BIMM 100 Lecture 11

BIMM 100 Lecture 11 - BIMM100: Lecture 11 ­...

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Unformatted text preview: BIMM100: Lecture 11 ­ chromosomes & invaders Reading: 226 ­236 Repe??ous DNA in the genome •  Simple sequence DNA repeats (SSRs): satellite repeats –  DNA “fingerprin?ng” •  Transposable DNA elements: mobile, interspersed repeats –  Overview –  Inser?on sequences –  Transposons –  Retrotransposons (with LTRs) –  Retrotransposons (w/o LTRs) –  Pseudogenes –  Their roles in disease (and evolu?on!) Simple sequence repeats (SSRs) Simple repeats comprise 3 ­6% of the human genome From Genes, Lewin et. al SSRs and DNA fingerprin?ng Minisatellite sequences are conserved, but their lengths are not! –  1 ­5kb regions, scaYered –  20 ­50 repeat units that each contain 14 ­100 bp sequences KEY? Everyone has a unique repeat “signature.” Repe??ous DNA: a closer look What are transposons? They comprise almost half of our genomic DNA! Really important in biology! A few reasons: 1.  Likely responsible for the evolu?on of the complex eukaryo?c genome 2.  Can cause mul?drug an?bio?c resistance! Ex: low levels of tetracyclines ENHANCE the frequency of transposi?on in some bacteria! 3.  Cause human diseases! Ex: hemophilias A & B, X ­linked SCID (there it is again!), porphyria, colon cancer, and dystrophin 4.  Powerful research tool! Use this technology to generate mutant organisms to study gene func?on. Transposi?ons come in two general “flavors” DNA intermediate: “cut and paste” technique ­ they move themselves around the genome RNA intermediate: “copy and paste” technique ­they copy themselves, and insert that new copy somewhere in the genome! Transposons are present in bacteria and eukaryotes A bacterial inser?on (IS) element (this is a DNA transposon) > 20 different ISs known inser?on is rare! (occurs in only 1 out of 105 ­107 cells per genera?on) causes random genomic integra?ons Inverted repeat is specific for each par?cular IS Protein codes for transposase, which moves the IS element to a new site. It is expressed at a low level, which is why IS elements have a low frequency of transposi?on Target site length is characteris?c (but not the sequence). Not part of the IS element! How does the bacterial transposi?on happen? Ds DNA cleavage by transposase It cuts between the inverted repeat and direct repeats to be “blunt” (this is the IS10 element) It cuts the direct repeat to be “s?cky” (this is in the target DNA) How does the bacterial transposi?on happen? Then, the transposase ligates the IS10 into the target sequence. How does the bacterial transposi?on happen? DNA polymerase (from the host cell) fills in the missing bps. Ligase (from the host cell) joins the ends. The 9bp target site repeats are conserved! Why? Because of the way that the transposase cuts them to be “s?cky” and then uses the host cell’s machinery to fill in the missing bases! Eukaryotes have transposable DNA elements, too •  First descrip?on? Barbara McClintock, in the 1940s and 1950s. –  Described the genome of maize, including concepts of transposi?on, recombina?on, and the roles of telomeres and centromeres. –  No one believed her. She stopped publishing results on transposi?on in 1953 due to extreme cri?cism of her work! –  Her research was finally more accepted in the 60’s and 70’s, and she was awarded a Nobel prize in 1983 for this research (the only woman ever to receive an unshared Nobel prize in Physiology or Medicine). Essen?al finding ­ important for crea?ng mutants (to discover the role of genes in organisms) •  Design a DNA transposable element (this is based on the Drosophila P element). •  If the inser?on causes an interes?ng phenotype, you can find out where the P element inserted in the genome (did it insert in a gene? An enhancer? A promoter?) •  You PCR amplify the DNA around the transposon to figure it out! DNA transposons can become “copied” in the genome as well The key? It happens during S phase, when a piece of DNA that has already been replicated inserts itself into a chuck of DNA that has not yet been replicated! RNA intermediates (retrotransposons) •  Mobile DNA that transposes through an RNA intermediate •  Two flavors: –  Long terminal repeat ­containing (LTRs) •  About 8% of our genome –  Lacking LTRs •  Most common in mammals Generic model of an LTR ­based retrotransposon LTR sequences are longer than DNA ­based transposons ­ also ­ NOT inverted repeats! Characteris?c of inserted retroviral DNA, and are cri?cal to the life cycle of retroviruses Encode all the proteins of retroviruses (RT, integrase, and other proteins). NOT coat proteins, though ­ these are not infec?ous because they can’t leave the cell! Retroviruses vs. retrotransposons Ty: yeast transposon Copia: Drosophila transposon Key difference? No env protein ­encoding regions! Genes, Lewin et al. Genera?ng retroviral genomic RNA from an integrated retroviral DNA 5’ LTR func?ons as a “promoter.” RNA pol starts transcribing at the R site. 3’ LTR tells the cellular machinery where to cleave the RNA transcript (processing) KEY: the transcript has incomplete LTRs! This is transported out of the nucleus and packaged into a virus par?cle (in a retrovirus) Aper being transcribed… •  The RNA genome is reverse transcribed to make dsDNA in the cytosol •  Then, the dsDNA (with LTR sequences) is transported back into the nucleus (in a complex that includes integrase ­ made by the virus). •  Integrase inserts the DNA into the genome •  CRITCAL to the life cycle of a retrovirus! –  Ensures that nothing is lost from an LTR transposon when it moves around The anima?on: how to prevent LTR loss Check this out ­ it will help you understand this complex process! How to make sure the LTRs aren’t