Transcript Document

Research Experience in Molecular Biotechnology & Genomics
Summer 2007
Center for Integrated Animal Genomics
Genetic diversity of the antiviral Mx gene in 14 diverse chicken lines
Patricia Davison1, Michael Kaiser2 and Susan J. Lamont2
1 Coe
College, Cedar Rapids IA, 2 Animal Science, Iowa State University, Ames IA
Objectives
Results
• Evaluate biodiversity of the Mx gene in 14 diverse chicken lines.
• Predict amino acid and protein variations in the Mx gene amplicon.
• PCR produced 330 base pairs of sequence, 230 bp of reliable sequence.
Abstract
•The first SNP at bp 131 of the amplicon consensus sequence, with variation
among lines (Tables 1, 2).
• Poly T chain of variable length and two SNPs (Fig. 1, Table 1).
• Aligns with Ser631Asn functional SNP (Ko et al., 2002; Tables 1, 2).
A natural method of combating Avian Influenza has been previously
reported in the form of a Ser631Asn mutation in the chicken Mx gene,
where the Asn allele confers viral resistance. The Mx gene of 14 unique,
genetically diverse chicken lines was PCR amplified and sequenced. The
sequences were aligned and compared to a reference sequence of Red jungle
fowl. Two SNPs were found between lines. The first SNP was determined to
be Ser631Asn SNP and was polymorphic among the ISU chicken lines as
well as within the HN(12,12) line. The second SNP was a silent mutation
found across lines. The polymorphism of the Ser631Asn SNP among the
chicken lines opens up future studies in phenotypic trials for antiviral
resistance.
• Sequence chromatograms of 8(15.1,15.1) line suggested polymorphism
in Ser631Asn SNP. Restriction enzyme digest with Hyp8I was used to
confirm homozygous Asn allele (Figs. 3, 4).
• The broilers were also cut with Hpy8I. Five samples were cut twice.
One sample was cut once (Fig. 6). One cut site is at Ser631Asn (Fig. 1).
• The Ser631Asn SNP is polymorphic in the HN(12,12) line.
Heterozygosity seen on sequence chromatograph and restriction
enzyme digest results for two individuals. One individual was
homozygous for the viral resistance allele (Figs. 5, 6).
Broiler Male
Leghorn Female
Spanish Female
Fayoumi Female
• The second SNP is a silent mutation at bp 258 of the amplicon consensus
sequence and aligns to 2159 SNP (Ko et al., 2002).
Introduction
Table 1: SNP genotype, Linkage Phase of Mx 13th Exon of Chicken Lines
• Avian influenza is a major concern to the poultry industry.
• Estimated losses of 200 million birds since 2003 from H5N1 viral strain
(MSNBC, 2007).
• Worries of human outbreak of H5N1 persist (CDC, 2007) with over 270
human cases since 2003 worldwide (MSNBC, 2007).
•The interferon induced Mx gene has been associated with viral resistance
in chickens (Ko et al., 2002; Fig 2).
• A single nucleotide polymorphism (SNP) in Mx produces a
acid Ser to Asn substitution (Ko et al., 2002).
• The Asn
Red Jungle Fowl (1)
Broiler
GHs(13,13)
GHs(6,6)
M(15.2,15.2)
M(5.1,5.1)
19(15.1,15.1)
Poly T (12th
intron)
- TTTTTTTTT
- TTTTTTTTT
- - - TTTTTTT
- TTTTTTTTT
- TTTTTTTTT
- TTTTTTTTT
TTTTTTTTTT
19(13,13)
Sp(21.1,21.1)
GH(1,1)
GH(15.1,15.1)
GH(13,13)
HN(15,15)
HN(12,12)
8(15.1,15.1)
Consensus
TTTTTTTTTT
- TTTTTTTTT
- - - TTTTTTT
- - - TTTTTTT
- - - TTTTTTT
- TTTTTTTTT
- TTTTTTTTT
- TTTTTTTTT
TTTTTTTTTT
Line
631st
amino
allele confers resistance to avian influenza virus.
