bi190 2005 sternberg analyzing and building chromosomes
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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
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a b
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