Immunogene ics (2004) 56: 170–177
DOI 10.1007/s00251-004-0675-0
ORIGINAL PAPER
Da id J. Lynn .Rowan Higgs .Susan Gaines .
Joanna Tie ney .Tha appel James .And ew T. Lloyd .
Ma io A. Fa es .G ace Mulcahy .Cliona O’Fa elly
Bioin o ma ic disco e y and ini ial cha ac e isa ion o nine no el
an imic obial pep ide genes in he chicken
Recei ed: 30 Janua y 2004 / Re ised: 22 Ma ch 2004 / Accep ed: 22 Ma ch 2004 / Published online: 18 May 2004
#Sp inge -Ve lag 2004
Abs ac An imic obial pep ides (AMPs) a e essen ial
componen s o inna e immuni y in a ange o species
omD osophila o humans and a e gene ally hough o
ac by dis up ing he memb ane in eg i y o mic obes. In
o de o disco e no el AMPs in he chicken, we ha e
implemen ed a bioin o ma ic app oach ha in ol es he
clus e ing o mo e han 420,000 chicken exp essed
sequence ags (ESTs). Simila i y sea ching o p o eins—
p edic ed o be encoded by hese EST clus e s— o
homology o known AMPs has esul ed in he in silico
iden i ica ion o ull-leng h sequences o se en no el
gallinacins (Gal-4 o Gal-10), a no el ca helicidin and a
no el li e -exp essed an imic obial pep ide 2 (LEAP-2) in
he chicken. Di e en ial gene exp ession o hese no el
genes has been demons a ed ac oss a panel o chicken
issues. An e olu iona y analysis o he gallinacin amily
has de ec ed si es—p ima ily in he ma u e AMP— ha a e
unde posi i e selec ion in hese molecules. The unc ional
implica ions o hese esul s a e discussed.
Keywo ds Chicken .De ensins .An imic obial pep ide .
Inna e immune sys em
In oduc ion
An imic obial pep ides (AMPs), essen ial componen s o
inna e hos de ence in species as di e se as plan s, lies
and mammals a e gene ally hough o ac by dis up ing
he memb ane in eg i y o mic obes (Kagan e al. 1990;
Sa chell e al. 2003). In an age when an ibio ic esis ance
is an inc easing p oblem, hese pep ides a e o in e es as
po en ial no el pha maceu ical agen s. In e eb a es, he e
a e wo majo amilies o AMPs: de ensins and ca he-
licidins. In mammals, α-de ensins and β-de ensins a e wo
s uc u ally dis inc ca ionic, cys eine- ich AMPs, which
di e in size and in he spacing o a six-cys eine s uc u al
mo i (Liu e al. 1997). α-de ensins a e unique o
mammals, bu β-de ensins a e much mo e widely
dis ibu ed and, in he chicken, ou β-de ensins, known
as gallinacins, ha e been desc ibed o da e (E ans e al.
1994; Ha wig e al. 1994; Zhao e al. 2001). Homologous
pep ides ha e also been desc ibed in he u key (E ans e
al. 1994; Zhao e al. 2001). Gal-1, Gal-1αand Gal-2 we e
isola ed om chicken he e ophils, while Gal-3 was shown
o be cons i u i ely exp essed in he epi helia o a ange o
issues and o be inducible in he achea ollowing
in ec ion (Zhao e al. 2001). Gallinacins exhibi a iable
ac i i y agains a numbe o G am-posi i e and G am-
nega i e bac e ia, and Gal-1 and Gal-1αa e ac i e agains
he yeas Candida albicans (E ans e al. 1995; Ha mon
1998).
