nylon membrane increased the intensity of the radioactive signals. Using the method reported here, increased signals of between 10 and 40 fold were observed, depending on the species of transcript tested. Moderately abundant as well as rare transcripts can easily be detected in as little as 5 micrograms total cellular RNA.

Animals↗
PubMed2002

Characterisation of six alpha-expansin genes in Gossypium hirsutum (upland cotton).

A genomic library screen and PCR-based strategies were employed to isolate six genes with sequence similarity to a cotton fibre-specific mRNA encoding an alpha-expansin. alpha-Expansins are cell wall proteins that facilitate cell wall extension by disruption of non-covalent bonds between wall components. The characterisation and expression analysis of these six novel genes ( GhExp1-GhExp6) is described. Four of them ( GhExp3- GhExp6) are expressed within multiple tissues of the cotton plant and two ( GhExp1 and GhExp2) give rise to transcripts that are specific to the developing cotton fibre. GhExp1 transcripts are highly abundant in the fibre, while transcripts for GhExp2 are detected at a low level. Cotton fibres are highly elongated cells of the ovule epidermis, and we envisage that GhExp1 may play an important role in cell wall extension during development.

Amino Acid Sequence↗
PubMed1997

Identification and transcriptional analysis of pseudorabies virus UL6 to UL12 genes.

We determined the nucleotide sequence of an 11059 bp fragment of the pseudorabies virus genome located in the right part of genomic BamHI fragment 3 and the adjacent part of BamHI fragment 6. Within this region eight open reading frames were identified whose deduced amino acid sequences exhibited homology to the UL6, UL7, UL8, UL8.5, UL9, UL10, UL11, and UL12 protein products of herpes simplex virus type 1. Transcriptional analyses indicated presence of 3'-coterminal mRNAs for genes UL8, UL8.5, and UL9 as well as for genes UL6 and UL7, respectively, while UL10 was represented by a very abundant unique transcript. Both gene arrangement and transcriptional organization within this region of the pseudorabies virus genome thus parallels the situation found in other alphaherpesviruses.

Amino Acid Sequence↗
PubMed1990

Stage- and cell-specific expression of the ornithine decarboxylase gene during rat and mouse spermatogenesis.

Ornithine decarboxylase (ODC) is an enzyme that has been shown to be induced in the growth, differentiation and proliferation of cells. We have used a cDNA probe to determine ODC mRNA levels in different stages of the cycle of rat and mouse seminiferous epithelium. For Northern and slot-blot hybridizations, RNA was isolated from microdissected staged seminiferous tubules. Cell-specific localization of ODC mRNA was studied by in situ hybridization. In the rat, in situ hybridization showed increasing mRNA levels during prophase of meiosis with the highest mRNA levels seen in late pachytene spermatocytes and step 3-5 spermatids. In the mouse, the mRNA levels increased in a similar fashion and the highest mRNA levels were found in step 1-8 spermatids. In the rat, Northern blot hybridizations revealed three molecular sizes of ODC mRNA: 2.2, 2.7 and 1.6 kb. The levels of all molecular sizes were highest in stages VII-VIII, and the lowest mRNA levels were seen in stage I of the seminiferous epithelial cycle. The level of the 2.2 kb transcript was low during stages XIII-I. In the mouse, the Northern blot hybridizations also showed three molecular sizes of ODC mRNA: 2.2 and 2.7 kb and very low levels of 1.6 kb transcript. The levels of the transcripts were steady throughout the cycle. In the mouse, the 2.2 kb transcript was more abundant than the 2.7 kb transcript indicating a species difference between rat and mouse in the usage of the two polyadenylation signals within the ODC gene.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1999

Cloning, genomic organization and expression of a putative human transmembrane protein related to the Caenorhabditis elegans M01F1.4 gene.

A novel human transcript CG-2 (C9ORF5), was isolated from the familial dysautonomia candidate region on 9q31 using a combination of cDNA selection and exon trapping. CG-2 was detected as a relatively abundant 8kb transcript in all adult and fetal tissues with the exception of adult thymus. Genomic analysis of CG-2 identified 18 exons that span more than 110kb. The gene encodes a 911-amino-acid protein with a predicted molecular weight of 101kDa and a hypothetical pI of 9.03. Sequence analysis of CG-2 indicates that it is likely to encode a transmembrane protein. Here, we assess CG-2 as a candidate for familial dysautonomia.

