The mammary gland-specific marsupial ELP and eutherian CTI share a common ancestral gene.

Abstract

The marsupial early lactation protein (ELP) gene is expressed in the mammary gland and the protein is secreted into milk during early lactation (Phase 2A). Mature ELP shares approximately 55.4% similarity with the colostrum-specific bovine colostrum trypsin inhibitor (CTI) protein. Although ELP and CTI both have a single bovine pancreatic trypsin inhibitor (BPTI)-Kunitz domain and are secreted only during the early lactation phases, their evolutionary history is yet to be investigated.
Tammar ELP was isolated from a genomic library and the fat-tailed dunnart and Southern koala ELP genes cloned from genomic DNA. The tammar ELP gene was expressed only in the mammary gland during late pregnancy (Phase 1) and early lactation (Phase 2A). The opossum and fat-tailed dunnart ELP and cow CTI transcripts were cloned from RNA isolated from the mammary gland and dog CTI from cells in colostrum. The putative mature ELP and CTI peptides shared 44.6%-62.2% similarity. In silico analyses identified the ELP and CTI genes in the other species examined and provided compelling evidence that they evolved from a common ancestral gene. In addition, whilst the eutherian CTI gene was conserved in the Laurasiatherian orders Carnivora and Cetartiodactyla, it had become a pseudogene in others. These data suggest that bovine CTI may be the ancestral gene of the Artiodactyla-specific, rapidly evolving chromosome 13 pancreatic trypsin inhibitor (PTI), spleen trypsin inhibitor (STI) and the five placenta-specific trophoblast Kunitz domain protein (TKDP1-5) genes.
Marsupial ELP and eutherian CTI evolved from an ancestral therian mammal gene before the divergence of marsupials and eutherians between 130 and 160 million years ago. The retention of the ELP gene in marsupials suggests that this early lactation-specific milk protein may have an important role in the immunologically naïve young of these species.

Full text

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The mammary gland-specific marsupial ELP and
eutherian CTI share a common ancestral gene
Elizabeth A Pharo1,2*, Alison A De Leo1,2, Marilyn B Renfree1,3, Peter C Thomson2,4, Christophe M Lefèvre1,2,5and
Kevin R Nicholas1,2,5
Abstract
Background: The marsupial early lactation protein (ELP) gene is expressed in the mammary gland and the protein is
secreted into milk during early lactation (Phase 2A). Mature ELP shares approximately 55.4% similarity with the
colostrum-specific bovine colostrum trypsin inhibitor (CTI) protein. Although ELP and CTI both have a single bovine
pancreatic trypsin inhibitor (BPTI)-Kunitz domain and are secreted only during the early lactation phases, their
evolutionary history is yet to be investigated.
Results: Tammar ELP was isolated from a genomic library and the fat-tailed dunnart and Southern koala ELP genes
cloned from genomic DNA. The tammar ELP gene was expressed only in the mammary gland during late
pregnancy (Phase 1) and early lactation (Phase 2A). The opossum and fat-tailed dunnart ELP and cow CTI transcripts
were cloned from RNA isolated from the mammary gland and dog CTI from cells in colostrum. The putative mature
ELP and CTI peptides shared 44.6%-62.2% similarity. In silico analyses identified the ELP and CTI genes in the other
species examined and provided compelling evidence that they evolved from a common ancestral gene. In addition,
whilst the eutherian CTI gene was conserved in the Laurasiatherian orders Carnivora and Cetartiodactyla, it had
become a pseudogene in others. These data suggest that bovine CTI may be the ancestral gene of the Artiodactyla-
specific, rapidly evolving chromosome 13 pancreatic trypsin inhibitor (PTI), spleen trypsin inhibitor (STI) and the five
placenta-specific trophoblast Kunitz domain protein (TKDP1-5) genes.
Conclusions: Marsupial ELP and eutherian CTI evolved from an ancestral therian mammal gene before the
divergence of marsupials and eutherians between 130 and 160 million years ago. The retention of the ELP gene in
marsupials suggests that this early lactation-specific milk protein may have an important role in the immunologically
naïve young of these species.
Background
Marsupials and eutherians diverged between 130 and 160
million years ago [1-3] and evolved very different
reproductive strategies [4-6]. Marsupials have an ultra-
short gestation ranging from 10.7 days for the stripe-faced
dunnart (Smithopsis macroura) [7] to 38 days for the long-
nosed potoroo (Potorous tridactylus) [8] and deliver an al-
tricial young [5].
Organogenesis is completed after birth supported by a
long and physiologically complex lactation, during which
there is an increase in maternal mammary gland size and
milk production, and there are dramatic changes in milk
composition [5,9-13]. In contrast, eutherians have a long
pregnancy during which maternal investment is high
[14,15]. During eutherian lactation, milk composition
remains relatively constant apart from the initial produc-
tion of colostrum 24–36 hr postpartum (pp) [16].