Ca helicidins a e a amily o highly di e se AMPs bu
a e all encoded by p ep opep ides con aining highly
D. J. Lynn .R. Higgs .S. Gaines .T. James .A. T. Lloyd .
C. O’Fa elly (*)
Educa ion and Resea ch Cen e, S . Vincen ’s Uni e si y
Hospi al,
4 Dublin, I eland
e-mail: [email p o ec ed]
Tel.: +353-1-2094940
Fax: +353-1-2838123
D. J. Lynn .R. Higgs
Depa men o Medicine, Uni e si y College Dublin,
4 Bel ield, Dublin, I eland
T. James
Moyne Ins i u e o P e en i e Medicine, T ini y College
Dublin,
2 Dublin, I eland
A. T. Lloyd
Depa men o Gene ics, T ini y College Dublin,
2 Dublin, I eland
J. Tie ney .G. Mulcahy
Depa men o Mic obiology and Pa asi ology, Facul y o
Ve e ina y Medicine, Uni e si y College Dublin,
4 Bel ield, Dublin, I eland
M. A. Fa es
Biology Depa men , Na ional Uni e si y o I eland,
Maynoo h, I eland
Conway Ins i u e, Uni e si y College Dublin,
4 Bel ield, Dublin, I eland
conse ed ca helin domains. To da e, ca helicidins ha e
only been desc ibed in mammals, including humans,
monkeys, ho ses, ca le, sheep, goa s, pigs, abbi s, mice
and guinea pig ( o e iew, see Ramana han e al. 2002).
Each species has a iable numbe s o ca helicidin genes;
a iodac yls in pa icula ha e high copy numbe s (Scocchi
e al. 1997), whe eas humans and mice ha e only one gene
copy each (Age be h e al. 1995; Cowland e al. 1995;
Gallo e al. 1997; La ick e al. 1995). Neu ophils a e a
pa icula ly ich sou ce o ca helicidins in a a ie y o
species. In humans, ca helicidins ha e also been ound o
be exp essed in se e al o he issues, including he es is
(Age be h e al. 1995; Malm e al. 2000), squamous
epi helia (F ohm Nilsson e al. 1999; Nize e al. 2001),
ai way epi helia (Bals e al. 1998), swea glands
(Mu akami e al. 2002b), sali a y glands (Mu akami e
al. 2002a) and colon (Hase e al. 2002). Ca helicidins ha e
a wide spec um o an imic obial ac i i y and ha e been
shown o be ac i e agains G am-nega i e and G am-
posi i e bac e ia (T a is e al. 2000), ungi (Shin e al.
2000) and en eloped i uses (Tamamu a e al. 1995).
Ac i i y agains a pa icula mic obe depends on he ype
o ma u e pep ide and he species om which i o igina es.
In his s udy, we desc ibe a bioin o ma ics app oach o
he iden i ica ion o no el AMPs in he chicken. This
me hod in ol es homology sea ching o clus e ed chicken
exp essed sequences ags (ESTs) by BLAST (Al schul e
al. 1997) and by he mo e sensi i e hidden Ma ko model
(HMM) p o ile sea ching (Eddy 1998). Ou app oach has
iden i ied eigh no el an imic obial pep ides, se en
gallinacins and one ca helicidin in he chicken. We show
ha all hese pep ides as well as li e -exp essed an imi-
c obial pep ide 2 (LEAP-2)—an AMP we ha e p e iously
iden i ied in chicken (Lynn e al. 2003)—a e exp essed a
he mRNA le el in a panel o chicken issues. Fu he -
mo e, an e olu iona y analysis o he gallinacin amily has
p o ided e idence ha ce ain amino acid si es in he
ac i e pep ide a e subjec o posi i e selec ion.
Ma e ials and me hods
F om he dbEST Web si e (h p://www.ncbi.nlm.nih.go /dbEST/),
422,426 chicken ESTs we e downloaded. P io o he clus e ing
s ep, epea sequences in he ESTs we e masked using Repea Mas-
ke (Smi and G een, unpublished), and con aminan sequences we e
emo ed using SeqClean (h p://www. ig .o g/ db/ gi/so wa e/) o
minimize alse clus e ing due o spu ious EST simila i y.
The ESTs we e hen clus e ed using The Ins i u e o Genomic
Resea ch Gene Indices clus e ing ools (TGICL) (Pe ea e al. 2003)
(a ailable om h p://www. ig .o g/ db/ gi/so wa e/). ESTs we e
clus e ed i hey sha ed mo e han 30 bp o a leas 95% iden i y. The
clus e ed con ig sequences a e a ailable a h p://www.bin .o g/
immunogene ics/con igs. a.
The sequences o each clus e we e pos -p ocessed wi h
ESTScan (Iseli e al. 1999). ESTScan de ec s and econs uc s
po en ial coding egions in ESTs, using a no el HMM me hod ha
can au oma ically co ec o ame-shi e o s. F om he coding
egions, he p edic ed p o ein can be de e mined (a ailable a h p://
www.bin .o g/immunogene ics/es scanp edic ions. a). A da abase o
known AMPs, as iden i ied by an SRS sea ch (h p://s s.ebi.ac.uk/)
o he Swissp o -T embl p o ein da abase was cons uc ed and is
a ailable a h p://e cbin o1.ucd.ie/APPDb/. All AMPs om his
da abase we e sea ched agains he clus e ed EST con igs and he
da abase o p o eins p edic ed by ESTScan om he EST clus e s
using he BLAST p og ams, wi h an E- alue cu -o o 0.001
(Al schul e al. 1997). Molecula weigh and ne cha ge o he
comple e chicken p o eins we e p edic ed using he P o Pa am ool
(h p://www.expasy.o g/ ools/p o pa am.h ml).
