Cdna Case Study

Cin1 exists as Cin1-S (short-form) and Cin1-L (long-form) isoforms as a result of alternative splicing of intron #6. Cin1-S differs from Cin1-L by lacking the C-terminal RhoGEF (DH)-PH domain. We previously generated a CIN1-S mutant strain by replacing the C-terminus with an NAT marker gene. Significantly, we have found that Cin1-S confers a survival advantage over WT in the mouse brain via co-infection. Further investigation will include constructing a mutant expressing the long isoform Cin1-L. We have generated a partial CIN1-L point-mutation allele linked to G418 resistance, but have yet to obtain the Cin1-L mutant through the conventional gene replacement method. Recently, we have developed a one-step CRISPR-CAS9 technique for mutagenesis in Cn. In this aim, we will generate the Cin1-L mutant, characterize its function, and perform co-infection assays. Moreover, we will perform dual RNA-Seq to profile whether there is differential gene expression in infection by Cin1-S and Cin1-L in order to address the mechanism associated with Cin1-S CNS survival

The dsRNA can be delivered to the worms in many ways, but in this particular experiment, the bacteria containing plasmid for dsRNA is fed to the worms. The dsRNA is able to move throughout cells in worms by the pore created by SID-1

Throughout the cells there are much type of Eukaryotic cells which make many types of cells and then they proceeds to the RNA, There is no complete data of this RNA present and the characteristic are very poorly unstated. The genetic information is directly represented by the RNA and it focuses on its synthesis, translation and modification it helps to understand the genome functions also. These observation are taken up together to form and define the functions and description related to genes (Bell, 2004). This observation tells about the range of expression and localization. As the technology is been improving day by day for the RNA profiling and the type of isolation made by the cells , the number of RNA has grown and

This study identifies conserved cis sequences as the primary determinant of RNA editing evolution, but does not rule out a role for either trans factors or Adar proteins. It also opens up future research into how cis sequence changes alter RNA editing level.

The Basic Local Alignment Search Tool (BLAST) was the final portion of this lab report. BLAST is provided courtesy of the National Center for Biotechnology Information (NCBI). After running the five chosen tests, students were given the two 16S rRNA sequences that were associated with their assigned culture. The BLAST results served to confirm or disprove the hypothesis of what the unknown may be. The BLAST program compared the given 16S rRNA sequence to a database of known sequences and searched for similarity. (10) This search tool is capable of comparing nucleotide or protein sequences to find statistical significance between the matches. A perfect match to the unknown sequence is indicated by a

This process consists of three major steps which are denaturing, annealing, and elongation. The cells are lysed before the denaturation step in order to obtain chromosomal DNA. In order to denature the DNA, or separate the two strands, the DNA is heated to 95 degrees Celsius. Once the denaturing is complete, the PCR tube is cooled to approximately 55 degrees Celsius during the transition to the annealing step. During this step, there are two primers: 27F and 1492R, which bind to the 16S gene on the strands of DNA. A high molar ratio of primers is used to ensure that there is attachment between the selected region of DNA and the primers as well as to ensure that the primers will hybridize. After the annealing step, elongation happens at around 75 degrees Celsius. A Master Mix is added during this step, which contains Taq polymerase and dNTPs. In order to replicate the sequence, Taq polymerase uses the dNTPs. Since the primer is on the 5’ end of the strand, the elongation is happening in the direction of the 3’ strand and the elongation is indefinite and these

The quantity of each product is then analyzed using qRT-PCR and the brains characterized to see if adding these genes has rescued the function of ADCK3 in the RNAi fly line

Risk-dependent lncRNAs transcripts significantly recaptured both neural tube-specific enhancers that were validated in-vivo and the neuroblastoma-susceptibility variations that were found by GWASs.

The lncRNAs transcripts will be given by the defined reference noncoding genome (Aim 1.1). An effect size of differential lncRNA transcript methylation (M_i) will be calculated for reference region i, similar as in Formula 2.

The RNA sequence in the anticodon region, as well as other parts of the transfer RNA molecule, are important

Mediation of lncRNA in gene expression can be concluded to tissue and cell type specifics. In most aspects it plays a vital

Currently, circRNAs consist of three subclasses based on their generating pathways (Figure 1). The most popular circRNA is the exon-derived circRNA (ecRNA), containing only exons and completely lacking introns [16]. Two mechanisms, including both exon skipping and direct backsplicing, have been proposed to explain ecRNA biogenesis [13,25,36]. In terms of exon skipping, the downstream exon turns around to link the upstream exon, producing a circular and exon-skipped transcript [13,37,38]. By contrast, the direct backsplicing first generates alternatively spliced RNA and a lariat intermediate regulated by the intron-pairing mechanism. The introns in the lariat are then removed by the canonical splicing process [7,21,39-41]. Recent evidence indicates that direct backsplicing, not exon skipping, may be the primary mechanism regulating ecRNA formation [42].

In recent years, the next-generation sequencing technology have been used as an powerfull tool by researchers to study cancer data, including cancer diagnosis, therapy, and prognosis. This technology enabled researchers to investigate all human genome transcripts, including non-coding versions. Recent investigations have revealed the association of non-coding RNAs (ncRNAs), including miRNAs and lncRNAs, with cancer pathogenesis, prognosis, survival rate and uses of these ncRNAs as cancer biomarker[22-25].

We will use a modified plasmid derived from pCAS9 that contains both the CAS9 endonuclease and the Pol III promoter for expressing gRNA. Transformation with two such plasmids, one expressing gRNA prior to the alternating splicing site and one posterior to the NEO marker in the presence of the 6 kb CIN1-L-NEO fragment as donor DNA will result in the replacement of the wild type CIN1 allele with the mutant one (CIN1-L) (Fig. 7). Once verified by RT-PCR and DNA sequencing, Cin1-L will be characterized in comparison with Cin1-S and WT (JEC21). We will test whether medium, temperature, and different physiological conditions present in the body (5% CO2 and hypoxia mimicking cobalt chloride) affect the expression of different isoforms through quantitative RT-PCR and Northern blotting analysis. Cin1-L transcripts will be measured with primers specific to the C-terminus sequence, while Cin1-S transcripts will be estimated through subtraction using a densitometer. Additional phenotype studies, including endocytosis of FM4-64 and actin distribution using rhodamine-conjugated phalloidin stain will also be performed.

To date, exRNA has not been used to investigate intracellular trafficking or pathogenesis in fungi. We first isolated EVs from the cin1 mutant and WT strains and extracted exRNA with Trizol. Since the cin1 mutant is drastically reduced in growth, 3x more culture volume than WT was necessary to obtain a yield comparable to that of WT. Using BGISEQ-500 technology, a total of 27,084,174 cleans reads was obtained from the cin1 mutant while 27,402,307 clean sRNA reads were obtained from WT (JEC21). The length of sRNA is between 18 nt and 30 nt. Initial sRNA annotation provided by BGI suggested that approximately same 96% of sRNA sequence was mapped to available sRNA database including miRBase and Rfam. This and other early reports such as annotation and prediction, miRNA and siRNA target prediction, and validation by expression will be re-analyzed with the help of collaborator Dr. Taylor. Through this effort, we will determine the role of Cin1 in expression and secretion of sRNA.