Isolation of the gene encoding yeast DNA polymerase I

A yeast genomic DNA expression library in lambda gt11 antibody prepared against yeast DNA polymerase I were used to isolate the gene encoding DNA polymerase I. The identity of the DNA polymerase I gene was determined by several criteria. First, the clone-encoded protein is immunologically related to DNA polymerase I. Second, cells containing the gene cloned in the high copy number plasmid YEp24 overproduce the polymerase activity 4- to 5-fold as measured in yeast extracts. Finally, insertion of the gene downstream from a bacteriophage T7 promoter allows synthesis of yeast DNA polymerase I in Escherichia coli. Gene disruption and Southern hybridization experiments show that the polymerase is encoded by an essential, single copy gene. Examination of the germinated spores containing the disrupted gene reveals a defect in nuclear division and a terminal phenotype typical of replication mutants.     

On the structure of heme d1. An isobacteriochlorin derivative as the prosthetic group of dissimilatory nitrite reductase?

Timkovich and co-workers have recently proposed a chlorin macrocycle structure for the heme d1 prosthetic group isolated from cytochrome cd1 of Pseudomonas aeruginosa and Paracoccus denitrificans (Timkovich, R., Cork, M. S., and Taylor, P. V. (1984) J. Biol. Chem. 259, 1577-1585; 15089-15093). However, this chlorin structure deduced by them is not entirely consistent with the spectral data. An alternative structure is proposed here based on the available spectral evidence. It is suggested that heme d1 in vivo is not a chlorin, but a dioxo-isobacteriochlorin having two adjacent pyrrole rings saturated.     

Sequence and translation of the murine coronavirus 5''-end genomic RNA reveals the N-terminal structure of the putative RNA polymerase.

A 28-kilodalton protein has been suggested to be the amino-terminal protein cleavage product of the putative coronavirus RNA polymerase (gene A) (M.R. Denison and S. Perlman, Virology 157:565-568, 1987). To elucidate the structure and mechanism of synthesis of this protein, the nucleotide sequence of the 5' 2.0 kilobases of the coronavirus mouse hepatitis virus strain JHM genome was determined. This sequence contains a single, long open reading frame and predicts a highly basic amino-terminal region. Cell-free translation of RNAs transcribed in vitro from DNAs containing gene A sequences in pT7 vectors yielded proteins initiated from the 5'-most optimal initiation codon at position 215 from the 5' end of the genome. The sequence preceding this initiation codon predicts the presence of a stable hairpin loop structure. The presence of an RNA secondary structure at the 5' end of the RNA genome is supported by the observation that gene A sequences were more efficiently translated in vitro when upstream noncoding sequences were removed. By comparing the translation products of virion genomic RNA and in vitro transcribed RNAs, we established that our clones encompassing the 5'-end mouse hepatitis virus genomic RNA encode the 28-kilodalton N-terminal cleavage product of the gene A protein. Possible cleavage sites for this protein are proposed.     

Circadian rhythm of intracellular protein synthesis signaling in rat cardiac and skeletal muscles

Intracellular signaling exhibits circadian variation in the suprachiasmatic nucleus and liver. However, it is unclear whether circadian regulation also extends to intracellular signaling pathways in the cardiac and skeletal muscles. Here, we examined circadian variation in the intracellular mammalian target of rapamycin (mTOR)/70 kDa ribosomal protein S6 kinase 1 (p70S6K) and extracellular signal-regulated kinase (ERK) pathways, which regulate protein synthesis in rat cardiac and skeletal muscles. Seven-week-old male Wistar rats were assigned to six groups: Zeitgeber time (ZT) 2, ZT6, ZT10, ZT14, ZT18, and ZT22 (ZT0, lights on; ZT12, lights off). The cardiac, plantaris, and soleus muscles were removed after a 12-h fasting period, and signal transducers involved in protein synthesis (mTOR, p70S6K, and ERK) were analyzed by western blotting. Circadian rhythms of signal transducers were observed in both cardiac (mTOR, p70S6K, and ERK) and plantaris (p70S6K and ERK) muscles (p<0.05), but not in the soleus muscle. In the cardiac muscle, the phosphorylation rate of mTOR was significantly higher at ZT6 (peak) than at ZT18 (bottom), and the phosphorylation rate of p70S6K was significantly higher at ZT2 (peak) than at ZT18 (bottom). In contrast, in the plantaris muscle, the phosphorylation rate of ERK was significantly lower at ZT2 (bottom) than at ZT18 (peak). Our data suggested that protein synthesis via mTOR/p70S6K and ERK signaling molecules exhibits circadian variation in rat cardiac and fast-type plantaris muscles.     

