Advantages of genome sequencing by long-read sequencer using SMRT technology in medical area

PacBio RS II is the first commercialized third-generation DNA sequencer able to sequence a single molecule DNA in real-time without amplification. PacBio RS II’s sequencing technology is novel and unique, enabling the direct observation of DNA synthesis by DNA polymerase. PacBio RS II confers four major advantages compared to other sequencing technologies: long read lengths, high consensus accuracy, a low degree of bias, and simultaneous capability of epigenetic characterization. These advantages surmount the obstacle of sequencing genomic regions such as high/low G+C, tandem repeat, and interspersed repeat regions. Moreover, PacBio RS II is ideal for whole genome sequencing, targeted sequencing, complex population analysis, RNA sequencing, and epigenetics characterization. With PacBio RS II, we have sequenced and analyzed the genomes of many species, from viruses to humans. Herein, we summarize and review some of our key genome sequencing projects, including full-length viral sequencing, complete bacterial genome and almost-complete plant genome assemblies, and long amplicon sequencing of a disease-associated gene region. We believe that PacBio RS II is not only an effective tool for use in the basic biological sciences but also in the medical/clinical setting.     

Intra- and interbreed genetic variations of mitochondrial DNA major non-coding regions in Japanese native dog breeds [Canis familiaris)

Mitochondrial DNA (mtDNA) major non-coding regions were amplified from 73 dogs of eight Japanese native dog breeds and from 21 dogs of 16 non-Japanese dog breeds by the polymerase chain reaction and their DNA sequences were determined. A total of 51 nucleotide positions within the non-coding region (969–972 base pairs) showed nucleotide variations of which 48 were caused by transition. These nucleotide substitutions were abundant in the region proximate to tRNA^Pro. In addition to the nucleotide substitutions, the dog mtDNA D-loop sequences had a heteroplasmic repetitive sequence (TACACGTÀCG) involving size variation. The DNA sequences of the non-coding region were classified into four different groups by phylogenetic analysis and the deepest branchpoints of this dog phylogeny was calculated to about 100 000 years before the present. Phylogenetic analysis showed that Japanese native dog breeds could not be clearly delimited as distinct breeds. Many haplotypes found in members of some clustering groups were seen in each dog breed, and interbreed nucleotide differences between Japanese dog breeds were almost the same as the intrabreed nucleotide diversities.     

Long term maintenance of an anther-derived haploid callus line of the Asiatic hybrid lily ‘Connecticut King’

A haploid callus line from anther cultures of the Asiatic hybrid lily ‘Connecticut King’ was maintained for a long term. The survival and growth of the haploid calluses were affected by auxins of picloram, α-naphthaleneacetic acid (NAA) and 2,4-dichlorophenoxyacetic acid (2,4-D) and temperatures of 25, 15 and 7 °C during culture. Picloram was more suitable for maintenance of the haploid calluses, whereas NAA and 2,4-D led to root and shoot formation from the haploid calluses. The best temperature for maintenance was 25 °C. About 90% of cells in calluses were maintained in haploid level during 60 weeks of subculture, and about 80% of cells were haploid in the calluses maintained over 2 years with the MS medium containing 4 μM picloram in the dark at 25 °C. This revised version was published online in June 2006 with corrections to the Cover Date.     

Subcellular distribution of hydrolase in germinating pea cotyledons

The subcellular distribution of hydrolases in germinating peacotyledons was investigated by differential and sucrose densitygradient centrifugations. Acid phosphatase was present in atleast two kinds of subcellular components. One was slightlyheavier than mitochondria, and the other was so light that itremained on the upper part of the gradient after sucrose densitygradient centrifugation. Protease was localized in the smooth-surfacedmicrosomal fraction which contained antimycin A-insensitiveNADH₂-cytochrome c reductase and no RNA. Aryl esterase existedin a soluble form in the cytoplasm. (Received July 22, 1971; )     

Degradation of thyroxine by microsomal particles from rat liver II. Purification of Fe3+-dependent particles and some of their properties

Particles, which catalyze the deiodination of thyroid hormones in the presence of Fe²⁺, have been purified about 25-fold with respect to the rat liver microsomes. This purification involves deoxycholate treatment, trypsin treatment and density gradient centrifugutions.The purified particles range from 1.019 to 1.063 in density and consist of 21% protein, 60% phospholipid and 19% neutral fat. No hemoprotein moiety is associated with the purified particles. The purified particles degrade not only l-thyroxine but also other iodinated thyronines and their derivatives, such as d-thyroxine, l-3′,3,5- triiodothyronine and tetraiodothyropropionic acid. They are inactive towards monoiodotyrosine and diiodotyrosine.Thyroxine is not metabolized in the drug hydroxylation system which involves cytochrome P-450.     

Purification and characterization of a gelatinase produced by fibroblasts from human gingiva

An endopeptidase which digests denatured collagen to small, dialysable fragments was purified 2675-fold from medium that had been conditioned by the culture of fibroblasts grown from explants of human gingiva. This enzyme was inhibited by chelating agents, but not by phenylmethylsulphonyl fluoride nor by N-ethylmaleimide, and is therefore probably a metalloproteinase. It showed no demonstrable activity against native collagen or ovalbumin, while alpha-casein was digested slowly, if at all. It therefore belongs to the group of enzymes which have been called tissue gelatinases. This gelatinase was secreted in a latent form or forms and could be activated by proteolysis with trypsin. The active enzyme had an apparent molecular weight of 69 000 (gel chromatography) or 72 000 (gel electrophoresis in sodium dodecyl sulphate) and an apparent isoelectric point of 4.15.     

Generation of a purely clonal defective interfering influenza virus

Defective interfering (DI) influenza viruses carry a large deletion in a gene segment that interferes with the replication of infectious virus; thus, such viruses have potential for antiviral therapy. However, because DI viruses cannot replicate autonomously without the aid of an infectious helper virus, clonal DI virus stocks that are not contaminated with helper virus have not yet been generated. To overcome this problem, we used reverse genetics to generate a clonal DI virus with a PB2 DI gene, amplified the clonal DI virus using a cell line stably expressing the PB2 protein, and confirmed its ability to interfere with infectious virus replication in vitro. Thus, our approach is suitable for obtaining purely clonal DI viruses, will contribute to the understanding of DI virus interference mechanisms and can be used to develop DI virus‐based antivirals.