However, most of theC. elegans phosphatase genes have not been well characterized for their biological functions, especially in thetissue-specific context.
I was curious as to why this was true., since C elegans cells are very well characterized. The case is, the genes are known, but the pathways aren't mapped. So scientists know of the genes functions, and expression patterns, but not specific tissue roles.
Oil Red O staining with fixed animals andquantification indicate that the germline rde-1 rescue strain has normal neutral lipids levels (Fig. 4C,D)
Oil Red O is a dye that stains lipids. This was performed to observe the level of lipids inside the germline. Tje results show us that fat storage was normal.
To quantitatively access the RNAi efficiency, we performed RT-qPCR experiments to measure mRNA levelsof tissue-specific genes cpr-1 (intestine) 37 , sqt-3 (epidermis) 38 and pat-4 (muscles) 39 , upon corresponding RNAitreatments.
RT qPCR measures mRNA levels to quantify RNAi efficiency. It uses molecular evidence of tissue specific knockdown and can detect partial reductions that aren't typically visible.
UNC-112 is a muscle dense body/M-linecomponent. Knockdown of unc-112 results in paralysis 33 . Unlike wild-type animals, majority of the germlinerde-1 rescue animals showed normal phenotypes upon corresponding RNAi treatments (
This confirms that somatic RNAi is inactive in the muscle.
imilar to wild-type animals, the germline rde-1 rescue strainshowed germline defects upon the gld-1 RNAi treatment with around 50% penetrance (Fig. 2A, Table 1). egg-5encodes a pseudo-tyrosine phosphatase in oocytes.
50% penetrance is expected for RNAi, and it confirms that RNAis active in the germline.
However, theRDE-1 deficiencies were either an E411K missense mutation 3 or a Q825Ochre nonsense mutation4 that is close tothe C terminus of the protein.
I just wanted to define what a missense mutation was, vs a nonsense mutation. According to google, a missense mution is when one amino acid is changed. A nonsense mutation is a premature stop codon.
In order to facilitate the genetic analysis in the C. elegans germline, we set out to create a tissue-specific RNAistrain that allows RNAi to be effective only in the germline
Animal RNAi cannot distinguish direct germline gene functions from somatic effects that are indirect. By restricting RAi to the germline, we can analuze he germline specific gene roles.
owever, the germline tissue is difficult forgenetic manipulations since transgenes created by traditional methods are usually silenced in the germlin
Germline activity silences foreign and repetitive DNA through RNA defense mechanisms (like piRNA pathways, RNA-mediated silencing, histone modifications.)
Researchers using C. elegans as a model have developed tools to performtissue-specific RNAi experiments 3–10
These experiments include epidermis, muscle, an intestine specific RNAi strains which are mentioned later in the article.
Researchers using C. elegans as a model have developed tools to performtissue-specific RNAi experiments 3–10 . The strategies usually involve tissue-specific promoters-driving transgenerescue of mutations that are essential for the RNAi machinery. rde-1, which encodes an Argonaute protein, func-tions cell autonomously to ensure RNAi capacity 11 . Therefore, tissue-specific promoters-driving rde-1 rescuestrains will allow RNAi to be effective in a tissue-specific manner
The strategy is to disable RNAi completely, and then restore the RNAi mechanism in one tissue using a tissue specific promoter;
Tomato golden mosaic virus (TGMV) is a bipartite gemini-virus with a genome of two circular molecules, A andB (
Is there a possibility this experiment could be performed with a monopartite gemini virus, in which it includes the functionality of both replication and movement?
This result suggestedthat the TGMV::su sequences did not integrate intochromosomal DNA at significant frequencies and/or thatany chromosomal form of TGMV::su was not sufficient tocause silencing of the endogenous su alleles.
This shows the virus may have potentially become a part of chromosomal DNA, but it was not nearly stable enough to be passed onto generations. The silencing trait is not an inheritable trait.
we suspect that a diffusible factor may beinvolved. Cell-to-cell movement of TGMV is known torequire the B component (Jeffrey et al., 1996). Althoughwe cannot rule out the formal possibility that single-stranded viral DNA may be moving cell-to-cell, we think itunlikely because there was no evidence of further viralmovement from the spots, even after 4 weeks
If this were possible, my assumption would be the virus has specialized movement proteins, in which allows the virus to systemically navigate the cells, and also trigger the cell to replicate the virus.
There are many examples of gene silencing mediated bystably integrated homologous DNA, or by homologousnuclear gene sequences carried by cytoplasmic RNAviruses
One example is the with the petunia plant (we discussed this in class), in which adding extra copies of a purple pigment genes resulted in the petunia silencing the pigment gene, and turning white.
The mutant Su allele cloned from N. tabacum has a singlemissense mutation causing an altered amino acid (Nguyen,1995). This polypeptide has been hypothesized to stablyinterfere with the function of the heteromeric magnesiumchelatase complex, causing a light green phenotype inheterozygous plants
I would very likely agree with this hypothesis, that the mutation of the amino acid sequence resulted in a different phenotype. I was surprised with the results, that the virus had not integrated into the chromosomal DNA. This may have to do with the cell recognizing the virus as foreign.
Replacement of the AR1 gene by foreignDNA, such as su, is known to attenuate symptoms
These symptoms include the natural disease system symptoms, including leaf curling or stunting. So, removing the coat protein does affect the the life of the plant.
The TGMV coat protein (AR1) is dispensable for replicationand movement in N. benthamiana
I found this interesting. It suggests that the coat protein, which is responsible for the viruses ability to spread, is not a necessary part for the individual virus' survival.
N. benthamiana plants were infected using particle gunbombardment to deliver cloned viral DNA. Wt TGMV pro-duced chlorotic
I was curious as to how particle bombardment works. Particle bombardment is the process of shooting gold or tungsten particles, coated with the virus (TGMV), into the plant tissue, with the goal of inserting the DNA into the plant nucleus. Gold and Tungsten are used because they are very dense particles, so their movement to the nucleus is very quick. They are also more inert, so they do not cause a chemical reaction inside the cell.
Several geminiviruses havebeen used to carry foreign genes in plants (
One example of this is Tomato Yellow Leaf Curl Virus (TYLCV). This virus was used in a study in 2010, in which researchers wanted to test the infectivity of the virus in tomato plants, via the "in-vitro inoculation method." This means the infectivity was tested on controlled, lab grown tomatoes.