Finishing the euchromatic sequence of the human genome

The sequence of the human genome encodes the genetic instructions for human physiology, as well as rich information about human evolution. In 2001, the International Human Genome Sequencing Consortium reported a draft sequence of the euchromatic portion of the human genome. Since then, the international collaboration has worked to convert this draft into a genome sequence with high accuracy and nearly complete coverage. Here, we report the result of this finishing process. The current genome sequence (Build 35) contains 2.85 billion nucleotides interrupted by only 341 gaps. It covers ∼99% of the euchromatic genome and is accurate to an error rate of ∼1 event per 100,000 bases. Many of the remaining euchromatic gaps are associated with segmental duplications and will require focused work with new methods. The near-complete sequence, the first for a vertebrate, greatly improves the precision of biological analyses of the human genome including studies of gene number, birth and death. Notably, the human enome seems to encode only 20,000-25,000 protein-coding genes. The genome sequence reported here should serve as a firm foundation for biomedical research in the decades ahead

Upregulation of type I collagen by TGF-β in mesangial cells is blocked by PPARγ activation

We found that peroxisome proliferator-activated receptor-γ (PPARγ) mRNA was reduced by 77% in glomeruli of diabetic mice. Because mesangial cells play an important role in diabetic nephropathy, we examined regulation of type I collagen expression by PPARγ and transforming growth factor-β1 (TGF-β1) in mouse mesangial cells in the presence of 6 and 25 mM glucose. Mesangial cells contained functionally active PPARγ. Exposure to 25 mM glucose resulted in reduced PPARγ expression and transcriptional activity, accompanied by increased type I collagen expression. Restoration of PPARγ activity to normal levels in cells cultured in 25 mM glucose, by transfection with a PPARγ expression construct and treatment with the PPARγ agonist troglitazone, returned type I collagen levels toward normal values. Activation of PPARγ by troglitazone also decreased type I collagen mRNA and blocked TGF-β1-mediated upregulation of type I collagen mRNA and protein. Moreover, PPARγ activation suppressed basal and activated TGF-β1 responses in mesangial cells. This action was blocked by transfection of cells with a dominant-negative PPARγ construct. In summary, PPARγ suppresses the increased type I collagen mRNA and protein expression mediated by TGF-β1 in mesangial cells

Cdc7-Dbf4 Regulates NDT80 Transcription as Well as Reductional Segregation during Budding Yeast Meiosis

In budding yeast, as in other eukaryotes, the Cdc7 protein kinase is important for initiation of DNA synthesis in vegetative cells. In addition, Cdc7 has crucial meiotic functions: it facilitates premeiotic DNA replication, and it is essential for the initiation of recombination. This work uses a chemical genetic approach to demonstrate that Cdc7 kinase has additional roles in meiosis. First, Cdc7 allows expression of NDT80, a meiosis-specific transcriptional activator required for the induction of genes involved in exit from pachytene, meiotic progression, and spore formation. Second, Cdc7 is necessary for recruitment of monopolin to sister kinetochores, and it is necessary for the reductional segregation occurring at meiosis I. The use of the same kinase to regulate several distinct meiosis-specific processes may be important for the coordination of these processes during meiosis

Guidelines for the use and interpretation of assays for monitoring autophagy (4th edition)

In 2008, we published the first set of guidelines for standardizing research in autophagy. Since then, this topic has received increasing attention, and many scientists have entered the field. Our knowledge base and relevant new technologies have also been expanding. Thus, it is important to formulate on a regular basis updated guidelines for monitoring autophagy in different organisms. Despite numerous reviews, there continues to be confusion regarding acceptable methods to evaluate autophagy, especially in multicellular eukaryotes. Here, we present a set of guidelines for investigators to select and interpret methods to examine autophagy and related processes, and for reviewers to provide realistic and reasonable critiques of reports that are focused on these processes. These guidelines are not meant to be a dogmatic set of rules, because the appropriateness of any assay largely depends on the question being asked and the system being used. Moreover, no individual assay is perfect for every situation, calling for the use of multiple techniques to properly monitor autophagy in each experimental setting. Finally, several core components of the autophagy machinery have been implicated in distinct autophagic processes (canonical and noncanonical autophagy), implying that genetic approaches to block autophagy should rely on targeting two or more autophagy-related genes that ideally participate in distinct steps of the pathway. Along similar lines, because multiple proteins involved in autophagy also regulate other cellular pathways including apoptosis, not all of them can be used as a specific marker for bona fide autophagic responses. Here, we critically discuss current methods of assessing autophagy and the information they can, or cannot, provide. Our ultimate goal is to encourage intellectual and technical innovation in the field