Adult↗
PubMed1997

NFI-B3, a novel transcriptional repressor of the nuclear factor I family, is generated by alternative RNA processing.

Nuclear factor I (NFI) proteins constitute a family of sequence-specific transcription factors whose functional diversity is generated through transcription from four different genes (NFI-A, NFI-B, NFI-C, and NFI-X), alternative RNA splicing, and protein heterodimerization. Here we describe a naturally truncated isoform, NFI-B3, which is derived from the human NFI-B gene, in addition to characterizing further human NFI-B1 and NFI-B2, two differentially spliced variants previously isolated from hamster and chicken. Although NFI-B1 and NFI-B2 proteins are translated from an 8. 7-kilobase message, the mRNA for NFI-B3 has a size of only 1.8 kilobases. The NFI-B3 message originates from the failure to excise the first intron downstream of the exons encoding the DNA binding domain and subsequent processing of this transcript at an intron-internal polyadenylation signal. The translation product includes the proposed DNA binding and dimerization domain and terminates after translation of two additional "intron" encoded codons. In SL-2 cells, which are void of endogenous NFI, NFI-B3 by itself had no effect on transcriptional regulation and failed to bind DNA. Coexpression of NFI-B3 with other isoforms of the NFI-B, -C, and -X family, however, led to a strong reduction of transcriptional activation compared with the expression of these factors alone. Gel shift analysis indicated that NFI-B3 disrupts the function of other NFI proteins by reducing their DNA binding activity by heterodimer formation. The efficiency of NFI-B3 heterodimers to bind to DNA correlated with the degree of transcriptional repression. The abundance of NFI-B transcripts varied significantly between different human cell lines and tissues, suggesting a potential involvement of these factors in the complex mechanisms that generate cell type specificity.

Alternative Splicing↗
PubMed1991

Comparison of viral nucleic acid intermediates at early and late stages of cauliflower mosaic virus infection suggests a feedback regulatory mechanism.

An important phase of the multiplication cycle of the pararetrovirus cauliflower mosaic virus (CaMV) is transcription of the viral minichromosome in the nucleus. Leaves of infected turnip plants at the vein clearing stage were found to contain a relatively low level of minichromosome DNA, and abundant viral transcripts and characteristic reverse transcription products. In contrast, at the much later stage of severe leaf chlorosis, an elevated level of minichromosome DNA but less RNA, especially the 35S RNA reverse transcription template, was observed. Changes in the composition of virus nucleic acid intermediates were also seen in roots and stems early, compared with late, in infection. A possible feedback mechanism controlling the level of viral minichromosome DNA and its importance in regulation of the CaMV multiplication cycle are discussed in the light of these observations.

Blotting, Northern↗
PubMed1991

Stress Responses in Alfalfa (Medicago sativa L.): X. Molecular Cloning and Expression of S-Adenosyl-l-Methionine:Caffeic Acid 3-O-Methyltransferase, a Key Enzyme of Lignin Biosynthesis.

S-Adenosyl-l-methionine:caffeic acid 3-O-methyltransferase (COMT, EC 2.1.1.6) catalyzes the conversion of caffeic acid to ferulic acid, a key step in the biosynthesis of lignin monomers. We have isolated a functionally active cDNA clone (pCOMT1) encoding alfalfa (Medicago sativa L.) COMT by immunoscreening a lambdaZAPII cDNA expression library with anti-(aspen COMT) antibodies. The derived amino acid sequence of pCOMT1 is 86% identical to that of COMT from aspen. Southern blot analysis indicates that COMT in alfalfa is encoded by at least two genes. Addition of an elicitor preparation from bakers' yeast to alfalfa cell suspension cultures resulted in a rapid accumulation of COMT transcripts, which reached a maximum level around 19 hours postelicitation. Northern blot analysis of total RNA from different organs of alfalfa plants at various developmental stages showed that COMT transcripts are most abundant in roots and stems. Transcripts encoding ATP: i-methionine-S-adenosyl transferase (AdoMet synthetase, EC 2.5.1.6), the enzyme responsible for the synthesis of the methyl donor for the COMT reaction, were coinduced with COMT transcripts in elicitor-treated cells and exhibited a similar pattern of expression to that of COMT in different organs of alfalfa plants at various stages of development.

