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PubMed2003

Molecular and genetic analysis of the Polycomb group gene Sex combs extra/Ring in Drosophila.

Polycomb group (PcG) proteins repress homeotic genes and other developmental regulatory genes in cells where these genes must remain inactive during development. In Drosophila and in vertebrates, PcG proteins exist in two distinct multiprotein complexes, the Esc/Eed-E(z) complex and PRC1. Drosophila PRC1 contains Polycomb, Posterior sexcombs and Polyhomeotic, the products of three PcG genes that are critically needed for PcG silencing. Formation of stable PRC1 requires Ring, the product of a gene for which no mutations have been described. Here, we show that Sex combs extra (Sce) encodes Ring and that Sce/Ring function is critically required for PcG silencing.

Amino Acid Sequence↗
PubMed2004

The histone methyltransferases Trithorax and Ash1 prevent transcriptional silencing by Polycomb group proteins.

Transcriptional on and off states of HOX genes and other developmental control genes are maintained by antagonistic regulators encoded by trithorax group (trxG) and Polycomb group (PcG) genes. The trxG proteins Ash1 and hTRX and the PcG repressor E(z) are histone methyltransferases (HMTases) that methylate distinct lysine residues in the N-terminal tail of histone H3. trxG proteins are generally thought to function as activators of HOX genes, but how histone methylation by Ash1 and Trx promotes HOX gene transcription is not clear. Here, we show that in ash1 and trx mutants expression of HOX genes is lost within their normal expression domains, but we find that, contrary to expectation, this expression is restored in ash1 and trx mutants that also lack PcG gene function. Moreover, such trxG PcG double mutants show severe misexpression of HOX genes and, hence, ectopic activation of HOX genes caused by the removal of PcG gene function also occurs in the absence of ash1 and trx function. Together, these results suggest that the Ash1 and Trx HMTases are not "coactivators" required for transcriptional activation of HOX genes, but function specifically as anti-repressors. We propose that histone methylation by Ash1 and Trx is required continuously throughout development to prevent inappropriate PcG silencing of HOX genes in cells in which they must stay transcriptionally active.

Animals↗
PubMed1982

Cloning of developmentally regulated flagellin genes from Caulobacter crescentus via immunoprecipitation of polyribosomes.

Immunoprecipitation of Caulobacter crescentus polyribosomes with antiflagellin antibody provided RNA for the synthesis of cDNA probes that were used to identify three specific EcoRI restriction fragments (6.8, 10, and 22 kilobases) in genomic digests of Caulobacter DNA. The RNA was present only in polyribosomes isolated from a time interval in the Caulobacter cell cycle that was coincident with flagellin polypeptide synthesis. The structural gene for Mr 27,500 flagellin polypeptide was assigned to a region of the 10-kilobase EcoRI restriction fragment by DNA sequence analysis. Analysis of mutants defective in motility further established a correlation between the Mr 27,500 flagellin gene and the flaE gene locus [Johnson, R. C. & Ely, B. (1979) J. Bacteriol. 137, 627-634]. The other EcoRI fragments that hybridize with the immunoprecipitated polyribosome-derived cDNA probe are also temporally regulated and have features that suggest they encode other polypeptides associated with the flagellum. Modifications were required to adapt the procedure of immunoprecipitation of polyribosomes for use with Caulobacter and should be applicable to the production of specific structural gene probes from other prokaryotic systems.

Bacteria↗
PubMed2005

Molecular cloning and characterization of an almond 9-hydroperoxide lyase, a new CYP74 targeted to lipid bodies.

Oxylipin metabolism represents one of many defence mechanisms employed by plants. It begins with the oxygenation of polyunsaturated fatty acids by lipoxygenases to form fatty acid hydroperoxides that are substrates for several enzymes, including specialized cytochrome P450s known as CYP74s. The targeting of a new CYP74, a 9-hydroperoxide lyase (HPL) from almonds, to the endomembrane system and lipid bodies, both as enzyme activity in almond seeds and as GFP fusions transiently expressed in tobacco protoplasts, is described. Such association of a CYP74 with lipid bodies has not been reported previously. Also described are the properties of a 9-HPL gene, the developmental regulation of its expression, the production and characterization of recombinant 9-HPL in Escherichia coli, and the developmental correlation between gene expression, enzyme activity, and the appearance of volatile C9 aldehydes from HPL action.