To sea ch o no el an imic obial p o eins ha could ha e been
missed by BLAST, we cons uc ed HMMs o a numbe o AMP
amilies. All he sequences anno a ed as ei he α-de ensins,
ca helicidins o hepcidins in he P am da abase (Ba eman e al.
2002)—a manually cu a ed and anno a ed collec ion o p o ein
amilies (h p://www.sange .ac.uk/So wa e/P am)—we e ex ac ed.
The gallinacin amily is no ep esen ed in he P am da abase, so all
known gallinacin sequences we e ex ac ed om he Na ional
Cen e o Bio echnology In o ma ion GenPep p o ein da abase.
These sequences included Gal-1 (P46156), Gal-1α(Q9DG59), Gal-
2 (P46158), Gal-3 (Q9DG58), u key he e ophil pep ide-1 (THP1)
(P80391), THP2 (P80392) and u key β-de ensin (TBD) (Q9DG57).
To c ea e an HMM p o ile o each amily, all cons i uen sequences
o AMPs we e aligned using he T-Co ee p og am (No edame e al.
2000), and hese alignmen s we e used as inpu o he HMMER,
e sion 2.1.1, sui e o p og ams (Eddy 1998) (h p://hmme .wus l.
edu/). The HMM o each amily was cons uc ed using he
Table 1 PCR p ime sequences and p edic ed p oduc leng hs
Ta ge mRNA
a
5′P ime 3′P ime P oduc
size (bp)
Gal-1 5′-GAAATGCTCAAGATTTCACCTCTG-3′5′-CCTTTATTCAGCAGAGAAAAGCAG-3′231
Gal-2 5′-GCATAAACACTTCATGAGTCCATC-3′5′-GAAGAAAGGCAGTGCAGAAGATA-3′166
Gal-3 5′-CCTTCTTCCTCTTGTTTCTCCAG-3′5′-ATCAACCTCATATGCTCTTCCAC-3′158
Gal-4 5′-GATCCTTTACCTGCTGCTGTCT-3′5′-TCCTCACACAGCAAGATTTTAGTC-3′185
Gal-5 5′-GATCCTTTACCTGCTGCTGTCT-3′5′-AGCAAGAGCCTATTCCATTGTTAC-3′176
Gal-6 5′-ATGAGAATCCTTTTCTTCCTTGTTGC-3′5′-TTAGGAGCTAGGTGCCCATTTGCAGC-3′201
Gal-7 5′-ATCGTGCTCCTCTTTGTGGCAGTTCA-3′5′-CTACAACCATCTACAGCAAGAATACT-3′171
Gal-8 5′-CTGTTCTCCTCTTCCTCTTCCAG-3′5′-AATCTTGGCACAGCAGTTTAACA-3′170
Gal-9 5′-ATGCAGATCCTGCCTCTCCTCTTTGCT-3′5′-TCAGGAATACCATCGGCTCCGGCAGCAGAA-3′201
Gal-10 5′-ATGAGGAACCTTTGTTTCGTGT-3′5′-TCAGGTCTTGGTGGGAGTTGGTG-3′198
LEAP-2 5′-CACCATGCACTGTTTGAAAATTATGGCA-3′5′-TCACTCGGAGGCCGTTCTAAGGAA-3′235
Ca helicidin 5′-CACCATGCTGAGCTGCTGGGTGCTGCTG-3′5′-TCACTTCTTCTTGATCGCCCGGTA-3′451
β-ac in 5′-GCGCTCGTTGTTGACA-3′5′-TCATCCCAGTTGGTGACA-3′206
a
Gal-Gallinacin, LEAP-2 li e -exp essed an imic obial pep ide 2
171
hmmbuild p og am, and hmmcalib a e was used o calib a e E- alue
sco es. The HMM p o iles we e hen used o sea ch agains he
da abase o p o eins p edic ed by ESTScan om he EST clus e s
using he hmmsea ch p og am.
