Development of Aspergillus oryzae thiA promoter as a tool for molecular biological studies

In filamentous fungi, the repertoire of promoters available for exogenous gene expression is limited. Here, we report the development and application of the thiamine-regulatable thiA promoter (PthiA) in Aspergillus oryzae as a tool for molecular biological studies. When PthiA was used to express the enhanced green fluorescent protein (EGFP) reporter, the fluorescence in the mycelia was either repressed or induced in the presence or absence of thiamine in the culture media, respectively. In addition, the expression level from the thiA promoter can be controlled by the concentration of external thiamine. Thiamine content in the media did not affect mycelial morphology, making the thiA promoter more useful compared with alcA and amyB promoters that depend on carbon source for regulation. Moreover, as the A. oryzae thiA promoter was also regulated by thiamine in A. nidulans, this promoter can be further applied as an inducible promoter in other Aspergilli.     

Genetic architecture of chalcone isomerase non-coding regions in common bean (Phaseolus vulgaris L.).

Sequence data for 2 non-coding regions of the chalcone isomerase gene were analyzed to study the genetic architecture of common bean (Phaseolus vulgaris L.). One region corresponded to the first 596 nucleotides (nt) of the 5'-untranslated region (UTR). The other region was the 710 nt intron 3. Data were collected from 67 genotypes representing both landraces and cultivars from the geographical range of the cultivated form of the species. Variability in the 5'-UTR region was represented by single nucleotide polymorphisms (SNPs), whereas intron 3 variation was due to a collection of SNPs and insertion-deletion events. Diversity was greater in the 5'-UTR (pi = 0.0175) than in intron 3 (pi = 0.0089). For each region, diversity was greater for genotypes of Middle American than Andean origin. A single recombination event was observed, and the hybridization pattern necessary to derive the recombinant genotypes supported the previous observation of an ancestral gene pool from which modern domesticated genotypes are derived. For both regions, a strongly supported Andean group was observed, whereas the presence of 2 Middle American subgroups was also supported. Although a significantly positive Tajima's D statistic was observed for the 5'-UTR for all genotypes, we conclude that this is more likely the result of a strong demographic effect and that balancing selection is occurring only among the Middle American genotypes.     

cis-acting sequences involved in exon selection in the chicken beta-tropomyosin gene.

The chicken beta-tropomyosin gene contains an internal pair of mutually exclusive exons (6A and 6B) that are selected in a tissue-specific manner. Exon 6A is incorporated in fibroblasts and smooth muscle cells, whereas exon 6B is skeletal muscle specific. In this study we show that two different regions in the intron between the two mutually exclusive exons are important for this specific selection in nonmuscle cells. Sequences in the 3' end of the intron have a negative effect in the recognition of the 3' splice site, while sequences in the 5' end of the intron have a positive effect in the recognition of the 5' splice site. First, sequences in exon 6B as well as in the intron upstream of exon 6B are both able to inhibit splicing when placed in a heterologous gene. The sequences in the polypyrimidine stretch region contribute to splicing inhibition of exons 5 or 6A to 6B through a mechanism independent of their implication in the previously described secondary structure around exon 6B. Second, we have identified a sequence of 30 nucleotides in the intron just downstream of exon 6A that is essential for the recognition of the 5' splice site of exon 6A. This is so even after introduction of a consensus sequence into the 5' splice site of this exon. Deletion of this sequence blocks splicing of exon 6A to 6B after formation of the presplicing complex. Taken together, these results suggest that both the mutually exclusive behavior and the choice between exons 6A and 6B of the chicken beta-tropomyosin gene are trans regulated.     

Exploring the role of 5' alternative splicing and of the 3'-untranslated region of cathepsin B mRNA

The cysteine peptidase cathepsin B is responsible for connective tissue breakdown in several diseases. The pathological expression of cathepsin B may depend on the structure of its mRNA. We investigated the translational efficiency of the cathepsin B mRNA untranslated regions (UTRs) using fusion constructs to green fluorescent protein (GFP) and luciferase. Transfection of fusion constructs with GFP and luciferase containing the full-length 5'-UTR, the variant lacking exon 2, and that lacking exons 2 and 3 into mammalian cells, resulted in modulation of the biosynthetic rate of cathepsin B in a cell-specific manner. Constructs missing these exons were biosynthetically more efficient than the full-length counterpart. Luciferase was cloned upstream of the 3'-UTR, downstream of the 5'-UTR, or sandwiched between the 5'- and the 3'-UTR. The UTRs of cathepsin B downregulated luciferase biosynthesis moderately when present individually, with the 3'-UTR being more efficient than the 5'-UTR, and downregulated it even more when present simultaneously. A truncated cathepsin B-GFP chimeric product derived from the 5'-UTR missing exons 2 and 3 induced cell death. The increased biosynthetic rate and abnormal trafficking of cathepsin B observed in pathologies such as cancer and osteoarthritis may depend on alternative splicing of pre-mRNA.     

