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Nilay Patel

            This presentation reports the initial findings of a functional analysis of a gene called the Drosophila nuclear transport factor-2-related (Dntf-2r).  Dr. Ruth Steward’s lab at the Waksman Institute studies the Rel pathway in Drosophila, which functions in establishing dorsoventral polarity in early embryos and the innate immune response via transcription factors called Rel proteins.  The Drosophila nuclear transport factor-2 (Dntf-2) is an essential component of the nuclear import machinery responsible for targeting Rel proteins to the nucleus [2].  It mediates the Ran gradient, which is the driving force of nuclear import [3].

            Dntf-2r is a recently discovered retroposed copy of Dntf-2 [1] whose function is not known, but which may act in the same pathway because it is so closely related to Dntf-2.  My objective was to determine if Dntf-2r is an essential gene through genetic crosses, PCR analysis, Southern blotting experiments, and sequencing.  Thus far, the P-element of a viable strain containing a P-element insertion site 32 bases upstream of the start of the gene was mobilized, and mutations causing lethality in homozygotes for the deficiency were isolated.  The lethal phenotype suggested that the gene is essential for viability.  To confirm this, PCR analysis was first employed to find any deletion in the Dntf-2r gene resulting from the P-element jump that would have caused the lethal mutation.  This line of testing, however, failed to show any deletion in homozygous lethal stocks.  Thus, Southern blotting experiments were next employed to detect genomic rearrangements in the Dntf-2r region.  This line of testing revealed a rearrangement in one of the mutants of Dntf-2r with a breakpoint mapping to a region downstream of the gene.  Attempts to sequence the rearranged gene area of this mutant and, thereby, conclusively determine that a deletion occurred in the Dntf-2r gene causing lethality in homozygotes for the mutation failed.  Thus, though Dntf-2r is likely to be an essential gene, this still cannot be said with total certainty.

            To continue this project, an RT-PCR analysis using another available viable strain containing a new P-element insertion site located within the coding region of the single exon of Dntf-2r could be performed to get a sense of the transcript levels of Dntf-2r in cells and reveal whether Dntf-2r is merely a pseudogene.

References

1.   Betran E, Thornton K, and Long M: Retroposed New Genes Out of the X in Drosophila. Genome Research  2002, 12:1854-1859.

2.   Bhattacharya A and Steward R: The Drosophila homolog of NTF-2, the nuclear transport factor-2, is essential for immune response. EMBO reports 2002,3:378-383. 

3.   http://www.marcsaric.de/doktor1.html

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