RESUMO
The scarcity of primordial germ cells (PGCs) in the developing mammalian embryo hampers robust biochemical analysis of the processes that underlie early germ cell formation. Here, we demonstrate that DAZL, a germ cell-specific RNA binding protein, is a robust PGC marker during in vitro germ cell development. Using Dazl-GFP reporter ESCs, we demonstrate that DAZL plays a central role in a large mRNA/protein interactive network that blocks the translation of core pluripotency factors, including Sox2 and Sall4, as well as of Suz12, a polycomb family member required for differentiation of pluripotent cells. Thus, DAZL limits both pluripotency and somatic differentiation in nascent PGCs. In addition, we observed that DAZL associates with mRNAs of key Caspases and similarly inhibits their translation. This elegant fail-safe mechanism ensures that, whereas loss of DAZL results in prolonged expression of pluripotency factors, teratoma formation is avoided due to the concomitant activation of the apoptotic cascade.
Assuntos
Apoptose/genética , Diferenciação Celular/genética , Embrião de Mamíferos/metabolismo , Células Germinativas/metabolismo , Proteínas de Ligação a RNA/genética , Animais , Animais Geneticamente Modificados , Células Cultivadas , Embrião de Mamíferos/citologia , Embrião de Mamíferos/embriologia , Células-Tronco Embrionárias/metabolismo , Perfilação da Expressão Gênica , Regulação da Expressão Gênica no Desenvolvimento , Redes Reguladoras de Genes , Immunoblotting , Proteínas Luminescentes/genética , Proteínas Luminescentes/metabolismo , Camundongos , Microscopia Confocal , Análise de Sequência com Séries de Oligonucleotídeos , Células-Tronco Pluripotentes/metabolismo , Interferência de RNA , Proteínas de Ligação a RNA/metabolismo , Reação em Cadeia da Polimerase Via Transcriptase ReversaRESUMO
The microbial diversity and metabolic potential of a methanogenic consortium residing in a 3785-liter anaerobic digester, fed with wastewater algae, was analyzed using 454 pyrosequencing technology. DNA was extracted from anaerobic sludge material and used in metagenomic analysis through PCR amplification of the methyl-coenzyme M reductase α subunit (mcrA) gene using primer sets ML, MCR, and ME. The majority of annotated mcrA sequences were assigned taxonomically to the genera Methanosaeta in the order Methanosarcinales. Methanogens from the genus Methanosaeta are obligate acetotrophs, suggesting this genus plays a dominant role in methane production from the analyzed fermentation sample. Numerous analyzed sequences within the algae fed anaerobic digester were unclassified and could not be assigned taxonomically. Relative amplicon frequencies were determined for each primer set to determine the utility of each in pyrosequencing. Primer sets ML and MCR performed better quantitatively (representing the large majority of analyzed sequences) than primer set ME. However, each of these primer sets was shown to provide a quantitatively unique community structure, and thus they are of equal importance in mcrA metagenomic analysis.
RESUMO
Lactobacillus helveticus R0052 is a bacterium used in commercial probiotic preparations. R0052 contains a small, cryptic plasmid comprised of eight open reading frames, four of which encode proteins of unknown function. Based on the sequence of the replication initiation protein RepA, pIR52-1 is a member of the recently described RepA_N family of Gram-positive theta-replicating plasmids. The repA gene of pIR52-1 is the minimal origin of replication for L. helveticus and other Lactobacillus hosts. Additionally, pIR52-1 belongs to a subgroup of the RepA_N plasmid family which have RepA proteins of high amino acid identity and a conserved, non-coding element upstream of repA which, in pIR52-1, is responsible for the control of plasmid copy number and contributes to plasmid maintenance.