Differentially expressed isoforms of the mouse retinoic acid receptor beta generated by usage of two promoters and alternative splicing.

Using anchored PCR, three different cDNA isoforms of the mouse retinoic acid receptor beta [mRAR-beta 1, mRAR-beta 2 (formerly mRAR-beta 0) and mRAR-beta 3], generated from the same gene by differential promoter usage and alternative splicing, were isolated. These three isoforms encode RAR proteins with different N-terminal A regions and identical B - F regions. The sequence encoding the first 59 amino acids of the mRAR-beta 3 A region is identical with the entire A region of mRAR-beta 1. However, the sequence of mRAR-beta 3 region A differs from that of mRAR-beta 1 by an additional 27 C-terminal amino acids encoded in an 81 nucleotide-long putative exon which is spliced in between the exons encoding the A and B regions of mRAR-beta 1. Both mRAR-beta 1 and beta 3 cDNAs differ entirely from mRAR-beta 2 in their 5'-untranslated (5'-UTR) and A region coding sequences. This N-terminal variability, in a region which was shown to be important for cell-type specific differential target gene trans-activation by other nuclear receptors, suggests that the three mRAR-beta isoforms may be functionally distinct. The conservation of RAR-beta isoform sequences from mouse to human, as seen by cross-hybridization on Southern blots or DNA sequence analysis, as well as their differential patterns of expression in various mouse tissues, corroborates this view. Additionally, the mRNA analysis data suggest that mRAR-beta 2, whose expression predominates in RA-treated embryonal carcinoma (EC) and embryonic stem (ES) cells, may be important during early stages of development. mRAR-beta 1 and beta 3, on the other hand, which are predominantly expressed in fetal and adult brain, may play some specific role in the development of the central nervous system.     

A highly stable duplex structure sequesters the 5' splice site region of hnRNP A1 alternative exon 7B.

Exon 7B in the hnRNP A1 pre-mRNA is alternatively spliced to yield A1 and A1(B), two proteins that differ in their ability to modulate 5' splice site selection. Sequencing the murine intron downstream of exon 7B revealed the existence of several regions of similarity to the corresponding human intron. In vitro splicing assays indicate that an 84-nt region (CE6IO) decreases splicing to the proximal 5' splice site in a pre-mRNA carrying the 5' splice sites of exon 7 and 7B. In vivo, the CE6IO element promotes exon 7B skipping in pre-mRNAs expressed from a mini-gene containing the hnRNP A1 alternative splicing unit. Using oligonucleotide-targeted RNase H cleavage assays, we provide support for the existence of highly stable base pairing interactions between CE6IO and the 5' splice site region of exon 7B. Duplex formation occurs in naked pre-mRNA, resists incubation in splicing extracts, and is associated with a reduction in the assembly of U1 snRNP-dependent complexes to the 5' splice site of exon 7B. Our results demonstrate that pre-mRNA secondary structure plays an important role in promoting exon 7B skipping in the A1 pre-mRNA.     

Characterization of regulatory intronic and exonic sequences involved in alternative splicing of scavenger receptor class B gene

Scavenger receptor class B type I (SR-BI) is a major receptor of the high-density lipoprotein that mediates cholesterol efflux and reverse cholesterol transport. Alternative splicing of the scavenger receptor class B (SR-B) gene is observed and different splice forms, SR-BI and scavenger receptor class B type II (SR-BII), have been shown to function and localize in distinct ways. We have previously shown that SR-B alternative splicing regulation is associated with splicing factor ASF/SF2. In this study, using a SR-B minigene as a model, we determined the critical regulatory regions in the upstream intron, intron 11, by serial deletion and mutation analyses. We also further characterized the regulatory elements in intron 11 as well as in the skipped exon, exon 12. Moreover, we studied the interactions of these elements with the splicing factor ASF/SF2. This study provides new insights into the mechanism of SR-B splicing and it is important for further study on the mechanism of SR-B alternative splicing regulation, such as its regulation by estrogen.