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PubMed2002

Fatty acid and lipid biosynthetic genes are expressed at constant molar ratios but different absolute levels during embryogenesis.

In plants, fatty acid and complex lipid synthesis requires the correct spatial and temporal activity of many gene products. Quantitative northern analysis showed that mRNA for the biotin carboxylase subunit of heteromeric acetyl-coenzyme A carboxylase, fatty acid synthase components (3-oxoacyl-acyl carrier protein [ACP] reductase, enoyl-ACP reductase, and acyl-ACP thioesterase), and stearoyl-ACP desaturase accumulate in a coordinate manner during Brassica napus embryogenesis. The mRNAs were present in a constant molar stoichiometric ratio. Transcript abundance of mRNAs for the catalytic proteins was found to be similar, whereas the number of ACP transcripts was approximately 7-fold higher. The peak of mRNA accumulation of all products was between 20 and 29 d after flowering; by 42 d after flowering, the steady-state levels of all transcripts fell to about 5% of their peak levels, which suggests that the mRNAs have similar stability and kinetics of synthesis. Biotin carboxylase was found to accumulate to a maximum of 59 fmol mg(-1) total RNA in embryos, which is in general agreement with the value of 170 fmol mg(-1) determined for Arabidopsis siliques (J.S. Ke, T.N. Wen, B.J. Nikolau, E.S. Wurtele [2000] Plant Physiol 122: 1057-1071). Embryos accumulated between 3- and 15-fold more transcripts per unit total RNA than young leaf tissue; the lower quantity of leaf 3-oxoacyl-ACP reductase mRNA was confirmed by reverse transcriptase-polymerase chain reaction. This is in conflict with analysis of B. napus transcripts using an Arabidopsis microarray (T. Girke, J. Todd, S. Ruuska, J. White, C. Benning, J. Ohlrogge [2000] Plant Physiol 124: 1570-1581) where similar leaf to seed levels of fatty acid synthase component mRNAs were reported.

3-Oxoacyl-(Acyl-Carrier-Protein) Reductase↗
PubMed1999

Spatially restricted expression of PlOtp, a Paracentrotus lividus orthopedia-related homeobox gene, is correlated with oral ectodermal patterning and skeletal morphogenesis in late-cleavage sea urchin embryos.

Several homeobox genes are expressed in the sea urchin embryo but their roles in development have yet to be elucidated. Of particular interest are homologues of homeobox genes that in mouse and Drosophila are involved in patterning the developing central nervous system (CNS). Here, we report the cloning of an orthopedia (Otp)-related gene from Paracentrotus lividus, PlOtp. Otp is a single copy zygotic gene that presents a unique and highly restricted expression pattern. Transcripts were first detected at the mid-gastrula stage in two pairs of oral ectoderm cells located in a ventrolateral position, overlying primary mesenchyme cell (PMC) clusters. Increases in both transcript abundance and the number of Otp-expressing cells were observed at prism and pluteus stages. Otp transcripts are symmetrically distributed in a few ectodermal cells of the oral field. Labelled cells were observed close to sites of active skeletal rod growth (tips of the budding oral and anal arms), and at the juxtaposition of stomodeum and foregut. Chemicals known to perturb PMC patterning along animal-vegetal and oral-aboral axes altered the pattern of Otp expression. Vegetalization by LiCl caused a shift in Otp-expressing cells toward the animal pole, adjacent to shifted PMC aggregates. Nickel treatment induced expression of the Otp gene in an increased number of ectodermal cells, which adopted a radialized pattern. Finally, ectopic expression of Otp mRNA affected patterning along the oral-aboral axis and caused skeletal abnormalities that resembled those exhibited by nickel-treated embryos. From these results, we conclude that the Otp homeodomain gene is involved in short-range cell signalling within the oral ectoderm for patterning the endoskeleton of the larva through epithelial-mesenchymal interactions.

Amino Acid Sequence↗
PubMed1996

Temporal patterns of gene expression of G1-S cyclins and cdks during the first and second mitotic cell cycles in mouse embryos.

