Splicing Flashcards
When were introns discovered, and by whom?
In 1977 by Richard J Roberts, and Phillip A Sharp
Roberts and Sharp discovered introns through their examination of the genetic material in which virus? Why?
Adenovirus (causes common cold)
Because it infect the cells of higher organisms, and its genome has many properties resembling those of the host cell.
ALSO has a simple structure
Why were Roberts and Sharp studying adenovirus specifically?
It infects the cells of higher organisms, and its genome has many similar properties to the host cell
It has a simple structure, so more readily studied
Why did Sharp and Roberts suspect that introns existed, when it was widely assumed that nuclear DNA was directly transferred to RNA?
Because the genome of many animals contained such large amounts of DNA that the possibility that it all encoded simple, bacteria-type genes seemed unlikely.
In addition, unusually long RNA was detected in the nucleus compared to the shorter mRNA that emerged in the cytoplasm
How many introns do vertebrates typically have per gene?
10
About ? of all mutations in the globin gees causing Beta-thalassemia in humans results from defects in?
1/4 …. splicing
Defects in splicing result in mutations in which genes that cause B-thalassemia?
Globin genes
When was alternative splicing first discovered?
1977
How do primary transcripts (ie pre-mRNA) differ from mRNA?
They are bigger
Why is pre-mRNA bigger than mRNA?
It contains introns
When was the first example of alternative splicing in a transcript from a normal, endogenous gene characterised?
1981
What was the first normal endogenous gene to have its alternative splicing characterised?
The mammalian gene encoding the thyroid hormone calcitonin
The primary transcript from the calcitonin-encoding gene contains how many exons?
6
The calcitonin-encoding gene produces a primary transcript containing 6 exons. It can be alternatively spliced to produce which mRNA products, and which exons do these contain?
Calcitonin mRNA: exons 1-4 (terminates after a polyA site in exon 4)
CGRP mRNA: exons 1-3 and 5-6 (SKIPS exon 4)
Calcitonin gene related peptide (CGRP) is synthesised from the calcitonin-encoding gene. How?
Alternative splicing: It skips exon 4 in the primary transcript to produce an mRNA containing exons 1-3 and 5-6.
The D.melanogaster gene, Dscam, is the ‘record holder’ for alternative splicing. How many splice variants may it have?
38,016 (approx. 38,000)
Which gene is thought to have the most splice variants? How many splice variants is it thought to have?
Dscam (a D.melanogaster gene) … 38,016 splice variants
Which 4 main techniques were used in the discovery of splicing?
1) Southern blot with cDNA probe
2) Electron microscopy of (viral) cDNA with genome
3) Nuclease S1 mapping of boundaries relative to restriction enzyme sites with probes from cloned genomic DNA
4) Northern blot probed with cDNA
As a result of alternative splicing, different mRNAs producing different proteins may be generated from the same adenovirus primary transcript. How are these mRNAs similar?
They share the same 5’ sequence, which is stitched together from three short non-protein-coding sequences known as the ‘tripartite leader’
What was R-loop mapping used for? What does it involve?
An experiment that elucidated that mRNA is formed from RNAs arising from several regions of the genome.
It involves incubating RNA with dsDNA containing a sequence complementary to the RNA. The RNA anneals to its complement, displacing a stretch of the non-complementary strand in the form of a loop.
Following the staining procedure used to visualise nucleic acids, the R-loop can be observed in the electron microscope.
Why can the R-loop be visualised by electron microscopy?
Because RNA-DNA and DNA-DNA duplexes appear thicker than single-stranded nucleic acids
In identifying the splice sites on pre-mRNA, why did mutating conserved sequences not work well with transfected genes?
It was hard to prove that low levels of expression were caused by a failure to splice, because:
1) the pre-mRNA degraded rather than accumulate
2) many conserved nucleotides had little effect.
Why might it be that mutating conserved nucleotides not work well in determining splice sites in pre-mRNA?
Perhaps because, in the absence of other choices, slow splicing at poor sites didn’t affect expression overall because there were other rate-limiting steps
Mutating conserved sequences (or sequences identified by deletions) did not work well with transfected genes when trying to determine the splice site. What experiments were carried out instead?
Splice site sequences were duplicated, so that they were in competition. Then one could be mutated systematically
