MCB 121 Lecture 4

MCB 121 Lecture 4 - MCB 121 Lecture 4 Outline I II III...

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MCB 121 – Lecture 4 Outline: I. Origins of replication a. ID of replicators (genetics) b. ID of origins (molecular) II. Fork movement a. Measure rate in vivo b. Chemotherapy III. Telomerase Origins of replication: In human, there are 30000 replicons. Take chromosome I => 260 mb only put one ori on this chromosome the rate of replication is 50nt/ sec. it takes 30 days to replicate this chromosome in bidirectional. Rational: A circular plasmid (double stranded DNA) => put it into budding yeast (that doesn’t have URA 3) * the plasmid contains marker (URA 3 gene) Before the transfer of the plasmid: cells are phonotypical “URA3-“ * They cannot grow on media that doesn’t have uracil * It’s a type of mutation: auxatroph; require something to grow * if the bacteria contain URA3 gene, they can grow on the media without uracil because they can simply make it themselves. Introduce the plasmid into the budding yeast: * the plasmid has the origin of replication on it. = allows this plasmid in the budding yeast to replicate. = cause this yeast cell to be URA3 + = the yeast cell then can successfully replicate by allowing uracil production in cell. = thus, multiple copies of plasmid are generated and transferred to each copies of the replicated yeast cell. = a colony of URA3+ yeast cells form. = This confirms the URA3+ genotype. When a plasmid that contains URA3+ but without origin is introduced into the budding yeast: = plasmid can’t be replicated. = no URA+ cells will be generated = no colony will form = because URA3+ plasmid is introduced to only one cell at one time! . This is how a phenotypic way can use to distinguish between two states of DNA. Genetic identification of replicators Basic Idea: Clone DNA segments randomly from a genome into a plasmid: 1. isolate genomic DNA 2. cut it with restriction enzyme (ex. EcoR1) = generate bunches of fragments of genome = each of the fragment has EcoR1 ends = which will facilitate the cloning into backbone vector (that’s going to be introduced into yeast) = each fragment has overhang of single stranded DNA
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3. Take a fragment and perform ligation to form a plasmid
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This note was uploaded on 05/09/2010 for the course MCB 121 taught by Professor Gasser during the Winter '09 term at UC Davis.

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MCB 121 Lecture 4 - MCB 121 Lecture 4 Outline I II III...

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