Cell-cycle progression in somatic cells is regulated by a family of cyclins and cyclindependent kinases (cdks) that form specific complexes as a function of cell-cycle progression. However, the transcript abundance of G1-S cyclins and cdks during the meiotic and mitotic cell cycles of mammalian embryos has not been previously reported. Using a reverse transcription-polymerase chain reaction (PCR) assay that detects changes in either mRNA abundance or polyadenylation state, we examined the relative levels of gene expression for the G1-S cyclins and cdks, as well as for p21, p27, and the retinoblastoma (Rb) gene in mouse oocytes, metaphase II-arrested eggs, and 1-2-cell embryos. The PCR products for cyclins D1, D3, and A, as well as cdk4, p21, and Rb, displayed similar levels in meiotically incompetent and competent oocytes, as well as in metaphase II-arrested eggs. The levels of PCR products for cyclin D2, p27, and two forms of cdk2 were similar in meiotically incompetent and competent oocytes but decreased during oocyte maturation. Finally, the level of PCR products for cyclin E and cdk2 gradually decreased during the progression from meiotically incompetent oocytes to metaphase II-arrested eggs. When the levels of PCR products for the G1-S regulatory genes were evaluated during the first and second mitotic cell cycles, four main patterns were found: 1) steady levels for cyclin A; 2) steady levels followed by a 2-3-fold increase during the G2 phase of the second mitotic cell cycle for cyclins D1, E, cdk2, and p21; 3) a transient increase during the S and/or G2 phases of the first mitotic cell cycle for p27, cyclin D3, and the two forms of cdk2; and 4) higher levels during the first cell cycle and then a decrease with lower levels during the second mitotic cell cycle for cyclin D2 and Rb. cdk4 expression displayed a combination of patterns 2 and 3. The increase in the amount of PCR product for the cdk4 gene during the first mitotic cell cycle was due to polyadenylation, whereas the increase in the amount of PCR product for cdk4, cdk2, and cyclins D1 and E in the second mitotic cell cycle was a product of activation of the embryonic genome.

Amanitins↗
PubMed2004

Melanopsin expression in the chick retina and pineal gland.

The chick retina and pineal gland exhibit circadian oscillations in biochemical and physiological processes in vivo and in vitro and entrain to 24 h LD cycles. However, the phototransductive mechanisms responsible for entrainment are largely unknown. Experiments utilizing mice lacking functional rods and cones reveal normal circadian responses to light. These studies also implicate a subset of photoresponsive cells in the ganglion cell layer (GCL) which express melanopsin (opn4). The current study investigates opn4 mRNA distribution and regulation in the chick. Opn4 mRNA is widespread in the central nervous system and peripheral tissues, with highest levels of transcript abundance in the pineal gland and retina. Opn4 mRNA is also regulated on a circadian basis in the pineal gland, reaching peak values of accumulation in the late subjective night. In the retina, opn4 is not robustly regulated at the transcriptional level, exhibiting a slight increase in mRNA abundance during subjective night. In situ hybridization (ISH) revealed opn4 mRNA in areas associated with phototransduction and the visual system, including the pineal gland and optic tectum of the brain, along with GCL, inner nuclear layer (INL), and photoreceptor layer (PL) of the retina. Opn4 is also present in cerebellum. Our results present an opsin-like gene located in two circadian oscillators associated with circadian phototransduction and melatonin biosynthesis that may play a role in entrainment of these tissues' clocks.

Animals↗
PubMed1995

The gene family encoding the fucoxanthin chlorophyll proteins from the brown alga Macrocystis pyrifera.

