Cloning our way to a perfect sequence

Kelsey Coates, Duquesne University

The Approach: In my first blog post, “FROM FEMALE TO MALE – MUD SNAILS TELL ALL!,” I described the goal of my research, to sequence isoforms of a hormone receptor called the Retinoid X Receptor (RXR) in the eastern mud snail.  

Mud snails all over a beach at Fort Johnson, SC.

These isoforms have yet to be sequenced in the mud snail! But what exactly is a DNA sequence? DNA is made of building blocks called nucleotides. A DNA sequence is the order of the nucleotides. A sequence like ACG could tell the organisms’ body to do one thing while a sequence like AGC could tell the organisms’ body to do another. A bit of the sequence has already been identified, but there is a gap in the sequence we are still trying to figure out.  

Theoretically, different chemicals or different concentrations of the same chemical can change the relative levels of the RXR isoforms. If this hypothesis is confirmed, mud snails can be used in the future to detect contaminants that affect marine organisms in the Charleston Harbor. Their patterns of isoform expression might suggest which seasonal contaminants are present in the environment where they live. For example, chemical one may trigger isoform A which has sequence ACG while chemical two may trigger isoform B which has sequence AGC.            

So how will we get these sequences? It starts with amplifying the known sequence of the mud snail that surrounds the isoform, including the mysterious gap. Amplification will be done by polymerase chain reaction (PCR) to ensure there are thousands of copies of the DNA to work with. After purification, the sequence is ready to be incorporated into a plasmid along with an antibiotic resistance component. Bacteria, like E. Coli, store their DNA in plasmid form compared to the double-helix form of humans.

Plates of E.Coli in the presence of Ampicillin set in the incubator.

Luckily for us, plasmids are easily manipulated and are reproduced rapidly in bacteria. E. Coli will be grown in the presence of the antibiotic ampicillin with the sequence we cloned into its DNA.  If the sequence is incorporated into the plasmid, the bacteria will have anti-biotic resistance and be able to grow on the ampicillin plates. The bacterial colonies with our plasmid will be PCR amplified. Then, after a final plasmid preparation, the samples from E. Coli can be sent to a lab that specializes in sequencing. Hopefully the lab will identify the gap and we will achieve our goal!

ACKNOWLEDGEMENTS

I would like to acknowledge Dr. Demetri Spyropoulos, Edwina Mathis, Dr. Bob Podolsky, The Fort Johnson REU Program, The Hollings Marine Lab, NOAA, and The Grice Marine Lab. This research was supported by the Fort Johnson REU Program, NSF DBI-1757899.

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Stirring up the sediment, are we opening Pandora’s box?

Samera Mulatu, Georgia Southern University

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The Approach: Would you believe if I told you that animals in the Charleston harbor are changing from female to male?! This process, known as imposex, occurs in marine snails when females develop male sex traits because they are exposed to harmful chemicals. One of my main goals in this project is to measure the rates of imposex in the Eastern mud snail (Tritia obsoleta, previously known as Ilyanassa obsoleta) within the Charleston Harbor to see if these rates increase over time due to the dredging of the harbor. There is a plan to begin dredging the Harbor later this fall, and the idea is that dredging will bring harmful chemicals in the sediment up into the water column. The data I am collecting now will be the imposex rates of the mud snail before the dredging brings up any harmful chemicals buried in the sediment of the harbor. However, we aren’t just collecting a bunch of snails and waiting for them to change sexes! No, there’s so much more to it than that!

As mentioned in my previous post, disruption of the Retinoid X Receptor (RXR) gene pathway is known to be central to inducing imposex in mud snails. By studying RXR we could learn a lot about what chemicals and how much of them are needed to induce imposex. However, the RXR gene for Tritia obsoleta has never been sequenced! So the first task in this project was to find the most closely related snails to the mud snail whose RXR sequences were already known. Primers were then designed based on these related RXR genes of known species. After this, mud snails were collected from the Charleston Harbor. 50 mud snails were collected that had a shell size of greater than 12 mM in height (to ensure that we were only using adults). The mud snails were dissected, and from different dissected parts RNA was then extracted to retrieve messenger RNA (mRNA). The mRNA was then reverse transcribed with reverse transcriptase enzyme into cDNA (‘reverse’ because DNA is usually transcribed into mRNA). The cDNA library generated represents all of the mRNAs in the mud snail tissue. The cDNA was then PCR amplified using the RXR-specific primers described above. Once the PCR products were obtained, they were column purified and sent off for sequencing!

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I was preparing the primers for purification. Picture taken by: Cheldina Jean

Once the mud snail RXR sequences are retrieved, we will distinguish them into the two types of RXR gene forms, isoforms a and b. Designing new primers specific to these RXR isoforms, we can determine the relative abundance of each isoform based on chemical (i.e. TBT, DOSS, or SPAN 80) exposure in the lab using adult females. Hopefully, my results will contribute to a better understanding of what effect the dredging of the harbor will have on imposex rates of the mud snail. Furthermore, if we see that dredging is harmful to mud snails, it is probably not healthy for consumable seafood and people, as well. Something that may be considered when making future plans of dredging not only in the Charleston Harbor but other waterways as well.

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Extracting the RNA of the mud snails. Picture taken by Samera Mulatu

I would like to give a big thank to Dr. Demetri Spyropoulos for guiding me in my research. Also to the Fort Johnson REU Program, NSF DBI- 1757899, for providing me with the funds to complete this project.

Related research

Hotchkiss, A.K, A.G.Leblanc, R.M. Sternberg. 2002. Synchronized expression of Retinoid X Receptor mRNA with Reproductive Tract Recrudescence in an Imposex- Susceptible Mollusc. Environ. Sci Technol. 42: 1345- 1351.

Ravitchandirane, V. S, M.Thangaraj. 2013. Phylogenetic Status of Babylonia Zeylanica (Family Babyloniidae) Based on 18S rRNA GENE FRAGMENT.Annals of West University of Timisoara, ser. Biology. 1(2): 135- 140.

Barron- Vivanco, B.S, D. Dominguez- Ojeda, I.M. Medina- Diaz, A.E. Rojas- Garcia, M.L. Robledo- Marenco. 2014. Exposure to tributyltin chloride induces penis and vas deferns development and increases RXR expression in females of the purple snail (Plicopurpura pansa). Invertebrate Survival Journal. 11: 204-2012.

Horiguchi, T., M. Morita, T. Nishikawa, Y. Ohta, H. Shiraishi. 2007. Retinoid X Receptor gene expression and protein content in tissues of the rock shell Thais clavigeraAquatic Toxicology. 84: 379-388.