BIO 344 1st Edition Lecture 23 Outline of Last Lecture I. Protein Turnovera. N-end ruleb. Ubiquitini. Degradation signalc. E3d. Proteosomee. P53i. Mdm2 and HAUSPOutline of Current Lecture I. Genome Structure and Transposable elementsa. piRNAb. mobile elementsII. Transposonsa. DNA onlyi. Cut and paste (nonreplicative)ii. replicativeb. Retroviral-like retrotransposonsc. Nonretroviral retrotransposonsIII. Activity of retrotransposible elements in our genomea. ALU, SVA, L1b. L1 retrotranscriptioni. SNPii. FGF4c. Suppressing LINEs Current LectureGenome Structure and Mobile Elements- piRNAs suppress retrotransposition in the germline- recall only 1-2% genome codes for proteino half is repetitive sequence—potentially mobile elementsTransposonsThese notes represent a detailed interpretation of the professor’s lecture. GradeBuddy is best used as a supplement to your own notes, not as a substitute.- DNA only—short repeats on ends, retroviral-like retrotransposons—long terminal repeats on ends, nonretroviral retrotransposons—poly A end repeats 5’ often truncated- DNA only transposable elementso Encode transposase= mobilizes the DNAo Many transposable elements also carry other functions like antibiotic resistance Phage infects bacteria, transposon carried, now can transfer antibiotic resistanceo 2 mechanisms of transposition Nonreplicated- Cut and paste, no change in copy number, typically staggered ends Replicative- Increases transposon number- Transcription can also interrupt an essential gene- Retroviral-like Transposonso RNA virus, moves via RNA intermediateo uses reverse transcriptase to make cDNA, duplicated to make dsDNA, DNA integrated into host chromosome, transcription, more RNA copies to make more cDNA- Nonretroviral retrotransposons—dominate human genomeo Move with RNA intermediateo 34% of genome made up of LINES and SINES LINE= long interspersed nuclear elements SINE= short interspersed nuclear elements o LINE= L1 Target site repeat-stop codons-Orf1-Orf2-AT rich poly A tail- Orf1= encodes RNA binding protein- Orf2= encodes RT and endonuclease 90% of L1 elements are missing variable amounds of sequence making them immobile Only L1 are autonomous ALU and SVA (SINEs) are nonautonomousActivity of Retrotransposible Elements in Our Genome- Human vs. chimp vs. orangutan o All have ALU, SVA, and L1 coming from common ancestorso At point of divergence can see difference—humans having most ALU, chimps having more L1, and orangutans having most L1 and little ALU These differences mean there must be transposition occurring- Transposition events are kept fairly inert, but there are ~1 in every 20-125 births- Even when inactive, can passively promote gene duplication- L1 retrotranscriptiono Requires proteins encoded by Orf1 and Orf2o mRNA L1 polypeptide emerging from ribosome Orf1 protein binds in cis to its own mRNA selects a target similar to polyA – TTAAAA nuclease cleaves target site and string of Ts will bind with polyA, 1st strandsynthesis, new L1 copy (see slide for an image)- Chondrodysplasia—short leggedness in dogso Correlation between short legs and SNP on chromosome 18 SNP= short nucleotide polymorphism- A one nucleotide difference among sequences for the same chromosome Short legged dogs have ~5kb insertion on chromosome 18o FGF4= growth factor gene on 18 In short legged dogs, extra copy Lacks intron and promoter but has a polyA tail Orf1 and Orf2 hijacks cellular RNA instead of L1 element FGF4 retrotransposon into new genomic locus, into an L1 element whose promoter now drives mis-expression of FGF4- Suppressing LINEso DNA is methylated to suppress transcriptiono Small PIWI-associated RNA (piRNAs) direct RNAi-like silencing pathway Act on transposons in a manner similar to siRNA PIWI are relatives of Argonauto piRNAs transcribed from clusters of piRNAs processed without DICER expressed predominately in germ line when demethylated DNA in males loss of PIWI=> depression, increase transposition, arrest of spermogenesis, infertility most encoded in dense clusters 20-100 kbp- get sense or antisense out of them- amplified in ping-pong cycle and targeted for degradationo ping pong= sense transcriptcleavage and methylationantisense transcript cleavage and methylation sense transcript cleavage and methylation… etc.o mechanism for keeping transposition
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