Molecular Genetics

Centromeres, central to segregation, can be identified.

Facts

• CEN sequences have been isolated by chromosome walking between two markers on opposite sides of the centromere in the genetic map.
• They have also been isolated by the use of genetic engineering of plasmid DNAs.
  • Wild-type yeast cells (LEU2) grow equally well with or without leucine.
  • Consider cells (leu2(YEP:LEU2) ) mutant in LEU2 and carrying a low copy number plasmid (linear with telomeres or circular, and containing ARS sequences) with a good copy of LEU2. They grow on leucine-deficient media. When grown on leucine-containing media, they loose the ability to grow on leucine-deficient media. This loss correlates with loss of plasmid.
  • Centromere sequences (CEN) added to such a LEU2 plasmid increase retention of the LEU2 gene in the absence of selective pressure.
• In primates, another approach was used. African green monkey cells were transfected with alpha satellite DNA. The DNA integrated into chromosomes. In subsequent cell divisions, many anaphase bridges and lagging chromosomes resulted.
• In Arabidopsis thaliana, tetrad analysis was used to identify regions for which only ditypes were found among tetrads (ref). These regions were thought to define the centromeres.

Interpretations

• The maintenance of a selectable phenotype in the absence of selection is a test for the presence of a CEN element. This is because low copy number yeast DNA plasmids are rapidly lost from most cells due to unequal segregation of the DNA at cell division.
• A CEN element causes equal segregation of the products of replication into daughter cells.
• More than one CEN element per chromosome interferes with proper segregation.

Further information

• A variety of methods have been used to test sequences for their CEN activity.
• Some of these methods are useful not only for the original isolation of a CEN sequence but also for analysis of the products of its mutagenesis.
• The studies allow comparisons of centromere structure.

This is page 1363 of Molecular Genetics by Ulrich Melcher, © 1997-9, 2001, 2003