Reduction of SOST gene promotes bone formation through the Wnt/β-catenin signalling pathway and compensates particle-induced osteolysis

The increase in bone resorption and/or the inhibition of bone regeneration caused by wear particles are the main causes of periprosthetic osteolysis. The SOST gene and Sclerostin, a protein synthesized by the SOST gene, are the characteristic marker of osteocytes and regulate bone formation and resorption. We aimed to verify whether the SOST gene was involved in osteolysis induced by titanium (Ti) particles and to investigate the effects of SOST reduction on osteolysis. The results showed osteolysis on the skull surface with an increase of sclerostin levels after treated with Ti particles. Similarly, sclerostin expression in MLO-Y4 osteocytes increased when treated with Ti particles in vitro. After reduction of SOST, local bone mineral density and bone volume increased, while number of lytic pores on the skull surface decreased and the erodibility of the skull surface was compensated. Histological analyses revealed that SOST reduction increased significantly alkaline phosphatase- (ALP) and osterix-positive expression on the skull surface which promoted bone formation. ALP activity and mineralization of MC3T3-E1 cells also increased in vitro when SOST was silenced, even if treated with Ti particles. In addition, Ti particles decreased β-catenin expression with an increase in sclerostin levels, in vivo and in vitro. Inversely, reduction of SOST expression increased β-catenin expression. In summary, our results suggested that reduction of SOST gene can activate the Wnt/β-catenin signalling pathway, promoting bone formation and compensated for bone loss induced by Ti particles. Thus, this study provided new perspectives in understanding the mechanisms of periprosthetic osteolysis.     

Physiological and genetic convergence supports hypoxia resistance in high-altitude songbirds

Skeletal muscle plays a central role in regulating glucose uptake and body metabolism; however, highland hypoxia is a severe challenge to aerobic metabolism in small endotherms. Therefore, understanding the physiological and genetic convergence of muscle hypoxia tolerance has a potential broad range of medical implications. Here we report and experimentally validate a common physiological mechanism across multiple high-altitude songbirds that improvement in insulin sensitivity contributes to glucose homeostasis, low oxygen consumption, and relative activity, and thus increases body weight. By contrast, low-altitude songbirds exhibit muscle loss, glucose intolerance, and increase energy expenditures under hypoxia. This adaptive mechanism is attributable to convergent missense mutations in the BNIP3L gene, and METTL8 gene that activates MEF2C expression in highlanders, which in turn increases hypoxia tolerance. Together, our findings from wild high-altitude songbirds suggest convergent physiological and genetic mechanisms of skeletal muscle in hypoxia resistance, which highlights the potentially medical implications of hypoxia-related metabolic diseases.     

Genomic Consequences of Long-Term Population Decline in Brown Eared Pheasant

Population genetic theory and empirical evidence indicate that deleterious alleles can be purged in small populations. However, this viewpoint remains controversial. It is unclear whether natural selection is powerful enough to purge deleterious mutations when wild populations continue to decline. Pheasants are terrestrial birds facing a long-term risk of extinction as a result of anthropogenic perturbations and exploitation. Nevertheless, there are scant genomics resources available for conservation management and planning. Here, we analyzed comparative population genomic data for the three extant isolated populations of Brown eared pheasant (Crossoptilon mantchuricum) in China. We showed that C. mantchuricum has low genome-wide diversity and a contracting effective population size because of persistent declines over the past 100,000 years. We compared genome-wide variation in C. mantchuricum with that of its closely related sister species, the Blue eared pheasant (C. auritum) for which the conservation concern is low. There were detrimental genetic consequences across all C. mantchuricum genomes including extended runs of homozygous sequences, slow rates of linkage disequilibrium decay, excessive loss-of-function mutations, and loss of adaptive genetic diversity at the major histocompatibility complex region. To the best of our knowledge, this study is the first to perform a comprehensive conservation genomic analysis on this threatened pheasant species. Moreover, we demonstrated that natural selection may not suffice to purge deleterious mutations in wild populations undergoing long-term decline. The findings of this study could facilitate conservation planning for threatened species and help recover their population size.     

Losartan Attenuates Insulin Resistance and Regulates Browning Phenomenon of White Adipose Tissue in ob/ob Mice

Insulin resistance (IR) is a villain role to the pathology of fatty liver diseases implicated in adipose tissue dysfunction, which is characterized by lipid droplets (LDs) accumulation and hypoxia-inducible factor 1α (HIF1α) related macrophage infiltration. HIF1α is required for its lipogenic actions in adipocytes, while and it regulates M1 and M2 polarization features of macrophages. Losartan has been shown to be an insulin sensitizer in obese states, actions involving in HIF1α signaling. However, the exact mechanisms accounting for these effects have not been fully elucidated. Therefore, GTT, ITT, and HOMA-IR were identified losartan alleviated IR signaling in obese mice. This alleviation may through inhibits HIF1α by suppressing STAT3-NF-κB signaling, which, in turn, revealed HIF1α-dependent decreases the angiogenesis pathway in adipose tissue, including regulation of VEGF and TGFβR2 levels. In white adipose tissue, a set of lipogenesis-related genes, Srebp1, Fas, and Scd-1 were markedly downregulated after losartan intervention, as well as reduced LDs size and LD-associated proteins, perilipin family proteins (PLINs) compared with obese mice. Losartan abolished macrophage infiltration with upregulation of M2 and inhibition of M1 macrophage markers in obese mice. Our data suggest that losartan attenuated obese-induced fatty liver, linked to alleviating inflammation in adipose tissues and a shift in M1/M2 macrophage balance. Furthermore, losartan might improve mitochondria biogenesis by upregulating SIRT1, PGC1α, UCP1, and mRNA of Tfam, Cd137, Tmem26, Ucp1 expression in white adipose tissue compared with the obese group. Taken together, losartan may improve IR and adipose dysfunction by inhibiting lipotoxicity and HIF1α pathways.     

Genome-wide programmable transcriptional memory by CRISPR-based epigenome editing

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