E olu iona y analysis o he gallinacins
A mul iple-sequence alignmen o he gallinacin amily o AMPs
(including he no el sequences and he homologous u key
sequences) was cons uc ed using he T-Co ee p og am (No edame
e al. 2000). A neighbo -joining phylogene ic ee was in e ed om
he p o ein alignmen using MEGA, e sion 2.1, wi h he Poisson
co ec ed model implemen ed (Kuma e al. 2001). One housand
boo s ap eplica es we e ca ied ou o es he signi icance o each
node in he ee. To cons uc an alignmen o he coding sequences,
he p o ein alignmen was used as a empla e and a ‘copygaps’Pe l
sc ip was used o align he DNA, main aining he gaps ha we e
p esen in he p o ein alignmen . Any columns in he DNA
alignmen ha had mo e han h ee gap cha ac e s we e emo ed.
The opology o he neighbo -joining ee and he DNA alignmen
we e used as inpu o he CODEML and CODEMLSITES p og ams
om he PAML package, e sion 3.12 (Yang 1997), o es o
e idence o posi i e selec ion du ing he e olu ion o he
gallinacins.
The p inciple in ol ed in such es s is o compa e he a es o
synonymous (d
S
) and non-synonymous (amino acid changing: d
N
)
changes among he DNA sequences. I amino acid changes a e
selec i ely neu al (i.e. mu a ions ha a e nei he ad an ageous o
dele e ious), hey will be ixed a he same a e as synonymous
mu a ions and ω a io (d
N
/d
S
)=1. ω alues >1 a e aken o indica e
ha amino acid changes a e accumula ing a a as e a e han is
accep able unde a neu al mu a ion model. Tha is o say, he a e o
amino acid changes (d
N
) signi ican ly exceeds he a e o
synonymous changes (d
S
) a he DNA le el. The CODEML
p og am es s o a iable selec i e p essu es among lineages in
he phylogeny by looking o signi ican di e ences in ω a ios. To
es o a iable selec i e p essu es among phylogene ic lineages,
he one- a io model, which assumes an equal ω a io o all b anches
in he phylogeny, was compa ed o he ee- a ios model, which
allows an independen ω a io o each b anch (Yang 1998; Yang
and Nielsen 1998). The esul o his p og am is a log-likelihood
alue o each model. To es which is he a ou ed model, he log-
likelihood alues o each model a e compa ed by a likelihood a io
es (LRT). Twice he log-likelihood di e ence be ween he wo
models is compa ed o a χ
2
dis ibu ion wi h n−1d , whe e nis he
numbe o b anches o he phylogeny. I a signi ican P- alue is
ob ained, i can be concluded ha he ee- a ios model is he
a ou ed model, and b anches on he phylogeny wi h ω alues >1
a e subjec o posi i e selec ion.
Posi i e selec ion in amino acid si es
Ano he way o looking o posi i e selec ion is o look o
signi ican a iabili y in ω a ios among amino acid si es in he
mul iple-sequence alignmen (Nielsen and Yang 1998). The
CODEMLSITES p og am de e mines whe he any o six p og es-
si ely mo e complex models o e olu ion a e signi ican ly be e a
explaining he obse ed a ia ion in he da ase (Yang e al. 2000).
The i s es compa es he models M0 and M3. Model M0 is an
e olu iona y model whe eby all he amino acid si es ha e a single ω
alue. This model is compa ed o M3, which classi ies he amino
acid si es in o one o h ee classes, wi h he p opo ion o si es
belonging o a pa icula class and he ω alues o each class o si e
es ima ed by CODEMLSITES om he da a. M3 is a es o amino
acid si es subjec o posi i e selec ion, as i allows o he p esence
o si es wi h ω>1.
The second es compa es he models M1 and M2. M1 is a model
o neu al e olu ion whe e amino acid si es can be conse ed (ω=0)
o neu ally e ol ing (ω=1). Model M2 is a es o selec ion, as i
allows o he p esence o si es whe e ωis a ee pa ame e and as
such can ha e a alue >1.
The inal es , which compa es he models M7 and M8, is he
mos s ingen es . M7 allows o si es wi h ω alues ha ollow a
βdis ibu ion o alues be ween ω= 0 and ω=1. Model M8 is he
same as M7 bu allows o he p esence o si es wi h ω>1, and
compa ing hese wo models is a es o selec ion.
