75 Integrating Membrane Transport with Male Gametophyte ... - TAIR

315 Finding Intron Sequences That Enhance Gene Expression
Alan Rose, Ian Korf
Molecular and Cellular Biology, University of California, 1 Shields Ave., Davis, CA 95616
Many introns increase gene expression in diverse organisms by mechanisms that are poorly understood but are unlike
enhancer elements. Because efficiently spliced introns differ widely in their ability to stimulate expression, splicing alone
is not sufficient for intron-mediated enhancement (IME), and there must be sequences that differ between introns that
determine the magnitude of enhancement. To locate these sequences, a series of deletion-containing and hybrid introns
were tested in a fusion between the TRP1 gene and GUS in transgenic Arabidopsis. All of the deletion-containing introns
stimulated mRNA accumulation as much as did the full-length intron. Similarly, hybrids in which either the 5' or 3' half
of a strongly enhancing intron were fused to the other half of a 'weak' intron appear to be as effective as the entire 'strong'
intron. Thus, enhancing sequences are redundant, dispersed, and more prevalent in some introns than others. To identify
sequences that fit these criteria, an algorithm was devised that determines how well a test intron matches the pentamer
sequence profile of all the introns in genes whose expression is predicted to be in the top 20% of Arabidopsis genes,
based on codon entropy. Three findings suggest that this algorithm (the IMEter) may be relevant to IME. 1) The score
generated by the IMEter is proportional to the degree to which each of six introns elevate TRP1:GUS expression. 2) Of
the 15 Arabidopsis introns shown by other labs to enhance expression, virtually all generate an IMEter score of 10 or
more, even though less than 2.8% of all Arabidopsis introns yield a score this high. 3) Average IMEter scores of introns
separated by their ordinal number drop from first introns to sixth introns and then level off. This pattern is in striking
agreement with the finding that the ability of an intron to stimulate mRNA accumulation declines with distance from
the promoter until it is lost entirely around 1 Kb from the start. We are testing the importance of the sequence CGAT,
identified using the IMEter, by asking if altering the abundance of this motif changes the enhancing ability of introns.
316 Genetic and Epigenetic Study of de novo Centromere Formation in Arabidopsis thaliana
Jay Shrestha, Song Luo, Daphne Preuss
University of Chicago
Centromeres play a critical role in ensuring proper segregation of chromosomes by specifying site for kinetochore
assembly and spindle attachment during cell division. Our lab is interested in studying the genetic and epigenetic
determinants at the centromere that specify its function in Arabidopsis thaliana. The primary question this study aims to
answer is: what genetic elements in the centromeric region of Arabidopsis thaliana are sufficient to confer centromeric
activity. To this end, DNA fragments from different regions of the Arabidopsis thaliana genome are being integrated
into the chromosome by Agrobacterium-mediated transformation. These DNA fragments include BAC clones carrying
centromeric DNA repeats, gene rich euchromatic sequences, heterochromatic DNA from the NOR, and the chromosome
4 knob. Following integration of these DNA fragments into the chromosome, de novo formation of centromeres at these
inserted sequences will be investigated by using various genetic, cell and molecular biological methods. The candidates
that show de novo centromere formation will be further analyzed for epigenetic changes at the new centromeric site.
Studying these candidate centromeric fragments can give us an insight into the size and sequence requirement for
centromere function in Arabidopsis thaliana.