Molecular Genetics (Andy Bailey 23-24) Flashcards
What are polyketides?
polyketides are complex organic compounds that are often biologically highly active. They’re produced by certain living organisms in order to impart to them some survival advantage.
- tenellin is used to kill insects and is made by condensation of acetate units and a polypeptide.
How were the genes for polyketide synthases found and how did they get the rest of the gene?
- designed degenerate primers
- amplified the genome
- cloned and sequenced the PCR product
- PCR product was from the right sort of gene but was only a small part of just one expected gene.
- The rest were found by using the PCR product to probe a Lambda genomic library
How was a Lambda genomic library and primer walking used to find the genes for polyketide synthases?
- sequences were fragmented and overlapping fragments were aligned to their correct order.
- the gene library can then be screened by hybridisation with a gene probe to identify clones.
- These sequences were then extended by primer walking (using the sequence to design a new primer over and over)
polyketide synthase genes can occur in clusters and are often co-located. How can we prove we are looking at the right set of genes?
Gene Disruption/Knockout:
- The target gene is replaced by a selectable marker (Note: homologous recombination).
- Fungi are haploid so we get an immediate phenotype.
- Loss of character of interest = successful disruption.
- Disruption of PKS (first step of the pathway) = complete loss of tenellin- like products
How did they find out which gene did which step?
- made a series of expression plasmids and built up the pathway one gene at a time in a different fungus (heterologous expression)
What did the Tenellin project show?
- by swapping around the domains betwen tenellin and DMB (different methylations and different ketides) different compounds were produced.
Why is RNA harder to engineer than DNA?
- Degrades easily
- tissue-specific expression (can’t plate out)
- can’t directly clone or sequence it (yet).
What is PepMV?
Pepino mosaic virus
- jumped host from a South American shrub to European tomatoes, threatening commercial tomato production.
- has 5 peptides: coat protein/ replicase/ TGBP1, 2 & 3
- in the Potex (Potato virus X) family so can use Potex primers to sequence its genome and find out how it jumped host.
How were infectious clones used to determine why the South American strain of PepMV is more aggressive than the European one?
- They mad various swaps and put them back together in yeast (great for overlapping fragments).
- Changing the coat protein of the European strain to the Chilean one was enough to change its virulence.
Whats a problem with infectious clones and how is this overcome?
Some viral cDNA is unstable in E.coli.
- overcome by adding plant introns, however, you can’t do in vitro transcription anymore as the introns won’t be spliced out.
How can agroinfiltration be used to study plant disease?
- agrobacterium with GFP tagged genes can be injected through stomata.
- T-DNA migrates to the nucleus and is transcribed by the 35s promoter.
- GFP tag can show how viruses are transmitted. we can then develop effective preventative methods.
