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2002
LSU-HHMI Summer Undergraduate Research Program |
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 Elizabeth
Robert (Notre Dame University) (David Donze, LSU Dept. of Biological
Sciences) Identification of Three Genes that Reduce
tRNA Heterochromatin Barrier Activity in Blocking Silencing
in Saccharomyces cervisae
The genomes of eukaryotes are organized into structurally distinct
domains termed heterochromatin and euchromatin. These two structural
domains possess a functional distinction; genes present within
the highly condensed heterochromatin remain largely inactive
or silenced while genes within regions of euchromatin are, in
general, transcriptionally active. Within genomic DNA there
are elements which function to separate the two structural domains.
Elements termed barriers block the spread of heterochromatin
into surrounding regions of euchromatin. Previous results identified
a unique tRNA gene in yeast that functions as a heterochromatin
barrier. This study was designed to identify genes which affect
the ability of this specific tRNA barrier to block silencing.
To carry out this study gene disruptions were introduced into
the yeast Saccharomyces cerevisiae by transforming the yeast
with a library of yeast genomic DNA containing random transposon
insertions. Integration of these fragments into the yeast genome
results in random mutagenesis of yeast open reading frames and
gene regulatory sequences. Subsequent screens of transformants
revealed mutants with reduced barrier function. Sequencing performed
on these mutants in order to determine the location of transposon
insertion identified three genes. The SAS5 gene encodes an acetyltransferase,
which suggests its role and effect on silencing since part of
the structural distinction between heterochromatin and euchromatin
is the acetylation state of the tails of the histone core proteins
of nucleosomes. Heterochromatin is hypoacetylated while euchromatin
is acetylated. The second identified protein, YTA7p, exhibits
homology to a protein involved in protein degradation while
the function of the third and final gene, YPLO33C, is currently
unknown. Mutations in these three genes allow silencing to propagate
beyond the tRNA barrier thus silencing nearby euchromatin.
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