17th Feb - Discovery of Splicing and the Essential Sequences Flashcards
Who elucidated that genes must encode the protein’s sequence?
Sanger
What is the information problem?
DNA couldn’t carry the information for protein sequences because in eukaryotes DNA was in the nucleus and proteins were made in the cytoplasm therefore there must be a messenger
When did Crick propose his central dogma?
1957
What is the Pa Ja Mo experiment?
Performed in 1959 by Pardee with Monod, it identified the cytoplasmic messenger as mRNA.
Used two strains of yeast Hfr and F-
- Hfr contained F, lacZ and lacI
- F- contained F-, lacZ- and lacI-
–> conjugation leading to F- production of lacZ
In 1976 what did southern blotting show to be a problem with mRNA?
Isolated fragments of mammalian mRNA cloned as cDNA didn’t match the genes in DNA
In 1977 what did electron microscopy show to be a problem with mRNA?
Examined viral cDNA with genome. Showed there were sequences of DNA in between the bits that hybridised to the DNA i.e. the RNA was discontinous with the genome
What did nuclease protection show in 1977 to be a problem with mRNA?
Nuclease S1 mapping of boundaries relative to restriction enzyme sites with probes from cloned genomic DNA, showed that genes were split.
How was it shown that genes were split by processing rather than jumping RNAP?
Northern blot showed that pre-mRNA contained all portions of DNA
How did they find that there was no specific sequence to each intron?
Rearranged introns and showed that splicing was still fine
How was the splicing signal identified?
Made a series of deletions to identify which sites were necessary for splicing. Identified:
- 6nt of introm from 5’ss
- 24nt of intron from 3’ss
- gap in the middle needed to be filled
Outline Eperon’s work from 1986
- Investigated whether splice sites can be ranked in a hierarchy of preferential use
- Calibrated against a constant reference site
- Choice between alternative splice sites contained in short oligonucleotides and the 5’ss of the 2nd intervening sequence (IVS-2) of the rabbit beta globin gene
- sequences vary in the 5’ splice site junction in the last 3nt of the exon and the first 6nt of the intron
- Sites of splicing were determined by S1 mapping
- The proportion of splicing at the test site was compared to the reference site and was estimated using laser densitometry of autoradiographs
- -> Splice site specific degradation is not a significant factor
- -> Importance of the secondary structure in masking the splice site
- -> Indicated the us ss’ was used preferentially
What is the conserved 5’ss?
GU
What is the 3’ss?
AG
What is the branchpoint?
A
