Bi Sternberg 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 Yeast yeast functional assays for (a) ARS (b) centromere (c) telomere

Szostak & Blackburn (1982) Tetrahymena rDNA: 21 kb small linear molecule Bam H1 + Bgl II Tel Bgl II Bam H1 LEU2+ ars1 Col E1 ori AmpR Of 15 Leu+ transformants, 14 were mitotically unstable; 1 integrant. ~20 copies per cell, and LINEAR! by R-Mapping HIS3+ CCCCAA GGGGTT

Clone a yeast telomere: T.TELLEU2yeast DNA yeast DNA, cut with Pvu I into ~2000 pieces 32 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+ CEN3 ARS1 Col E1 ori AmpR TEL transform yeast Plate 100 colonies/plate replica plate to -Leu count fraction Leu+ plasmid%cellsSegr.copies/cell with plasmidFreq. circle circleCEN YLp4CEN YLp TEL inhibits CEN function; yeast chr are fold longer Pedigree analysis to determine the segregation frequency, the fraction of divisions at which only one daughter receives the plasmid

Telomere Repeats of Some Organisms NB – 3’ overhang is G-rich

human artificial chromosomes [Harrington….Willard, 1997) linear, mitotically stable CEN TEL ORI transfect in alpha-satellite, TTAGGG (TEL), selectable marker and obtain a minichromosome. After 240 generations with no selection, % had the mini 99.5% 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 Double-strand break repair model

Transposon-induced Gap repair ab b b b a a a ab ba ba ab