As wi h he es o posi i e selec ion among lineages, CODEML-
SITES es ima es a log-likelihood alue o each model. To es
which a e he a ou ed models, he log-likelihood alues o M0
e sus M3, M1 e sus M2 and M7 e sus M8 we e compa ed by
LRTs. Pos e io Bayesian p obabili ies we e calcula ed o de e mine
which amino si es belong o which si e classes (Nielsen and Yang
1998). I signi ican a iabili y is e ealed, hen hose si es, which
ha e ω>1 and high pos e io p obabili ies, a e likely o be unde
posi i e, di e si ying selec ion.
Exp ession o AMPs in chicken issue
One-day-old male chickens (Cobb 500 b oile ) we e pu chased om
he Knocknaga m Ha che y, hen housed in a loo pen in he
Biomedical Facili y, Uni e si y College Dublin, Bel ield, Dublin,
I eland. En i onmen al empe a u e was kep a a cons an 25°C.
Animals we e ed comme cial coccidios a - ee s a e /g owe a ion
and wa e ad lib. One bi d was sac i iced a 3 weeks o age by
in a enous pen oba bi one sodium inocula ion. The issues we e
quickly dissec ed, squeezed be ween Wha man il e pape o
emo e excess blood, insed in saline and snap ozen in liquid
ni ogen. Tissues we e s o ed a −80°C un il p ocessed u he .
Following pul e isa ion o he issues using a Mik o-Dismemb a o
U (B. B aun Bio ech In e na ional), o al cellula RNA was pu i ied
using he RNeasy Ki (Qiagen, Wes Sussex, UK) acco ding o he
manu ac u e ’s ecommenda ions. Spec ophome ic analysis was
pe o med in o de o assess he quan i y and quali y o o al RNA.
Single-s anded cDNA was syn hesised om 1 μg RNA using oligo-
dT p ime (P omega, Madison, Wis.) and Omnisc ip (Qiagen). The
AMP-speci ic cDNAs we e ampli ied by PCR using Taq polyme ase
(Qiagen) and p ime s designed in e nally om he coding sequence
o Gal-1 o Gal-10, LEAP-2, ca helicidin and β-ac in. Thi y cycles
(94°C o 30 s, 55°C o 30 s and 72°C o 30 s) we e used o
ampli ica ion. PCR p oduc s we e sepa a ed by elec opho esis on
e hidium b omide-s ained 2% aga ose gels and isualised using
Eagle Eye (S a agene, La Jolla, Cali .). A lis o PCR p ime
sequences and p oduc leng hs a e shown in Table 1.
cDNA cloning
Posi i e issues om he abo e exp ession panel we e chosen as
sou ce ma e ial o each speci ic gene. The cDNA was ampli ied as
desc ibed abo e bu using P u DNA polyme ase (P omega) and
gene-speci ic p ime s wi h CACC o e hangs ups eam o he s a
codon, hus p o iding he complemen a y sequence necessa y o
di ec ional cloning. The ampli ied cDNA was pu i ied (GenElu e
PCR Clean-up Ki , Sigma, S . Louis, Mo.), and liga ed in o he
pcDNA 3.1 cloning ec o (In i ogen, G oningen, The Ne he -
lands). Cloned plasmids we e sequenced (Ad anced Bio echnology
Cen e, London, UK), using ec o -speci ic p ime s and compa ed
o he EST consensus sequence.
Resul s
In he absence o comple e genome sequence, ESTs a e a
ich sou ce o no el sequence in o ma ion. By de ini ion,
ESTs a e sho , e o -p one sequences. Clus e ing o ESTs
ha a e likely o be encoded by he same mRNA educes
he edundancy in he EST da abase, imp o es he
172
sequence quali y and inc eases he sequence co e age o a
pa icula clus e .
By implemen ing a bioin o ma ic app oach ha in-
ol es he clus e ing o mo e han 420,000 chicken ESTs,
we ha e iden i ied eigh no el AMPs in he chicken.
Clus e ing o hese ESTs esul ed in he gene a ion o
34,819 chicken con igs and om his, 29,344 coding
sequences we e p edic ed. Gi en es ima es o he gene
numbe in human o 30,000 (Pennisi 2003), we expec o
ha e a leas pa ial sequence in o ma ion o mos chicken
genes. TBLASTN sea ches (which sea ches p o ein
que ies agains a nucleo ide da abase) o known AMPs
agains he clus e ed EST con igs iden i ied i e con igs
wi h homology o he β-de ensins, which we ha e named
Gal-4 o Gal-8. These sea ches also iden i ied a no el
chicken ca helicidin. BLASTP sea ches (which sea ches
p o ein que ies agains a p o ein da abase ansla ed in all
six eading ames) o p o eins, p edic ed by ESTScan o
be encoded by hese EST clus e s, ailed o iden i y any
o he gallinacins o ca helicidins.
