RESUMEN
Little is known about gene action in the preimplantation events that initiate mammalian development. Based on cDNA collections made from each stage from egg to blastocyst, 25438 3'-ESTs were derived, and represent 9718 genes, half of them novel. Thus, a considerable fraction of mammalian genes is dedicated to embryonic expression. This study reveals profound changes in gene expression that include the transient induction of transcripts at each stage. These results raise the possibility that development is driven by the action of a series of stage-specific expressed genes. The new genes, 798 of them placed on the mouse genetic map, provide entry points for analyses of human and mouse developmental disorders.
Asunto(s)
Desarrollo Embrionario/genética , Expresión Génica , Animales , Mapeo Cromosómico , ADN Complementario , Etiquetas de Secuencia Expresada , Femenino , Biblioteca de Genes , Humanos , Masculino , Ratones , Ratones Endogámicos C57BL , EmbarazoRESUMEN
Two novel mouse genes and one novel human gene that define distinctive eukaryotic nucleotide-binding proteins (NUBP) and are related to the mrp gene of prokaryotes are characterized. Phylogenetic analyses of the genes, encoding a short form (Nubp2) and a long form (Nubp1) of NUBP, clearly establish them as a new NUBP/MRP gene family that is well conserved throughout phylogeny. In addition to conserved ATP/GTP-binding motifs A (P-loop) and A', members of this family share at least two highly conserved sequence motifs, NUBP/MRP motifs alpha and beta. Only one type of NUBP/MRP gene has been observed thus far in prokaryotes, but there are two types in eukaryotes. One group includes mouse Nubp1, human NBP, yeast NBP35, and Caenorhabditis elegans F10G8.6 and is characterized by a unique N-terminal sequence with four cysteine residues that is lacking in the other group, which includes mouse Nubp2, human NUBP2, and yeast YIA3w. Northern blot analyses of the two mouse genes show distinctive patterns consistent with this classification. Mouse Nubp2 is mapped to the t-complex region of mouse Chromosome 17, whereas Nubp1 is mapped to the proximal region of mouse Chromosome 16. Interestingly, both regions are syntenic with human chromosome 16p13.1-p13.3, suggesting that a chromosomal breakage between Nubp2 and Nubp1 probably occurred during the evolution of mouse chromosomes.
Asunto(s)
Proteínas de Unión al GTP/genética , Familia de Multigenes , Nucleótidos/metabolismo , Secuencia de Aminoácidos , Animales , Secuencia de Bases , Caenorhabditis elegans/genética , Proteínas Portadoras/genética , Mapeo Cromosómico , Secuencia de Consenso , Cartilla de ADN/genética , ADN Complementario/genética , Humanos , Péptidos y Proteínas de Señalización Intracelular , Ratones , Datos de Secuencia Molecular , Filogenia , Saccharomyces cerevisiae/genética , Homología de Secuencia de Aminoácido , Especificidad de la EspecieRESUMEN
Mammalian embryos can only survive if they attach to the uterus (implantation) and establish proper maternal-fetal interactions. To understand this complex implantation pathway, we have initiated genomic analysis with a systematic study of the cohort of genes expressed in extraembryonic cells that are derived from the conceptus and play a major role in this process. A total of 2103 cDNAs from the extraembryonic portion of 7.5-day post-conception mouse embryos yielded 3186 expressed sequence tags, approximately 40% of which were novel to the sequence databases. Furthermore, when 155 of the cDNA clones with no homology to previously detected genes were genetically mapped, apparent clustering of these expressed genes was detected in subregions of chromosomes 2, 7, 9 and 17, with 6.5% of the observed genes localized in the t-complex region of chromosome 17, which represents only approximately 1.5% of the mouse genome. In contrast, X-linked genes were under-represented. Semi-quantitative RT-PCR analyses of the mapped genes demonstrated that one third of the genes were expressed solely in extraembryonic tissue and an additional one third of the genes were expressed predominantly in the extraembryonic tissues. The over-representation of extraembryonic-expressed genes in dosage-sensitive autosomal imprinted regions and under-representation on the dosage-compensated X chromosome may reflect a need for tight quantitative control of expression during development.