• A section of the Mx gene’s 13th exon (Fig 1) of 14 genetically diverse
chicken lines were sequenced.
• The sequences were scrutinized for the presence of SNPs, including the
functional mutation.
Figure 2. Mx gene activation and activity
Viral infection
Poly T Functional SNP
type
bp 131
0
CAAGTAA
0
CAAGTAA
-2
CAAATAA
0
CAAATAA
0
CAAGTAA
0
CAAGTAA
+1
CAAGTAA
+1
0
-2
-2
-2
0
0
0
+1
CAAGTAA
CAAATAA
CAAATAA
CAAATAA
CAAATAA
CAAGTAA
CAARTAA
CAAATAA
CAARTAA
Silent SNP
bp 258
CTCCAGG
CTCCAGG
CTCCAGG
CTCCAGG
CTCCAGG
CTCCAGG
CTCCAGG
Linkage
phase
0GG
0GG
-2AG
0AG
0GG
0GG
+1GG
CTCCAGG
CTCCAGG
CTCCAAG
CTCCAAG
CTCCAAG
CTCCAGG
CTCCAGG
CTCCAAG
CTCCARG
+1GG
0AG
-2AA
-2AA
-2AA
0GG
0RG
0AA
n/a
Poly T Type: 0= 9 Ts; +1= 10 Ts; -2= 7 Ts
Figure 3. Putative polymorphism
of silent SNP in 8(15.1,15.1)
Figure 4. BpmI restriction enzyme digest
of silent Mx SNP for selected chicken lines
- Level of response depends on amount of virus present
Lane assignment: 1= undigested amplicon; 2-4= HN(15,15)- G, cut; 5-7=
GH (13,13)- A, not cut; 8-10=Line 8(15.1,15.1), not cut; Line 8 putative
SNP.
Mx gene activated
Mx gene in chickens- GTPase activity
Figure 5. Heterozygous
Ser631Asn SNP in HN(12,12)
Figure 6. Hpy8I restriction enzyme digest of
functional Mx SNP of selected chicken lines
Outcome
• The PCR amplicon was quality tested on a 1.5% agarose gel, purified
with ExoSapIT and sequenced at the ISU DNA Facility.
• Sequence data was processed with Seaquencher and aligned with BioEdit.
• Restriction enzyme digests with BpmI and Hpy8I were used to investigate
possible SNPs.
• Digestion patterns were evaluated via 1.5% agarose gel.
• Chicken lines with the A allele at the functional mutation site at bp 131
code for the amino acid Asn instead of Ser (Tables 1, 2).
•This mutation has been previously shown to confer resistance to
viruses, such as avian influenza (Ko et al., 2002).
• Allelic variation of Mx gene opens possibility of viral resistance in
some of these chicken lines.
• May be unresolved PCR or sequencing artifacts.
• May be a genuine line difference, supported by congenic line set
consensus of Poly T region.
• Explanations of errant polymorphism in HN(12,12): DNA sample
contamination or sampling, record keeping or mating error.
• Ser631Asn polymorphism in HN(12,12) is not experimental error
because of agreement among: sequence chromatographs, restriction
enzyme results, and SNP Chip database.
• The second broiler RE recognition site undetected in sequence data.
References
Materials and Methods
• PCR amplification of the Mx 13th exon, from 12th intron to the 3’ UTR.
Forward primer: GAATAGCAACTCCATACCGTG (Livant et al.
2007). The reverse primer was developed by E. Livant and S. Ewald
of Auburn University and shared with us. The exact sequence will be
released after they have completed their studies with this primer.
Discussion
• Second cut site is probably at the end of the sequence, where it’s poor
quality. Fits the small fragment that couldn’t be seen on gel (Fig. 6).