Due o hei small size and poo sequence conse a ion,
sea ching o no el AMPs by con en ional homology
sea ch ools such as BLAST (Al schul e al. 1997) may
mean ha signi ican hi s a e missed. Howe e , he
p esence o conse ed mo i s makes hese pep ides good
candida es o HMM p o ile sea ching. An HMM p o ile
is a p obabilis ic model o a p o ein amily mul iple-
sequence alignmen , which uses posi ion-speci ic sco es o
indica e he likelihood o each amino acid occu ing in
each posi ion in he alignmen (Eddy 1998). To sea ch o
no el AMPs ha could be missed by BLAST, we
cons uc ed HMM p o iles o a numbe o AMP amilies,
including α-de ensins, gallinacins, ca helicidins and
hepcidins. The HMM p o iles we e hen used o sea ch
agains he da abase o p o eins p edic ed by ESTScan
om he EST clus e s. This me hod iden i ied wo
addi ional gallinacins (Gal-9 and Gal-10). Fu he mo e,
his app oach also led o he iden i ica ion o a gallinacin-
like sequence ha has unusual cys eine spacing. The e is
e idence om o he species ha β-de ensins wi h
al e na i e cys eine mo i s a e s ill ac i e as AMPs
(Maxwell e al. 2003).
We ha e cloned and sequenced all he no el AMPs
iden i ied in his s udy and submi ed he sequences o
GenBank. The accession numbe s and p ope ies o he
p edic ed encoded p o eins a e summa ised in Table 2. All
he sequences we e in ag eemen wi h he EST p edic ions,
excep o Gal-10, which had a single synonymous change
a base posi ion 159 om C o T. We ha e examined he
exp ession o he known gallinacins (Gal-1–Gal-3), he
no el gallinacins (Gal-4–Gal-10), ca helicidin and LEAP-
2 in a panel o 21 di e en issues om a heal hy 3-week-
old chicken (Fig. 4). These issues co e he diges i e
sys em, he espi a o y sys em, he geni o-u ina y sys em
and se e al o he a eas o he chicken ana omy.
The known gallinacins Gal-1 and Gal-2 a e exp essed
s ongly in he bone ma ow and he lung, as has been
p e iously shown (Zhao e al. 2001). Howe e , we ha e
also shown s ong exp ession o Gal-1 and Gal-2 in he
es is, mode a e exp ession in he bu sa and in es ine and
low exp ession in he cloaca, gall bladde , b ain and
panc eas. Gal-2 is also exp essed a low le els in he
achea, ai sacs and spleen. The hi d known gallinacin,
Gal-3, was exp essed in he ongue and bone ma ow, as
has p e iously been shown (Zhao e al. 2001), howe e ,
we ound no Gal-3 exp ession in o he issues.
The no el gallinacins (Gal-4–Gal-10) exhibi a iable
exp ession ac oss mos o he issues examined, wi h
di e en gallinacins being exp essed in di e en issues.
The phylogene ically ela ed Gal-4 and Gal-5 show a
simila pa e n o exp ession, as does Gal-7, wi h all h ee
being highly exp essed in he bone ma ow and es is. Gal-
8 is also s ongly exp essed in he es is, and along wi h
Gal-6 shows e y s ong exp ession in he li e , gall
bladde and kidneys. Gal-9 is he only no el gallinacin o
be exp essed in he ongue and also shows low exp ession
in he oesophagus, achea, b ain and bone ma ow whils
Gal-10 shows low exp ession in he la ge in es ine,
kidneys and es is.
In addi ion o LEAP-2 being highly exp essed in he
li e , simila le els o exp ession we e ound in he
Fig. 1 Neighbo -joined ee o he gallinacin amily o AMPs.