Asunto(s)
Cromosomas/genética , Ectodermo/metabolismo , Embrión de Mamíferos/metabolismo , Desarrollo Embrionario y Fetal/genética , Péptidos y Proteínas de Señalización Intracelular , Proteínas Asociadas a Microtúbulos , Proteínas Nucleares/genética , Cromosoma X/genética , Animales , Mapeo Cromosómico , ADN Complementario/química , ADN Complementario/genética , Interpretación Estadística de Datos , Etiquetas de Secuencia Expresada , Femenino , Expresión Génica , Regulación del Desarrollo de la Expresión Génica , Biblioteca de Genes , Genes/genética , Marcadores Genéticos , Genoma , Edad Gestacional , Ratones , Ratones Endogámicos C57BL , Datos de Secuencia Molecular , Embarazo , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Transcripción Genética , Ubiquitina-Proteína Ligasas , Región del Complejo T del GenomaRESUMEN
We have shown previously that a GC-rich element (GGGGCGGGGTGGGGGG) conferring epidermal growth factor (EGF) responsiveness to the human gastrin promoter binds Sp1 and additional undefined complexes. A rat GH4 cell line expression library was screened by using a multimer of the gastrin EGF response element, and three overlapping cDNA clones were identified. The full-length rat cDNA encoded an 89-kDa zinc finger protein (ZBP-89) that was 89% identical to a 49-kDa human factor, ht(beta), that binds a GTGGG/CACCC element in T-cell receptor promoters. The conservation of amino acids between the zinc fingers indicates that ZBP-89 is a member of the C2H2 zinc finger family subclass typified by the Drosophila Krüppel protein. ZBP-89 is ubiquitously expressed in normal adult tissues. It binds specifically to the gastrin EGF response element and inhibits EGF induction of the gastrin promoter. Collectively, these results demonstrate that ZBP-89 functions as a repressor of basal and inducible expression of the gastrin gene.
Asunto(s)
Proteínas de Unión al ADN/genética , Factor de Crecimiento Epidérmico/metabolismo , Gastrinas/genética , Regulación de la Expresión Génica , Proteínas Represoras , Factores de Transcripción/genética , Dedos de Zinc/genética , Secuencia de Aminoácidos , Animales , Secuencia de Bases , Línea Celular , Factor de Crecimiento Epidérmico/genética , Gastrinas/metabolismo , Humanos , Factores de Transcripción de Tipo Kruppel , Datos de Secuencia Molecular , Regiones Promotoras Genéticas/genética , RatasRESUMEN
Hyaluronic acid (HA) and its synthesis were studied in intact Swiss 3T3 mouse fibroblasts and isolated membranes. HA chains in culture medium, attached to cells and in isolated membranes, were determined to possess average M(r) values of 5.2 x 10(6), 1.8 x 10(6) and 0.14 x 10(6) respectively. Log cells were determined to possess 680,000 HA molecules/cell, and to release 120,000 HA chains/h. The time required for intact cells to synthesize and release a complete HA chain was approximately 4 h, with elongation proceeding at a rate of 57 dimers/min. The amount of cell-associated HA of various cell populations correlated strongly with their rate of HA release into culture media and with the HA synthetase activity determined for their membranes. Prevention of protein synthesis with cycloheximide decreased the rate of HA synthesis of log cells and HA synthetase activity of isolated membranes by 50% within 2-3 h. Because of the similarity between the biological lifetime of HA synthetase and the time required to synthesize a HA chain, we propose a model where each synthetase makes only one HA chain; after synthesis of a complete HA chain, HA synthetase activity is terminated as its HA chain is released from the cell.