Lecture 18+19 - Hypoacetylated tails are binding sites for SIR3 and SIR4 which bind to telomere region that lacks nucleosomes M ultiple copies of RAP1

Multiple copies of RAP1 bind to a single repeated sequence at telomere region that lacks nucleosomes SIR3 and SIR4 bind to RAP1 and SIR2 binds to SIR4 SIR2 deacetylates the histone tails neighbouring repeated RAP1 binding site Hypoacetylated tails are binding sites for SIR3 and SIR4 which bind to SIR2  Neighbouring histones are deacetylated Process is repeated and results in spreading of region of hypoacetylated histones with associated SIR2, SIR3 and SIR4 Interactions between complexes of SIR2, SIR3 and SIR$ cause the chromatin to condense and telomeres to associate  Higher order chromatin structure generated sterically blocks other proteins from interacting with the underlying DNA Lecture 18+19 Chromatin Structure + Chromatin Modification and Gene Regulation Heterochromatin Interphase: heterochromatin/euchromatin Heterochromatin : Condensed chromatin • No or very little gene expression • Dynamic  can spread or retract Important function: • Structure of centromere • Structure of telomeres • Gene silencing • Protection for mobile elements Elements required for replication and stable inheritance of chromosomes 1. Origin of replication 2. Centromere 3. Two telomeres Assays can be used to identify origins or replications, centromere and telomere sequences. Origin of Replication 1 | Pa g e

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-Transfect yeast leu - cells with circular plasmids containing LEU - Yeast can grow in medium lacking leucine only if LEU gene is present  If only the plasmid is present there is selection -leu - is transformed to LEU + by LEU gene on plasmid -Yeast cells missing leucine cannot grow in a medium without leucine -Yeast cells contain ARSs that act as replication origins -Insertion of a plasmid with ARS allows it to replicated in yeast cells Conclusion: 1. ARS required for plasmid replication 2. Mitotic segregation is faulty if no CEN present Centromere 2 | Pa g e

CEN : Centromeric sequence -Randomly broken pieces of genomic yeast DNA inserted into plasmids containing ARS and LEU -Transfected cells produce larger colonies -High rate of mitotic segregation • If ORI and centromere sequence are present then you have a very good growth • Everything divides with normal chromosomes and segregated properly • Only good for circular chromosomes Conclusion : Cells transfected with CEN have a higher mitotic segregation Telomeres 3 | Pa g e

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-Cut circular plasmid with restriction enzyme to produce linear plasmid -No LEU + colonies unless telomeric sequence present at their ends -Addition of TEL sequences to end of linear DNA gives linearlized plasmids the ability to replicate as new chromosomes Conclusion: 1. Linear plasmid lacking TEL is unstable 2. Linear plasmids containing ARS and CEN behave like normal chromosomes if genomic fragment TEL is added at both ends 3.