Journal Article↗
PubMed2003

Microarray assessment of fibronectin, collagen and integrin expression and the role of fibronectin-collagen coating in the growth of normal, SV40 T-antigen-immortalised and malignant human oral keratinocytes.

Extracellular matrix proteins affect the growth and survival of epithelial tissues. Accordingly, surface coating with fibronectin and collagen is a common practice for promoting keratinocyte culture. In this study, the expression of fibronectin and collagen-related factors, including integrins, by normal (NOK), SV40 T-antigen-immortalised (SVpgC2a) and malignant (SqCC/Y1) human oral keratinocytes, under standardised, serum-free conditions, was investigated by using microarray analysis. Cell growth was also studied in the presence and absence of a matrix consisting of human fibronectin and bovine collagen type I (FN-COL). Fibronectin transcripts were abundant in all cells, whereas 16 of 29 collagen chains and 14 of 24 integrin subunits were variably detected. With regard to both the expression level and the number of transcripts, higher collagen and lower integrin expression was observed in SVpgC2a cells than in NOKs and SqCC/Y1 cells. The cell types differed with regard to colony-forming efficiency and the rate and kinetics of growth at high cell density. For all cell types, FN-COL coating consistently stimulated cell migration, without influencing growth in mass culture or clonal density. The results demonstrate the transcription of genes associated with the formation and function of fibronectin and collagen in oral epithelium, and variably altered expression patterns in transformed states, and show that keratinocyte lines can be successfully transferred without the stimulus from extracellular FN-COL.

Antigens, Polyomavirus Transforming↗
PubMed1995

Calbindin-D9K gene expression in rat chorioallantoic placenta is not regulated by 1,25-dihydroxyvitamin D3.

The aim of this study was to investigate whether 1,25-dihydroxyvitamin D3 (1,25(OH)2D3), the active metabolite of vitamin D, regulates the expression of rat placental calbindin-D9K mRNA. One group of rats (-D) was fed a vitamin D-deficient diet before and during pregnancy, whereas a second group (+D) was fed a vitamin D-replete diet over the same period. Animals were killed on d 21 of gestation, and plasma concentrations of 1,25(OH)2D3 for +D and -D animals were significantly (p < 0.05) different (260 +/- 78 and 39 +/- 8 pM in maternal plasma and 122 +/- 39 and 42 +/- 10 pM in fetal plasma, respectively; mean +/- SE, n = 4-5). Vitamin D deficiency had no effect on placental weight, fetal weight, fetal ashed weight, fetal calcium accretion, or the maternofetal calcium gradient. Hybridization of RNA from maternal duodena (used as a positive control tissue) and placentas of +D and -D rats with a rat calbindin-D9K cDNA revealed a single 0.6-kb transcript in both tissues. The abundance of this transcript was markedly lower (p = 0.06) in the duodena of -D compared with +D rats (mean change -68 +/- 9%) but there was no difference between the placentas of the two groups (mean change +13 +/- 22%). These changes were significantly (p < 0.05) different between the two tissues and the response of each tissue to vitamin D deficiency was significantly different (p < 0.01). These data indicate that 1,25(OH)2D3 does not regulate the expression of calbindin-D9K mRNA in rat placenta.

Allantois↗
PubMed2004

Vasoactive intestinal peptide (VIP) stimulates cortisol secretion from the H295 human adrenocortical tumour cell line via VPAC1 receptors.

Vasoactive intestinal peptide (VIP) shows a wide tissue distribution and exerts numerous physiological actions. VIP was shown in a dose-dependent manner to increase cortisol secretion in the NCI-H295R human adrenocortical carcinoma (H295) cell line (threshold dose 3.3x10(-10) M, maximal dose 10(-7) M), coupled with a parallel increase in cAMP accumulation. Receptor-specific agonists were employed to determine which of the two known VIP receptor subtypes was involved in cortisol secretion. Treatment with the VPAC1 receptor agonist, [K(15), R(16), L(27)]VIP(1-7)/GRF(8-27), produced a dose-dependent increase in H295 cell cortisol secretion (threshold dose 10(-11) M, maximal dose 10(-7) M) similar to that seen with VIP. Meanwhile, the high-affinity VPAC2 receptor agonist, RO-25-1553, failed to stimulate significantly cortisol or cAMP production from H295 cells. Inhibition of VIP-mediated H295 cell cortisol secretion by PG97-269, a competitive VPAC1-specific antagonist, produced parallel shifts of the dose-response curve and a Schild regression slope of 0.99, indicating competitive inhibition at a single receptor subtype. VIP is known also to interact with the PAC1 receptor, albeit with lower affinity (EC(50) of approximately 200 nM) than the homologous ligand, PACAP (EC(50) of approximately 0.5 nM). PACAP stimulated cortisol secretion from H295 cells (EC(50) of 0.3 nM), suggesting the presence of functional PAC1 receptors. However, stimulation of cortisol secretion by nanomolar concentrations of VIP (EC(50) of 5 nM), coupled with real-time PCR estimation that VPAC1 receptor transcripts appear 1000-fold more abundant than PAC1 transcripts in H295 cells, makes it unlikely that VIP signals via PAC1 receptors. Together, these data suggest that VIP directly stimulates cortisol secretion from H295 cells via activation of the VPAC1 receptor subtype.

