49 research outputs found
Nanopore sequencing and assembly of a human genome with ultra-long reads
We report the sequencing and assembly of a reference genome for the human GM12878 Utah/Ceph cell line using the MinION (Oxford Nanopore Technologies) nanopore sequencer. 91.2 Gb of sequence data, representing ~30× theoretical coverage, were produced. Reference-based alignment enabled detection of large structural variants and epigenetic modifications. De novo assembly of nanopore reads alone yielded a contiguous assembly (NG50 ~3 Mb). Next, we developed a protocol to generate ultra-long reads (N50 > 100kb, up to 882 kb). Incorporating an additional 5×-coverage of these data more than doubled the assembly contiguity (NG50 ~6.4 Mb). The final assembled genome was 2,867 million bases in size, covering 85.8% of the reference. Assembly accuracy, after incorporating complementary short-read sequencing data, exceeded 99.8%. Ultra-long reads enabled assembly and phasing of the 4 Mb major histocompatibility complex (MHC) locus in its entirety, measurement of telomere repeat length and closure of gaps in the reference human genome assembly GRCh38
Inhibitory effects of antisense oligonucleotides on the expression of procollagen type III gene in mouse hepatic stellate cells transformed by simian virus 40
Persistent activation of NF-kB in cultured rat hepatic stellate cells involves the induction of potentially novel rel-like factors and prolonged changes in the expression of IkB family proteins.
Rat hepatic stellate cells (HSC) cultured in serum-containing medium underwent a rapid (3-hour) classical induction of p50:p65 and p65:p65 nuclear factor-kappa B (NF-kappa B) dimers, Subsequent culturing was associated with prolonged expression of active p50:p65 and persistent induction of a high-mobility NF-kappa B DNA binding complex consisting of potentially novel Rel-like protein(s). Formation of the latter complex was competed for by specific double-stranded oligonucleotides, was up-regulated by treatment of HSCs with tumor necrosis factor alpha (TNF-alpha), and was maintained at basal levels of expression by a soluble HSC-derived factor. An NF-kappa B-responsive CAT reporter gene was highly active in early cultured HSCs but was also trans-activated at a lower but significant level in longer-term cultured cells and could be completely suppressed by expression of dominant negative I kappa B-alpha. Physiological significance of the lower persistent NF-kappa B activities was also demonstrated by the ability of long-term cultured HSCs to support the activity of the NF-kappa B-dependent human intercellular adhesion molecule-1 (ICAM-1) promoter. Freshly isolated HSCs expressed high levels of I kappa B-alpha and I kappa B-beta. Culture activation was accompanied by a long-term reduction in levels of I kappa B-alpha With no detectable expression in the nuclear fraction of cells, under these conditions p50:p65 was detected in the nucleus. I kappa B-beta expression was transiently reduced and, upon replenishment, was associated with appearance of a lower-mobility I kappa B-beta antibody-reactive species. Bcl3 expression was absent in freshly isolated HSC but was induced during culturing and became a persistent feature of the activated HSC, Inhibition of NF-kappa B DNA binding activity by gliotoxin was associated with increased numbers of apoptotic cells. We suggest that activation of NF-kappa B in cultured HSC is required for expression of specific genes associated with the activated phenotype such as ICAM-1 and may be antiapoptotic for rat HSCs.</p
Persistent activation of NF-kB in cultured rat hepatic stellate cells involves the induction of potentially novel rel-like factors and prolonged changes in the expression of IkB family proteins.
Rat hepatic stellate cells (HSC) cultured in serum-containing medium underwent a rapid (3-hour) classical induction of p50:p65 and p65:p65 nuclear factor-kappa B (NF-kappa B) dimers, Subsequent culturing was associated with prolonged expression of active p50:p65 and persistent induction of a high-mobility NF-kappa B DNA binding complex consisting of potentially novel Rel-like protein(s). Formation of the latter complex was competed for by specific double-stranded oligonucleotides, was up-regulated by treatment of HSCs with tumor necrosis factor alpha (TNF-alpha), and was maintained at basal levels of expression by a soluble HSC-derived factor. An NF-kappa B-responsive CAT reporter gene was highly active in early cultured HSCs but was also trans-activated at a lower but significant level in longer-term cultured cells and could be completely suppressed by expression of dominant negative I kappa B-alpha. Physiological significance of the lower persistent NF-kappa B activities was also demonstrated by the ability of long-term cultured HSCs to support the activity of the NF-kappa B-dependent human intercellular adhesion molecule-1 (ICAM-1) promoter. Freshly isolated HSCs expressed high levels of I kappa B-alpha and I kappa B-beta. Culture activation was accompanied by a long-term reduction in levels of I kappa B-alpha With no detectable expression in the nuclear fraction of cells, under these conditions p50:p65 was detected in the nucleus. I kappa B-beta expression was transiently reduced and, upon replenishment, was associated with appearance of a lower-mobility I kappa B-beta antibody-reactive species. Bcl3 expression was absent in freshly isolated HSC but was induced during culturing and became a persistent feature of the activated HSC, Inhibition of NF-kappa B DNA binding activity by gliotoxin was associated with increased numbers of apoptotic cells. We suggest that activation of NF-kappa B in cultured HSC is required for expression of specific genes associated with the activated phenotype such as ICAM-1 and may be antiapoptotic for rat HSCs.</p