RESUMO
A sequence element within plasmid pBR322 has a cis-acting negative effect on the expression of a cloned Ad gene in transient expression assays. The negative element is located between the PvuII and Tth111I restriction sites on pBR322 (nt 2068-2223). This element was also shown to be responsible for the decrease in focus number, when plasmids carrying Ad2 E1 genes were used to transform baby rat kidney cells. In a similar manner, this element diminished the number of G418-resistant cell foci, when plasmids containing the neo marker gene under the control of E1a promoter were used. Plasmid stability in transfected cells was not modified by deletion of this cis-acting negative element.
Assuntos
Adenoviridae/genética , Regulação Viral da Expressão Gênica , Vetores Genéticos , Proteínas Oncogênicas Virais/genética , Plasmídeos , Proteínas Recombinantes de Fusão/biossíntese , Sequências Reguladoras de Ácido Nucleico , Proteínas Precoces de Adenovirus , Células HeLa , Humanos , Proteínas Oncogênicas Virais/biossíntese , TransfecçãoRESUMO
We have isolated the gene from Saccharomyces cerevisiae encoding an alpha-mannosidase of unique specificity which catalyzes the removal of one mannose residue from Man9GlcNAc to produce a single isomer of Man8GlcNAc (Jelinek-Kelly, S., and Herscovics, A. (1988) J. Biol. Chem. 263, 14757-14763). Amino acid sequence information was obtained and corresponding degenerate oligonucleotide primers were synthesized for polymerase chain reactions on yeast genomic DNA. The labeled polymerase chain reaction products were used to screen a S. cerevisiae genomic library in YEp24, and positive clones of different lengths with similar restriction maps were isolated. A 4.6-kilobase fragment which hybridized with the probes was sequenced. It contained a 1650-base pair open reading frame encoding peptide sequences corresponding to the amino acid sequences of the purified alpha-mannosidase. The gene, designated MNS1, encodes a 549-amino acid polypeptide of calculated molecular size 63,017 Da produced by an mRNA species of approximately 1.7 kilobases. The protein possesses a putative noncleavable signal sequence near its N-terminal region which probably acts as a transmembrane domain. It has three potential N-glycosylation sites and a calcium-binding consensus sequence. Its amino acid sequence is homologous to the recently isolated cDNA from rabbit liver alpha-1,2 mannosidase which can transform Man9GlcNAc to Man5GlcNAc (Moremen, K. W., Schutzbach, J. S., Forsee, W. T., Neame, P., Bishoff, J., Lodish, H. F., and Robbins, P. W. (1990) Glycoconjugate J. 7, 401). Overexpression of the MNS1 gene caused an 8-10-fold increase in specific alpha-mannosidase activity. Disruption of the MNS1 gene resulted in undetectable specific alpha-mannosidase activity but no apparent effect on growth. These results demonstrate that MNS1 is the structural gene for the specific alpha-mannosidase and that its activity is not essential for viability.
Assuntos
Genes Fúngicos , Glicoproteínas/biossíntese , Manosidases/genética , Saccharomyces cerevisiae/metabolismo , Sequência de Aminoácidos , Sequência de Bases , Northern Blotting , Southern Blotting , Sequência de Carboidratos , Clonagem Molecular , DNA Fúngico/genética , Glicoproteínas/genética , Glicoproteínas/isolamento & purificação , Dados de Sequência Molecular , Plasmídeos , Poli A/análise , Reação em Cadeia da Polimerase , RNA/análise , RNA Mensageiro , Mapeamento por Restrição , Saccharomyces cerevisiae/enzimologia , Saccharomyces cerevisiae/genéticaRESUMO
The nucleotide sequence of the adenovirus serotype 3 E2A early promoter has been determined. In contrast to Ad2, the Ad3 E2A early promoter possessed only one TATA-like box and one nuclear transcription factor E2F binding site and lacked the silencer sequences; however, as in Ad2, the ATF binding site was present. Moreover, the Ad3 E2A promoter harbored a protein binding sequence recognized by the SP1 factor. By transient expression analysis in HeLa cells, we demonstrated that the E1A gene products of Ad3 and Ad2 stimulated Ad3 E2A transcription. In competition experiments, the Ad3 E2A promoter was used in preference to the Ad2 E2A promoter.