Expression analysis of dihydroflavonol 4-reductase genes in Petunia hybrida.

Dihydroflavonol 4-reductase (DFR) genes from Rosa chinensis (Asn type) and Calibrachoa hybrida (Asp type), driven by a CaMV 35S promoter, were integrated into the petunia (Petunia hybrida) cultivar 9702. Exogenous DFR gene expression characteristics were similar to flower-color changes, and effects on anthocyanin concentration were observed in both types of DFR gene transformants. Expression analysis showed that exogenous DFR genes were expressed in all of the tissues, but the expression levels were significantly different. However, both of them exhibited a high expression level in petals that were starting to open. The introgression of DFR genes may significantly change DFR enzyme activity. Anthocyanin ultra-performance liquid chromatography results showed that anthocyanin concentrations changed according to DFR enzyme activity. Therefore, the change in flower color was probably the result of a DFR enzyme change. Pelargonidin 3-O-glucoside was found in two different transgenic petunias, indicating that both CaDFR and RoDFR could catalyze dihydrokaempferol. Our results also suggest that transgenic petunias with DFR gene of Asp type could biosynthesize pelargonidin 3-O-glucoside.

Increased expression of prostaglandin reductase 1 in hepatocellular carcinomas from clinical cases and experimental tumors in rats.

To identify novel tumor-associated proteins, we analyzed the protein expression patterns from experimental hepatocellular carcinoma (HCC) that were induced using hepatocarcinogenesis models in rats. Rats were subjected to two previously described protocols of hepatocarcinogenesis using diethylnitrosamine as a carcinogen: the alternative Solt-Farber (aS&F) protocol, which induces HCC within 9 months, and Schiffer's model, which induces cirrhosis and multifocal HCC within 18 weeks. The patterns of protein expression from tumors and normal liver tissue were examined by SDS-PAGE and the bands identified at 33-34 kDa were analyzed by mass spectrometry. The prostaglandin reductase 1 (PTGR1) showed the highest number of peptides, with a confidence of level >99%. The increased expression of PTGR1 in tumors was confirmed in these two models by Western blotting and by increase in alkenal/one oxidoreductase activity (25-fold higher than normal liver). In addition, the gene expression level of Ptgr1, as measured by qRT-PCR, was increased during cancer development in a time-dependent manner (200-fold higher than normal liver). Furthermore, PTGR1 was detected in the cytoplasm of neoplastic cells in rat tumors and in 12 human HCC cases by immunohistochemistry. These analyses were performed by comparing the expression of PTGR1 to that of two well-known markers of hepatocarcinoma, Glutathione S-transferase pi 1 (GSTP1) in rats and glypican-3 in humans. The increased expression and activity of PTGR1 in liver carcinogenesis encourage further research aimed at understanding the metabolic role of PTGR1 in HCC and its potential application for human cancer diagnosis and treatment.

Aldose-6-phosphate reductase from apple leaves: Importance of the quaternary structure for enzyme activity.

Aldose-6-phosphate reductase (A6PRase) is a key enzyme for glucitol biosynthesis in plants from the Rosaceae family. To gain on molecular tools for enzymological studies, we developed an accurate system for the heterologous expression of A6PRase from apple leaves. The recombinant enzyme was expressed with a His-tag alternatively placed in the N- or C-terminus, thus allowing the one-step protein purification by immobilized metal affinity chromatography. Both, the N- and the C-term tagged enzymes exhibited similar affinity toward substrates, although the k(cat) of the latter enzyme was 80-fold lower than that having the His-tag in the N-term. Gel filtration chromatography showed different oligomeric structures arranged by the N- (dimer) and the C-term (monomer) tagged enzymes. These results, reinforced by homology modeling studies, point out the relevance of the C-term domain in the structure of A6PRase to conform an enzyme having optimal specific activity and the proper quaternary structure.

Homogeneous recombinant Mycobacterium tuberculosis shikimate dehydrogenase production: an essential step towards target-based drug design.

Mycobacterium tuberculosis shikimate dehydrogenase (MtbSD) catalyzes the forth reaction in the shikimate pathway. Here we describe production of K69A, K69H, K69I, K69Q, D105A, and D105N mutant proteins. Screening of several conditions was performed to optimize MtbSD production yield, and an improved purification protocol to obtain homogeneous MtbSD is presented. The rational design of new antitubercular drugs hinges on the availability of M. tuberculosis proteins. Our results show that optimization of expression, disruption, and purification protocols resulted in a higher yield of functional MtbSD enzyme.

Selection of an Escherichia coli host that expresses mutant forms of Mycobacterium tuberculosis 2-trans enoyl-ACP(CoA) reductase and 3-ketoacyl-ACP(CoA) reductase enzymes.

Tuberculosis (TB) still remains a worldwide health concern. Efforts to understand the complex biology of Mycobacterium tuberculosis, the causative agent of TB, are important for new antitubercular drug development. Despite the completion of the genome sequence and the development of new genetic tools to manipulate this organism, the availability of sufficient amounts of mycobacterial proteins still remains an essential and laborious step to study the biochemical features of this pathogen. The T7-RNA polymerase-based pET system has been largely employed to express mycobacterial proteins in Escherichia coli, but it presents some limitations. To overcome problems with unstable expression of an M. tuberculosis inhA-encoded enoyl reductase mutant protein and lack of expression of two mabA-encoded ketoacyl reductase mutants, a sub-population of E. coli BL21(DE3) host cells was selected from a small-opaque colony. This empirically selected host, named BL21(DE3)NH, allowed stable expression of these mutant proteins. Although the mechanism that led the BL21(DE3)NH host to express the recombinant mutant proteins remains unknown, the persistent phenotype points to a stable genetic switch. This genetic alteration resulted in a tight control of the highly processive T7 RNA polymerase. Moreover, the absolute requirement for IPTG to obtain protein expression in the BL21(DE3)NH host cells suggests that no inherent defect in the transcriptional activity of the T7 promoter is present. Empirical host selection requires no further genetic manipulation of recombinant plasmids and may represent a means of obtaining tailor-made E. coli strains that overcome toxic effects associated with heterologous protein expression.

Genetic differentiation within and between species of the Drosophila willistoni group.

We describe allelic variation at 28 loci in six Caribbean populations of four sympatric species of Drosophila. Within any one species the allelic frequencies are very similar from population to population, although there is evidence of local as well as regional genetic differentiation. The genetic distance is greater between populations from different islands than between populations of the same island. When the allelic frequencies are compared between different species, a remarkable pattern appears. In any pair of species nearly half of the loci have essentially identical allelic frequencies, while nearly the other half of the loci have different alleles and in different frequencies. The loci with nearly identical allelic frequencies are different when different pairs of species are compared. The patterns of allelic variation within and between species are inconsistent with the hypothesis that the variation is adaptively neutral. Migration or mutation cannot explain the patterns of genetic variation, either. Balancing natural selection is the main process maintaining protein polymorphisms in natural populations.