Animals↗
PubMed2001

Correlation of gene expression of ten drug efflux proteins of the ATP-binding cassette transporter family in normal human jejunum and in human intestinal epithelial Caco-2 cell monolayers.

This investigation describes the expression and interindividual variability in transcript levels of multiple drug efflux systems in the human jejunum and compares the expression profiles in these cells with that of the commonly used Caco-2 cell drug absorption model. Transcript levels of ten-drug efflux proteins of the ATP-binding cassette (ABC) transporter family [MDR1, MDR3, ABCB5, MRP1-6, and breast cancer resistance protein (BCRP)], lung resistance-related protein (LRP), and CYP3A4 were determined using quantitative polymerase chain reaction in jejunal biopsies from 13 healthy human subjects and in Caco-2 cells. All genes except ABCB5 were expressed, and transcript levels varied between individuals only by a factor of 2 to 3. Surprisingly, BCRP and MRP2 transcripts were more abundant in jejunum than MDR1 transcripts. Jejunal transcript levels of the different ABC transporters spanned a range of three log units with the rank order: BCRP approximately MRP2 > MDR1 approximately MRP3 approximately MRP6 approximately MRP5 approximately MRP1 > MRP4 > MDR3. Furthermore, transcript levels of 9 of 10 ABC transporters correlated well between jejunum and Caco-2 cells (r2 = 0.90). However, BCRP exhibited a 100-fold lower transcript level in Caco-2 cells compared with jejunum. Thus, the expression of a number of efflux protein transcripts in jejunum are equal to, or even higher than, that of MDR1, suggesting that the roles of these proteins (in particular BCRP and MRP2) in intestinal drug efflux have been underestimated. Also, we tentatively conclude that the Caco-2 cell line is a useful model of jejunal drug efflux, if the low expression of BCRP is taken into account.

ATP-Binding Cassette Transporters↗
PubMed1997

bldA-dependent expression of the Streptomyces exfoliatus M11 lipase gene (lipA) is mediated by the product of a contiguous gene, lipR, encoding a putative transcriptional activator.

Extracellular lipase synthesis by Streptomyces lividans 66 carrying the cloned lipase gene (lipA) from Streptomyces exfoliatus M11 was found to be growth phase dependent, since lipase was secreted into the medium mainly during the stationary phase; S1 nuclease protection experiments revealed abundant lipA transcripts in RNA preparations obtained during the stationary phase but not in those obtained during exponential growth. Transcription from the lipA promoter was dependent on the presence of lipR, a contiguous downstream gene with a very high guanine-plus-cytosine content (80.2%). The deduced lipR product consists of a protein of 934 amino acids that shows similarity to known transcriptional activators and has a strong helix-turn-helix motif at its C terminus; this motif is part of a domain homologous to DNA-binding domains of bacterial regulators of the UhpA/LuxR superfamily. The lipR sequence revealed the presence of a leucine residue, encoded by the rare TTA codon, which caused bldA dependence of lipA transcription in Streptomyces coelicolor A3(2); replacement of the TTA codon by the alternate CTC leucine codon alleviated bidA dependence but not the apparent growth phase-dependent regulation of lipA transcription. When lipR expression was induced in a controlled fashion during the exponential growth phase, by placing it under the inducible tipA promoter, lipase synthesis was shifted to the exponential growth phase, indicating that the timing of lipR expression, and not its bldA dependence, is the main cause for stationary-phase transcription of lipA.

