Allelic Variation in Biomphalaria glabrata

1. Allelic Variation in Biomphalariaglabrata Jordan Peterschmidt Dr. Michael Blouin Zoology Department

2. Background • Biomphalaria glabrata • Vector for Schistosoma • Freshwater snail • South America and Africa • 1316R inbred lines

3. Background • Schistosomiasis • Chronic, tropical disease • 300,000 deaths per year • Infects over 2 million • Second greatest socioeconomically devastating parasitic disease • Effects

4. Schistosoma mansoni

5. Resistance in snails • Cu/Zn Superoxide dismutase (SOD1) • 2O2− + 2H+ → O2 + H2O2 • Blood cells: Hemocytes • Encapsulate and respiratory burst

6. Allelic variation • Three alleles in population: A, B, and C

7. 100 90 80 70 60 50 40 30 20 10 0 • Allelic variation • Resistance C < A < B % individuals not infected Genotype

8. Allelic variation • Three alleles in population: A, B, and C • B allele over expressed • Known to differ in size of first intron

9. B allele ~4kbp • Allelic variation 1stintron ~2kbp A and C allele ~6kbp 1stintron ~4kbp ~2kbp

10. B allele ~4kbp • Allelic variation • Complete sequence of B allele known • A and C incomplete 1stintron ~2kbp A and C allele ~6kbp 1stintron ~4kbp ~2kbp

11. Goal • Understand the functional differences between alleles by: • Completing full exon and intron sequence of A and C alleles at SOD1 locus

12. Hypothesis • Alleles differ in: • Exon sequence and/or • Regulating properties of first intron sequence in A and C alleles

13. Methods • DNA extraction • Genotype individual • Identified by indel within fourth intron

14. Indel • Insertion/deletion # base pairs A B C B C

15. Methods • DNA extraction from head-foot • Genotyped individuals • Identified by insertion within fourth intron • Designed primers from known B sequence • PCR and long PCR • Sequence and align PCR products

16. Results • Sequenced entire A and C alleles at SOD1 locus • First intron incomplete • Unable to amplify first intron in C allele

17. A allele ex1 in1 ex2 in2 ex3 in3 ex4 n4 ex5 Diagram Key Hole in contig Single strand Double strand

18. C allele ex1 in1 ex2 in2 ex3 in3 ex4 in4 ex5 Diagram Key Hole in contig Single strand Double strand

19. Coding sequence EXON 1: B ATGGTCAAAGCAGTTTGCGTTTTAAGTCCAGGCTCTGGAACTGGCATTACTGGAACCATTACTTTTACCCAGGAA A .....................................C..................................... C .....................................C..................................... M V K A V C V L S P G S G T G I T G T I T F T Q E Glycine to Alalnine EXON 2: B AAAGCAGGGGACTGTACAGTGGTTTCTGGTAAAGTATCAGGCCTTGCTCCAGGTAATCATGGATTTCATATCCATCAATTTGGTGACTACTCAAATG A ................................................................................................. C ........A..............................................................T......................... K A G D C T V V S G K V S G L A P G N H G F H I H Q F G D Y S N EXON 3: B GGGTGTATTAGTGCAGGTGCTCACTTTAACCCAGCAAACAAGAATCATGGAGGCCCTTGTGATACAGAAAG A ..C..............C..................................................... C ..C..............C..................................................... G C I S A G A H F N P A N K N H G G P C D T E EXON 4: B AGACATGTTGGTGATTTAGGTAACATTGTGGCTGGTGATGATGGTGTTGCTGATGTTAGTATCAAAGATCAACAAATTTCTTTGATTGGAGAAAATTCTATCATTGGTCGCAGCTTAGTG A ........................................................................................................................ C .....................................................C..........................A....................................... R H V G D L G N I V A G D D G V A D V S I K D Q Q I S L I G E N S I I G R S L V EXON 5: B GTGCATGATAAAGAGGATGATCTCGGCAAGGGAGGCAATGAAGAAAGTCTTAAAACAGGAAATGCTGGACCTCGCTTAGCTTGTGGTGTCATTGGTATCACTGTGTAA A ................................................................................. C ................................................................................. V H D K E D D L G K G G N E E S L K T G N A G P R L A C G V I G I T V V

20. Discussion • Exons • Introns • Expect many differences • Greatest difference in first intron • Difficulty amplifying first intron in C allele

21. Future work • Complete first intron • Understand full extent of variation • Analyze first intron • Examine for transcription regulating factors that affect gene expression

22. Acknowledgements • Dr. Michael Blouin • Kaitlin Bonner • HHMI • URISC