The tammar wallaby (Macropus eugenii) has a 26.5-day
pregnancy after embryonic diapause [17]. After giving
birth, the tammar produces milk for ~300 days until the
young is weaned. Phase 1 of lactation is comprised of
mammary development during pregnancy and lactogen-
esis around parturition. At birth, the altricial young
(~400 mg) attaches to one of the four teats [5,9,13,18].
Lactation proceeds only in the sucked gland, whilst the
remaining three glands regress [5,9]. The young remains
permanently attached to the teat from the day of birth
* Correspondence: epharo@unimelb.edu.au
1Department of Zoology, The University of Melbourne, Melbourne, Victoria
3010, Australia.
2Cooperative Research Centre for Innovative Dairy Products
Full list of author information is available at the end of the article
© 2012 Pharo et al.; licensee BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative
Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and
reproduction in any medium, provided the original work is properly cited.
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RESEARCH ARTICLE Open Access

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until day 100 pp (Phase 2A) followed by detachment
from the teat and a period of intermittent sucking while
confined in the pouch between days 100–200 pp (Phase
2B) [5,13,18]. The final phase is from day 200 to at least
day 300 when the young suckles variably and begins to
graze as well as maintaining a milk intake (Phase 3) [18].
These phases are highly correlated with changes in milk
composition and mammary gland gene expression
[10,13,19]. Milk protein genes such as α-lactalbumin, β-
lactoglobulin (LGB), α-casein, β-casein and κ-casein are
induced at parturition and expressed throughout lacta-
tion, whilst others are expressed and secreted in a phase-
specific manner [13]. Early lactation protein (ELP) is
expressed during Phase 2A only [13,20,21], whey acidic
protein (WAP) is Phase 2B-specific [22] and late lacta-
tion protein A and B are characteristic to late Phase 2B/
Phase 3 and Phase 3 respectively [23,24].
The ELP gene was first identified in an Australian mar-
supial, the brushtail possum (Trichosurus vulpecula)
[25]. ELP encodes a small precursor protein with a single
bovine pancreatic trypsin inhibitor (BPTI)-Kunitz do-
main characteristic to serine protease inhibitors. ELP is
secreted in milk in multiple isoforms, which include an
~8 kDa peptide and a heavily N-glycosylated protein
(~16 kDa) [25]. ELP was later identified in the tammar
[13,20,21,26], the stripe-faced and fat-tailed dunnarts
(Sminthopsis macroura and Sminthopsis crassicaudata
respectively) and the South American grey short-tailed
opossum (Monodelphis domestica) [27] (Refer to Add-
itional file 1: Table S1 for the species in which the puta-
tive functional ELP/CTI gene, transcript and protein
have been identified). Marsupial ELP expression is lim-
ited to the early phase of lactation [13,20,21,27,28] at the
time the mother produces milk for an immunologically
naïve young [29,30]. During this period, the tammar
young is permanently attached to the teat and protected
by humoral (passive) immunity acquired from its
mother’s milk and its own innate immunity [18,30].
Whilst an ELP orthologue is yet to be identified in
eutherians, tammar and possum ELP share ~37% similar-
ity with bovine colostrum trypsin inhibitor (CTI) [20,25].
CTI was discovered by chance in bovine colostrum over
60 years ago [31]. Putative CTI proteins with trypsin in-
hibitor activity were subsequently isolated from colos-
trum of the pig [32], cat, sheep, goat, dog, reindeer,
ferret and Blue fox [33], but were not found in equine
colostrum [34]. These glycosylated proteins inhibited
serine endopeptidases such as trypsin, pepsin and
chymotrypsin [31,32,35]. However, of these putative CTI
proteins, only bovine CTI has been sequenced (Add-
itional file 1: Table S1) and found to contain a Kunitz do-
main which generally indicates serine protease inhi-
bitor activity (see below) [36]. Laskowski and Laskowski
[31] hypothesisedthat bovineCTI protected
immunoglobulins against proteolysis during the crucial
period of immunoglobulin transfer from cow to calf via
colostrum. However, its function is yet to be determined.
Although CTI and ELP are expressed in early milk, bo-
vine CTI secretion is brief (~1-2 days) [31,37], but mar-
supial ELP expression is prolonged (up to 100 days pp)
[20,21,25,28]. However, their secretion in milk is corre-
lated with the period of immuno-incompetence in the
young [29,31].
The Kunitz domain was thought to have evolved over
500 million years ago [38] and is now ubiquitous in
mammals, reptiles, birds, plants, insects, nematodes,
venoms from snakes, spiders, cone snails and sea ane-
mones and in viruses and bacteria [39-42]. The arche-
typal protein of the Kunitz domain and the BPTI-Kunitz
family I2, clan IB of serine endopeptidase inhibitors in
the MEROPS database [43,44] is the much studied bo-
vine pancreatic trypsin inhibitor, also known as aprotinin
(reviewed in [45]). The Kunitz domain is characterised
by six conserved cysteine residues which form three di-
sulphide bonds, producing a compact, globular protein
of α+β folds [43,46,47]. Serine endopeptidase inhibition
occurs through the binding of the P1reactive site residue
within the ‘binding loop’ of the Kunitz domain to a
serine residue within the catalytic cleft of the protease
[47,48]. This is a reversible, tight-binding, 1:1 interaction
[44,48]. Furthermore, the Kunitz domain P1 residue
determines protease-specificity [39,47].