Amino Acid Sequence↗
PubMed1988

Expression of SPARC/osteonectin transcript in murine embryos and gonads.

A cDNA clone, p2-4, was isolated from mouse teratocarcinoma-derived parietal endoderm-like cells and used to analyze expression of the corresponding transcript during mouse embryogenesis. Nucleotide-sequence analysis revealed extensive homology between this clone and SPARC/osteonectin cDNA cloned from mouse parietal endoderm and bovine bone cells. The SPARC/osteonectin transcript became more abundant when embryonal carcinoma (EC) cells differentiated into parietal endoderm-like cells. In embryos, the transcript began to appear in the embryo proper on day 11 and continued to be expressed throughout the gestation period. The transcript was also present in extraembryonic membranes and placenta from days 9 and 11 onward, respectively. Thus, expression of the transcript was regulated during differentiation of EC cells and during embryogenesis. In adult mice, several non-bone tissues, including testis, also expressed the transcript. Analysis of germ-cell-deficient mice indicated that non-germ-cell components of the testis expressed the transcript. Analysis of mouse testicular cell lines further suggested that the transcript was abundant in Sertoli cells and Leydig cells. Cumulus oophorus cells that envelope the ovulated egg also expressed high levels of the transcript.

Animals↗
PubMed2006

Timecourse microarray analyses reveal global changes in gene expression of susceptible Glycine max (soybean) roots during infection by Heterodera glycines (soybean cyst nematode).

Changes in gene expression within roots of Glycine max (soybean), cv. Kent, susceptible to infection by Heterodera glycines (the soybean cyst nematode [SCN]), at 6, 12, and 24 h, and 2, 4, 6, and 8 days post-inoculation were monitored using microarrays containing more than 6,000 cDNA inserts. Replicate, independent biological samples were examined at each time point. Gene expression was analyzed statistically using T-tests, ANOVA, clustering algorithms, and online analytical processing (OLAP). These analyses allow the user to query the data in several ways without importing the data into third-party software. RT-PCR confirmed that WRKY6 transcription factor, trehalose phosphate synthase, EIF4a, Skp1, and CLB1 were differentially induced across most time-points. Other genes induced across most timepoints included lipoxygenase, calmodulin, phospholipase C, metallothionein-like protein, and chalcone reductase. RT-PCR demonstrated enhanced expression during the first 12 h of infection for Kunitz trypsin inhibitor and sucrose synthase. The stress-related gene, SAM-22, phospholipase D and 12-oxophytodienoate reductase were also induced at the early time-points. At 6 and 8 dpi there was an abundance of transcripts expressed that encoded genes involved in transcription and protein synthesis. Some of those genes included ribosomal proteins, and initiation and elongation factors. Several genes involved in carbon metabolism and transport were also more abundant. Those genes included glyceraldehyde 3-phosphate dehydrogenase, fructose-bisphosphate aldolase and sucrose synthase. These results identified specific changes in gene transcript levels triggered by infection of susceptible soybean roots by SCN.

Animals↗
PubMed2000

Gamma-radiation-induced growth arrest and apoptosis in p53-null lymphoma cells is accompanied by modest transcriptional changes in many genes.

Damage to DNA produces cell cycle arrest, apoptosis, or both. The response in cells with p53 tumor suppressor function involves transcriptional changes, but whether that holds for cells lacking active p53, as in most tumors, is not known. Better characterization of the DNA damage response in tumors lacking p53 function is relevant to cytotoxic therapy. We have explored whether gamma-irradiated p53-null mouse T lymphoma cells undergo marked changes in transcription. Their arrest in G2/M prior to apoptosis required transcription. Transcripts whose abundance altered on irradiation were sought by subtractive hybridization, and 1010 candidate clones from two oppositely enriched cDNA populations were sequenced. Hybridization revealed small (<3-fold) increases or decreases in the transcripts of more than 15 genes, including some implicated in cell cycle control (e.g., BTG, Bap1) or apoptosis (e.g., STAT1, calpain), but no marked changes like those associated with other forms of T-cell death. Moreover, the expression of some critical apoptosis regulators, such as Bcl-2 family members, did not change. Hence, the G2/M arrest and apoptosis in the irradiated p53-null lymphoma appears to involve modest expression changes for many genes, but post-transcriptional alterations may be more critical.

Animals↗