Invertebrate Biotechnology Flashcards
What is a transgenic insect?
Insects are made transgenic when one of more DNA sequences from other organisms are inserted into their genome
What is the accepted method of making transgenic insects?
Splicing the DNA intended for insertion into a mobile element also known as a jumping gene
What is a jumping gene?
A segment of DNA that can be integrated at many different sites along a chromosome
Describe the first stable transformation of an insect
1982
Drosophila
P transposable element used as the vector for transformation
Why are Drosophila used as the model system?
Have well characterised genetics
Have a short life cycle (10 days at room temp)
Easy to culture to large numbers
Easy to screen for effects of chemical mutagens
What are the disadvantages of using Drosophila?
Have poorly understood digestive biochemistry
What are mobile genetic elements?
A type of genetic material that can move around within a genome, can be transferred from one species, rearrange genes in the host genome, can cause mutations in protein coding regions (alter protein function) or replicon to another and play a distinct role in evolution
Are found in all organisms and in humans ~50% of the genome is thought to be MGEs
What are some examples of mobile genetic elements?
Transposons
Retrotransposons
DNA transposons
Insertion sequences
Plasmids
Bacteriophage elements
Group I and II introns
Who discovered transposons?
Barbara McClintock who found that maize plants had higher than expected mutation rates due to their different colours
Transposons were detected by their genetic effects as re-insertions can cause mutations
Mazie has 10 types of transposons
What is a transposon?
A short (3-4 kb) DNA sequence that can change position within a genome, sometimes creating or reversing mutations and as such can alter a cell’s genome size
Are classified into different families according to their sequence similarities
Contribute to a large part of plant genomes
What are the 2 classes of transposons?
Class I (retrotransposons/copy and paste)
Class II (cut and paste)
Describe class I transposons
Require RT
Can be autonomous (contain the sequence to encode its own transposase thus moves itself)
Can be non-autonomous (lack the gene for transposase or RT so must borrow these proteins from another element in order to relocate thus requires an active element to move and as such are more stable)
Describe class II transposons
Encode a transposase for excision and insertion
Can be autonomous and non-autonomous
Describe autonomous transposons
Autonomous elements contain a gene for transposase endonuclease that allows excision and re-insertion
Describe non-autonomous transposons
Are in 2 parts in the host genome
The transposase cuts the mobile element out upstream
The mobile element contains terminal repeats
What are the 3 schools of thought surrounding the origins of transposons?
Originated in the last universal common ancestor
Arisen independently multiple times
Arisen once and then spread to other kingdoms by horizontal gene transfer
What is the link between viruses and transposons?
Are found in all major branches of life
May confer some benefits on hosts but are generally considered selfish DNA parasites
Like viruses they live within the genome of cellular organisms, share features in their genome structure and their biochemical abilities
Speculation transposons and viruses may share a common ancestor
What is the link between bacteria and transposons?
Excessive transposon activity can destroy a genome so many organisms including bacteria have developed mechanisms to reduce this activity
i.e. high rates of gene deletion to remove transposons and viruses from their genomes
Transposons also may act as mutators in bacteria
How do eukaryotes manage transposon activity?
May have developed RNAi
In C. elegans nematode some genes required for RNAi reduce transposon activity
What is the link between transposons and vertebrates?
They may have been co-opted by the vertebrate immune system as a means of producing antibody diversity
What are P elements?
A family of transposons in Drosophila
First appeared in the species in the mid 20th century and have since spread through every population
Describe the first use of P elements to create Drosophila mutants
1982 Rubin and Spralding
Artificial P elements used to insert genes into Drosophila by injecting the embryo thus exogeneous DNA sequences introduced into the Drosophila germ line
A rosy transposon (ry1) was constructed by inserting a chromosomal fragment containing the wild-type rosy gene (red eye colour) into a P element
This transformed between 20-50% of the injected embryos
The transformants contained 1 or 2 copies of chromosomally integrated intact ry1 that were stably inherited in subsequent generations
Transformed flies had the visible genetic defect in the mutant host strain and were permanently corrected by the transferred gene
Why aren’t P elements used outside of Drosophila?
Attempts to genetically transform non-drosopholid insects with P elements were not successful as it is not a universal transposon (Handler et al, 1993)