A transcriptional activator, AoXlnR, controls the expression of genes encoding xylanolytic enzymes in Aspergillus oryzae

By deletion across the promoter region of the xynF1 gene encoding the major Aspergillus oryzae xylanase, a 53-bp DNA fragment containing the XlnR binding sequence GGCTAAA as well as two similar sequences was shown to confer xylan inducibility on the gene. Complementary and genomic DNAs encoding the Aspergillus niger xlnR homologous gene, abbreviated AoxlnR, were cloned from A. oryzae and sequenced. AoXlnR comprised 971 amino acids with a zinc binuclear cluster domain at the N-terminal region and revealed 77.5% identity to the A. niger XlnR. Recombinant AoXlnR protein encompassing the zinc cluster region of the N-terminal part bound to both the consensus binding sequence and its cognate sequence, GGCTGA, with an approximately 10 times lower affinity. GGCTA/GA is more appropriate as the XlnR consensus binding sequence. Both sequences functioned independently in vivo in XlnR-mediating induction of the xynF1 gene. This was further confirmed by using an AoxlnR disruptant. Neither the xynF1 nor the xylA gene was expressed in the disruptant, suggesting that the xylan-inducible genes in A. oryzae may also be controlled in the same manner as described for A. niger.     

Molecular cloning, characterization, and expression analysis of the xynF3 gene from Aspergillus oryzae

The gene encoding xylanase F3 (xynF3) was isolated from a genomic library of Aspergillus oryzae KBN616, used for making shoyu koji. The structural part of xynF3 was found to be 1468 bp. The nucleotide sequence of cDNA amplified by RT-PCR showed that the open reading frame of xynF3 was interrupted by ten short introns and encoded 323 amino acids. Direct N-terminal amino acid sequencing showed that the precursor of XynF3 had a signal peptide of 22 amino acids. The predicted amino acid sequence of XynF3 has strong similarity to other family 10 xylanases from fungi. The xynF3 gene was successfully overexpressed in A. oryzae and the XynF3 was purified. The molecular mass of XynF3 estimated on sodium dodecyl sulfate-polyacrylamide gel electrophoresis was 32,000. This was almost the same as the molecular mass of 32,437 calculated from the deduced amino acid sequence. The purified XynF3 showed an optimum activity at pH 5.0 and 58 degrees C. It had a Km of 6.5 mg/ml and a Vmax of 435 micromol x min(-1) x mg(-1) when birch wood xylan was used as a substrate. Expression of the xynF3 gene was analyzed using an Escherichia coli beta-glucuronidase gene as a reporter. The result indicated that xynF3 is expressed in the medium containing wheat bran as a carbon source.     

Escherichia coli lacZ gene as a biochemical and histochemical marker in plant cells

Several lacZ chimeric genes were constructed by fusing the truncated lacZ sequence of Escherichia coli to N-terminal sequences of few other genes. Promoters used to direct expression of the chimeric genes were the promoter for 35S RNA of cauliflower mosaic virus (P35S) as well as those of the small subunit gene of ribulose bisphosphate carboxylase and the octopine synthase gene. These constructs were introduced into tobacco cells using a Ti plasmid of Agrobacterium tumefaciens, and beta-galactosidase activity in uncloned and cloned calli derived from the crown galls were examined. The results showed that the P35S-linked lacZ chimeric gene is expressed very efficiently. When slices of the crown gall carrying this chimeric gene were placed on plates containing indicator XGal, localized areas of the outgrowth turned deep blue, whereas no such areas were found in the crown gall having promoter-less lacZ. Calli from galls containing this construct expressed beta-galactosidase activity at an eight-fold higher level (approx. 7000 units/mg protein) than the endogenous activity (approx. 900 units/mg protein). Some of the calli displayed over 20-fold higher activity. Actively growing mini calli expressing activity higher than 4000 units/mg protein dyed deep blue on XGal agar medium such that they were distinguishable from calli having no lacZ. Half of the uncloned P35S-lacZ transformant calli showed activity higher than this level. These results indicate that the lacZ gene linked to a strong promoter such as P35S is useful as a biochemical and histochemical marker gene in plant cells.     

