Transcriptional activation of retrotransposons alters the expression of adjacent genes in wheat

Kim Tae Hyung

2005. 01. 08

Khalil Kashkush, Moshe Feldman & Avraham A. Levy

Department of Plant Sciences, The Weizmann Institute of Science, Rehovot 76100, Israel

  HERVs Expression and Structure Analysis System

Alternative promoter of intergenic

Levy lab’s research interests

• Transposons We are focusing on the Ac/Ds elements; on their origin, on their regulation and on the intermediates of transposition.

• DSB repair We have studied the repair of DSBs generated by transposons, and we showed that these breaks can be repaired by homologous recombination between ectopic sequences. We have studied the repair of breaks done in vitro and characterized the end joining products. In general, we found the DSB repair in plants is error-prone. Accurate repair via homologous recombination is a less prominent DSB repair pathway in plants than non-homologous end-joining.

• Polyploidy-induced DNA rearrangements We have shown, in collaboration with Prof. Moshe Feldman, that certain DNA sequences that were isolated by microdissection of wheat chromosomes undergo rapid elimination as a result of polyploidy. We are currently investigating the underlying mechanism of this polyploidy-induced sequence elimination.

• Genetic tools We have developed a miniature tomato cultivar with a rapid growth cycle as a model system for forward and reverse genetics in tomato (Meissner et al., 1997; Meissner et al. 2000). We are using this cultivar for large scale mutagenesis via transposon, EMS andfast-neutron. This work was initiated under the auspices of the Israeli plant genome center. We have shown that the bacterial genes, RuvC stimulates homologous recombination in plants. We have isolated hyper-recombinogenic mutants and we are testing whether these mutants and plants expressing bacterial genes can be used for improving the efficiency of gene targteting.

Analysis of the Wis2-1A retrotransposon activation

Ae. Sharonensis (SlSI)T.monococcum ssp. aegilopodes (AmAm)

Amphiploid (BBAA)

Northern

cDNA-AFLP

SouthernControl (18S rDNA probe)8kb band

Internal Wis 2-1A fragment (RT)

More than 1000 sites

cDNA-retrotransposon display

Negative control

Putative activation

Putative silencing

Amphiploid vs Diploids360 transcripts

26/360 7%10 of 26 only appeared in the amphiploid 16 of 26 were only present in both parents

Molecular characterization of chimeric transcripts

22/26(gel purified & sequenced)

Similarity to Psal

8 new transcripts (Wis23, 29, 30, 31, 35, 36, 37, 39) were unknown sequence.6 (Wis38, 40, 24, 28, 43, 44) corresponded to wheat or barley ESTs with no known homology.6 ( Wis25, 34, 41, 42, 26, 32) were similar to previously characterized genes.

In most cases, the LTR was positioned in the 3’UTR downstream of the neighboring gene

Expression of transcripts flanking LTRs

RNase treatment

silencing

activation

Quantitative RT-PCR (12 cycles)

Promoter activity of the 5’LTR with TSS in U3 region

Induction of the iojap-like antisense transcript from the 5’ LTR

Quantitative RT-PCR(12 cycles)

The level of expression was 156 times higher in the amphiploid than in the diploid parents

Discussions

• Disruption of normal gene expression• Generate new patterns of gene expression• Generate antisense RNA for neighboring elements• Help maintain silence of the gene family

• Alternative promoter• Intragenic analysis • Effective of Coding capacity• Tissue specificity

>50kb