quiz 8? Flashcards
What 2 bits of experimental evidence presented in class suggests silencing was not due to a defect in the construct itself?
Normal levels of mRNA in nuclease silenced in plants
Silencing happened several weeks after germination
Did they see silencing in the original transgenic that they recovered after transformation
no
How did they show, or how could they have shown, that silencing did not arise from a mutation that occurred at the time of integration? (this question has multiple answers, I suggest you think of more than 1.)
Sequencing
RT-PCR
Western blot
How did they show, or how should they have shown, that the effect they saw was not due to transcriptional silencing? (again, this question has several answers, one very direct, and one or more that might be inferred.)
Anazlyzed mRNA levels in nucleus
Check promoter for methylation
Was the npt gene affected by this silencing also? What does this tell you about the kind of silencing occurring there?
No - seedling only grew on media with functional npt gene
This means npt gene OE isnt the cause
In the study of viroid expression carried out by Wassenegger, what aspect of viroid biology leads to gene silencing? Is it (more than one may apply)…
a. the fact that they are pathogens;
b. the fact that they are circular;
c. the fact that they are single stranded;
d. the fact that we inserted a copy of them in the genome;
e. the fact that it was self-replicating;
f. the fact that they accumulated to very large levels in the nucleus?
e. the fact that it was self-replicating;
f. the fact that they accumulated to very large levels in the nucleus?
Would methylation of this region normally affect transcription?
No because methylation of promoter typically cilences transcription
Why isn’t it possible to impose silencing of an actively transcribing gene made from a concatamer of a mutant (non-self-processing) viroid genome?
It doesnt produce enough RNA copies to bind to DNA like self processing viroids do
- If a transgene also shows transcriptional silencing, should we conclude (more than one may apply)…
a. that it was introduced on a viroid;
b. that it did not encode a protein;
c. that the RNA must have circularized;
d. that it had to be self-replicating;
e. that there was a copy of it in the genome;
f. that it was initially expressed, before it triggered silencing;
g. that the cell recognized it did not belong there?
that it had to be self-replicating;
That the cell recognized it did not belong there
Transcriptional silencing can only be caused if transgene
Self replicates to high # in nucleus
High copy number of RNA trapped in nucleus
Or cell recognize it didn’t belong there
Do plants use this form of silencing to protect themselves during normal viroid infections?
no, viroids circularize outside nucleus so no accumulation in nucleus to trigger silencing
If a gene is methylated in its original host, will we be able to detect this methylation in a cloned copy of the gene?
No – DNA polymerase does not distinguish between methylated/unmethylated bases and the clone would assume hosts methylation pattern
What is the role of transcriptional silencing during the lifecycle of an unengineered organism?
Transcriptional silencing is naturally a cells mechanism to turn off certain genes permanently this is utilized a lot in developing embryos and young or when rna accumulates in nucleus or when abnormal rna is formed
What is the role of post-transcriptional silencing in an unengineered organism?
Post transcriptional silencing is used to control regulate gene expression
How can we identify the methylation state of a gene
by digesting it with a methyl sensitive restriction enzyme
Does the method you described identify all possible methylation sites in the gene? Does it identify the ones that cause silencing??
No – it would only identify methylation of the specific CCmGG site if a C was methylated elsewhere we couldn’t ID it
