RESUMEN
Spermatogenesis is a complex process that occurs in successive mitotic, meiotic and post-meiotic phases and involves a highly regulated selective gene-expression pattern. However, this process has not been well characterised at the gene expression level due to the absence of germinal cell lines. We previously demonstrated that the rat skeletal muscle-specific gene for the glycolytic enzyme phosphoglycerate mutase is also specifically expressed in meiotic and haploid male germ cells from testis (12). To analyse the promoter elements that regulate the transcription of the phosphoglycerate mutase m gene (pgam-m)during spermatogenesis, we developed transgenic mice for a construct containing 1.3 kb from the pgam-m promoter linked to the Escherichia coli LacZ gene. RNA analysis by retrotranscription and PCR amplification of transgene expression showed transcriptional activity in the testis with a pattern during testis development that was identical to the endogenous gene. The transgene was also active in skeletal muscle but not in the adult heart in all the transgenic lines analysed. Collectively, these studies demonstrate that the 1.3 kb pgam-m promoter contains sufficient sequences to specify temporally regulated testis-specific expression as well as skeletal-muscle expression.
Asunto(s)
Músculo Esquelético/enzimología , Fosfoglicerato Mutasa/genética , Testículo/enzimología , Animales , Secuencia de Bases , ADN/genética , Cartilla de ADN/genética , Expresión Génica , Regulación del Desarrollo de la Expresión Génica , Regulación Enzimológica de la Expresión Génica , Humanos , Operón Lac , Masculino , Ratones , Ratones Transgénicos , Datos de Secuencia Molecular , Regiones Promotoras Genéticas , ARN Mensajero/genética , ARN Mensajero/metabolismo , Ratas , Espermatogénesis/genética , Espermatogénesis/fisiología , Testículo/crecimiento & desarrolloRESUMEN
We have previously demonstrated that maize (Zea mays) 2,3-bisphosphoglycerate-independent phosphoglycerate mutase (PGAM-i) is not related to 2,3-bisphosphoglycerate-dependent phosphoglycerate mutase. With the aid of specific anti-maize PGAM-i antibodies, we demonstrate here the presence of a closely related PGAM-i in other plants. We also describe the isolation and sequencing of a cDNA-encoding almond (Prunus amygdalus) PGAM-i that further demonstrates this relationship among plant PGAM-i. A search of the major databases for related sequences allowed us to identify some novel PGAM-i from different sources: plants (Arabidopsis thaliana, Oryza sativa and Antithamniom sp.), monera (Escherichia coli, Bacillus subtilis and Bacillus megaterium) and animals (Caenorhabditis elegans). All of these amino acid sequences share a high degree of homology with plant PGAM-i. These observations suggest that the PGAM-i from several biological kingdoms constitute a family of protein different from other proteins with related enzymatic function and arose from a common ancestral gene that has diverged throughout its evolution.
Asunto(s)
Fosfoglicerato Mutasa/genética , Secuencia de Aminoácidos , Animales , Bacterias , Evolución Biológica , Secuencia Conservada , ADN Complementario/genética , ADN Complementario/aislamiento & purificación , Datos de Secuencia Molecular , Plantas , Alineación de Secuencia , Análisis de SecuenciaRESUMEN
Spermatogenesis is a dramatic differentiation process which involves very selective but poorly characterized gene-expression patterns. To gain insight into this process, we have investigated the expression during spermatogenesis of the genes that encode phosphoglycerate mutase, an essential glycolytic enzyme for the spermatozoa energy supply. By using cDNA and genomic probes we demonstrate the presence in testis of a mRNA corresponding to the muscle-specific phosphoglycerate mutase which shows a longer poly(A) tail. This muscle-specific gene is submitted to developmental regulation during testis maturation and begins to be expressed at postnatal day 22, when germ cells start to enter into meiosis. Northern blot and in situ hybridization experiments show that in contrast to what happens during skeletal-muscle differentiation, PGAM-M gene expression during spermatogenesis is not coupled to constitutive phosphoglycerate mutase (PGAM-B) gene repression. Thus, the muscle-specific PGAM-M gene constitutes a meiotic gene and therefore represents a very interesting model to study differential tissue-specific gene expression.