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.     

A putative GDP–GTP exchange factor is required for development of the excretory cell in Caenorhabditis elegans

The Caenorhabditis elegans excretory cell extends tubular processes, called canals, along the basolateral surface of the epidermis. Mutations in the exc-5 gene cause tubulocystic defects in this canal. Ultrastructural analysis suggests that exc-5 is required for the proper placement of cytoskeletal elements at the apical epithelial surface. exc-5 encodes a protein homologous to guanine nucleotide exchange factors and contains motif architecture similar to that of FGD1, which is responsible for faciogenital dysplasia. exc-5 interacts genetically with mig-2, which encodes Rho GTPase. These results suggest that EXC-5 controls the structural organization of the excretory canal by regulating Rho family GTPase activities.     

Correction to: Herding mechanisms to maintain the cohesion of a harem group: two interaction phases during herding

Journal of Ethology - The article Herding mechanisms to maintain the cohesion of a harem group.     

On the Stability of Clathrate Hydrates Encaging Polar Guest Molecules: Contrast in the Hydrogen Bonds of Methylamine and Methanol Hydrates

Abstract

The stability of clathrate hydrates encaging highly polar guests has been investigated in order to explain the experimental observation that some amines form clathrate hydrates but alcohols act as inhibitor to hydrate formation. We choose methylamine and methanol as guest species and examine the stable structure, at which the total potential energy has a minimum value. At the local minima of those two hydrates, the potential energies of water-water and guest-water, and their hydrogen bonded networks are compared. It is found that methanol does not retain the host lattice structure, while the host-network structure is kept in the presence of methylamine. It is shown that the difference in the magnitude of the partial charge on the hydrogen atom between the hydroxyl and amino groups plays a much more significant role on the stability of both clathrate hydrates than the difference in molecular geometry. This is supported from the result of a methylamine-like model that has the same partial charges on the atoms in the hydrophilic site as methanol.     

Punctin, a novel ADAMTS-like molecule, ADAMTSL-1, in extracellular matrix.

Punctin (ADAMTSL-1) is a secreted molecule resembling members of the ADAMTS family of proteases. Punctin lacks the pro-metalloprotease and the disintegrin-like domain typical of this family but contains other ADAMTS domains in precise order including four thrombospondin type I repeats. Punctin is the product of a distinct gene on human chromosome 9p21-22 and mouse chromosome 4 that is expressed in adult skeletal muscle. His-tagged punctin expressed in stably transfected High-Five(TM) insect cells was purified to apparent homogeneity by Ni-chromatography of conditioned medium. The NH(2) terminus is not blocked and has the sequence EEDRD and so forth as determined by Edman degradation, demonstrating signal peptidase processing. Recombinant epitope-tagged punctin has a calculated mass of 59,991 Da but exhibits major molecular species of 61970 +/- 6 Da and 62131 +/- 5 Da as measured by liquid chromatography electrospray mass spectrometry. Punctin is a glycoprotein based on carbohydrate staining and liquid chromatography electrospray mass spectrometry glycopeptide analysis. Glycosylation occurs at a single N-linked site as demonstrated by altered electrophoretic migration of punctin expressed in the presence of tunicamycin A. Punctin contains disulfide bonds based on antibody accessibility and electrophoretic migration under reducing versus nonreducing conditions. Rotary shadowing demonstrates that punctin is hatchet-shaped having a globular region attached to a short stem. In transfected COS-1 cells, punctin is deposited in the cell substratum in a punctate fashion and is excluded from focal contacts. Punctin is the first member of a novel family of ADAMTS-like proteins that may have important functions in the extracellular matrix.     

Enhanced synthesis of type IV collagen in cultured arterial smooth muscle cells associated with phenotypic modulation by dimethyl sulfoxide

The effect of dimethyl sulfoxide (DMSO) on synthesis of basement membrane collagen in cultured smooth muscle cells was evaluated. DMSO promoted phenotypic modulation of cells from the synthetic state to the contractile state accompanied by formation of basement membranes. By immunofluorescence using monospecific antibody against type IV collagen, type IV collagen was identified not only in the cell cytoplasms but intensely along the cell surfaces in the cultures treated with DMSO for 7 days, as compared with untreated cultures. Electron microscopic immunohistochemistry revealed the presence of type IV collagen both in the basement membrane region and in the rough endoplasmic reticulum of DMSO-treated cells. Such an enhancement of type IV collagen synthesis appears to be expressed as a result of the phenotypic changes of smooth muscle cells to the contractile state modulated by DMSO.     

Primary structures of the mRNAs encoding the rat precursors for bradykinin and T-kinin. Structural relationship of kininogens with major acute phase protein and alpha 1-cysteine proteinase inhibitor

Three types of cloned cDNA sequences for rat low molecular weight prekininogens were isolated and determined by molecular cloning and sequence analysis. The deduced amino acid sequences indicated that one, termed K-prekininogen, represents the counterpart of the known low molecular weight prekininogen present in other mammals, while the other two, called T-prekininogens, contain a novel T-kinin sequence which was recently identified from rat plasma. Although T- and K-prekininogens are highly homologous with each other, both of the T-prekininogens contain methionine, instead of arginine or lysine, as an amino acid preceding T-kinin and exhibit two consecutive amino acid deletions in the preceding region of T-kinin as compared with K-prekininogen. The former finding accounts for the previous observation of strong resistance of T-kininogens to cleavage with trypsin or kallikreins, while the latter finding has been explained by the structural analysis of genomic clones in which T-kinin-coding exon is contracted at its intron junction. A partial nucleotide sequence reported recently for the rat major acute phase protein (alpha 1-MAP) mRNA was found to be extremely related to the corresponding portion of the rat T-prekininogen mRNA. Furthermore, consistent with the previous report of the structural identity of major acute phase protein and alpha 1-cysteine proteinase inhibitor, kininogen closely resembles not only the former but also the latter in the amino acid compositions. The interrelationship among the triad of these proteins has been discussed.     

Molecular cloning of a non-isopeptide-selective human endothelin receptor.

We isolated several complementary DNA (cDNA) clones encoding a non-isopeptide-selective human endothelin receptor (ETBR) from a human placenta cDNA library. The clones, different in the length of their 3'-untranslated regions, encoded the same 442-amino acid protein with a transmembrane topology similar to that of other G protein-coupled receptors. The rank order of the binding of ET isopeptides (ET-1, ET-2 and ET-3) to the receptor expressed in COS-7 cells was ET-1 = ET-2 = ET-3. Northern blot analysis identified three mRNA species, 4.3 kb, 2.7 kb and 1.7 kb in size, probably generated by their use of alternative polyadenylation sites. These mRNAs were expressed in a wide variety of human tissues, at the highest level in the brain and at a significant level in cultured endothelial cells.