RESUMO
The gene coding for an extracellular lipase of Bacillus licheniformis was cloned using PCR techniques. The sequence corresponding to the mature lipase was subcloned into the pET 20b(+) expression vector to construct a recombinant lipase protein containing 6 histidine residues at the C-terminal. High-level expression of the lipase by Escherichia coli cells harbouring the lipase gene-containing expression vector was observed upon induction with IPTG at 30 degrees C. A one step purification of the recombinant lipase was achieved with Ni-NTA resin. The specific activity of the purified enzyme was 130 units/mg with p-nitrophenyl-palmitate as substrate. The enzyme showed maximum activity at pH 10-11.5 and was remarkably stable at alkaline pH values up to 12. The enzyme was active toward p-nitrophenyl esters of short to long chains fatty acids but with a marked preference for esters with C(6) and C(8) acyl groups. The amino acid sequence of the lipase shows striking similarities to lipases from Bacillus subtilis and Bacillus pumilus. Based on the amino acid identity and biochemical characteristics, we propose that Bacillus lipases be classified into two distinct subfamilies of their own.
RESUMO
In the pro-alpha1(I) collagen gene a number of cis-regulatory elements, which interact with a variety of trans-acting factors, are present in the promoter and first intron. We have undertaken a comprehensive study of Sp1, Ap1, and Ap2 binding in the region spanning -442 to +1697 nt. DNase I footprinting analysis revealed these factors bind with varying affinities to some of the potential sites: Sp1 binds to 16 of 34 potential sites, Ap2 binds to 22 of 40 potential binding sites, and Ap1 binds to its only potential site. The Sp1 sites were mostly clustered in the intron region, while the Ap2 sites were clustered in the promoter region. Transmission electron microscopic analysis of DNA-protein complexes not only confirmed these results, but also clearly showed that heterologous and/or homologous protein-protein interactions between Sp1 and/or Ap2 bring the promoter and intron in contact with each other, with the resulting looping out of the intervening DNA. This strongly suggests that the DNA-looping model is an explanation for the orientation preference of the enhancing element in the first intron as these interactions possibly create an optimum environment for the binding of the rest of the transcriptional machinery.
Assuntos
Proteínas de Ligação a DNA/metabolismo , DNA/metabolismo , Pró-Colágeno/genética , Elementos de Resposta/genética , Fator de Transcrição Sp1/metabolismo , Fator de Transcrição AP-1/metabolismo , Fatores de Transcrição/metabolismo , Sequência de Bases , Sítios de Ligação , Linhagem Celular , DNA/química , DNA/genética , DNA/ultraestrutura , Pegada de DNA , Sondas de DNA/química , Sondas de DNA/genética , Sondas de DNA/metabolismo , Sondas de DNA/ultraestrutura , Proteínas de Ligação a DNA/ultraestrutura , Desoxirribonuclease I/metabolismo , Regulação da Expressão Gênica , Humanos , Íntrons/genética , Microscopia Eletrônica , Modelos Genéticos , Conformação de Ácido Nucleico , Regiões Promotoras Genéticas/genética , Ligação Proteica , Deleção de Sequência/genética , Fator de Transcrição Sp1/ultraestrutura , Termodinâmica , Fator de Transcrição AP-2 , Fatores de Transcrição/ultraestruturaRESUMO
A chimeric gene was constructed in which sequences between 253 base pairs (bp) upstream of the start of transcription of the human pro-alpha 1(I) collagen gene and 117 bp downstream of that site were fused to the human alpha 1-globin gene, at a site immediately upstream of the globin initiation codon. Expression of this and subsequent chimeric gene constructions was investigated in microinjected Xenopus laevis oocytes. Presence of 253 bp of pro-alpha 1(I) collagen gene promoter sequences, containing a CAAT box and a TATA box, resulted in a relatively modest level of expression of the linked globin sequences. Lengthening of the pro-alpha 1(I) collagen gene promoter region to include 2400 bp of upstream sequences, increased transcription of the marker gene to some extent. Strong activation of transcription was obtained when a 782-bp fragment of the first intron of the collagen gene was introduced in chimeric constructions containing only 240 bp of the collagen promoter. An enhancing effect of the intron segment on expression of the marker gene was observed from downstream or upstream positions relative to the initiation site of transcription. Sequencing of the intron segment revealed the presence of four decanucleotide consensus sites for binding of the constitutive transcription factor Sp1, as well as an enhancer "core" motif. Our experiments also showed that the cis-acting region strongly enhance the activity of the pro-alpha 1(I) promoter in transfected fibroblasts. On the basis of these observations we conclude that the segment broadly located between +700 and +1300 in the first intron of the human pro-alpha 1(I) collagen gene contains cis-acting sequences with an enhancer effect on transcription of the gene.