lost: the nuts and bolts Retroviral ­specific cellular tRNA binds the primer binding site (PBS). This is in the virion! Direct repeat sequence Comes into the cell via a retrovirus! With RT! (viral) With Rnase H (cellular) How to make sure the LTRs aren’t lost Dissociates and aYaches to other end of the LTR ­ they are complementary! RT (viral) How to make sure the LTRs aren’t lost RNAse H (cellular) RT (viral) How to make sure the LTRs aren’t lost Rnase H (cellular) All performed by the ac?vity of RT (from the virus)! Essen?al for gene?c diversity! •  LTR regions can homologously recombine! –  The recombina?on deletes the retroviral inser?ons in between them, but keeps the LTRs –  Likely leads to chromosomal DNA rearrangement that lead to exon and gene duplica?ons –  Also probably led to evolu?on of new genes with combina?ons of different exons (we’ll get to this later on) How do the Ty and Copia elements work? The big ques?on? Do these elements work through an RNA intermediate? The observa?on: transposi?on occurs, but at a very low rate. Hypothesis: If the amount of Ty mRNA was increased, transposi?on would increase… How do the Ty and Copia elements work? The observa?on: Adding galactose to the media increased the mRNA synthesis, which increased transposi?on The conclusion: mRNA synthesis increases transposi?on. Next ques?on: was this through an mRNA intermediate? How do the Ty and Copia elements work? The experiment: add in another intron into the gene. The ques?on: will this intron be incorporated into the element when it is copied? Or will it be removed? How do the Ty and Copia elements work? The observa?on: the transposed element lacked the exogenous intron. The conclusion: the primary transcript was spliced (how the intron was removed) ­ transposi?on of this element likely occurs through an mRNA intermediate! THIS IS DIFFERENT FROM DNA TRANSPOSONS ­ they do have introns that are within the transposase gene, indica?ng that they do not transpose via an RNA intermediate! More than just LTRs… Genes, Lewin et al. What about non ­LTR retrotransposons? Genes, Lewin et al. Non ­retroviral transposons •  Two classes: –  long interspersed elements (LINEs) •  About 6kb •  Abundant in mammals •  Three types (L1, L2, L3) –  L1 transpose into our genome (no func?onal L2 or L3 anymore) –  Short interspersed elements (SINEs) •  About 300bp •  Abundant in mammals Generalized structure of a non ­retroviral transposon (ex: LINE) Direct repeats! ORF: open reading frame. ORF1: RNA binding protein ORF2: region of homology to RT and LTR transposons. Has exonuclease ac?vity LINEs are important in human disease L1 LINE ­ most common in humans (makes up 15% of our genome ­ over 600,000 copies)! Most of these copies have muta?ons! 1:600 muta?ons that cause significant diseases in humans (like certain forms of muscular dystrophy and hemophilia) are caused by muta?ons in L1 LINEs and SINE transposi?ons related to LINEs. Usually these muta2ons are in the proteins coded for by these elements! The process? Key points: ORF2 nicks cellular DNA at the right spot to hybridize LINE RNA RT (by ORF2) makes another copy of the LINE RNA The process? Key points: ORF2 switches to the single ­stranded DNA region from the chromosome as a template Cellular enzymes degrade RNA and extend the DNA strand. The 5’ and 3’ ends are ligated. Steps 6 &7: Probably happens in the same way that Okazaki fragment primers are removed and ligated during replicaCon! KEY point: no integrase needed (like retrotransposons) ­ this occurs in the nucleus ­ not the cytosol! Short interspersed elements •  •  •  •  Comprise about 13% of the human genome 100 ­400 bp Don’t encode protein ­ transcribed by RNA pol Occur at 1.6 million sites in the genome –  1.1 million of these are Alu elements (cut by the restric?on enzyme AluI). •  Sequence homology with cytosolic RNA involved in signaling •  Also associated with human disease! FYI: many LINEs and SINEs are truncated •  Because they use RT, many LINEs and SINEs are truncated at their 5’ ends •  These are probably not transposed, as they are lacking important sequences for their transposi?on •  Only 0.01 % of LINEs in the human genome are fully func?onal and full ­length (only about 60 ­100!) •  S?ll essen?al for diversity because of their role in homologous recombina?on! Yet another retrotransposed RNA: “processed pseudogenes” •  These are copies of mRNAs that have been integrated into chromosomal DNA –  No introns –  No flanking sequences that correspond to their sequence –  So, they appear to be retrotransposed copies of mRNA that was spliced, polyadenylated, and inserted into the genome –  Flanked by short direct repeats, suppor?ng this hypothesis Processed pseudogenes How “copies” of mRNAs might get integrated into your genomic DNA Genes, Lewin et al. Mobile DNA elements are important in the evolu?on and expansion of our genome KEY: mobile DNA elements can serve as “recombina?on sites” to mobilize adjacent DNA RESULT: duplica?on of exons & exon shuffling (inser?on of certain protein domains into other proteins, conferring them some advantage), modifica?on of enhancer elements, etc. Take home message? •  Mobile DNA elements are not “selfish DNA” •  Contributed profoundly to the evolu?on of higher organisms by promo?ng: –  Genera?on of gene families through gene duplica?on –  Crea?on of new genes through exon shuffling –  Forma?on of complex regulatory regions for more control of gene expression in complicated organisms •  Uses? Gene therapy! ...
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This note was uploaded on 10/12/2011 for the course BIMM 100 taught by Professor Pasquinelli during the Summer '06 term at UCSD.

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