Sequence and promoter analysis of the highly expressed TEF gene of the filamentous fungus Ashbya gossypii

Ashbya gossypii carries only a single gene (TEF) coding for the abundant translation elongation factor 1. Cloning and sequencing of this gene and deletion analysis of the promoter region revealed an extremely high degree of similarity with the well studied TEF genes of the yeast Saccharomyces cerevisiae including promoter upstream activation sequence (UAS) elements. The open reading frames in both species are 458 codons long and show 88.6% identity at the DNA level and 93.7% identity at the protein level. A short DNA segment in the promoter, between nucleotides -268 and -213 upstream of the ATG start codon, is essential for high-level expression of the A. gossypii TEF gene. It carries two sequences, GCCCATACAT and ATCCATACAT, with high homology to the UAS_rpg sequence of S. cerevisiae, which is an essential promoter element in genes coding for highly expressed components of the translational apparatus. UAS_rpg sequences are binding sites for the S. cerevisiae protein TUF, also called RAP1 or GRF1. In gel retardation with A. gossypii protein extracts we demonstrated specific protein binding to the short TEF promoter segment carrying the UAS_rpg homologous sequences.     

Sequence-specific interactions of a maize factor with a GC-rich repeat in the phosphoenolpyruvate carboxylase gene

Summary A plant nuclear protein PEP-I, which binds specifically to the promoter region of the phosphoenolpyruvate carboxylase (PEPC) gene, was identified. Methylation interference analysis and DNA binding assays using synthetic oligonucleotides revealed that PEP-I binds to GC-rich elements. These elements are directly repeated sequences in the promoter region of the PEPC gene and we have suggested that they may be cis-regulatory element of this gene. The consensus sequence of the element is CCCTCTCCACATCC and the CTCC is essential for binding of PEP-I. PEP-I is present in the nuclear extracts of green leaves, where the PEPC gene is expressed. However, no binding was detected in tissues where the PEPC gene is not expressed in vivo, such as roots or etiolated leaves. Thus, PEP-1 is the first factor identified in plants which has different binding activity in light-grown compared with dark-grown tissue. PEP-I binding is also tissue-specific, suggesting that PEP-1 may function to coordinate PEPC gene expression with respect to light and tissue specificity. This report describes the identification and characterization of the sequences required for PEP-1 binding.     

人SATB1基因5′上游序列的转录激活功能分析

在对人SATB1基因进行生物信息学分析的基础上 ,采用PCR技术 ,扩增人基因组DNA中SATB1基因 5′上游序列的 - 2 95 5～ - 9片段 ,构建了 3个分别由SATB1基因 5′上游 - 2 95 5～ - 9,- 172 7～ - 9和 - 76 0～ - 9序列片段驱动的报告载体 -pGL3 SP2 94 6 luc ,pGL3 SP1718 luc和pGL3 SP75 1 luc ,分别瞬时转染JurkatT ,K5 6 2 ,U937和HeLa细胞 ,通过测定荧光素酶的表达活性 ,观察SATB1基因 5′上游序列片段 3个删除突变体在不同细胞内活性的差异 .结果显示 ,SATB1上游序列- 2 95 5 - 9在 4种细胞中的转录激活能力为U937>JurkatT >K5 6 2 ,在HeLa细胞中基本无激活 ,提示SATB1的转录激活可能具有一定的细胞类型特异性 .3种 5′删除突变体转录激活性由大至小顺序为 - 76 0 - 9>- 2 95 5 - 9>- 172 7 - 9,提示SATB1的核心启动子可能存在于其 5′上游序列的- 76 0至 - 9bp区域中 .     

Identification of regulatory sequences in the type 1 plasminogen activator inhibitor gene responsive to transforming growth factor beta.

