Lookin’ Fly

gel extraction irl

We don’t always look this cool, but when we do, we’re doing a gel extraction.

Hey everyone, this is Connor McLoughlin (’17) and Rachel Wigmore (’18) and we’re working in Dr. Hiraizumi’s lab this summer. The main focus of our research is the genetic basis for the expression of a class of enzymes called dipeptidases which breaks down small peptides into amino acids. Why is this research relevant? In diseases such as Alzheimer’s, Crohn’s, and Celiacs, symptoms are correlated with low dipeptidase activity. By better understanding the genetics of dipeptidase expression, we may learn more about their connection to human diseases.

To accomplish this, we study the dipeptidase B gene (Dip-B) in Drosophila melanogaster, or the fruit fly, as a model system. Specifically we use the strains NC25 III and CL55; NC25III  has DIP-B enzymatic activity that is only one-tenth that of CL55.

We’re focusing on two possibilities for the difference in enzymatic activity between the strains. First, NC25 III produces fewer messenger RNA (mRNA) that is translated into protein. Second, the two strains produce the same number of enzyme molecules but DIP-B of NC25 III is catalytically less active than that of CL55. How do we address these possibilities?

For the first possibility, an efficient and practical method to quantify Dip-B mRNA transcripts was needed. To do this, a process called Reverse Transcription- Polymerase Chain Reaction was used to make many copies of cDNA (copy DNA) from mRNA. The quantity of cDNA produced is a function of the quantity of mRNA in the samples. To compare the samples, a quantitative densitometric assay was developed. The cDNA was subjected to agarose gel electrophoresis, which separates DNA fragments by size in a matrix of electrical gradient, with a stain specific for DNA. The separated cDNA is imaged using a ChemiDoc machine, which detects the stain intensity by a densitometric scan. Densitometric intensity is a function of DNA quantity. The protocol for the densitometric quantitative assay using an external standard is under refinement but results are promising, as shown below.

goodjel         Screen Shot 2017-06-09 at 2.12.47 PM

The second possibility for the difference in activity levels is that NC25III might have a less active DIP-B enzyme in comparison to CL55. Will Ueckermann, a former member of the research lab, obtained the coding sequence for Dip-B in CL55 and NC25III.  Compared to the gene sequence in the NCBI database, both strains contained SNPs or single base mutations. Will’s findings suggest that NC25III strain has a non-conservative missense mutation which changes the hydrophilic amino acid serine into a hydrophobic amino acid isoleucine. This change can very well affect the structure of the DIP-B enzyme molecule. We are sequencing more independent samples to confirm the sequence data, so stay tuned until the end of summer for further updates!

In eukaryotes (such as plants, fungi, Drosophila melanogaster, and humans), non-coding sequences (introns) are spliced out during transcription of DNA into mRNA. However, sometimes some introns are retained to generate different mRNA molecules (mRNA isoforms). This process, referred to as alternative splicing, is not unusual and Drosophila melanogaster is not an exception.

Dip-B gene has been reported to produce several mRNA isoforms. Four of the isoforms (A, B, C, and D) are documented in the NCBI database; our lab found evidence for two more isoforms called E, and A/C that are diagrammed below. What makes Dip-B different from conventional alternative splicing is that the coding region for all mRNA isoforms are identical, but the mRNA isoforms vary in the sequence that precedes the coding region called the 5’ Untranslated Region (5’-UTR).

isforms UTRs

Isoforms A and C contain identical transcription initiation site, but what makes them interesting is the presence of introns in their 5’-UTR. Presence of introns in the 5’UTR in eukaryotic genes is not  uncommon, with 4000/14000 Drosophila genes and 35% of human genes having this feature. The function of introns in 5′ UTR is unknown, but it sure would be interesting to learn more about them during research here!

We have performed many experiments involving PCR amplification of isoforms A and C and gel imaging of PCR products. There have been good days…

6-2 Actin Gelgreen invert 2

Actin is the external standard of Connor’s Quantitative Densitometric Assay, so we need a lot of pure actin, shown in this gel.

…and not so good days.

5-31-actin-gelgreen-redo-b.jpg

An attempt to image Isoforms A and C.

Our main goals for the rest of the summer include sequencing non-coding regions, specifically the 5′ UTR of the Dip-B gene, as well as to refine the protocol for a densitometric quantitative assay. We hope to isolate more Dip-B DNA to be sent out for sequencing to confirm the existence of the SNPs Will found.

 

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