RESUMO
The structural gene of Hepatitis B virus surface protein (HBsAg) was introduced into a plasmid capable of autonomous replication and selection in both the yeast Saccharomyces cerevisiae and E. coli. In this plasmid transcription of the HBsAg is initiated by the 5'-flanking sequence of the yeast 3-phosphoglycerate kinase (PGK) gene and terminated by the 3'-flanking region of the yeast TRP1 gene. Yeast cells containing this plasmid produce a new major species of mRNA of 1200 nucleotides in length coding for HBsAg. Viral surface antigen is made in nonglycosylated form at a level of about 1-2 percent of total yeast protein. A small fraction of this polypeptide (2-5 percent) is found in aggregated form upon yeast cell disruption by glass beads. This material is similar in size, density, and shape to the 22nm particle, isolated from the plasma of human hepatitis carriers, and induced comparable levels of HBsAg antibodies in mice when compared with the natural particle.
Assuntos
Antígenos de Superfície da Hepatite B/genética , Saccharomyces cerevisiae/genética , Cisteína/metabolismo , Regulação da Expressão Gênica , Antígenos de Superfície da Hepatite B/imunologia , Metionina/metabolismo , Peso Molecular , Saccharomyces cerevisiae/ultraestruturaRESUMO
The DNA sequence of the gene for the yeast glycolytic enzyme, 3-phosphoglycerate kinase (PGK), has been obtained by sequencing part of a 3.1 kbp HindIII fragment obtained from the yeast genome. The structural gene sequence corresponds to a reading frame of 1251 bp coding for 416 amino acids with no intervening DNA sequences. The amino acid sequence is approximately 65 percent homologous with human and horse PGK protein sequences and is in general agreement with the published protein sequence for yeast PGK. As for other highly expressed structural genes in yeast, the coding sequence is highly codon biased with 95 percent of the amino acids coded for by a select 25 codons (out of 61 possible). Besides structural DNA sequence, 291 bp of 5'-flanking sequence and 286 bp of 3'-flanking sequence were determined. Transcription starts 36 nucleotides upstream from the translational start and stops 86-93 nucleotides downstream from the translational stop. These results suggest a non-polyadenylated mRNA length of 1373 to 1380 nucleotides, which is consistent with the observed length of 1500 nucleotides for polyadenylated PGK mRNA. A sequence TATATATAAA is found at 145 nucleotides upstream from the translational start. This sequence resembles the TATAAA box that is possibly associated with RNA polymerase II binding.
Assuntos
Genes , Fosfoglicerato Quinase/genética , Saccharomyces cerevisiae/enzimologia , Sequência de Aminoácidos , Sequência de Bases , Enzimas de Restrição do DNA , Plasmídeos , RNA Mensageiro/genética , Saccharomyces cerevisiae/genética , Transcrição GênicaRESUMO
A DNA sequence coding for mature human leukocyte interferon D (LeIF-D) was linked with DNA fragments of the 5'-flanking sequences of the Saccharomyces cerevisiae (yeast) alcohol dehydrogenase I gene in a plasmid capable of autonomous replication and selection in both yeast and Escherichia coli. Yeast cells transformed by these plasmids synthesize up to 1 x 10(6) molecules of biologically active LeIF-D per cell.