Regulation of the human type 1 plasminogen activator inhibitor (PAI-1) promoter by transforming growth factor-beta (TGF beta) was studied. An 800-base pair fragment from the PAI-1 promoter and 5'-flanking region was fused to the firefly luciferase reporter gene and transfected into Hep3B human hepatoma cells. Treatment of the cells with TGF beta induced luciferase activity by more than 50-fold. Transfection studies using constructs with 5' or 3' deletions through this region revealed that two sequences were important in the TGF beta response. The first sequence was located in the proximal promoter (-49 to -87) and mediated an 11-fold induction with TGF beta, while the second more distal region (-636 to -740) contained two sequences which together mediated a 50-fold or greater response. Sequence comparison indicated that both of the responsive regions contained sequences with high homology to the AP-1 consensus binding site. Moreover, gel retardation analysis experiments demonstrated that both sequences bound a common nuclear protein, and that an oligonucleotide containing a consensus AP-1 sequence was able to compete for the binding of this common protein. Thus, the response of the PAI-1 gene to TGF beta is mediated by at least two separate regions, and both of these regions contain DNA sequences homologous to the AP-1 binding site.     

Nucleotide sequence of the 5' flanking region responsible for the enhancement of the expression of yeast enolase 1 gene

Several plasmids carrying different length of the 5' flanking region of yeast (Saccharomyces cerevisiae) enolase 1 gene (ENO1) which is fused in frame to the Escherichia coli lacZ gene were constructed by recombination in vitro. Promoter activity of ENO1 was assayed by measuring beta-galactosidase activity of the fused gene product. Comparison of the promoter activity of these plasmids suggests that the sequences required for a strong promoter activity lie within the DNA segment -724 to -353 base pairs (bp) upstream from the start of ENO1 coding sequence. The nucleotide sequence of this region was determined.     

GATA and Ets cis-acting sequences mediate megakaryocyte-specific expression.

The human glycoprotein IIB (GPIIB) gene is expressed only in megakaryocytes, and its promoter displays cell type specificity. We show that this specificity involved two cis-acting sequences. The first one, located at -55, contains a GATA binding site. Point mutations that abolish protein binding on this site decrease the activity of the GPIIB promoter but do not affect its tissue specificity. The second one, located at -40, contains an Ets consensus sequence, and we show that Ets-1 or Ets-2 protein can interact with this -40 GPIIB sequence. Point mutations that impair Ets binding decrease the activity of the GPIIB promoter to the same extent as do mutations that abolish GATA binding. A GPIIB 40-bp DNA fragment containing the GATA and Ets binding sites can confer activity to a heterologous promoter in megakaryocytic cells. This activity is independent of the GPIIB DNA fragment orientation, and mutations on each binding site result in decreased activity. Using cotransfection assays, we show that c-Ets-1 and human GATA1 can transactive the GPIIB promoter in HeLa cells and can act additively. Northern (RNA) blot analysis indicates that the ets-1 mRNA level is increased during megakaryocyte-induced differentiation of erythrocytic/megakaryocytic cell lines. Gel retardation assays show that the same GATA-Ets association is found in the human GPIIB enhancer and the rat platelet factor 4 promoter, the other two characterized regulatory regions of megakaryocyte-specific genes. These results indicate that GATA and Ets cis-acting sequences are an important determinant of megakaryocytic specific gene expression.     

Sequence and promoter analysis of the highly expressed TEF gene of the filamentous fungus Ashbya gossypii

Ashbya gossypii carries only a single gene (TEF) coding for the abundant translation elongation factor 1α. Cloning and sequencing of this gene and deletion analysis of the promoter region revealed an extremely high degree of similarity with the well studied TEF genes of the yeast Saccharomyces cerevisiae including promoter upstream activation sequence (UAS) elements. The open reading frames in both species are 458 codons long and show 88.6% identity at the DNA level and 93.7% identity at the protein level. A short DNA segment in the promoter, between nucleotides -268 and -213 upstream of the ATG start codon, is essential for high-level expression of the A. gossypii TEF gene. It carries two sequences, GCCCATACAT and ATCCATACAT, with high homology to the UAS_rpg sequence of S. cerevisiae, which is an essential promoter element in genes coding for highly expressed components of the translational apparatus. UAS_rpg sequences are binding sites for the S. cerevisiae protein TUF, also called RAP1 or GRF1. In gel retardation with A. gossypii protein extracts we demonstrated specific protein binding to the short TEF promoter segment carrying the UAS_rpg homologous sequences.