Biochemical and Biophysical Research Communications
Pax-2 interacts with RB and reverses its repression on the promoter of Rig-1, a Robo member
Section snippets
Materials and methods
Primer extension assay. One μg mRNA, extracted from the E11.5 hindbrain–spinal cord of E11.5 mouse embryos, was used as the template. An antisense primer (30 mer), complementary to the 41–70 bp of the 5′-untranslated region, was labeled with γ-[32P]ATP. The 105 cpm antisense primer was mixed with mRNA and first strand cDNA was synthesized using Superscript II reverse transcriptase (Gibco). The samples were loaded onto a 6% polyacrylamide/7M urea gel and the gel was processed for
Determination of the transcription initiation site of the Rig-1 gene
Sequence analysis of the Rig-1 cDNA clones revealed that the Rig-1 transcript has a long 5′-untranslated region. To determine the transcription initiation site of the Rig-1 gene, a primer extension assay was performed. A major product with the expected size of 70 bases was detected, confirming that the Rig-1 transcript has a 928 bp 5′-untranslated region (Fig. 1, lane 6). Besides, the broad smear near position 60 in the primer extension assay (Fig. 1, lane 6) suggested the presence of an
Discussion
To the best of our knowledge, this is the first report showing the colocalization of a Pax family member and an RB-regulated protein. Furthermore, Pax-2's interaction with RB, as well as its modulation of RB's transcriptional repressor activity, provides new insight into the transcriptional activities of the Pax gene family members.
Acknowledgements
We thank Dr. Gregory Dressler for the Pax-2 expression constructs. We are grateful to Elvira Gerbino for helping with the luciferase assay and Wills Renthal for critical reading. The GenBank accession number for the 2.3 kb Rig-1 promoter region is AF532971.
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Present address: Department of Biological Chemistry, and Department of Developmental and Cell Biology, University of California, Irvine.