Cons uc ed using MEGA, e sion 2.1 (Poisson co ec ed model,
1,000 boo s ap eplica es). B anches wi h less han 50% boo s ap
suppo ha e been collapsed. GAL1–10 Gallinacins (Gal-) 1–10,
GAL1A Gal-1α,THP u key he e ophil pep ide, TBD u key β-
de ensin, BD07 MOUSE mouse β-de ensin 7 (Q91V70)
Table 2 GenBank accession numbe s and p ope ies o no el
AMPs
Name Accession numbe Leng h (aa) M
Ne cha ge
Gal-4 AY534892 67 7.5 +8
Gal-5 AY534893 67 7.6 +7
Gal-6 AY534894 67 7.3 +3
Gal-7 AY534895 63 7.2 +8
Gal-8 AY534896 68 7.1 +2
Gal-9 AY534897 66 7.4 +4
Gal-10 AY534898 65 7.2 +2
LEAP-2 AY534899 76 8.8 +8
Ca helicidin AY534900 148 16.1 +2
173
in es ine, gall bladde and kidneys. These esul s a e
consis en wi h s udies o human LEAP-2, which is also
exp essed in he li e , kidney and colon (K ause e al.
2003). Chicken ca helicidin is exp essed ac oss a wide
a ie y o issues, bu shows pa icula ly high le els o
exp ession in he bu sa, es is and bone ma ow, and is he
only no el AMP o show exp ession in he gizza d.
E olu iona y analysis o he gallinacins
We ha e pe o med an e olu iona y analysis o he
gallinacin amily and ha e de ec ed si es ha a e unde
posi i e selec ion in hese molecules. A neighbo -joining
phylogene ic ee was econs uc ed om he amino acid
alignmen (Fig. 1). This ee opology was used in he
subsequen analyses o de ec adap i e e olu ion. To es
o a iable ω a ios among phylogene ic lineages, he one-
a io model (Goldman and Yang 1994)—which assumes
he same ω a io o all lineages—was compa ed using he
LRT o he ee- a io model (Yang 1998), which assumes
an independen ω a io o each b anch. The ee- a io
model is no signi ican ly be e han he one- a io model
(P>0.5) bu does, howe e , p edic a iable ω alues
among lineages, some o which a e g ea e han 1 (Fig. 2).
Since he LRT did no e eal a signi ican di e ence, we
canno conclude ha he e is e idence o posi i e selec ion
among he gallinacin lineages.
To es o posi i e selec ion a indi idual amino acid
si es, LRTs we e ca ied ou be ween model M0 and M3,
M1 and M2, and M7 and M8. All 3 models (M2, M3 and
M8) which allow o selec ion (Table 3) a e signi ican ly
a ou ed o e he o he models (P<0.001) in all cases
(Table 4). Gallinacins a e encoded as p ep opep ides ha
a e p o eoly ically clea ed o elease he C- e minal AMP.
All o he si es p edic ed o be subjec o posi i e selec ion
a e loca ed in he ma u e AMP and no in he p ep opep-
ide egion (Fig. 3a), sugges ing unc ional signi icance.
The si es p edic ed o be subjec o posi i e selec ion ha e
been displayed supe imposed on he h ee-dimensional
s uc u e o mouse β-de ensin 7, a ela ed molecule o
which a h ee-dimensional s uc u e is a ailable (Fig. 3b).
The si es unde posi i e selec ion occu h oughou he
molecule and no pa icula pa e n o clus e ing o si es is
disce nable.
Discussion
We ha e applied a bioin o ma ics app oach ha in ol es
he clus e ing o mo e han 420,000 ESTs o he iden i-
ica ion o no el AMPs in he chicken. This app oach has
Table 3 E idence o adap i e e olu ion among si es in chicken gallinacins. ℓLog-likelihood alue o model, d
N
/d
S
Ra io o a es o
synonymous (d
S
) and non-synonymous (amino acid changing: d
N
) changes among he DNA sequences
Model ℓd
N
/d
S
Posi i ely Selec ed Si es
M0—one- a io −2,662.94 0.5834
M1—neu al −2,581.48 0.8923
M2—selec ion −2,541.32 2.5815 25,29,30,33,34,39,42,45,47,49,52,54,55,57,58,63,65
M3—disc e e −2,516.42 1.2094 21,23,24,25,26,27,28,29,30,32,33,34,35,39,42,45,46, 47,48,49,52,54,55,56,57,58,61,62,63,64,65
M7—β−2,531.96 0.6170
M8—βand ω−2,516.42 1.2987 25,29,30,42,45,52,57,63,65
Fig. 2 Phylogeny o gallinacins. B anch leng hs we e es ima ed by
maximum likelihood unde he ee- a io model, which assumes an
independen ω alue o each b anch. B anches wi h no ω alues
shown had alues=∞.ω alues >1 a e shown in bold ace
Table 4 Likelihood a io es o de ec adap i e e olu ion
Models 2Δℓχ
2
alue d P- alue
M1 e sus M2 2(−2581.48–2541.32) 80.32 2 <0.001
M0 e sus M3 2(−2662.94–2541.32) 243.24 4 <0.001
M7 e sus M8 2(−2531.96–2516.42) 31.08 2 <0.001
174

Fig. 3 a Si es p edic ed o be unde posi i e selec ion in he
gallinacins. Si es p edic ed o be unde posi i e selec ion a e
highligh ed in he mul iple-sequence alignmen . Si es shown in ed
a e hose si es p edic ed o be unde posi i e selec ion (model M8).
