A New Brood

Hello again! I am Will Ueckermann, and this is the account of how I have spent my second summer in the Hiraizumi lab so far. This summer’s research has been a continuation of last summer’s, where I am working on a few projects. Our research focus is on the functional and sequence variation of the 5′ untranslated regions (5’UTRs) of genes in Drosophila melanogaster, the common fruit fly. Our particular focus is on the Dipeptidase B gene (Dip-B), which codes for one of the three dipeptidases found in D. melanogaster. Dipeptidases hydrolyze dipeptides, compounds that consist of two amino acids bound together. They are found in all organisms, from bacteria to humans, making them a good model for examining mechanisms of gene regulation. While 5’ UTRs do not code for any part of the protein, they are still very important due to their impact in protein translation. The DNA sequence within 5’UTRs can contain regions referred to as pseudointrons that are often spliced out. Although the study of pseudointrons is still in its infancy, it is known that 35% of all human transcripts have them.

We use two strains of D. melanogaster in our lab: CL55 and NC25III. CL55 exhibits a wild-type level of DIP-B enzymatic activity while NC25III expresses significantly low enzymatic activity. The ongoing project for the past few summers has been to determine the molecular, biochemical, and genetic basis for this difference.

IMG_0497

One of the bottles used to maintain a population of a fly strain.

My experiments utilize three general procedures: nucleotide extraction and purification, polymerase chain reaction (PCR), and gel electrophoresis. Nucleotide extraction is the isolation of DNA or RNA from samples of specific fly strains. This entails collecting a certain number of flies, grinding them up into a slurry, rupturing their cells and then cleaning out all of the other cell fragments, leaving only DNA or RNA in the prepared sample. The nucleotide samples can serve as material for several experiments involving PCR.

IMG_0498

Anesthetized flies, ready for sorting for nucleotide extraction.

PCR is used to amplify a specific subset of the nucleotide sample which represents a nucleotide sequence that can be specifically studied, such as the coding sequence of a particular gene. The procedure involves a pair of primers to target the desired sequence to be amplified, and a DNA polymerase enzyme to synthesize a new sequence defined by the boundaries of those primers. In effect, by repetitive cycling through the targeted synthesis reactions, millions of copies of the desired DNA sequence can be generated.

IMG_0496

A thermocycler used for PCR, with the lid open, ready for an experiment.

Gel electrophoresis of the PCR products is conducted to confirm fidelity of the reaction and presence of the predicted products. This involves subjecting the nucleic acid sample to an electrical gradient through a flat agarose matrix. Because DNA is negatively charged under electrophoresis conditions, molecules migrate through the pores of the agarose gel as a function of their size. DNA molecules are stained with a fluorescent dye and bands of differing sizes can be visualized against DNA reference markers of known size.

Gel Chamber

Electrophoretic separation of PCR products in progress. The orange dye front migrates ahead of all nucleotide samples and serves as a visual indicator for completion of electrophoresis.

Perhaps our most exciting progress is the discovery of dipeptidase B mRNA isoform E. When the Dip B gene is transcribed into messenger RNA, four mRNA isoforms that differ in sequence and length are produced: isoforms A, B, C, D. Isoforms B and D result from different sites for initiation of transcription, while isoforms A and C share the same transcription initiation site. The only difference between isoforms A and C is the size of their pseudointrons that are spliced out to produce these mRNAs. These two isoforms have the same 5’ splice junction for the pseudointronic splicing, but they differ in the location of the 3’ splice junction, leading to differences in molecular lengths that can be detected and identified by agarose gel electrophoresis.

isoform-layout

Differences in size and initiation site for the five Dip-B mRNA isoforms.

The pursuit of isoform E began last summer was based on an observation of an unexpected PCR product generated by a previous student who had designed a PCR primer combination to amplify portions of isoforms A and C. In addition to the two predicted PCR products, two other molecules were generated, one of which was unspliced RNA, perhaps a pre-mRNA for isoforms A and C. However, there was another PCR product that was larger in size than the other two isoforms, but smaller than the unspliced RNA. After hypothesizing that this may have been an uncharacterized and unreported isoform, I began designing primers to specifically amplify it from samples of Drosophila mRNA. Working from the assumption that the possible isoform shared its 5’ splice junction with isoforms A and C, I designed a series of primers that bridged the gap defined by the pseudointron sequence, which would specifically amplify only this putative isoform. After several candidate primers were tested, one primer was found that generated a PCR product from total mRNA in the same size range as it was designed to. This was our first real support for the presence of isoform E.

Fast forward to this summer, and we are now working to isolate isoform E by PCR amplification so that the resulting product could be sequenced. The first set of PCR experiments was completed last week to confirm the repeatability of last year’s results and to eliminate the possibility of genetic contamination. The experiments were also conducted to see if the putative isoform was present in both males and females of both strains. While each PCR experiment did yield a product, there were some interesting results, such as PCR products from female samples being smaller than those from male samples. The difference was repeatable under varying electrophoretic conditions. In order to determine the nature of this difference, the PCR products will need to be sequenced.

Another project that we have been working on is the isolation of the gene sequence of Dip-B from genomic DNA of CL55 and NC25III. Numerous attempts to PCR amplify the genomic gene sequence were made over the course of a year, involving many primer combinations with different DNA polymerases. This summer, I finally seem to have found a primer combination that can amplify the entire gene sequence from genomic DNA using Taq DNA polymerase. Interestingly, the Dip-B gene sequence for NC25III seems to be smaller than that of CL55, suggesting a deletion within the Dip-B gene for NC25III. To determine if the deletion is associated with the exonic or coding sequence, these PCR products will be isolated and sequenced.

gel_image

Gel image of PCR amplified Dip-B gene sequence from genomic DNA.  Lanes one and three correspond to CL55 PCR product, and lanes two and four correspond to that of NC25III.

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