Title: Evaluation of the impact of RNA preservation methods of spiders for de novo transcriptome assembly Kono N, Nakamura H, Ito Y, Tomita M, Arakawa K Ref: Mol Ecol Resour, 16:662, 2016 : PubMed
With advances in high-throughput sequencing technologies, de novo transcriptome sequencing and assembly has become a cost-effective method to obtain comprehensive genetic information of a species of interest, especially in nonmodel species with large genomes such as spiders. However, high-quality RNA is essential for successful sequencing, and sample preservation conditions require careful consideration for the effective storage of field-collected samples. To this end, we report a streamlined feasibility study of various storage conditions and their effects on de novo transcriptome assembly results. The storage parameters considered include temperatures ranging from room temperature to -80 degrees C; preservatives, including ethanol, RNAlater, TRIzol and RNAlater-ICE; and sample submersion states. As a result, intact RNA was extracted and assembly was successful when samples were preserved at low temperatures regardless of the type of preservative used. The assemblies as well as the gene expression profiles were shown to be robust to RNA degradation, when 30 million 150-bp paired-end reads are obtained. The parameters for sample storage, RNA extraction, library preparation, sequencing and in silico assembly considered in this work provide a guideline for the study of field-collected samples of spiders.
Remineralization of organic matter in deep-sea sediments is important in oceanic biogeochemical cycles, and bacteria play a major role in this process. Shewanella violacea DSS12 is a psychrophilic and piezophilic gamma-proteobacterium that was isolated from the surface layer of deep sea sediment at a depth of 5110 m. Here, we report the complete genome sequence of S. violacea and comparative analysis with the genome of S. oneidensis MR-1, isolated from sediments of a freshwater lake. Unlike S. oneidensis, this deep-sea Shewanella possesses very few terminal reductases for anaerobic respiration and no c-type cytochromes or outer membrane proteins involved in respiratory Fe(iii) reduction, which is characteristic of most Shewanella species. Instead, the S. violacea genome contains more terminal oxidases for aerobic respiration and a much greater number of putative secreted proteases and polysaccharases, in particular, for hydrolysis of collagen, cellulose and chitin, than are encoded in S. oneidensis. Transporters and assimilatory reductases for nitrate and nitrite, and nitric oxide-detoxifying mechanisms (flavohemoglobin and flavorubredoxin) are found in S. violacea. Comparative analysis of the S. violacea genome revealed the respiratory adaptation of this bacterium to aerobiosis, leading to predominantly aerobic oxidation of organic matter in surface sediments, as well as its ability to efficiently use diverse organic matter and to assimilate inorganic nitrogen as a survival strategy in the nutrient-poor deep-sea floor.
Only a small proportion of the mouse genome is transcribed into mature messenger RNA transcripts. There is an international collaborative effort to identify all full-length mRNA transcripts from the mouse, and to ensure that each is represented in a physical collection of clones. Here we report the manual annotation of 60,770 full-length mouse complementary DNA sequences. These are clustered into 33,409 'transcriptional units', contributing 90.1% of a newly established mouse transcriptome database. Of these transcriptional units, 4,258 are new protein-coding and 11,665 are new non-coding messages, indicating that non-coding RNA is a major component of the transcriptome. 41% of all transcriptional units showed evidence of alternative splicing. In protein-coding transcripts, 79% of splice variations altered the protein product. Whole-transcriptome analyses resulted in the identification of 2,431 sense-antisense pairs. The present work, completely supported by physical clones, provides the most comprehensive survey of a mammalian transcriptome so far, and is a valuable resource for functional genomics.
