RESUMEN
Prohibition is a recently identified antiproliferative protein whose exact role in the cell is under investigation. To determine the human chromosomal location of the prohibition gene (PHB) and whether this site corresponds to that of any suspected tumor suppressor gene, we have analyzed DNA from three sources by hybridization analysis: mouse--human hybrid cell lines, hybrid cell lines containing portions of human chromosomes, and human metaphase chromosomes in situ. All three techniques confirm a location in the region 17q21-q22, a region genetically linked to early-onset human breast cancer. Further analysis will be required to establish the significance of this relationship; Southern hybridizations show a polymorphic EcoRI site that may be useful for this purpose.
Asunto(s)
Cromosomas Humanos Par 17 , Polimorfismo de Longitud del Fragmento de Restricción , Proteínas/genética , Proteínas Represoras , Southern Blotting , Bandeo Cromosómico , Mapeo Cromosómico , Femenino , Humanos , Masculino , Linaje , ProhibitinasRESUMEN
Genes that act inside the cell to negatively regulate proliferation are of great interest because of their implications for such processes as development and cancer, but these genes have been difficult to clone. This report details the cloning and analysis of cDNA for prohibitin, a novel mammalian antiproliferative protein. Microinjection of synthetic prohibitin mRNA blocks entry into S phase in both normal fibroblasts and HeLa cells. Microinjection of an antisense oligonucleotide stimulates entry into S phase. By sequence comparison, the prohibitin gene appears to be the mammalian analog of Cc, a Drosophila gene that is vital for normal development.
Asunto(s)
Ciclo Celular , Proteínas/genética , Proteínas Represoras , Secuencia de Aminoácidos , Animales , Secuencia de Bases , Northern Blotting , Clonación Molecular , ADN/biosíntesis , ADN/genética , Genes , Células HeLa , Humanos , Hígado/fisiología , Microinyecciones , Datos de Secuencia Molecular , Prohibitinas , Biosíntesis de Proteínas , ARN Mensajero/genética , Ratas , Homología de Secuencia de Ácido Nucleico , Transcripción GenéticaRESUMEN
Studies of chromosome loss in inherited cancers, of fusions between proliferating and quiescent cells, and of microinjection of RNA from quiescent cells into proliferation competent cells have all provided evidence for antiproliferative genes in mammalian cells. In this report, we describe a partial cDNA clone isolated on the basis of its preferential hybridization to RNA from normal versus regenerating rat liver. The corresponding mRNA, enriched by hybrid selection, was microinjected into normal human diploid fibroblasts in cell culture, resulting in a 53% decrease in the fraction of nuclei incorporating tritiated thymidine. This mRNA is 2 kb in size and is expressed in eight tissues examined.
Asunto(s)
Clonación Molecular , Replicación del ADN , ADN/aislamiento & purificación , Regeneración Hepática , Hígado/metabolismo , ARN Mensajero/genética , Animales , Northern Blotting , Línea Celular , ADN/genética , Humanos , Masculino , Microinyecciones , Hibridación de Ácido Nucleico , Plásmidos , Poli A/aislamiento & purificación , ARN/aislamiento & purificación , Ratas , Ratas EndogámicasRESUMEN
Poly(A) RNAs from normal rat liver and senescent human fibroblasts appear to have more antiproliferative activity than RNAs from regenerating rat liver and early passage human fibroblasts. We have screened two rat liver and one human liver library by differential hybridization and isolated four candidate cDNAs for this antiproliferative activity; one is fibronectin and three others do not match to any sequence in the mammalian portion of the GENBANK database. We are currently testing the antiproliferative nature of these cDNAs by microinjection of hybrid-selected RNA, and we describe an alternative strategy for cloning such genes based on construction of a cDNA library in an RNA expression vector.
Asunto(s)
División Celular , Genes , Animales , Clonación Molecular , Fibronectinas/farmacología , Biblioteca de Genes , Pruebas Genéticas , Humanos , Microinyecciones , Poli A , ARN Mensajero/aislamiento & purificación , Ratas , Ratas EndogámicasRESUMEN
The chloroperoxidase gene from the filamentous fungus Caldariomyces fumago has been isolated within a 16.3-kilobase insert in the vector lambda EMBL3. The DNA sequence of the gene and its immediate flanking regions has been determined, and the start site of transcription has been mapped by primer extension.