Bi1902005Sternberg

Analyzing and Building Chromosomes

Three types of DNA sequence required for eukaryotic chromosomes

Replication origin (many per chromosome) functions in interphase to duplicate DNA.

Centromere (one per chromosome) functions in mitosis for chromosome segregation.

Telomeres (two per chromosome) cap the chromosome ends.

The kinetochore forms in mitosis at the centromere

The primary constriction occurs at the centromere of a mitotic chromosome

In Situ Hybridization of Human Chromosomes With a Probe Against the

Telomere Repeat

Selection of Centromere DNA in Budding Yeastyeast functional assays for (a) ARS (b) centromere (c) telomere

Selection of Centromere DNA in Budding Yeastyeast functional assays for (a) ARS (b) centromere (c) telomere

Szostak & Blackburn (1982)

Tetrahymena rDNA: 21 kb small linear molecule

Bam H1Bam H1

+Bgl II

Tel

TelTel

Bgl IIBgl II Bam H1Bam H1

LEU2+ars1Col E1 oriAmpR

Of 15 Leu+ transformants, 14 were mitotically unstable; 1integrant. ~20 copies per cell, and LINEAR! by R-Mapping

HIS3+

CCCCAA

GGGGTT

Clone a yeast telomere:

T.TEL LEU2 yeast DNA

yeast DNA, cut with Pvu I into ~2000 pieces32 should be telomeres (1/65)

Of 60 colonies, 56 had plasmid (mitotically unstable)Pick 3 that had a small single Pvu I fragment---A yeast TEL

Pvu I

5´ ... C G A T^C G ... 3´ 3´ ... G C^T A G C ... 5´

Murray & Szostak

YLp4

LEU2+CEN3ARS1Col E1 oriAmpR

TEL

TEL

transform yeast

Plate 100 colonies/platereplica plate to -Leucount fraction Leu+

plasmid %cells Segr. copies/cellwith plasmid Freq.

circle - 5 0.34 50

circle CEN 90 0.02 1

YLp4 CEN 61 0.11 15

YLp30 - 72 0.16 50

TEL inhibits CEN function; yeast chr are 10-100 fold longer

Pedigree analysis todetermine the segregationfrequency, the fraction of divisions at which only one daughter receivesthe plasmid

Telomere Repeats of Some Organisms

NB – 3’ overhang is G-rich

human artificial chromosomes[Harrington….Willard, 1997)

linear, mitotically stableCENTELORI

transfect in alpha-satellite, TTAGGG (TEL), selectable markerand obtain a minichromosome.After 240 generations with no selection, 29-100% had the mini99.5% segregation frequency.

Tandem repeats of alpha satellite DNA constitute a primate centromere

[Schueler, Higgins, Rudd, Gustashaw and Willard, Science (2001)]

(A) Chromosome rearrangements define the CEN as (171bp)12

(B) Artificial chromosomes with 85 Kb alpha satellite DNA (red) bind the centromere protein CENP-E (green). Overlap of both is yellow.

Centromere Sequences Differ Between Organisms

Epigenetic model: something else besides DNA sequence may define a centromere

Analysis of CEN Mutations by DNA Sequence Replacement in Budding Yeast

(Rothstein)

ds replacement DNA

ds chromosomal DNA homology homology

ds chromosomal DNA with replacement

invasion

invasion

Double-strand break repair model

Transposon-induced Gap repaira b

b

b

b

a

a

a

a b

ba

ba

a b