Genome-Wide Identification and Analysis of the Metallothionein Genes in Oryza Genus

Metallothionein (MT) proteins are low molecular mass, cysteine-rich, and metal-binding proteins that play an important role in maintaining metal homeostasis and stress response. However, the evolutionary relationships and functional differentiation of MT in the Oryza genus remain unclear. Here we identified 53 MT genes from six Oryza genera, including O. sativa ssp. japonica, O. rufipogon, O. sativa ssp. indica, O. nivara, O. glumaepatula, and O. barthii. The MT genes were clustered into four groups based on phylogenetic analysis. MT genes are unevenly distributed on chromosomes; almost half of the MT genes were clustered on chromosome 12, which may result from a fragment duplication containing the MT genes on chromosome 12. Five pairs of segmental duplication events and ten pairs of tandem duplication events were found in the rice MT family. The Ka/Ks values of the fifteen duplicated MT genes indicated that the duplicated MT genes were under a strong negative selection during evolution. Next, combining the promoter activity assay with gene expression analysis revealed different expression patterns of MT genes. In addition, the expression of OsMT genes was induced under different stresses, including NaCl, CdCl2, ABA, and MeJ treatments. Additionally, we found that OsMT genes were mainly located in chloroplasts. These results imply that OsMT genes play different roles in response to these stresses. All results provide important insights into the evolution of the MT gene family in the Oryza genus, and will be helpful to further study the function of MT genes.

RESVERATROL INHIBITS EXPRESSION OF CANCER-SPECIFIC PENTOSE PHOSPHATE PATHWAY ENZYME TKTL1

Objective: The objective of this study was to identify cancer-specific metabolic enzyme Transketolase-like 1 (TKTL1) from pentose phosphate pathway as target of resveratrol, a naturally occurring nutraceutical.Methods: Methylthiazolyldiphenyl-tetrazolium bromide assay and trypan blue assay were used for the estimation of growth and proliferation. Reactive oxygen species (ROS) was estimated using 2’-7’-Dichlorodihydrofluorescein diacetate while reduced glutathione (GSH) was estimated using commercially available kit. Promoter activity, reverse transcription polymerase chain reaction, and western blotting were used for expression analysis.Results: Resveratrol treatment in HeLa cells inhibited proliferation, promoted ROS, and reduced intracellular GSH levels. In TKTL1 promoter activity assay, we found that resveratrol treatment directly inhibited promoter activity of TKTL1. Resveratrol inhibited both mRNA and protein expression of TKTL1 in a dose-dependent manner.Conclusion: This is the first report where we show that resveratrol inhibits cancer-specific isoform TKTL1 as one of its targets to elicit its anticancer activity.

Gene cloning, structural gene and promoter identification, and active assay of the phosphatidylcholine synthase of Pseudomonas sp. strain 593

Pseudomonas sp. strain 593, a soil bacterium, is able to use exogenous choline to synthesize phosphatidylcholine via phosphatidylcholine synthase (Pcs). A 2020 bp DNA fragment that hybridized to a Pcs probe was cloned. This fragment contained a large open reading frame (ORF) with two potential ATG start sites that would encode for 293 and 231 amino acid proteins. Fragments containing the two ORFs encoded Pcs when they were inserted into the expression vector pET23a and expressed under the control of the T7 promoter in Escherichia coli BL21(DE3) pLysS. However, when the two ORFs were inserted into the cloning vector pMD18-T and expressed without control of the plasmid promoter in E. coli DH5α, only the larger clone exhibited Pcs activity. This suggested that the larger fragment contained a native promoter driving expression of the smaller ORF. A promoter activity assay, in which DNA fragments were inserted into the promoter-probe plasmid pCB182 and β-galactosidase activity of E. coli transformants was tested, demonstrated that a promoter is indeed present in the DNA region. All results together indicate that the 696 bp ORF, not the larger 897 bp ORF, encodes the Pcs in Pseudomonas sp. strain 593 and carries a promoter in front of its 5′ terminus.

Global Effect of Indole-3-Acetic Acid Biosynthesis on Multiple Virulence Factors of Erwinia chrysanthemi 3937

ABSTRACT Production of the plant hormone indole-3-acetic acid (IAA) is widespread among plant-associated microorganisms. The non-gall-forming phytopathogen Erwinia chrysanthemi 3937 (strain Ech3937) possesses iaaM (ASAP16562) and iaaH (ASAP16563) gene homologues. In this work, the null knockout iaaM mutant strain Ech138 was constructed. The IAA production by Ech138 was reduced in M9 minimal medium supplemented with l-tryptophan. Compared with wild-type Ech3937, Ech138 exhibited reduced ability to produce local maceration, but its multiplication in Saintpaulia ionantha was unaffected. The pectate lyase production of Ech138 was diminished. Compared with wild-type Ech3937, the expression levels of an oligogalacturonate lyase gene, ogl, and three endopectate lyase genes, pelD, pelI, and pelL, were reduced in Ech138 as determined by a green fluorescent protein-based fluorescence-activated cell sorting promoter activity assay. In addition, the transcription of type III secretion system (T3SS) genes, dspE (a putative T3SS effector) and hrpN (T3SS harpin), was found to be diminished in the iaaM mutant Ech138. Compared with Ech3937, reduced expression of hrpL (a T3SS alternative sigma factor) and gacA but increased expression of rsmA in Ech138 was also observed, suggesting that the regulation of T3SS and pectate lyase genes by IAA biosynthesis might be partially due to the posttranscriptional regulation of the Gac-Rsm regulatory pathway.

Dual regulation of a polyethylene glycol degradative operon by AraC-type and GalR-type regulators in Sphingopyxis macrogoltabida strain 103

The genes for polyethylene glycol (PEG) catabolism (pegB, C, D, A and E) in Sphingopyxis macrogoltabida strain 103 were shown to form a PEG-inducible operon. The pegR gene, encoding an AraC-type regulator in the downstream area of the operon, is transcribed in the reverse direction. The transcription start sites of the operon were mapped, and three putative σ 70-type promoter sites were identified in the pegB, pegA and pegR promoters. A promoter activity assay showed that the pegB promoter was induced by PEG and oligomeric ethylene glycols, whereas the pegA and pegR promoters were induced by PEG. Deletion analysis of the pegB promoter indicated that the region containing the activator-binding motif of an AraC/XylS-type regulator was required for transcription of the pegBCDAE operon. Gel retardation assays demonstrated the specific binding of PegR to the pegB promoter. Transcriptional fusion studies of pegR with pegA and pegB promoters suggested that PegR regulates the expression of the pegBCDAE operon positively through its binding to the pegB promoter, but PegR does not bind to the pegA promoter. Two specific binding proteins for the pegA promoter were purified and identified as a GalR-type regulator and an H2A histone fragment (histone-like protein, HU). The binding motif of a GalR/LacI-type regulator was found in the pegA and pegR promoters. These results suggested the dual regulation of the pegBCDAE operon through the pegB promoter by an AraC-type regulator, PegR (PEG-independent), and through the pegA and pegR promoters by a GalR/LacI-type regulator together with HU (PEG-dependent).