Yusuke Hokii et al. (2004) East Asia Worm Meeting "Isolation of novel small RNAs from Caenorhabditis elegans"

Masayosi Shimoyama, Akira Muto, Yusuke Hokii, Chisato Ushida, Akiyosi Kubo, Risa Arai

[East Asia Worm Meeting, 2004]

We isolated novel small RNAs from C. elegans by using gel electrophoresis techniques. Sequences of the separated RNAs were determined by cloning and sequencing their cDNAs. RNAs of about 50 to 1,000 nt in length were prepared from the mixed stage worms and separated by the denaturing gel electrophoresis. 32 bands were detected and sequences of 107 cDNAs from the bands were determined. Eighty-seven cDNAs corresponded to parts of the known ncRNA gene sequences such as rRNAs, tRNAs and U snRNAs. Nine cDNAs had parts of exon sequences and eight had parts of intron sequences of protein coding genes. The remaining 3 cDNAs revealed sequences corresponded to the intergenic sequences of genome. RNAs smaller than 50 nt in length were also separated on the denaturing gel and fifteen bands were detected. Although the purified RNAs from the 15 bands were subjected to the enzymatic sequencing method of Donis-Keller, clear sequence could not be obtained. This is probably because each band contains more than one RNA species, since more than 100 species of tiny RNAs (19-23 nt) are detectable by Northern blotting (Ambros et al., 2003 and Lim et al., 2003). This small RNA fraction was further separated by a two dimensional (2D) gel electrophoresis, which resolved about 100 spots. By using this 2D-gel electrophoresis, we compared the expression pattern of embryonic small RNAs with small RNAs prepared from the mixed stage worms. Remarkable difference between the two was observed. The spots were classified into three groups, 1) spots which are detected only in the embryonic RNA preparation, 2) spots which are detected only in the mixed stage worm RNA preparation, 3) spots which are detected both in the embyonic and mixed stage worm RNA preparations. Each group had 85, 51 and 54 spots, respectively. Several cDNA seqeuences, which contained novel small non-coding RNA candidates, were obtained from each group.

Yasuda H et al. (1988) Worm Breeder's Gazette "a tubulin tale from Toyohashi: two genes one polypedtide."

Siddiqui SS, Yasuda H

[Worm Breeder's Gazette, 1988]

Previously we have described our efforts to clone tubulin genes by direct screening of C. elegans expression libraries using nerve specific anti-tubulin antibodies(WBG 10, 1:59, 1987). For alpha- tubulin genes, 14 lambda gt11 clones were identified with a monoclonal antibody 3A5 (kindly given by M. Fuller), which is specific for alpha- tubulin and stains C. elegans neurons. Two of the 14 clones were subcloned in pUC 118, and sequenced by dideoxy method of Sanger and Coulson. One of these clones SQ#TbA77 (insert size 1.0 Kb) contained a DNA which is 79% homologous to the chicken alpha-tubulin DNA sequence; and its amino acid sequence deduced from DNA sequence is 88% homologous to the C-terminal region of the chicken alpha-tubulin. The second putative clone on sequencing revealed DNA that has no homology with tubulin DNA. Similarly, we have identified 15 lambda gt11 clones using E6B6, ( kind gift of T. Arai) a monoclonal antibody which is specific for alpha-tubulin and also stains C. elegans nervous system. Three of the 15 clones were subcloned and sequenced as stated above. One of these SQ#TbB03 has a sequence 76% homologous to the chicken tubulin DNA. We compared the nucleotide sequence of SQ#TbB03 with Nw#B8, the only other -tubulin cloned and sequenced in C. elegans (L. Gremke, Ph.D. Thesis,1987). The two nucleotide sequences and the deduced amino acid sequence is given below. It is quite remarkable that the two -tubulin genes have 100% homology at the level of amino acid sequence. Currently, we are trying to get the complete DNA sequence data of the tubulins identified by SQ#TbA77 and SQ#TbB03. [See Figure 1]

Currey, Heather N et al. (2020) MicroPubl Biol

Liachko, Nicole F, Currey, Heather N, Malinkevich, Anna, Melquist, Penny

[MicroPubl Biol, 2020]

Aggregates of the protein tau are the hallmark of several neurodegenerative diseases including Alzheimers disease, frontotemporal lobar degeneration (FTLD-tau), progressive supranuclear palsy (PSP), corticobasal degeneration (CBD), Picks disease, and chronic traumatic encephalopathy (CTE) (VandeVrede, Boxer et al. 2020). Mutations in the gene coding for tau, MAPT, can cause FTLD-tau, directly linking tau dysfunction with disease (Dickson, Kouri et al. 2011). Another protein, TDP-43, comprises aggregates which are the primary hallmark of amyotrophic lateral sclerosis (ALS) and frontotemporal lobar degeneration (FTLD-TDP), and mutations in the gene coding for TDP-43, TARDBP, can cause disease (Kawakami, Arai et al. 2019). To model tau or TDP-43 proteinopathies, transgenic C. elegans have been generated that express the full-length human protein pan-neuronally. These worms exhibit significant uncoordinated movement on plates and impaired thrashing in liquid (Kraemer, Zhang et al. 2003; Liachko, Guthrie et al. 2010). However, tau- and TDP-43- expressing worms are not paralyzed; they still move their heads and have some motility on the plate (coiling, crawling with tail-drag, head swinging) which are not captured in standard crawling or thrashing assays. To assay differences in total activity, we used a WMicroTracker ARENA System (Phylumtech, AR and InVivo Biosystems, USA). The ARENA captures population level activity data by relying on optical interferometry, which uses a large array of infrared LED microbeams to detect both the movement and position of worms on a culture plate. Disruption of an LED microbeam by worm movement is recorded by repeat scans of the 6-well culture plate, and allows for real-time processing. The software identifies changes in the location of disrupted beams between scans and assigns an activity score based on differences identified between each consecutive scan (Simonetta SH). Both tau- and TDP-43- expressing worms had significantly less activity per minute than N2 (Figure 1). Further, we found the ARENA- assessed activity data recapitulated the relative severity of phenotypes among the strains as measured by motility assays. For example, both CK10 (tau V337M) and CK144 (tau WT) have significantly uncoordinated movement when crawling or thrashing in liquid, with CK144 having worse motility than CK10, due to its much higher burden of total tau protein expressed (Kraemer, Zhang et al. 2003). Likewise, CK410 (TDP-43 WT) worms have slightly impaired motility compared with N2 when crawling on a plate, CK423 (TDP-43 M337V) are severely uncoordinated, and CK426 (TDP-43 A315T) have the most severe uncoordinated phenotype. The relative toxicities of these strains stem from the effects of the mutations, as TDP-43 protein expression is relatively even among these transgenic strains (Liachko, Guthrie et al. 2010). Interestingly, the ARENA captures activity of these severely uncoordinated worms that move poorly in motility assays such as crawling on an NGM plate or thrashing in liquid (Kraemer, Zhang et al. 2003; Liachko, Guthrie et al. 2010). Therefore, ARENA assessment of aggregate activity may be a more accurate metric for capturing non-locomotor movement of C. elegans that are severely uncoordinated.