RESUMO
Human tissue-type plasminogen activator (t-PA) cDNA was cloned from uterine tissue and engineered in expression vectors for production in mouse C127 cells. The vectors consisted of the bovine papilloma virus-1 (BPV-1) genome and t-PA transcriptional unit with a mouse metallothionein (MT-1) promoter at the 5' end and MT-1 genomic sequences or SV40 early introns and polyadenylation signals at the 3' end. Analysis of the expression vectors transfected into cells revealed that t-PA is expressed 100- to 200-fold more with an intronless vector utilizing the SV40 polyadenylation signal than with other, intron-containing vectors. RNA analysis of stable cell lines indicated that t-PA expression levels correlated with mRNA abundance. DNA copy number and transcriptional rate of the MT-1 promoter remained constant in cell lines transformed by different BPV expression vectors. Uterine t-PA produced by recombinant DNA means was enzymatically active and similar in properties to Bowes melanoma t-PA.
Assuntos
Papillomavirus Bovino 1/genética , Vetores Genéticos , Papillomaviridae/genética , Ativador de Plasminogênio Tecidual/genética , Útero/enzimologia , Animais , Sequência de Bases , Clonagem Molecular , DNA/genética , Feminino , Regulação da Expressão Gênica , Humanos , Técnicas de Imunoadsorção , Camundongos , Dados de Sequência Molecular , Poli A/genética , Splicing de RNA , Ativador de Plasminogênio Tecidual/imunologia , Transfecção , Útero/fisiologiaRESUMO
We have inserted the cDNAs coding for both polypeptide subunits, alpha and beta, of human choriogonadotropin (hCG) into a single simian virus 40 expression vector in such a way that they replace the viral VP2 and VP1 coding sequences, respectively. Monkey cells infected with this virus and the appropriate helper virus produce dimeric hCG. hCG produced in this system was shown to chromatograph identically to standard hCG preparations on gel filtration and to be biologically active in the mouse uterine weight assay.