Title: Gene analysis of genomic DNA from stored serum by polymerase chain reaction: identification of three missense mutations in patients with cholinesterasemia and ABO genotyping Hidaka K, Watanabe Y, Tomita M, Ueda N, Higashi M, Minatogawa Y, Iuchi I Ref: Clinica Chimica Acta, 303:61, 2001 : PubMed
We established a method to determine the butyrylcholinesterase genotype associated with a BCHE deficiency directly using multiple PCR from stored serum, which was stored at -70 degrees C for more than 30 years. PCR products from sera of six propositi were used for DNA sequence analysis. All of these BChE variants were characterized by a single nucleotide substitution. Four of them were homozygotes and demonstrated a C-->T single nucleotide point mutation at codon 100 from CCA (Pro) to TCA (Ser). The fifth case was a heterozygote of this mutation. The remaining one was a compound heterozygote showing a T-->C transition mutation at codon 203 from TCA (Ser) to CCA (Pro) and a G-->C transversion mutation at codon 365 from GGA (Gly) to CGA (Arg). Furthermore we developed a method to determine the ABO genotype from the same serum. These results indicated that serum is useful as a starting material for amplification of genomic DNA when fresh blood samples are not available.
The RIKEN Mouse Gene Encyclopaedia Project, a systematic approach to determining the full coding potential of the mouse genome, involves collection and sequencing of full-length complementary DNAs and physical mapping of the corresponding genes to the mouse genome. We organized an international functional annotation meeting (FANTOM) to annotate the first 21,076 cDNAs to be analysed in this project. Here we describe the first RIKEN clone collection, which is one of the largest described for any organism. Analysis of these cDNAs extends known gene families and identifies new ones.
The present symposium during Brain 99 was convened to explore the current aspects of the neural (extrinsic and intrinsic) and chemical control of the microvasculature in the brain with specific relevance to stimuli and rapid flow responses. N. Suzuki demonstrated the presence of neurokinin-1 receptors along the axons of vasoactive intestinal polypeptide-containing cerebrovascular parasympathetic nerves. Since the receptors were activated by substance P, calcitonin gene-related peptide and neurokinin released from coexisting sensory nerve fibers, the parasympathetic (vasodilating) fibers could effect rapid local flow increases. N. Suzuki, however, considered this as part of an elaborate defensive network protecting the brain from invasions by noxious substances. E. Hamel discussed the responses of the microvessels to neurotransmitters and suggested that nitric oxide (NO) released from intrinsic neurons may serve as a relay in the flow activation responses by intracerebral cholinergic fibers originating in the basal forebrain nuclei. D. Busija summarized a vasodilating system of activated N-methyl-D-asparate receptors located on neurons involving Ca influx-NO production, and activated ATP-sensitive potassium channels located in the vascular system. According to Busija, such interactions were disrupted during hypoxia and ischemia due to cyclooxygenase-derived superoxide anion. M. Lauritzen observed a 10 times larger increase in blood flow on stimulation of the climbing nerve as compared with that following the parallel nerve stimulation. The former transmitters are considered by him to be NO and K, and the latter NO and adenosine. Each speaker singled out NO as a common mediator for the microvasculature in the rapid local flow increases.
Six serum samples with no detectable butyrylcholinesterase (BCHE) activity had been stored at -70 degrees C for more than 10 years. These sera were used for amplification of BCHE gene using polymerase chain reaction (PCR) and for nucleotide sequence analysis. Five of them demonstrated a C-->T transition at codon 100 (CCA-->TCA), resulting in a Pro-->Ser substitution. The other one was a compound heterozygote as revealed a T-->C transition mutation at codon 203 from TCA (Ser) to CCA (Pro) and G-->C transversion at codon 365 from GGA (Gly) to CGA (Arg). These results showed sera stored in a freezer could be used as a starting material for amplification of genomic DNA when it is not possible to obtain fresh blood samples.
A patient (64-year-old, male) with familial cholinesterasemia caused by BChE deficiency was studied. DNA sequence analysis of all exons identified a point mutation, an A-->G transition at codon 128, resulting in a Tyr-->Cys substitution. The propositus showed extremely low BChE activity, but his other family members (three individuals) showed from intermediate to normal BChE activity. An immunological method revealed the absence of BChE protein in serum of the propositus. Both PCR primer introduced restriction analysis (PCR-PIRA) and sequence analysis revealed all three family members to be heterozygotes for this mutation.