Six members of a multigene family encoding polypeptide constituents of the fucoxanthin, chlorophyll a/c protein complex from female gametophytes of the brown alga Macrocystis pyrifera have been cloned and characterized. The deduced amino acid sequences are very similar to those of fucoxanthin chlorophyll binding proteins (Fcp) from the diatom Phaeodactylum tricornutum and exhibit limited homology to chlorophyll a/b binding (Cab) polypeptides from higher plants. The primary translation products from the M. pyrifera fcp genes are synthesized as higher molecular weight precursors that are processed prior to their assembly into the Fcp complex. The presumed N-terminal 40-amino acid presequence of the Fcp precursor polypeptide has features resembling that of a signal sequence. This presequence may be required for the protein to transverse the endoplasmic reticulum that surrounds the plastid in brown algae. A subsequent targeting step would be required for the protein to cross the double membrane of the plastid envelope. M. pyrifera fcp transcripts are of two sizes, 1.2 and 1.6 kb. The size difference is accounted for by the length of the 3' untranslated region, which can be up to 1000 bases. Transcript abundance's of members of the fcp gene family are dependent on light quantity, light quality, or both. Transcript levels of one gene increased approximately five- to tenfold in thalli grown in low intensity relative to high intensity white or blue light. Transcripts from this gene also significantly increase in red light relative to blue light at equivalent light intensities.

Amino Acid Sequence↗
PubMed2003

MCB-mediated regulation of cell cycle-specific cdc22+ transcription in fission yeast.

The cdc22+ gene of the fission yeast, Schizosaccharomyces pombe, encodes the large subunit of ribonucleotide reductase, and is periodically expressed during the mitotic cell cycle, transcript abundance reaching a maximum at the G1-S boundary. This regulation of expression is controlled by a transcription factor complex called DSC1, which binds to MCB motifs (ACGCGT) present in the promoter of cdc22+. cdc22+ has a complex pattern of MCBs, including two clusters of four motifs each, one of which is located within the transcribed region. We show that both clusters of MCBs contribute to the regulation of cdc22+ expression during the cell cycle, each having a different role. The MCB cluster within the transcribed region has the major role in regulating cdc22+, as its removal results in loss of transcription. The upstream cluster, instead, controls cell cycle-specific transcription through a negative function, as its removal results in expression of cdc22+ throughout the cell cycle. Both MCB clusters bind DSC1. We show that the interaction of DSC1 with the MCB cluster within the transcribed region has a high "on-off" rate, suggesting a mechanism by which DSC1 could activate expression, and still allow RNA polymerase to pass during transcription. Finally, we show that both clusters are orientation-dependent in their function. The significance of these results, in the context of MCB-mediated regulation of G1-S expression in fission yeast, is discussed.

Base Sequence↗
PubMed2025

Landscape and m6A post-transcriptional regulation of soybean proteome.

The soybean is a critical source of vegetable protein, but its proteome remains undercharacterized. Here, we quantify 12,855 proteins across 14 soybean organs using 4D data-independent acquisition mass spectrometry (4D-DIA-MS), creating the most extensive soybean proteome dataset to date. Organ-specific protein expression and co-expression analyses highlight functional specificity with significant differences in protein-transcript abundance across organs. We also map N6-methyladenosine (m6A) modifications, identifying their key role in post-transcriptional protein regulation. Integrative analysis of the proteome and m6A methylome identifies a novel regulator in m6A methylation. This comprehensive proteomic and m6A landscape advances our understanding of soybean biology and provides a valuable resource for crop improvement.

Glycine max↗
PubMed1988

Expression and developmental control of platelet-derived growth factor A-chain and B-chain/Sis genes in rat aortic smooth muscle cells.

Cultured arterial smooth muscle cells (SMC) can produce platelet-derived growth factor (PDGF)-like molecules. This property raises the possibility that SMC-derived PDGFs function as autocrine/paracrine regulators in the formation and maintenance of the artery wall. In this study we have asked if levels of mRNAs directing synthesis of PDGF are modulated in aortic SMC during postnatal development. We report here that genes encoding PDGF A- and B-chain precursors are expressed at similar low levels in intact aortas from newborn and adult rats. Marked differences in regulation of transcript abundance of these genes were revealed when aortic SMC were grown in cell culture. PDGF B-chain transcripts accumulated in passaged newborn rat SMC but not adult rat SMC, whereas PDGF A-chain RNA was found in comparable amounts in SMC from both age groups. Similarly, SMC from newborn rats secreted at least 60-fold more PDGF-like activity into conditioned medium than did adult rat SMC. PDGF B-chain transcripts in newborn rat aortic SMC are short-lived and increased 5-fold by 3 hr after treatment with cycloheximide. In contrast, PDGF A-chain transcripts are more stable, and their constitutive levels were generally unaffected by cycloheximide. These results show that PDGF A- and B-chain genes are transcribed in the normal rat aorta and provide evidence for age-related change in the control of PDGF B-chain gene expression in aortic SMC. Independent regulation of transcript levels in cultured SMC leaves open the possibility that PDGFs of different composition (AA, AB, BB) play different roles in normal function of the artery wall.