Since its evolution, the Kunitz domain has been incor-
porated into many different genes [43,44]. In general,
each domain is encoded by a single exon [43,49]. Some
genes encode proteins with a single Kunitz domain, e.g.
ELP, CTI, PTI, spleen trypsin inhibitor (STI), the five
trophoblast Kunitz domain protein genes (TKDP1-5) and
serine protease inhibitor Kunitz-type-3 (SPINT3) and
SPINT4. These genes, apart from the TKDPs, have 3
exons. The first exon encodes the signal- and pro-pep-
tide, the second, a single Kunitz domain and the third, a
short C-terminus. However, the TKDPs have a variable
number of unique N domains inserted between the sig-
nal peptide and the Kunitz domain-encoding exon
[50,51]. Genes that encode multiple Kunitz domains in-
clude: hepatocyte growth factor activator inhibitor 1 and
2, also known as SPINT1 and SPINT2 respectively (two
domains), tissue factor pathway inhibitor 1 and 2 (three
domains); with up to 12 domains in the Ac-KPI-1 I
nematode (Ancylostoma caninum) protein [38,43,44]. In
addition, the Kunitz domain has been integrated into
multi-domain proteins, some of which include: the colla-
gen α3(VI), α1(VII) and α1(XXVIII) chains, WFDC6 and
WFDC8, amyloid beta A4 protein, α1-microglobulin/
bikunin precursor (AMBP), SPINLW1 [serine peptidase
inhibitor-like, with Kunitz and WAP domains 1 (eppin)]
and the WAP, follistatin/kazal, immunoglobulin, Kunitz
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and netrin domain containing (WFIKKN)1 and 2 pro-
teins [39]. Furthermore, each domain within a multi-
Kunitz domain protein, may exhibit different protease
activity, such as for the three tandemly repeated domains
within both tissue factor pathway inhibitor 1 and 2
[43,44,52].
The early lactation/colostrum-specific expression of
ELP/CTI suggests these Kunitz domain-encoding genes
may play an important role in the neonate. The sequen-
cing of the tammar genome [53], in addition to the avail-
ability of numerous vertebrate genomes including one
other marsupial, the opossum, a monotreme, the platy-
pus, many eutherians, birds (chicken, Zebra finch), fish
(Zebrafish, Japanese medaka, Three-spine stickleback,
Tiger and Green spotted puffers), amphibian (African
clawed frog) and reptile (Green anole lizard), provides an
invaluable resource with which to investigate the evolu-
tion of these genes. We used a comparative genomics ap-
proachbased upon bioinformatics
cloning of cDNA and genomic DNA to characterise the
marsupial ELP and eutherian CTI genes and investigate
their evolutionary history.
andPCR-based
Results
ELP/CTI evolved from a common ancestral gene
To determine whether the marsupial ELP gene was
present in other species, we used multiple approaches.
We cloned the ELP genes of the koala and fat-tailed dun-
nart and isolated tammar ELP from a genomic library.
ELP/CTI transcripts were cloned from the mammary
gland of the cow, opossum and fat-tailed dunnart and
the dog CTI transcript was cloned from epithelial cells
isolated from canine colostrum. We performed BLAST
searches of genomic databases (Ensembl, Release 62,
April 2011 [49], NCBI GenBank nr and WGS [54] and
UCSC [55]), using a cut-off of E-value≤1e-8 (nucleo-
tides) and E-value≤1e-17 (proteins). To further refine
the identification of ELP/CTI orthologues based upon
protein sequence, we also compared gene structures
(where possible) to identify genes with a similar three-
exon structure to ELP/CTI. Based upon these methods,
no genes orthologous to marsupial ELP/eutherian CTI
were present in fish (Zebrafish, Tiger and green spotted
puffers, Three-spined stickleback), birds (chicken, zebra
finch), amphibian (African clawed frog), reptile (Green
anole lizard), monotreme (platypus), nor sea squirts, fruit
fly, nematode (Caenorhabditis elegans) or yeast. How-
ever, many of the current genomes available provide only
low sequence coverage (e.g. anole lizard, 2x; green spot-
ted pufferfish, 2.5x; chicken, zebra finch and platypus,
6x; elephant, 7x). Many assemblies are also incomplete
(contain gaps) and may contain incorrect assemblies.
Hence it is possible that ELP/CTI orthologues may be
identified within these genomes with future improve-
ments in sequence coverage and assemblies.
The CTI gene was present in the Laurasiatherian orders
Cetartiodactyla (cow, pig, common bottle-nosed dolphin)
and Carnivora (dog, cat, Giant panda). However, based
upon current genome assemblies, it is a pseudogene in
Afrotheria, Xenarthra, Euarchontoglires and the Laura-
siatherian orders Chiroptera and Perissodactyla.
