Current Issue : October - December Volume : 2012 Issue Number : 4 Articles : 4 Articles
Methanosarcina mazei is one of the model organisms for the methanogenic order Methanosarcinales whose metabolism has been\r\nstudied in detail. However, the genetic toolbox is still limited. This study was aimed at widening the scope of utilizable methods\r\nin this group of organisms. (i) Proteins specific to methanogens are oftentimes difficult to produce in E. coli. However, a protein\r\nproduction system is not available for methanogens. Here we present an inducible system to produce Strep-tagged proteins in Ms.\r\nmazei. The promoter p1687, which directs the transcription of methyl transferases that demethylate methylamines, was cloned\r\ninto plasmid pWM321 and its activity was determined by monitoring �Ÿ-glucuronidase production. The promoter was inactive\r\nduring growth on methanol but was rapidly activated when trimethylamine was added to the medium. The gene encoding the\r\n�Ÿ-glucuronidase from E. coli was fused to a Strep-tag and was cloned downstream of the p1687 promoter. The protein was\r\noverproduced in Ms. mazei and was purified in an active form by affinity chromatography. (ii) Puromycin is currently the only\r\nantibiotic used as a selectable marker in Ms. mazei and its relatives. We established neomycin resistance as a second selectable\r\nmarker by designing a plasmid that confers neomycin resistance in Ms. mazei....
The prevalence of type 2 diabetes in the United States is projected to double or triple by 2050. We reasoned that the\r\ngenes that modulate insulin production might be new targets for diabetes therapeutics. Therefore, we developed an\r\nsiRNA screening system to identify genes important for the activity of the insulin promoter in beta cells. We created a\r\nsubclone of the MIN6 mouse pancreatic beta cell line that expresses destabilized GFP under the control of a 362 base pair\r\nfragment of the human insulin promoter and the mCherry red fluorescent protein under the control of the constitutively\r\nactive rous sarcoma virus promoter. The ratio of the GFP to mCherry fluorescence of a cell indicates its insulin promoter\r\nactivity. As G protein coupled receptors (GPCRs) have emerged as novel targets for diabetes therapies, we used this cell\r\nline to screen an siRNA library targeting all known mouse GPCRs. We identified several known GPCR regulators of insulin\r\nsecretion as regulators of the insulin promoter. One of the top positive regulators was Gpr27, an orphan GPCR with no\r\nknown role in beta cell function. We show that knockdown of Gpr27 reduces endogenous mouse insulin promoter activity\r\nand glucose stimulated insulin secretion. Furthermore, we show that Pdx1 is important for Gpr27�s effect on the insulin\r\npromoter and insulin secretion. Finally, the over-expression of Gpr27 in 293T cells increases inositol phosphate levels,\r\nwhile knockdown of Gpr27 in MIN6 cells reduces inositol phosphate levels, suggesting this orphan GPCR might couple to\r\nGq/11. In summary, we demonstrate a MIN6-based siRNA screening system that allows rapid identification of novel\r\npositive and negative regulators of the insulin promoter. Using this system, we identify Gpr27 as a positive regulator of\r\ninsulin production....
Islet transplantation is a promising therapy for patients with type 1 diabetes that can provide moment-to-moment metabolic\r\ncontrol of glucose and allow them to achieve insulin independence. However, two major problems need to be overcome: (1)\r\ndetrimental immune responses, including inflammation induced by the islet isolation/transplantation procedure, recurrence\r\nautoimmunity, and allorejection, can cause graft loss and (2) inadequate numbers of organ donors. Several gene therapy\r\napproaches and pharmaceutical treatments have been demonstrated to prolong the survival of pancreatic islet grafts in animal\r\nmodels; however, the clinical applications need to be investigated further. In addition, for an alternative source of pancreatic �Ÿ-cell\r\nreplacement therapy, the ex vivo generation of insulin-secreting cells from diverse origins of stem/progenitor cells has become an\r\nattractive option in regenerative medicine. This paper focuses on the genetic manipulation of islets during transplantation therapy\r\nand summarizes current strategies to obtain functional insulin-secreting cells from stem/progenitor cells...
The origin and evolution of the ribosome is central to our understanding of the cellular world. Most hypotheses posit that\r\nthe ribosome originated in the peptidyl transferase center of the large ribosomal subunit. However, these proposals do not\r\nlink protein synthesis to RNA recognition and do not use a phylogenetic comparative framework to study ribosomal\r\nevolution. Here we infer evolution of the structural components of the ribosome. Phylogenetic methods widely used in\r\nmorphometrics are applied directly to RNA structures of thousands of molecules and to a census of protein structures in\r\nhundreds of genomes. We find that components of the small subunit involved in ribosomal processivity evolved earlier than\r\nthe catalytic peptidyl transferase center responsible for protein synthesis. Remarkably, subunit RNA and proteins coevolved,\r\nstarting with interactions between the oldest proteins (S12 and S17) and the oldest substructure (the ribosomal ratchet) in\r\nthe small subunit and ending with the rise of a modern multi-subunit ribosome. Ancestral ribonucleoprotein components\r\nshow similarities to in vitro evolved RNA replicase ribozymes and protein structures in extant replication machinery. Our\r\nstudy therefore provides important clues about the chicken-or-egg dilemma associated with the central dogma of\r\nmolecular biology by showing that ribosomal history is driven by the gradual structural accretion of protein and RNA\r\nstructures. Most importantly, results suggest that functionally important and conserved regions of the ribosome were\r\nrecruited and could be relics of an ancient ribonucleoprotein world...
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