Pos e io p obabili ies o hese si es a e all g ea e han 0.95. Si es
shown in blue a e he si es ha 100% conse ed ac oss all
ope a ional axonomic uni s (OTUs). The ma u e AMP o Gal-1
is highligh ed. bThe s uc u e o he ma u e mouse β-de ensin 7
(PDB en y = 1E4T) was displayed using RasMol, e sion 2.7.2.1
(h p://www.open asmol.o g/so wa e/ asmol/). Si es shown in ed
a e hose si es p edic ed o be unde posi i e selec ion in he
gallinacins. Si es shown in blue a e he si es ha 100% conse ed
ac oss all OTUs
Fig. 4 Exp ession o Gal-1–
Gal-10, ca helicidin (Ca h) and
li e -exp essed an imic obial
pep ide 2 (LEAP-2) in a panel o
issues om a heal hy chicken. 1
ongue, 2oesophagus, 3p o-
en iculus, 4c op, 5gizza d, 6
li e , 7small in es ine, 8
LARGE in es ine, 9cloaca, 10
bu sa o Fab icius, 11 gall blad-
de , 12 achea,13 lung, 14 ai
sacs, 15 b ain, 16 skin, 17
kidney, 18 spleen, 19 panc eas,
20 es is, 21 bone ma ow
175
iden i ied nine no el AMPs, se en o which a e
gallinacins, one a ca helicidin and one a LEAP-2 (Lynn
e al. 2003). We ha e shown he di e en ial exp ession o
hese genes in a panel o issues om a single chicken and
ha e cloned and sequenced he mRNAs encoding hese
no el AMPs. Iden i ica ion o hese AMPs in he chicken
will aid in he s udy o he inna e immune esponse o he
chicken, economically an impo an species. Mo eo e ,
hese no el AMPs may be exploi ed o he de elopmen
o new he apeu ic agen s o economically signi ican
chicken diseases such as coccidiosis, which esul s in a
loss o he wo ld poul y indus y ha is es ima ed a $700
million annually. These AMPs could be po en ially
de eloped as na u al al e na i es o he a i icial an ibio ics
ha a e commonly ed o chickens and which a e o
g owing public conce n.
In his s udy, we ha e also de ec ed posi i e selec ion a
se e al amino acid si es loca ed in he ac i e an imic obial
pep ide egion o he gallinacin amily o an imic obial
pep ides. I is likely ha as bi ds e ol ed o occupy new
niches, hey we e aced wi h new anges o mic obial
pa hogens. E olu ion o an imic obial pep ides wi h new
sensi i i ies capable o a ge ing no el in ec ious agen s
would con e a selec i e ad an age. The e is expe imen al
e idence ha gallinacin pep ides a e di e se in hei
po ency agains di e en pa hogens. Fo example, Gal-1
and Gal-1αa e ac i e agains he yeas Candida albicans,
whe eas he o he s es ed so a do no show ac i i y
(E ans e al. 1995; Ha mon 1998). Ou esul s indica e
ha gallinacins ha e been subjec o adap i e e olu ion o
inc ease he s uc u al and unc ional di e si y o his
p o ein amily. This is an e ec i e esponse in an a ms
ace agains an inc easing di e si y o mic obial pa ho-
gens.
Acknowledgemen s This esea ch was suppo ed by he Food
Ins i u ional Resea ch Measu e g an no. 01/R&D/D/135 om he
I ish Depa men o Ag icul u e, Food and Ru al De elopmen . All
expe imen s desc ibed in his manusc ip comply wi h he cu en
laws o he Republic o I eland. Au ho s Da id Lynn and Rowan
Higgs con ibu ed equally o his wo k.
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