The mammalian ELP/CTI gene was composed of 3
exons and 2 introns (Figure 1). The marsupial ELP gene
ranged from ~1.4 kb for the koala to ~4.8 kb for the stripe
faced dunnart, whilst eutherian ELP spanned from ~2.5 kb
for the panda to ~3.8 kb for the pig. ELP exon 1 and 2
sizes respectively were highly conserved across all mam-
mals (Figure 1). Exon 1 encoded the putative signal pep-
tide and the first four amino acids at the N-terminus of
the protein. The 216 bp exon 2 (with the exception of the
koala, 210 bp) encoded the remainder of the N-terminal
region, plus a single BPTI-Kunitz domain towards its 3'-
end. ELP/CTI exon 3 differed most and encoded a
maximum of seven amino acids. The ELP/CTI transcripts
(putative translation start site to the polyadenylation sig-
nal, inclusive) were short. Marsupial ELP and eutherian
CTI transcripts ranged from 425–447 bp and 416–428 bp
respectively and shared 56.1%-63.6% similarity at the nu-
cleotide level (Additional file 2: Figure S1; Additional file
3: Tables S2A, S2B). A highly conserved marsupial-specific
region (87%-100%) was also identified within the ELP 3'-
UTR (nt 420–475, Additional file 2: Figure S1; Additional
file 3: Table S2C).
Based upon signal peptide analysis [56], the putative
ELP/CTI peptides identified in this study were predicted
to be secreted in milk, as for tammar and possum ELP
and bovine CTI [20,25,26,31]. The mature ELP and CTI
peptides shared 44.6%-62.2% similarity (Table 1; Add-
itional file 4: Table S3A). In addition, the conservation of
the two Kunitz domain motifs in all species suggested
they may inhibit the S1 family of serine endopeptidases
like many other members of the BPTI-Kunitz family
[43,44]. The BPTI KUNITZ 2 motif [C1-C6, C2-C4 and
C3-C5, Prosite: PS00280] indicates the 3 disulphide
bonds which determine the structure of the domain (Fig-
ure 2). This motif spanned the entire 51 amino acid
Kunitz domain (aa 23–73, C23-C73, C32-C56 and C48-
C69, Figure 2). The second shorter motif BPTI KUNITZ
1 [F-x(2)-{I}-G-C-x(6)-[FY]-x(5)-C; where x represents
any residue, those within square brackets are permitted,
but those within curly brackets are not, Prosite:
PS00280] was located within BPTI KUNITZ 2 (aa 51–69,
Figure 2). A putative trypsin interaction site within the
Kunitz domain (from KU NCBI cd00109) [57], is also
depicted (aa 30–34, 36, Figure 2).
Conserved amino acid residues within a protein pro-
vide an indication of sites essential for its structure and
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biological function. Comparison of the marsupial ELP
and eutherian CTI precursor proteins showed that the
signal peptide (57.1%-81.0% similarity), the 51 aa BPTI
KUNITZ 2 motif (54.9%-68.6%), plus the shorter 19 aa
BPTI KUNITZ 1 motif within it (63.2%-73.7%) were con-
served. However, the 20–22 residue linear chain of the
mature ELP/CTI N-terminus had marsupial-specific and
eutherian-specific homology (59.1%-100%, Table 1; Add-
itional file 4: Tables S3B, S3C, S3D, S3E). Conservation
of the short (3–10 residue) C-terminus was variable
(Additional file 4: Table S3F). This was in part due to the
use of different stop codons in ELP/CTI transcripts
across divergent species. The opossum and dunnart ELP
proteins were truncated at the end of exon 2, with the
stop codon encoded by one nucleotide in exon 2 and
two in exon 3 (nt 323–325 inclusive; Additional file 2:
Figure S1). For all other species, two different stop
codons within exon 3 were used. For the panda, cat and
dog, the TAA stop codon (nt 333–335) was used. How-
ever, for the pig, cow, dolphin and the remainder of the
marsupials, the equivalent TGA stop codon (nt 344–346
inclusive) was used.