Amino Acid Sequence↗
PubMed1996

Human tumor rejection antigens MAGE.

Characterization of the MAGE genes has facilitated a molecular approach to identification of the genes encoding tumor-rejection antigens expressed on human cancer cells. MAGE proteins are normal tissue antigens compartmentalized in testicular cells that play an important role in the early phase of spermatogenesis. MAGE-1, -2, -3, -4, and -6 genes are preferentially expressed in many different cancers at both the mRNA and protein levels. More than half of human cancers of various histologic type express at least one of these MAGE genes. Demethylation induces MAGE antigens in cells, suggesting that MAGE genes are important developmentally regulated genes under methylating control. Thus, genetic instability in cells causing loss of this methylating control could result in the preferential expression of MAGE genes in cancer cells. Therefore, MAGE gene products may be appropriate target molecules for development of new cancer vaccine.

Amino Acid Sequence↗
PubMed1984

Developmental regulation of gene expression in wheat embryos. Molecular cloning of a DNA sequence encoding the early-methionine-labelled (Em) polypeptide.

The early-methionine-labelled (Em) polypeptide is the most abundant single polypeptide found in the cytosolic fraction (30 000 X g supernatant) of dry wheat embryos. Synthesis of this polypeptide is readily detectable during the earliest stages (0-3 h) of wheat embryo germination, by labelling with [35S]methionine. Thereafter, synthesis of the Em polypeptide declines rapidly. A DNA sequence encoding a portion of this polypeptide has been isolated by the molecular cloning of DNA complementary to the messenger RNA from dry wheat embryos. The identity of this cloned sequence has been confirmed by hybrid-selected translation of the Em messenger, the product being analysed by one- and two-dimensional polyacrylamide gel electrophoresis, and by analysis of its partial proteolytic digestion products. Gel blot hybridisation of RNA isolated from embryos at successive stages of germination with this cloned DNA sequence confirms that the decline in the synthesis of the Em polypeptide, detected by labelling in vivo, is a consequence of the degradation of its corresponding messenger RNA.

Cloning, Molecular↗
PubMed1990

The spoIIJ gene, which regulates early developmental steps in Bacillus subtilis, belongs to a class of environmentally responsive genes.

The Bacillus subtilis spoIIJ locus is defined by a Tn917 insertion which leads to an oligosporogenous phenotype. Here we show that this mutation severely decreases transcription of spoIIA, spoIIE, and spoIIG, three operons involved in asymmetric septation, the earliest morphological event of sporulation. A 14.3-kilobase region overlapping the site of the spoIIJ::Tn917 insertion was cloned and the exact location of the spoIIJ gene was defined with various integrative plasmids carrying subfragments of that region. DNA sequencing established that spoIIJ is a monocistronic locus encoding a 606-amino-acid polypeptide which contains a canonical "transmitter" domain, indicating that spoIIJ is a new member of the "sensor" class of signal-transducing systems in bacteria. Thus, spoIIj, which is transcribed during vegetative growth, presumably under the control of sigma H, encodes a protein that could interact with major regulators of early sporulation stages, such as SpoOA and/or SpoOF.

Bacillus subtilis↗
PubMed2001

The polycomb-group gene Ezh2 is required for early mouse development.