-Inhibition of viral infection and replication
•Inbred chicken lines (99% inbred); 1 Spanish, 2 Fayoumi (Egyptian),
10 Leghorn lines. Broiler line is outbred (Zhou and Lamont 1999).
• SNPs and poly T chain are in linkage phase in congenic line sets (Table 1).
• HN(12,12) birds are inbred full sibs; Polymorphisms are unexpected.
Interferons produced by infected cell
• Purified DNA of 3 individuals from each of 13 inbred lines and 6
broiler individuals.
• Illumina SNP data show that HN(12,12) line has heterozygotes in 5 of 12
SNPs in a 2 mb section on each side of the Mx gene (data not shown).
• The Poly T variable length is a phenomenon noted in Livant et al. (2007).
-influenza, VSV, etc
- Protein found mainly in cytoplasm
- At least one allele has antiviral activity
• Sequence chromatorgams of 8(15.1,15.1) also suggested polymorphism
for the silent SNP. BpmI restriction enzyme digestion confirmed
homozygous A allele (Figs. 3, 4).
Lane assignment: 1= undigested amplicon; 2-4= GHs(6,6)- A, uncut; 5-7=
19(15.1,15.1)- G, cut; 8-10= Line 8(15.1,15.1)- uncut; 11-13= HN(12,12)- 11
and 13: heterozygous, 12: uncut; 14-19= Broilers: 14,15,17,18,19- cut twice;
16- cut once.
(1)- Red Jungle Fowl sequence obtained from Ensembl.
http://www.ensembl.org/Gallus_gallus/geneview?gene=ENSGALG00000016142#ENSGALT00
000025999.
Table 2: Predicted Amino acid sequence and 631 amino acids of Mx gene 13th
exon amplicon of chicken lines
Key Facts About Avian Influenza (Bird Flu) and Avian Influenza A (H5N1) Virus. 2007.
http://www.cdc.gov/flu/avian/gen-info/facts.htm. accessed 6/11/07.
Line
Red Jungle
Fowl (1)
Broiler
19(15.1,15.1)
GHs(13,13)
GHs(6,6)
M(15.2,15.2)
M(5.1,5.1)
19(13,13)
Sp(21.1,21.1)
GH(1,1)
GH(15.1,15.1)
GH(13,13)
HN(15,15)
HN(12,12)
8(15.1,15.1)
Ko,et.al. 2002. Genome Res. 12: 595-601.
Amino acid sequence
GASKRLSNQIPLIILSTVLHDFGNYLQTSMLHLLQGKEEINYLLQEDHEAANQQK
Livant, et.al. 2007. An. Gen. 38: 177-179.
--S----------------------------------------------------------S----------------------------------------------------------N----------------------------------------------------------N----------------------------------------------------------S----------------------------------------------------------S----------------------------------------------------------S----------------------------------------------------------N----------------------------------------------------------N----------------------------------------------------------N----------------------------------------------------------N----------------------------------------------------------S----------------------------------------------------------X----------------------------------------------------------N---------------------------------------------------------
No sign of deadly avian flu found in U.S.: U.S. Geological Survey detects only mild strain of
virus in 75,000 wild birds. 2007. http://www.msnbc.msn.com/id/17169305/. Accessed 7/25/07.
Questions and Answers About Avian Influenza (Bird Flu) and Avian Influenza A (H5N1) Virus.
2007. http://www.cdc.gov/flu/avian/gen-info/qa.htm. accessed 6/11/07.
Zhou, H. and S.J. Lamont. 1999. An. Gen. 30: 256-264.
Acknowledgements
Advice about technical procedures and downstream primer sequence was generously provided by E. Livant
and S. Ewald of Auburn University. Sara Beth Sherrill, Jason Hasenstein and Jennifer Cheeseman for
advice and help. Dr. Rothschild’s lab group, for the use of the computer and Seaquencher program.
Author contacts: Patricia Davison- [email protected]. Susan Lamont: [email protected].
S= Ser; N= Asn; X= polymorphic
Program supported by the National Science Foundation Research Experience for Undergraduates
DBI-0552371