Surprisingly, there was little conservation of the amino
acid residue type (physiochemical properties) at the P1
Pig
(3,771 bp)
76 216
2,849494
136
Koala
(1,387 bp)
Opossum
(4,497 bp)
Dog
(2,578 bp)
Cow
(2,907 bp)
Tammar
(4,268 bp)
Cat
(3,283 bp)
Dolphin
(2,720 bp)
76
73
73
73
76
76
76 216
216
216
216
216
216
210 142
158
718 314
2,219 1,831
146
3,522 311
2,346 511
134
133
1,671 491
136
124
1,788694
1,785 507
ELP/CTI
0
24531 kb
Ex1 Ex2 Ex3
Dunnart FT
(4,726 bp)
73 216
73216
154
154
3233,960
4,069306
Dunnart SF
(4,818 bp)
Panda
(2,561 bp)
1,645 490
76 216134
Figure 1 Structure of the marsupial ELP and eutherian CTI genes. The ELP/CTI genes of the stripe-faced (SF) dunnart (Sminthopsis macroura)
[GenBank: AC186006], fat-tailed (FT) dunnart (Sminthopsis crassicaudata) [GenBank: JN191336], koala (Phascolarctos cinereus) [GenBank: JN191337],
opossum (Monodelphis domestica) [GenBank: BK008085], tammar (Macropus eugenii) [GenBank: JN191335], cat (Felis catus, Abyssinian domestic cat)
[GenBank: BK008083], cow (Bos Taurus, Hereford Breed) [Ensembl: ENSBTAG00000016127], dog (Canis familiaris, Boxer breed) [GenBank: BK008082],
dolphin (Tursiops truncatus) [GenBank: BK008086], pig (Sus scrofa domestica) [Ensembl: F1SD34_PIG (ENSSSCG00000007398)] and Giant panda
(Ailuropoda melanoleuca) [GenBank: BK008084] have 3 exons and 2 introns. Gene size is indicated within brackets and refers to the number of
nucleotides from the putative translation start (ATG, exon 1) to the polyadenylation signal (AATAAA, inclusive, exon 3). Exons are colour-coded:
exon 1 (green rectangle), the Kunitz domain-encoding exon 2 (blue) and exon 3 (red) and exon size is indicated in bold text. Intron sizes are
italicised. The horizontal scale bar indicates the relative sizes of the ELP/CTI genes (kb), with the putative translation start site (ATG) of all
sequences aligned with the origin (0 kb). Genes are drawn approximately to scale.
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reactive site within the Kunitz domain (residue 33,
Figure 2). Although the P1residue type (basic amino acid
with a positively charged side chain) was conserved
amongst eutherians: K (lysine) for the pig, cow and dol-
phin and R (arginine) for the cat, dog and panda, this
was not so for marsupials. The opossum and possum
ELP P1residue was acidic with a negatively charged side
chain (D, aspartate). However, the P1residue for tammar
(S, serine) and the koala and dunnarts (N, asparagine)
was polar with uncharged side chains.
Although P1residues differed, all ELP/CTI peptides were
predicted to be N-glycosylated at asparagine-42, consistent
for bovine CTI [58] and therefore should be larger than
their predicted masses (8.6 to 9.6 kDa, data not shown).
Selective pressure acting upon marsupial ELP and
eutherian CTI
The evolutionary selection pressure acting upon different
regions of the protein-coding marsupial ELP and euther-
ian CTI transcripts was determined by dN/dS analysis
(Table 2). The dN/dS ratio measures the number of non-
synonymous changes per non-synonymous site (those
which produce amino acid substitutions) compared to
the number of synonymous changes per synonymous site
(no amino acid change) [59,60]. A ratio of dN/dS=1
suggests a neutral condition, with nucleotide changes ac-
cumulating in the absence of selection pressure, i.e. both
dN and dS occur at the same rates. dN/dS<1 indicates
purifying selection, with amino acid changes not tolerated.
Table 1 Homology between and within the marsupial ELP and eutherian CTI peptides1
Species comparisonsSignal peptide
Marsupial ELP
85 - 95%
Mature peptideN-terminus Kunitz motif2 (51 aa) Kunitz motif1 (19 aa)C-terminus
67.5 - 100% 59.1 - 100%76.5 - 100% 84.2 - 100%20 - 100%
Eutherian CTI
57.1 - 90.5%70.7 - 88.6% 59.1 - 90.9%76.5 - 94.1%84.2- 100% 40 - 83.3%
Marsupial ELP vs Eutherian CTI
57.1 - 81.0%44.6 - 62.2%18.2 -59.1% 54.9 - 68.6% 63.2 - 73.7% 10 - 60%
Pairwise amino acids similarities were calculated using MatGAT 2.01 (BLOSUM62 matrix).
1Refer to Additional file 4: Tables S3 for individual species comparisons.
1 11 2131 41
FYNSTSAECELFMYGGCQGNANNFETTAICRRVCNPPDTKVKNG
51 61 81
: *:*: ** * * **.** *:*:: * : *.
