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Lecture 21

Biology 2581B Lecture 21: Lec 21 - Trans. Elements

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Biology 2581B
David R Smith

Lecture 21 – Transposable Elements and Variations in Chromosome Numbers Transposable elements - Transposable elements = jumping genes - Discovered by Barbara McClintock in maize o First one to discover transposable elements – saw through the colours of the kernels - First evidence by Marcus Roades in 1930s and described in detail by Barbara McClintock in maize from 1940 onwards o Earlier evidence from Roades but McClintock developed the field of transposable elements - They exist in many organism including mammals, insects, plants…… o Don’t only exist in maize Transposable elements in the human genome Classification – two different major classes - Retrotransposons – retroposons o Transpose via reverse transcription of an RNA intermediate o Use RNA intermediate to jump from one location to another in the genome - Transposons o Transpose DNA directly without an RNA intermediate Transposable elements - Many different types some with cool names: mariner, gypsy, looper, springer, hopper, stalker, invader, diver, frogger o Refer to different types of transposable elements and all have something to do with moving - Can be 50 bp to 10 kb in length (small or big) - Differences in copy number exist between individuals or in a species - Different copy numbers: one to 100 thousands “selfish” o Selfish: transposable elements carry information to jump from one location to another within their own coding sequence (self-sufficient) o Carrying only information that allows their self-perpetuation - Transposable elements have been around for a long time and some gained a function - There are examples where they have functions in the host o Maintain telomere length in Drosophila o Gained other types of functions at times In the human genome - 45% of the total genome is linked to a sequence associated with transposable elements - Large amount of transposable elements makeup genomes or CAN make up genomes - 2 largest groups: LINEs, SINEs - Non-LTR (long terminal repeats) o LINEs: long interspersed elements o L1: app. 20000 copies, 6.4 kb o SINEs: short interspersed elements ▪ Huge number of copies within the given genome because they are short o Alu: app. 300000 copies, 0.28 kb - LTR: long terminal repeats - Transposable elements make up a huge proportion of sequences and impact genomes - Impacted on o Evolution of the genome o Genome size and structure o Gene expression - Something can jump into a promoter and impact the expression of protein sequences o If it jumps into intron or exon, it can impact the protein sequence Copia from Drosophila - Polytene chromosomes from Drosophila glands o Use them because we can visualize things very easily - In situ hybridization with a probe for copia sequences o Made a probe for the transposable element (copia) o In situ hybridization: variation of southern blot - Binds to sequences that are identical in that sequence and identifies where copia element are located in the genome - Two different strains from different geographic locations o 2 different insects coming from different geographical locations o Relatives haven’t seen each other – takes long for flies to change locations - There are many generations between the two flies and changes in the genome occurred o Track the difference by looking where copia transposable elements are located o Determine the copy number of the transposable elements - The more different the copy number is, and the more different the location that the element is found in between the two individuals o = The longer they’ve been geographically kept apart o Do not have interchanges of genetic material - Use them to visualize what happens to genomes in a particular species - 30-50 copies each – but often in different positions - Position of TE have shifted relative to genes - Use TEs to judge/estimate how closely related 2 individuals are if they’re the same species o Track the number of copies and location of a TE. o If they’re quite different, the 2 individuals probably aren’t closely related Retrotransposoms - Two types of retrotransposons: LTRs and non-LTRs (long term repeats) - Have reverse transcriptase gene - Non-LTRs: o Have a poly A tail at the end (like mRNAs that add it after transcription) - LTRs: o Sequence of the gene is surrounded by long terminal repeats (direct repeats) - LTRs o Encode a reverse transcriptase gene for jumping - Reverse transcriptase is coded in retrotransposons o DNA polymerase o Uses RNA as a template o Synthesizes cDNA - Retrotranpsosons can encode other proteins as well o I.e. endonucleases found in coding sequence of retrotransposons - Use an RNA intermediate: an experiment was done to determine that there was an RNA intermediate required for the jumping mechanism of retrotransposons - Grow and replicate the transposons in yeast - Insert a yeast transposable element into the plasmid (yeast retrotransposon) - Changed the coding sequence of the retrotransposon and introduced an intron sequence from another yeast gene - Put yeast intron into the reverse transcriptase sequence so the sequence is non-functional - Track the intron - DNA copy contains intron - RNA copy lacks intron - When transcriptase is made, the yeast introns sequences are apart of the primary transcript - Host organism will recognize the intron sequence and splice them out - Left with mRNA that does not have intron sequences anymore - mRNA is translated it into reverse transcriptase - Protein can prepare the transposon to jump into a different location in the genome - Makes sequences that can get integrated into the yeast genome - The new location where the intron jumped into: o Analyze the sequence of the retrotransposon o It does not contain an intron any longer o If it jumped directly from a DNA locus to another DNA locus, it should’ve carried the intron with it because we inserted the DNA into the sequence of the retrotransposon o New location does not have intron ▪ Went through an RNA intermediate, intron was spliced and removed then the piece was inserted into a new location - If intron was present in the new location, there would be no RNA intermediate - Using splicing event to track what happens to the transposition event How retrotransposons can move - Have direct repeats and LTRs - Outside of the retrotransposon sequences, there are repeat bases that are not part of the retrotransposon sequence itself - Transcribe and translate the reverse transcriptase o It can make another strand that is replaced and made into a double stranded piece of DNA o Reverse transcriptase reverse transcribes its own mRNA to make cDNA to then be inserted somewhere else in the genome - Double stranded piece gets inserted into another location in the genome - A gap is formed at the new location o Unknown what protein or enzyme is involved in the gap o Some retrotransposons encode an endonuclease that can be involved in this process - Gap has sticky ends (what happens when you cut something with a restriction enzyme) - Have one of the strands attached to the left and the other end to the right side - Retrotransposon inserts in between the two strands - Sequences that were once represented on one side are represented at both sides where the retrotransposon gets inserted - Repair system: host polymerases extend the strands and fill them in - Completes the insertion – have no gaps left and retrotransposon is inserted into a new site - Sequences are duplicated on the outside of the insertion event o The duplicated regions are now different o There is no target sequence involved = sequence can be different - Duplication occurs because of the opening of the gap and the insertion in between - What moves is the mRNA that is made from the cDNA - This a generic view of transposition – basic components - Details might differ from TE to TE (depends on the transposable element) - Specificity of target sequences o No specificity of the target sequence usually but some do have specificity - May or may not have involvement of other proteins - Generally, retrotransposon increase the frequency of the copy number of the transposable elements because you keep the original copy - Frequency of transposition is different for different elements depending on the factors involved  copy number Transposon structure - Go directly from DNA to DNA without RNA intermediate - Structure is different than retrotransposon - Code for a transposase, not a reverse transcripase - Have repeats but they are INVERTED REPEATS o Sequence of the repeats is related to the type of transposon - Repeats can differ from transposon to transposon - Transposase gene gets transcribed and translated into a protein - Transposase is a protein o Recognizes inverted repeats only and they must be of a specific sequence o Recognition process: enzyme is introducing cuts at the end of the transposon ▪ Between the transposon and the genomic sequence - Because of the breaks, it excises the elements that are in between the two different breaks How transposons can move - What happens is analogous to retrotransposons - Transposon must insert into another site - Sequences surrounding are duplicated on each side of the insertion site o Different from genomic sites o Sticky ends with things inserted into the middle - You have a gap when you remove something o There is a hole where that transposon originally existed - Must fix the gap because it is not viable and can be lethal - Gap gets widened by exonucleases that recognize these situations - Repair mechanisms come in to fill in the gap - Example: P element - Transposase recognizes a repeat sequence - When it is jumping, the new location does not have a target sequence o Random insertion – does not require a certain insertion sequence to get inserted into a location - RECONGITION IS APART OF THE TRASOSON SEQUENCE o Transposon has repats on the outside – they are the target sequence for the transposase o THEY ARE APART OF THE TRANSPOSON NOT APART OF THE TRANSPOASE ▪ The repeat goes with the transposon o Cut OUTSIDE the repeat sequence - Clone stuff in between transposon because the enzyme recognizes specific sequences and does not care what is in between o It will move things in between the sequence - Put transposase on another vector o Move things randomly into the genome - Learn from transposon that it has inverted repeats on the outside o Put inverted repeats around things we want to move o Have transposase present and it will move things for us o Repeats must be specific for that specific transposase - FIRST POSSIBILTY: homologues that contain the transposon sequence - Diploid organisms have homologues o One where the transposon has jumped from and the homologue with a transposon sequence at the same site - Repair mechanisms align the homologous sequences - Other enzymes (polymerase) come along and use the homologous sequences to fill in the gap = other strand is made - If there is a
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