RESUMEN
Expression of the avian apoVLDLII gene is liver specific and completely dependent on estrogen. Previous analyses of protein binding sites in the apoVLDLII promoter revealed interactions between liver-enriched and ubiquitous factors at a location, site F', between nucleotides -229 and -260 relative to the major transcriptional start site. Site-directed mutagenesis of G residues contacted by these factors decreased expression from the promoter approximately 5-fold in the avian hepatoma cell line LMH2A. We have used this site to screen a cDNA expression library constructed from day 9 embryonic liver RNA. One of the two DNA binding factors isolated is a novel homeodomain protein. With the exception of the homeodomain itself, which is atypically located close to the protein N-terminus, the factor displays little similarity to any known DNA binding protein. Its homeodomain is most similar to that of the maize protein Knotted-1, while the most closely related vertebrate domain is that of the human proto-oncoprotein Pbx1. We demonstrate that the DNA binding specificity of the factor is consistent with its involvement in the ubiquitous complex formed with site F' and that it is capable of suppressing expression from the apoVLDLII promoter in short-term transfection experiments. These studies, combined with its DNA binding specificity, the tissue distribution of its mRNA and its developmental regulation, suggest a role as a negative regulator of gene expression in non-hepatic tissues and in the liver early during embryogenesis.
Asunto(s)
Proteínas de Homeodominio/aislamiento & purificación , Secuencia de Aminoácidos , Animales , Apolipoproteínas/genética , Secuencia de Bases , Sitios de Unión , Pollos , Clonación Molecular , ADN/genética , ADN/metabolismo , ADN Complementario/genética , Femenino , Regulación del Desarrollo de la Expresión Génica , Proteínas de Homeodominio/genética , Lipoproteínas VLDL/genética , Masculino , Datos de Secuencia Molecular , Regiones Promotoras Genéticas , ARN Mensajero/genética , ARN Mensajero/metabolismo , Homología de Secuencia de Aminoácido , Distribución TisularRESUMEN
We have constructed two new mini-Mu derivatives, pMRfP and pBEf, that combine the properties of known mini-Mu vectors and the advantages of the replication origin (orifd) of filamentous phage fd. Mini-Mu pMRfP consists of the left (850 bp) and the right (216 bp) ends of the Mu genome, orifd, packaging signal of fd, and the gene conferring resistance to chloramphenicol. The second mini-Mu, termed pBEf, carries the left end of Mu (1001 bp), which contains the so-called internal activation sequence (enhancer of transposition), required for a higher frequency of transposition, the right end (116 bp) and the gene conferring resistance to kanamycin. These new mini-Mu vectors are suitable for in vivo cloning with the ability of single-stranded DNA preparation using one of the helper phages (M13K07, rv1, IR1, R408) and with a large cloning capacity (the size of the cloned fragment can be up to 35 kb). They can also be used as the hoppers (a transposable ori that can be turned on or off depending on the presence of the fd gene 2 product). Thus, these mini-Mu derivatives can be employed as vectors for in vivo cloning, and as regulated transposons or mobile replicons.