BPTI KUNITZ 2 (PS50279)
51 residues
#
P
1-'P
1
Exon 1 Exon 2 Exon 3
BPTI KUNITZ 1 (PS00280)
F-x(2)-{I}-G-C-x(6)-[FY]-x(5)-C
+-------------------------------------------------+
+-----------------------+
--
CCC
-- -------- #-------------- ------- ------------ --- --
--
----
C
+--------------------+
--
Pig
Dolphin
Cow
Dog
Panda
Cat
Tammar
Possum
Koala
DunnartSF MKFT-
DunnartFT MKFT-
Opossum
M
MKFSLFLALCFLLGLVGITSLEKASAHLRQEAFQELSQTLPVLCQLPPGKGPCRGRFYRY
FYNSTSSACEP
FYNSTSSECEHFIYGGCQGNANNFETTEICLKICKPPETR
FSNSTSSECEHFTYGGCQGNANDFETTEICSRICKPPETG
FYNSTAHECEHFTYGGCRGNANNFETTEMCLKVCKPPGTR
-
----
----
---- MKFSLFLALCFP
MKFT-
MKFT-
MKFT-IVALCFALSLAGLTSSEKLSD--
IIALCFAFSLAGMTSSEKLLDQIPVNSLENPSRLVPALCQLSPQRGNCNDNIRRY
IIALFFAFSLAGMTSSEKLLDQIPMNSLENPSRLVPALCQLSPQRGNCNDNIRRY
IVALCFALGLAGITSSEEVLEQNPLNTQENPVPLVLPLC
-
-------
-------
-------MKFT-
**:: :** :* *::*: :: .: :* ** * :*
----
LLPPERGNCDSLNLRY
MKLSLSLALCLTLCLPGMASSGKTLASLKQEASQELFQTPPALCQLPPVGGPCKASLRRY
MKLSRLLALCLTLCLVGLASSGKTSANLQQEASQELLQTPPALCQLPAVRGPCKASLHRY
KLSCLLALCLTPCLVGLASSGETSDNLKQEASQDLFQTPPDLCQLPQARGPCKAALLRY
MKFSPFLALCFLLCLVGISSSEKASAHLKHEAPRELSQALPAMCQLRPAKGPCRGLFYRY
FCLVGIASSEKTSAHLEREAPQELLQTLPALCRLPPVEGPCRGRFYRY
IVALYFALSLAGMTSSEKCLDQIQVNSLENLSLLVPSLCLLPPVRGNCSSQILHY
IIALCLALSLVGMTSSEKLLDRIRANSLENLSRLVPSLCLLPSGRGNCDSQILRY
HVNSLENPYQLVPSLCLLSPARGNCNSQTLRY
C
--
C
Signal peptide
Trypsin interaction site
from KU (NCBI cd00109)
71
FYNSTSIECEPFTYGGCQGNANNFETTEICVRVCKPPETKVKSS
FTYGGCQGNDNNFETTEMCLRICQPPETEDKS
FYNTTSRTCETFIYSGCNGNRNNFNSEEYCLKTCRRNKNRNNNN
FYNATSHTCEVFLYSGCNGNGNNFDSLECCLKTCRLNKYRNNN
FYNTTSRTCEAFIYSGCHGNGNNFDSLQCCLKTCRPNKNRNDNN
YYNTTSRICEEFIYTGCNGNGNNFDSVECCLKTCKLN
YYNTTSRICEEFIYTGCNGNGNNFDSVECCLKTCKLN
FYNSTSRLCEAFIYSGCNGNGNNFDTVECCLKTCRPN
Figure 2 Alignment of the marsupial ELP and eutherian CTI precursor proteins. The nucleotide sequences of the ELP/CTI mRNA transcripts
of the following species were conceptually translated and aligned with ClustalW2: tammar [GenBank: JN191338; UniProtKB/Swiss-Prot: O62845
(mature protein)], brushtail possum [GenBank: U34208], fat-tailed dunnart (FT) [GenBank: JN191339], opossum [GenBank: JN191340], cow (Holstein-
Friesian breed) [GenBank: JN191341] and dog [GenBank: JN191342]. The stripe-faced dunnart (SF) [GenBank: AC186006], koala [GenBank:
JN191337], cat [GenBank: BK008083], pig [Ensembl: F1SD34_PIG (ENSSSCT00000008098)], dolphin [GenBank: BK008086], and panda [GenBank:
BK008084] ELP/CTI genes were conceptually spliced based upon conserved splice sites and translated. Amino acid residues are numbered based
upon the start (N-terminus) of the mature ELP/CTI peptides. Black shading indicates nucleotide residues common to at least 10 of the species and
grey, the remainder that differ. The six conserved cysteine residues (C1-C6, C2-C4 and C3-C5), which form the three disulphide bonds and
produce a globular protein are shaded red. Teal shading indicates amino acids common to marsupials and blue, those common to eutherians.
The location of exons is indicated by arrows. The predicted signal peptides are boxed (blue). The BPTI KUNITZ 1 and 2 motifs are indicated (green
and red bars respectively) and the putative trypsin interaction site from the KU motif (NCBI cd00109) is depicted by orange triangles. The putative
P1and P1' reactive site residues are shaded yellow and purple respectively. Italicised asparagine (N) residues indicate predicted sites of post-
translational N-glycosylation. Conservation between groups of amino acids with strongly similar properties, i.e., scoring>0.5 in the Gonnet PAM
250 matrix is indicated (:). Conservation between groups of amino acids with weakly similar properties (scoring<0.5 in the Gonnet PAM 250
matrix) is also noted (.). Gaps within the alignment are indicated (−).
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In contrast, dN/dS>1 is indicative of positive Darwinian
selection for amino acid changes [59,61].