Age Factors↗
PubMed1991

The secretin gene: evolutionary history, alternative splicing, and developmental regulation.

The gene encoding the hormone secretin has been isolated and structurally characterized. The transcriptional unit is divided into four exons spanning 813 nucleotides. Comparison of the rat secretin gene to the other members of the glucagon-secretin gene family reveals that similarities are restricted to the exons encoding the biologically active peptides. Analysis of RNA from porcine intestine indicates that at least two transcripts are generated from the porcine secretin gene as a result of differential splicing. The longer and more abundant transcript appears to be identical to a previously isolated cDNA, which encodes a precursor that includes a 72-amino acid C-terminal extension peptide. The shorter transcript does not contain the third exon and, as a result, encodes only 44 residues beyond the C terminus of secretin. The amino acid sequence deduced from the shorter transcript is identical to a precursor form of secretin recently isolated from porcine duodenum [Gafvelin, G., Jornvall, H. & Mutt, V. (1990) Proc. Natl. Acad. Sci. USA 87, 6781-6785]. Developmental studies reveal that both secretin mRNA and peptide levels in the intestine are highest just before birth, prior to the onset of gastric acid secretion and feeding. This observation implies that secretin biosynthesis in developing animals is controlled independently of the principal factors known to regulate secretin release in adult animals.

Amino Acid Sequence↗
PubMed1986

Developmentally regulated RNA transcripts coding for alcohol dehydrogenase in Drosophila affinidisjuncta.

The organization of the gene coding for alcohol dehydrogenase (Adh) in Drosophila affinidisjuncta has been determined by physically mapping Adh RNA transcripts to cloned genomic DNA. Two distinct transcript types accumulate with developmental specificity. Because only a single genomic Adh locus is detected in D. affinidisjuncta, and since all Adh transcripts appear to be identical except at their termini, the two Adh RNA types are products of the same gene. One type of transcript, abundant in adults, contains a small 5' terminal exon that is completely lacking in the other type of transcript, which accumulates in larvae. This 5' end difference suggests that the D. affinidisjuncta Adh gene, like the homologous gene from the distantly related species D. melanogaster, is expressed from two promoters. According to the transcription map, these D. affinidisjuncta promoters are separated by approximately 560 base pairs of genomic DNA sequence. D. affinidisjuncta Adh transcripts also resemble D. melanogaster Adh transcripts in both their overall organization and their developmental distribution. Multiple 3' ends are responsible for the size heterogeneity of both types of D. affinidisjuncta Adh RNA, and some of these also appear with stage specificity.

Alcohol Dehydrogenase↗
PubMed1991

Epstein-Barr virus infection in oral hairy leukoplakia: virus replication in the absence of a detectable latent phase.

