Allelic Variation in
Biomphalaria glabrata
Jordan Peterschmidt
Dr. Michael Blouin
Zoology Department
Background
 Biomphalaria
glabrata
◦ Vector for Schistosoma
◦ Freshwater snail
◦ South America and Africa
◦ 1316R inbred lines
Background
 Schistosomiasis
◦ Chronic, tropical disease
◦ 300,000 deaths per year
◦ Infects over 2 million
◦ Second greatest socioeconomically
devastating parasitic disease
◦ Effects
 Schistosoma
mansoni
 Resistance
in snails
◦ Cu/Zn Superoxide dismutase (SOD1)
◦ 2O2− + 2H+ → O2 + H2O2
◦ Blood cells: Hemocytes
◦ Encapsulate and respiratory burst
 Allelic
variation
◦ Three alleles in population: A, B, and C
 Allelic
variation
◦ Resistance
C <A < B
100
90
80
70
% individuals
not infected
60
50
40
30
20
10
0
Genotype
 Allelic
variation
◦ Three alleles in population: A, B, and C
◦ B allele over expressed
◦ Known to differ in size of first intron
 Allelic
variation
B allele ~4kbp
1st intron ~2kbp
A and C allele ~6kbp
1st intron ~4kbp
~2kbp
 Allelic
variation
B allele ~4kbp
1st intron ~2kbp
◦ Complete sequence of B allele known
A and C allele ~6kbp
1st intron ~4kbp
~2kbp
◦ A and C incomplete
Goal

Understand the functional differences
between alleles by:
◦ Completing full exon and intron
sequence of A and C alleles at SOD1
locus
Hypothesis

Alleles differ in:
◦ Exon sequence and/or
◦ Regulating properties of first intron
sequence in A and C alleles
Methods
DNA extraction
 Genotype individual
◦ Identified by indel within fourth intron

 Indel
◦ Insertion/deletion
# base pairs
A
B
C
C
B
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

Results
Sequenced entire A and C alleles at
SOD1 locus
◦ First intron incomplete
 Unable to amplify first intron in C allele

A allele
ex1
in1
Diagram Key
Hole in contig
Single strand
Double strand
ex2
in2
ex3 in3 ex4
n4
ex5
C allele
ex1
in1
Diagram Key
Hole in contig
Single strand
Double strand
ex2
in2
ex3 in3 ex4
in4
ex5
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
Discussion
 Exons
 Introns
Expect many differences
 Greatest difference in first intron
 Difficulty amplifying first intron in C
allele

Future work
 Complete
first intron
◦ Understand full extent of variation
 Analyze
first intron
◦ Examine for transcription regulating
factors that affect gene expression
Acknowledgements
Dr. Michael Blouin
 Kaitlin Bonner
 HHMI
 URISC