The protein-coding marsupial ELP and eutherian CTI
transcripts and regions within them generally exhibited a
trend towards purifying selection, with a dN/dS ratio <1
(Table 2). However, based upon codon-based Z-tests, only
the eutherian CTI BPTI KUNITZ 1 motif (57 nt encoding
19 amino acids) was found to be undergoing purifying se-
lection (p<0.05). Although the regions encoding the mar-
supial BPTI KUNITZ 1 motif (p=0.103) and the
marsupialand eutherian
(p=0.101 and p=0.105 respectively) exhibited a strong
trend towards purifying selection, the test values (dN<dS)
were not significant. This tendency was also consistent for
the putative trypsin interaction site. In contrast, three
regions of the ELP/CTI transcripts showed a trend towards
positive selection (dN/dS>1). These included the regions
encoding the ELP/CTI N-terminus and the eutherian CTI
signal peptide. However, based upon codon-based Z-tests
(dN>dS), only the eutherian CTI signal peptide (p<0.05)
was undergoing positive selection.
BPTI KUNITZ2 motifs
Marsupial ELP and eutherian CTI share common flanking
genes
In order to confirm that the marsupial ELP and eutherian
CTI genes were orthologous, we characterised the location
and arrangement of ELP/CTI and its flanking genes. We
used fluorescence in situ hybridisation to map tammar ELP
to chromosome 1q (Figure 3). The ELP/CTI gene was
located on a syntenic segment in the marsupial (stripe-faced
dunnart [27] and opossum) and eutherian genomes [49,55]
and was generally flanked by one or both of the single-copy
genes phosphatidyl inositol glycan, class T (PIGT) and WAP
four disulphide core domain 2 (WFDC2), confirming they
were true orthologues (Figure 4).
The PIGT-WFDC2 region of bovine chromosome 13
(~74.51-75.14 Mb) was unique. Bovine CTI was adjacent to
PIGT, but there was an insertion of ~602 kb between the
CTI and WFDC2 genes [49,55] (data not shown). This re-
gion included 7 Artiodactyla-specific Kunitz domain-en-
coding genes including PTI, STI, plus the five placenta-
specific TKDP1-TKDP5 genes inclusive [50,63]. Further-
more, the SPINLW1 gene which contains both a Kunitz
and a WAP domain and the eutherian-specific SPINT4
gene were located a further ~38 kb and ~90 kb respectively
downstream from WFDC2 [49,55] (data not shown). As
mentioned previously, these genes, with the exception of
SPINLW1 and the TKDPs, share a similar 3-exon structure.
However, the TKDPs differ due to the likely “exonisation”
of an intron and its subsequent duplication to produce a
variable number of tripartite N-domains between the exon
encoding the signal peptide and the Kunitz domain [50,51].
CTI has been lost in some eutherians
Using the canine sequence as the basis for mVISTA
comparative analysis [64], the region between the PIGT
Table 2 Average rates of synonymous (dS) and non-synonymous (dN) substitutions occurring in marsupial ELP and
eutherian CTI
ELP/CTI protein-coding
region
dNSE dSSE dN/dSRatio (a) Neutral
selection test
(dN6¼dS)+*
0.256 (NS{)
(b) Purifying
selection test
(dN<dS)+*
(c) Positive
selection test
(dN>dS)+*
Precursor protein
Marsupials
0.145 0.0220.1900.033 0.763
0.117 (NS) 1.000 (NS)
Eutherians
0.1940.0260.225 0.0330.862 0.232 (NS) 0.472 (NS)1.000 (NS)
Mature protein
Marsupials
0.1660.0260.185 0.0360.897 0.653 (NS)0.334 (NS)1.000 (NS)
Eutherians
0.1860.028 0.2420.039 0.786 0.273 (NS)0.130 (NS) 1.000 (NS)
Signal peptide
Marsupials
0.071 0.0290.2260.094 0.3140.133 (NS) 0.064 (NS)1.000 (NS)
Eutherians
0.2250.0720.165 0.0691.360.451 (NS) 1.000 (NS) 0.224 (NS)
N-terminus
Marsupials
0.2400.064 0.116 0.0482.070.064 (NS) 1.000 (NS) 0.041*
Eutherians
0.2420.0500.2240.065 1.080.842 (NS) 1.000 (NS)0.424 (NS)
BTPI KUNITZ 2#
Marsupials
0.146 0.0310.224 0.0520.651 0.215 (NS) 0.101 (NS) 1.000 (NS)
Eutherians
0.1620.0350.243 0.054 0.6670.200 (NS) 0.105 (NS)1.000 (NS)
BPTI KUNITZ 1~
Marsupials
0.095 0.0300.2230.0980.426 0.212 (NS)0.103 (NS) 1.000 (NS)
Eutherians
0.0660.0260.2640.1100.2500.122 (NS) 0.046* 1.000 (NS)
Trypsin interaction site^
Marsupials
0.2300.136 0.323 0.1810.712 0.740 (NS)0.363 (NS) 1.000 (NS)
Eutherians
0.1750.093 0.2280.1310.768 0.689 (NS)0.345 (NS)1.000 (NS)
#PS50279 153 nt, 51 aa.
~PS00280 57 nt, 19 aa.
^18 nt, 6 aa site from KU (NCBI cd00109).
+Codon based Z-tests in MEGA5.
*p<0.05.
{NS not significant.