Epstein-Barr virus (EBV) infects both B lymphocytes and oropharyngeal epithelium, and it has been argued that the true reservoir of virus persistence in vivo is the self-renewing basal epithelial compartment. The identification of oral hairy leukoplakia (HL) of AIDS patients as a clinically apparent focus of EBV replication in lingual epithelium therefore provides a means of studying the EBV-epithelial cell interaction in situ. Replicative EBV DNA and productive cycle antigens are restricted to the upper, more differentiated epithelial layers in HL, and here we have applied highly sensitive in situ hybridization and immunohistological methods to examine the lower basal/suprabasal layers for evidence of latent EBV infection. We could not detect EBV DNA in these layers using an in situ DNA hybridization protocol which, on reference B cell lines, detected 1 viral genome/cell. Likewise, using sensitive in situ RNA hybridization for both the small non-polyadenylated EBER RNAs (abundant transcripts seen in all known forms of EBV latency) and the latent membrane protein (LMP) mRNA (the most abundant viral mRNA in B lymphoblastoid cell lines), the basal/suprabasal cells in HL were consistently negative; immunohistological staining with specific monoclonal antibodies also gave no evidence of latently infected LMP-positive cells. When the biopsy extracts were analysed by immunoblotting with selected human antisera, in addition to abundant productive cycle antigens, a band of constant size (66K) was observed which also reacted with immunopurified antibodies monospecific for one of the latency-associated nuclear antigens, EBNA 1; the cellular origin of this EBNA 1 could not be ascertained, but it is possible that in HL the protein is expressed during the productive cycle. The absence of demonstrable EBV latency in the basal/suprabasal cells of HL suggests that this is purely a virus replicative lesion which is sustained by continual re-infection of the maturing epithelium, not by the maturation of latently infected cells from the basal compartment.

Antigens, Viral↗
PubMed2006

Laser capture microdissection of plant cells from tape-transferred paraffin sections promotes recovery of structurally intact RNA for global gene profiling.

Laser capture microdissection and related technologies permit the harvest of individual cells and cell types. Isolation of either nucleic acids or proteins from laser-captured cells supports such downstream applications as the construction of cell-specific cDNA libraries and the profiling of expressed genes and proteins. The success of these endeavors is dependent upon the yield, purity and structural integrity of the macromolecules derived from harvested cells. Here, we report protocols that promote the isolation of structurally intact RNA from laser-captured cells of paraffin-embedded tissues. The use of a tape transfer system that obviates the need to wet paraffin sections prior to slide mounting significantly increases RNA structural quality. Integrity is assessed directly via electrophoretic separation of picogram-nanogram levels of total RNA isolated from multiple cell types, including those comprising Arabidopsis ovules, replums and stamen abscission zones. RNA prepared from specialized cells within siliques provided targets for profiling the Arabidopsis genome during replum cell development. Digital northern analysis of transcripts expressed near the threshold of the system's ability to score signal presence suggests that low-abundance transcripts representing as little as approximately 0.002% of total mRNA can be reliably detected. Microarray data reveal a significant shift from primary cell-wall metabolism to lignin biosynthesis in replum tissues during fruit maturation.

Arabidopsis↗
PubMed1997

An open reading frame for a protein involved in cytochrome c biogenesis is split into two parts in Brassica mitochondria.

A plant mitochondrial gene with sequence similarity to ccl1, a bacterial gene involved in cytochrome c biogenesis, occurs as a single open reading frame in wheat, Oenothera, carrot and Marchantia mtDNAs. In Brassica napus "Polima" or Brassica campestris mtDNA, however, this open reading frame is split into two segments that are located 60 kb apart on the master-circle form of the genome. Although transcripts of the split ORF are edited in a manner similar to that of a functional gene, transcripts that span both portions of the ORF are not evident in gel-blot hybridization experiments. Low-abundance transcripts that span both portions of the split ORF can be detected by RT-PCR, but these contain an additional 54-bp sequence, inserted between the two segments, that is unrelated to the corresponding sequences of other plants. Since this additional sequence introduces an in-frame stop codon, no transcripts have been found that could be translated to yield a protein product of a size similar to that present in other plant species. An antiserum directed against the product of the corresponding wheat gene detects polypeptides of similar size in wheat and Brassica mitochondria. This antiserum, however, immunoprecipitates a labelled polypeptide from the products of wheat, but not Brassica, in organello protein synthesis. Gel-blot analysis of total Brassica DNA indicates that sequences capable of hybridizing with the ORF are present in the nuclear genome. We suggest that the functional form of the Brassica gene may reside in the nucleus.

