A Knockout of a Caenorhabditis elegans Gene ZK563.2 and the Potential Relation to Human Fanconi Syndrome

Nicholas J. MatyiDepartment of Biology York College of PA

Project SummaryFanconi syndrome is a renal failure kidney disease that causes the body to lose amino acids, glucose, uric acid, phosphate and bicarbonates through urine (Fathallah-Shaykh, S.,2014). Phosphate in particular leads to rickets/osteomalacia (Fathallah-Shaykh, S., 2014). Current treatments involve kidney transplants, and taking more supplements that are being lost. Some of these supplements are phosphate, vitamin D, and citrate. Caenorhabditis elegans has a gene potentially linked to this on of the supplement losses cause by the disease. There is a possible connection to a gene called ZK563.2, which is a sodium phosphate cotransporter 2B. By using and Inductive Coupled Plasma with a Mass Spectrometer finding the difference in phosphorous levels between a mutant and a wild type will determine if the gene is linked to the disease. By linking this disease to c. elegans it could lead to finding a better treatment option that could cure us of this disease.

Hypothesis- The gene for a phosphate transport protein

2B (ZK563.2) is located in the excretory tract of Caenorhabditis elegans.

- The mutant c. elegans will have lower phosphorus levels than the wild type showing signs of Fanconi syndrome.

- When placed on a medium with enriched phosphate the levels phosphate loss of the mutant will return to normal levels since there will be more phosphate available for other channels. - Any change occurred to the mutants will

revert back the wild type when the mutants are placed on the phosphate enriched plate.

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Introduction- C. elegans is commonly used because they

contain common genes that are also found in humans.

- Online Mendelian Inheritance in Man (OMIM) has inferred that Fanconi syndrome may be linked to the c. elegans gene ZK563.2 a sodium phosphate cotransporter (Shaye, D.D. 2011).

- This has not been tested yet in this specie and lead to a better understanding of the disease. By knocking out the gene in c. elegans one will be able to determine if this gene is linked to this disease by causing phosphate loss.

- Low levels of manganese were measured by using a

laser ablation- Inductively Coupled Plasma Mass Spectrometer (LA-ICP-MS) to detect changes in c. elegans (Brinkhaus, S.G., 2014). Phosphorus can be tested as well since this protocol can detect low levels of elements.- The human antalog for this gene SLC34A1 has

been located in the kidneys (Larsson,M. 2011).- Klo-1 and klo-2 is a gene found in c. elegans that

is expressed in the excretory channel and these genes are associated with human diseases of hypophosmatemic

rickets (Polanska, U.M. 2011). - A renal tubular kidney disease failure (Bartter

disease) chloride channel gene (clh-1) has been knocked out and when placed onto a medium with enriched chloride c elegans regained normal body width (Petalcorin, M.I. 1999).

Review of Literature- SLC34A1 is a important factor in renal phosphate absorption which is found in humans and mice (Iwaki, T. 2008).- When this gene was turned off the

mice were not able to reabsorb phosphate effectively and suffered from hypophosphatemia and hyperphosphaturia (Iwaki,T. 2008). - When the Klo-1 and klo-2 genes were

knocked out defects in excretory channel development and function were observed (Polanska, U.M.2011).

- One observation that they found was that

due to an under expressed klo-1 (expressed in intestine and excretory) that late in the L1 stage and then dies due to a non-functioning excretory canal (Polanska,U.M. 2011). - A disease related to another renal

failure kidney disease was tested and found morphological changes occurred (Petalcorin, M.I. 1999).

- When these mutant worms were placed onto a plate with enriched chloride they regained normal structure. (Petalcorin, M.I. 1999).

- An ICP-MS was used to measure the amount of free iron in c. elegans (Pate, K.T. 2006).

- A more sophisticated LA-ICP-MS was used in another study to determine the differences in manganese (Brinkhaus, S.G., 2014).

- They created a new method and are able to measure manganese signals as low as 4m (Brinkhaus, S.G., 2014).

Transform E.coli OP50 bacteria with plasmid

containing the ZK563.2 gene

Grow transformed E.coli on an

ampicillin resistant plate then place c. elegans worms that are in the L4 stage

on

Record any changes that

occurred to the knockout worms

and observe expression of gene

with GFP.

Run an RTPCR to confirm

knockout in mutants

Run an LA-ICP-MS to

confirm lost of

phosphate

Place worms on a plate with enriched phosphate and

observe any physical changes and then run an LA-ICP-MS to see an increase in

phosphate pick up

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Research Design

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Theoretical Results

- The mutant worm will have lower phosphorous levels than the wild type worm due to the worm not being able to reabsorb some phosphate.

- The loss of phosphate is due to the knockout of ZK563.2 showing a link to Human Fanconi Syndrome since the lost of phosphate is a symptom of this diseaseWorks Cited

- Brinkhaus, S.G., Bornhorst, J., Chakroborty S., Wehe C.A., Niehaus, R., Reifschneider, O., Aschner, M. and Karst

U. 2014. Elemental bioimaging of manganese uptake in C. elegans. Metallomics. 6: 617-621- Fathallah-Shaykh, S. and Spitzer, A. Fanconi

Syndrom. Medscape http://emedicine.medscape.com/article/981774-overview

- Larsson, M., Morland, C., Poblete-Naredo, I., Biber, J., Danbolt, N.C., and Gundersen, V. The Sodium- Dependent Inorganic Phosphate Transporter SlC34A1 (NAPi-lla) Is Not Localized in the Mouse Brain. Journal of Histochemistry and cytochemistry. 59:807-812

- Iwaki, T., Sandoval-Cooper, M.J., Tenenhouse, H.S., and Castellino, F.J. 2008. A Missense Mutation in the Sodium Phosphate Co-transporter Slc34A1 Impairs Phosphate Homeostais. Journal of the American Society of Nephrology 19: 1753-1762

- Pate, K.T., Rangel, N.A., Fraser, B. Clement, M.H.S. and Srinivasan, C. 2006. Measruing “Free” Iron Levels in Caenorhabditis Elegans Using Low Temperature Fe(III) Electron Paramagnetic Resonance Spectroscopy. Anal BioChem 358:199-207

- Petalcorin, M.I.R., Oka, T., Koga, M., Ogura, K., Wada, Y., Ohshima, Y. and Futai, M. 1999. Disruption of clh-1, a chloride channel gene, results in a wider body of Caenorhabditis elegans Journal of Molcecular Biology. 294:347-355

- Polanska, U.M., Edwards, E., Fernig,D.G., and Kinneven, T.K. 2011. The Cooperation of FGF Receptor and Koltho is Involved in Excretory Channel Development and Regulation of Metabolic Homeostasis in Caneorhabditis elegans. Journal of Biological Chemistry 286:5657-5666

- Shaye, D.D. and Greenwald I. 2011. OrthoList: a compendium of C. elegans genes with human othologs. Plos One. 9:DOI: 10.1371/annotation/f5ffb738-a176-4a43-b0e0-249cdea45fe0

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AcknowledgmentsI would like to thank Dr. Kaltreider for his advice and help. I also appreciate all the knowledge my professors had provided me over my time here.

Create a plasmid by inserting the ZK563.2 sequence into a L4440

vector with GFP

Discussion

Control Mutant0.00

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Figure 2. Differences of phosphorous levelsin the control N2 wild type worm and ZK563.2

knockout after being placed on a plate withincreased phosphorous. Data is based on

theoretical numbers.

Control mutant0.00

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Figure 1. Based on theoretical dataphosphrorous levels from a control N2 wild typeworm and the ZK563.2 knockout worm.