Polycomb-group (Pc-G) genes are required for the stable repression of the homeotic selector genes and other developmentally regulated genes, presumably through the modulation of chromatin domains. Among the Drosophila Pc-G genes, Enhancer of zeste [E(z)] merits special consideration since it represents one of the Pc-G genes most conserved through evolution. In addition, the E(Z) protein family contains the SET domain, which has recently been linked with histone methyltransferase (HMTase) activity. Although E(Z)-related proteins have not (yet) been directly associated with HMTase activity, mammalian Ezh2 is a member of a histone deacetylase complex. To investigate its in vivo function, we generated mice deficient for Ezh2. The Ezh2 null mutation results in lethality at early stages of mouse development. Ezh2 mutant mice either cease developing after implantation or initiate but fail to complete gastrulation. Moreover, Ezh2-deficient blastocysts display an impaired potential for outgrowth, preventing the establishment of Ezh2-null embryonic stem cells. Interestingly, Ezh2 is up-regulated upon fertilization and remains highly expressed at the preimplantation stages of mouse development. Together, these data suggest an essential role for Ezh2 during early mouse development and genetically link Ezh2 with eed and YY1, the only other early-acting Pc-G genes.

Animals↗
PubMed1985

Developmentally regulated cytokeratin gene in Xenopus laevis.

We have determined the sequence of cloned cDNAs derived from a 1,665-nucleotide mRNA which transiently accumulates during Xenopus laevis embryogenesis. Computer analysis of the deduced amino acid sequence revealed that this mRNA encodes a 47-kilodalton type I intermediate filament subunit, i.e., a cytokeratin. As is common to all intermediate filament subunits so far examined, the predicted polypeptide, named XK70, contains N- and C-terminal domains flanking a central alpha-helical rod domain. The overall amino acid homology between XK70 and a human 50-kilodalton type I keratin is 47%; homology within the alpha-helical domain is 57%. The N-terminal domain, which is not completely contained in our cDNAs, is basic, contains 42% serine plus alanine, and includes five copies of a six-amino-acid repeating unit. The C-terminal domain has a high alpha-helical content and contains a region with sequence homology to the C-terminal domains of other type I and type III intermediate filament proteins. We suggest that different keratin filament subtypes may have different functional roles during amphibian oogenesis and embryogenesis.

Amino Acid Sequence↗
PubMed1986

Developmental expression of genes for the stereoidogenic enzymes P450scc (20,22-desmolase), P450c17 (17 alpha-hydroxylase/17,20-lyase), and P450c21 (21-hydroxylase) in the human fetus.

Fetal adrenal steroidogenesis is required for the production of placental estrogen, and fetal testicular steroidogenesis is required for the development of male external genitalia. We studied the ontogeny and tissue specificity of expression of the genes for three steroidogenic enzymes: P450scc (the cholesterol side-chain cleavage enzyme), P450c17 (17 alpha-hydroxylase/17,20-lyase), and P450c21 (21-hydroxylase) in the human fetus. RNA from fetal tissues was probed with homologous human P450scc, P450c17, and P450c21 cDNAs cloned in our laboratory. At 20-21 weeks gestation, P450scc mRNA was most abundant in the adrenal, followed by testis, placenta, and ovary. P450c17 mRNA was also most abundant in the adrenal, followed by testis and ovary, but was undetectable in the placenta. P450c21 mRNA was detected only in the adrenal. None of these mRNAs was detected in kidney, liver, spleen, intestine, or muscle. Twenty-two fetal testis samples (13-25.8 weeks gestation) were studied. P450scc and P450c17 mRNAs were most abundant at 14-16 weeks and diminished to 35 and 19% of their peak values, respectively, by 20-25.8 weeks. Ovarian P450scc and P450c17 mRNAs were present, respectively, in only 6.2% and 1.8% of the maximum amount in the testis and did not vary detectably from 14.9 to 21.5 weeks gestation. The testicular and ovarian steroidogenic enzyme mRNA data correlate well with previously reported changes in gonadal steroidogenesis with gestational age. The presence of P450scc mRNA, but not P450c17 mRNA, in the placenta indicates that the placenta is able to initiate the synthesis of some steroid hormones, but is not able to synthesize estrogen de novo. Since P450c21 was found only in the adrenal, the extraadrenal 21-hydroxylation of progesterone to deoxycorticosterone, a common event in the fetus, is probably mediated by an enzyme(s) other than P450c21.