Amino Acid Sequence↗
PubMed1978

The transcription map of mouse mitochondrial DNA.

Nine transcripts complementary to mouse L cell mitochondrial DNA have been detected, sized and mapped to restriction fragments using the method of Berk and Sharp (1977). RNA isolated from L cell mitochondria was hybridized to 32P-labeled, cloned L cell mitochondrial DNA restriction fragments in 70% formamide under conditions 5 degrees C above the melting temperature of the DNA-DNA duplex, but approximately 15 degrees C below the melting temperature of the RNA-DNA duplex. The heteroduplexed material was then treated with the single-strand-specific nuclease S1, whick cleaves the single-stranded DNA not protected by RNA-DNA duplex formation into oligonucleotides and leaves intact 32P-labeled, single-stranded DNA replicas complementary to the transcripts. The single-stranded DNA replicas were then resolved and sized by alkaline agarose gel electrophoresis. Hybridization to strand-separated, 32P-labeled L cell mitochondria DNA restriction fragments under the same conditions showed that all nine transcripts hybridized exclusively to the heavy strand (H strand) of restriction fragments isolated as the dense strand from alkaline CsCl gradients, indicating that all stable transcripts 300 bases or longer detected by this technique originate from genes on the H strand. The two most abundant transcripts homologous to mitochondrial DNA map adjacent to the origin of replication. This result is consistent with map positions assigned to the large and small mitochondrial ribosomal RNAs isolated from Xenopus laevis and HeLa cells. Six of the other seven transcripts map continuously in approximately 40% of the genome. Only one transcript of 950 bases maps in the first quadrant of the genome as defined by the origin and direction of mitochondrial DNA replication, and it does not lie within the D loop region. The genetic function of the remaining 75% of this region of the genome is yet to be determined.

DNA Replication↗
PubMed1994

The Lighten up (Lip) gene of Drosophila melanogaster, a modifier of retroelement expression, position effect variegation and white locus insertion alleles.

We are interested in identifying single gene mutations that are involved in trans-acting dosage regulation in order to understand further the role of such genes in aneuploid syndromes, various types of dosage compensation as well as in regulatory mechanisms. The Lighten up (Lip) gene in Drosophila melanogaster was identified in a mutagenic screen to detect dominant second site modifiers of white-blood (wbl), a retrotransposon induced allele of the white eye color locus. Lip specifically enhances the phenotype of wbl as well as a subset of other retroelement insertion alleles of white, including the copia-induced allele, white-apricot (wa), and six alleles caused by insertion of I elements. We isolated six alleles of Lip which are all recessive lethal, although phenotypically additive heteroallelic escapers were recovered in some combinations. Lip also suppresses position effect variegation, indicating that it may have a role in chromatin configuration. Additionally, Lip modifies the total transcript abundance of both the blood and copia retrotransposons, having an inverse effect on the steady state level of blood transcripts, while showing a non-additive effect on copia RNA.

Alleles↗
PubMed2004

Furanocoumarin biosynthesis in Ammi majus L. Cloning of bergaptol O-methyltransferase.

Plants belonging to the Apiaceae or Rutaceae accumulate methoxylated psoralens, such as bergapten or xanthotoxin, as the final products of their furanocoumarin biosynthesis, and the rate of accumulation depends on environmental and other cues. Distinct O-methyltransferase activities had been reported to methylate bergaptol to bergapten and xanthotoxol to xanthotoxin, from induced cell cultures of Ruta graveolens, Petroselinum crispum and Ammi majus. Bergaptol 5-O-methyltransferase (BMT) cDNA was cloned from dark-grown Ammi majus L. cells treated with a crude fungal elicitor. The translated polypeptide of 38.7 kDa, composed of 354 amino acids, revealed considerable sequence similarity to heterologous caffeic acid 3-O-methyltransferases (COMTs). For homologous comparison, COMT was cloned from A. majus plants and shown to share 64% identity and about 79% similarity with the BMT sequence at the polypeptide level. Functional expression of both enzymes in Escherichia coli revealed that the BMT activity in the bacterial extracts was labile and rapidly lost on purification, whereas the COMT activity remained stable. Furthermore, the recombinant AmBMT, which was most active in potassium phosphate buffer of pH 8 at 42 degrees C, showed narrow substrate specificity for bergaptol (Km SAM 6.5 micro m; Km Bergaptol 2.8 micro m) when assayed with a variety of substrates, including xanthotoxol, while the AmCOMT accepted 5-hydroxyferulic acid, esculetin and other substrates. Dark-grown A. majus cells expressed significant BMT activity which nevertheless increased sevenfold within 8 h upon the addition of elicitor and reached a transient maximum at 8-11 h, whereas the COMT activity was rather low and did not respond to the elicitation. Complementary Northern blotting revealed that the BMT transcript abundance increased to a maximum at 7 h, while only a weak constitutive signal was observed for the COMT transcript. The AmBMT sequence thus represents a novel database accession specific for the biosynthesis of psoralens.

