GENE EXPRESSION OF THE YEAST PLASMID 2 MICRON CIRCLE

Project: Research project

Description

The yeast plasmid, 2 micron circle, and hybrid plasmids derived
from it are more stable than most plasmids of similar size
constructed from chromosomal ARS sequences. Previous studies
in this and other laboratories have suggested that this can be
attributed to a plasmid encoded system for maintenance of a
high, regulated copy number and for equipartitioning of plasmids
in mitotic and meiotic cell divisions. Our current efforts are
focused on identifying the plasmid components responsible for
these systems and elucidating their regulatory interactions. We have shown that equipartitioning of plasmid molecules
requires a cis-acting sequence, REP3, and two plasmid genes,
REP1 and REP2. We have purified the REP1 protein and shown
that it is a component of the yeast nuclear matrix. Further, we
have found that it undergoes calcium induced aggregation and
binds specifically to discrete plasmid DNA sequences. We have
also raised antibodies to the REP2 protein and identified it in
extracts of plasmid-bearing yeast cells. We plan to continue
these studies by precisely defining the nature of the interaction
between the components of this system - namely, REP1 protein,
REP2 protein, specific 2 micron sequences, and the nuclear
matrix - and by delineating in more detail the characteristics of
the partitioning process. The FLP protein of 2 micron circle catalyzes site-specific
recombination between two repeated segments present on the
plasmid. We have shown that this system enables the plasmid to
increase its mean intracellular copy number in yeast cells
growing under non-selective conditions by a FLP-induced
transient shift in the mode of replication from theta to double
rolling circle. We are pursuing this system by identifying
intermediates in amplification. In addition, we are developing it
as a system to promote directed plasmid and chromosomal
rearrangements in yeast for use in addressing various novel
questions of recombination and cell structure. Evidence exists to indicate that 2 micron circle controls its copy
level through autogenous regulation of expression of its various
products. We are using beta-galactosidase gene fusions to the
four 2 micron circle genes in conjunction with constructs
consisting of promoter fusions to the 2 micron circle coding
regions to provide a complete description of 2 micron circle's
regulatory circuitry.
StatusFinished
Effective start/end date9/1/848/31/93

Funding

  • National Institutes of Health
  • National Institutes of Health
  • National Institutes of Health
  • National Institutes of Health
  • National Institutes of Health: $189,346.00
  • National Institutes of Health
  • National Institutes of Health

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Plasmids
Yeasts
Gene Expression
Proteins
Genetic Recombination
Genes
Nuclear Matrix
Gene Fusion
beta-Galactosidase
Cell Division
Replication Origin
Maintenance
DNA Replication
Calcium