Sara Smolinski

 

Determination of Hdc Transcription Initiation in the Drosophila Genome Using 5-RACE.

The study of basic gene structure can allow the identification of unique regulatory mechanisms involved in the expression of a gene.  Histidine decarboxylase (Hdc) is an enzyme that synthesizes histamine, the neurotransmitter used by photoreceptors in Drosophila melanogaster and many other invertebrates.  Mutations in the Hdc gene cause flies to be functionally blind and exhibit other behavioral deficits, indicating that histamine may be used by a variety of processes in the fly.  While the sequence of four different Hdc mutants has recently been elucidated, the 5' and 3' ends of the Hdc transcript have not been conclusively identified.  To complete the molecular structure of the Hdc gene, both the 5 and 3 ends of the transcript need to be identified.  To map the 5 end of the transcript, a 5 RACE system (Rapid Amplification of cDNA Ends) was used to determine the transcription initiation site of the Hdc gene.  Focusing on previously identified initiation sites established by several groups, three primers were developed to obtain the 5' RACE product through reverse transcription and two ensuing polymerase chain reaction (PCR) steps.  Isolation of mRNA from fly heads of the Oregon-R wild-type strain was accomplished and used as the template for these 5-RACE reactions.  The product of the final PCR step was determined to be 470 base pairs (bp) in length, suggesting that only one transcription initiation site for this gene exists.  The length of this fragment suggests also that transcription initiation occurs in the general area that was earlier predicted.  This 470 bp fragment is currently being cloned, after which it will be subjected to sequence analysis, which should indicate where transcription initiates in the Hdc gene. 

Faculty Mentor: Martin Burg

Page last modified July 14, 2009