5-Methoxypsoralen↗
PubMed2001

Developmental expression of cytochrome P450 aromatase genes (CYP19a and CYP19b) in zebrafish fry (Danio rerio).

Cytochrome P450 aromatase (CYP19) is the terminal enzyme in the steroidogenic pathway that converts androgens (e.g., testosterone) into estrogens (e.g., estradiol). Regulation of this gene dictates the ratio of androgens to estrogens; therefore, appropriate expression of this enzyme is critical for reproduction as well as being pivotal in sex differentiation for most vertebrates. It is assumed that most vertebrates have a single CYP19 gene that is regulated by multiple tissue-specific promoter regions. However, the zebrafish (Danio rerio) has two genes (CYP19a and CYP19b), each encoding a significantly different protein and possessing its own regulatory mechanism. The primary purpose of this study was to determine the pattern of expression of each of the CYP19 genes in the developing zebrafish. A fluorescent-based method of real-time, quantitative RT-PCR provided the sensitivity and specificity to determine transcript abundance in single embryos/juveniles harvested at days 0 through 41 days post-fertilization (dpf), which encompasses the developmental events of sex determination and gonadal differentiation. CYP19 transcripts could be detected as early as 3 or 4 dpf, (CYP19a and CYP19b, respectively) and peak abundance was detected on day five. In general, the CYP19 genes differed significantly in the ontogeny of their expression. In most cases, the gonadal form of CYP19 (CYP19a) was more abundant than the brain form (CYP19b); however, unlike CYP19a, the pattern of CYP19b expression could be clearly segregated into two populations, suggesting an association with sex differentiation. Pharmacological steroids (ethinylestradiol and 17 alpha-methyltestosterone) enhanced the expression of the CYP19b gene at all three days examined (4, 6, and 10 dpf). These data suggest that the timely and appropriate expression of CYP19 is important in development and that the expression of CYP19b (the "extra-gonadal" form) may be associated with sexual differentiation if not sexual determination. J. Exp. Zool. 290:475-483, 2001.

Animals↗
PubMed2004

An empirical Bayesian significance test of cDNA library data.

Automated high-throughput sequencing of cDNA clones from numerous libraries has generated a wealth of information about both genome sequence and relative transcript abundances. A common statistical challenge in the analysis of library sequences is to infer whether there is differential expression for the same transcript under two different conditions, such as normal and diseased tissue. In contrast to the continuously variable intensity measurements from microarray experiments, data from cDNA library sequencing presents itself as a discrete count of the incidence of some clone or transcript in a finite sample. In this paper, we first propose a statistical model for data generated from cDNA library sequencing efforts. The model is based on the Poisson mixed with generalized inverse Gaussian (PGIG), introduced by Sichel (1971, 1975). PGIG has been used in modeling population abundance, ecological studies, word frequencies in publications, etc. Using data from the literature, we show that the proposed model provides a good fit to the observed data. Using this new model for cDNA library data, we developed an empirical Bayesian significance test (EBST) for inferring the statistical significance of differential gene expression from discrete data.

Bayes Theorem↗