Aldehyde-Lyases↗
PubMed2006

Thyroid hormone-mediated negative transcriptional regulation of Necdin expression.

Unliganded thyroid hormone receptors (apoTRs) repress transcription of hormone-activated genes by recruiting corepressors to the promoters. In contrast, on promoters containing so-called negative thyroid hormone response elements (nTREs), apoTRs activate transcription. A number of different molecular mechanisms have been described as to how apoTRs activate transcription varying with the target gene of the study. Here we demonstrate that thyroid hormone regulates the transcription of the Necdin gene, a developmentally regulated candidate gene for the genomic imprinting-associated neurobehavioural disorder, Prader-Willi syndrome. ApoTRs activate Necdin expression through an nTRE in its promoter, downstream of the transcription start site. The nTRE of the Necdin gene resembles the nTREs of the TSHbeta genes of the hypothalamus-pituitary-thyroid axis in the sequence, position in the promoter, and mode of activation. We show that this group of nTRE-driven genes shares the requirements for binding of the retinoic X receptor and nuclear receptor corepressor/silencing mediator of retinoid and thyroid hormone receptors (NCoR/SMRT) for full ligand-independent activation, whereas there is no need for association of the p160 family of coactivators. In accordance with the requirement for corepressors, Necdin expression is influenced by deacetylase activity, suggesting that histone deacetylases and corepressors as well could function as activators of transcription, depending on the promoter context.

Animals↗
PubMed2000

[New approaches in developmental genetics and gene therapy: xenotransplantation of Drosophila embryonic nerve cells into the brain of vertebrate animals].

The author's studies dealing with xenotransplantations of the embryonic nervous tissue into the brain of amphibian and mammals are reviewed. Drosophila nerve cells have been shown to survive inside the brain, differentiate, form ganglion-resembling structure, and come into synaptic contact with the host tissue. The embryonic nerve cells of transgenic Delta mutants of Drosophila melanogaster carrying a gene of bacterial galactosidase (lacZ) were also transplanted into the brain of adult rats and then identified histologically by X-gal staining of brain sections for the lacZ-gene product. The xenografts were shown to survive in the host brain for at least 2-3 weeks, after which they were attacked by macrophages. No glial scar tissue formed around the site of the xenograft. Cotransplantation of Drosophila embryonic nerve cells and the homologous embryonic nerve tissue was favorable for homograft survival and development, because formation of the glial scar was blocked, whereas homograft vascularization and differentiation of its nerve cells were stimulated. The results obtained are of interest with regard to neurosurgery, because they may be used to prevent formation of glial scar, an important factor in successful neurotransplantation.

Amphibians↗
PubMed1992

Functional analysis of the human platelet-derived growth factor A-chain promoter region.

The platelet-derived growth factor (PDGF) A-chain gene is a developmentally regulated gene that is expressed in high levels in a limited number of normal and transformed cell lines and in cells stimulated by cytokines, including PDGF itself. We have now analyzed potential regulatory elements in 3.6 kilobase pairs (kb) of the 5'-flanking sequences of the human PDGF A-chain gene using reporter gene constructs and transient transfection analyses. The region between base pairs (bp) -618 and +392 (relative to the transcription initiation site) is sufficient for optimal promoter activity. A highly G + C region containing three contiguous Sp1 binding sites between bp -150 and -33 contributes over 80% of promotor activity. DNase I footprinting analyses indicates that Sp1 binds to and protects over 57 bp of this G + C region. A functional serum response element is located within bp -477 and -468 and positively regulates induction of PDGF A by PDGF. A negative regulatory (silencer) element is located from -1.9 to -0.9 kb. The results suggest that the major constitutive expression of the PDGF A-chain gene requires a highly G + C-rich region containing three Sp1 binding sites and that induction of the PDGF A-chain gene by PDGF is mediated by a SRE located at bp -477 to -468.

Base Sequence↗