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and WFDC2 genes was examined using the available
genome assemblies - which have variable sequence
coverage, contain gaps and may contain misassembled
sequences. Whilst the ELP/CTI gene was present in
some mammals, it appeared to have become a disrupted
pseudogene in others such as the African Savanna ele-
phant and human (Figure 5). Exon 1 of the elephant and
human CTI genes (signal- and pro-peptide) was present,
but exon 2 (Kunitz domain) and exon 3 (C-terminus)
were absent (red boxes, Figure 5), suggesting they had
been excised or transposed, whilst the horse and mouse
CTI genes initially appeared intact.
A closer examination of the nucleotide sequence be-
tween PIGT and WFDC2 in these and other species using
the Ensembl and UCSC genome databases revealed that
different mutations had most likely disrupted the CTI gene.
Exon 1 was disrupted in the elephant, Hoffmann's two-
toed sloth (Choloepus hoffmanni), armadillo (Dasypus
novemcinctus), human and other primates and horse, with
exon 2 (Kunitz domain) also excised for these species,
apart from the horse. Additional file 5: Figure S2A (i)
depicts a nucleotide alignment of the functional/protein-
coding dog CTI exon 1 compared with the putative dis-
rupted CTI exon 1 of the elephant, sloth, human and
horse. Additional file 5: Figure S2A (ii) shows the trans-
lated sequences to highlight mutations and/or deletions
within the signal peptide region of CTI. The deletion of
two nucleotides within human CTI exon 1 would produce
a frame-shift (as depicted by the +1 and +2 reading
frames). CTI exon 2 of the mouse, rat, large flying fox
(Pteropus vampyrus) and horse also appeared to have been
disrupted by deletions resulting in frame-shifts when com-
pared to the functional/protein-coding dog CTI exon 2.
The disruption of the protein-coding region of equine CTI
exons 1 and 2 by at least one mutation and one deletion
respectively would produce a frame-shift, suggested these
were a recent occurrence (Additional file 5: Figure S2B
(ii)).
Transposable elements within the ELP/CTI genes
Transposable elements integrate randomly into the gen-
ome, so the probability of the same element(s) integrat-
ing independently into orthologous positions in different
species is extremely low. They therefore act as genetic
markers and can be used to determine the phylogenetic
relationship between genes and species [65]. Further evi-
dence that marsupial ELP and eutherian CTI evolved
from a common ancestral gene was provided by CEN-
SOR retrotransposon analysis [66] (Additional file 6: Fig-
ure S3). Retroelements of conserved fragment size and
orientation were located within the PIGT-ELP/CTI re-
gion. However, the elephant and human which appear to
have lost CTI exons 2 and 3, had also lost retrotranspo-
sons in the corresponding region, but gained a MER5A
element.
Bovine CTI, PTI, STI and the TKDPs share a common
ancestral gene
The location of the 8 Kunitz-domain encoding genes (in-
cluding CTI) on bovine chromosome 13 between the PIGT
and WFDC2 genes and the Artiodactyla-specific distribu-
tion of PTI, STI and TKDP1-5 (cow and sheep [51,63]) sug-
gested they may have evolved from CTI. This hypothesis
was supported by phylogenetic analysis of the protein-cod-
ing regions of the mammalian ELP/CTI, bovine PTI, STI
and TKDP1-5 transcripts, with bovine SLPI used as an out-
group root (SLPI omitted, Figure 6). Several different meth-
ods in PHYLIP were used to determine the evolutionary
relationships. These included the character-based max-
imum-likelihood (with/without a molecular clock) and
maximum parsimony, as well as distance-based analysis
(Fitch-Margoliash tree method using the Kimura distance
model of nucleotide substitution). Trees were evaluated
using the bootstrap method (100 replicates). Of the algo-
rithms used, the maximum likelihood method using a mo-
lecular clock assumption, which assumes a constant
evolutionary rate for all species, produced a tree with the
highest bootstrap values. Huttley and colleagues [67] have
shown that the eutherian nucleotide substitution rates are
~30% slower than for marsupials. However, all methods
produced consensus trees which consistently separated the
19 sequences into the two groups depicted (Figure 6). The
hypothesis that bovine CTI was the ancestral gene for
1q
1q
Figure 3 Localisation of the tammar ELP gene to Macropus
eugenii chromosome 1q using FISH.
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\bovine PTI, STI and TKDP1-5 was supported by both
an alignment of precursor proteins and phylogenetic
analysis of CTI, PTI, STI, TKDP1-5 and the SPINT4
protein-coding transcripts (Additional file 7: Figure S4;
Additional file 8: Figure S5). Interestingly, the size of
the Kunitz domain-encoding exon varied. Whilst the
bovine CTI exon was 216 bp, those of the TKDPs were
196 bp, with 192 bp for PTI and STI and 175 bp for
SPINT4. Furthermore, apart from CTI and SPINT4,
none of the Kunitz domains were predicted to be N-
glycosylated. Additional evidence of the evolutionary
history of the CTI, PTI, STI and TKDP1-5 genes was
provided by mVISTA (Additional file 9: Figures S6A
and S5B (i-viii) and CENSOR analysis (Additional file
10: Figure S7; Additional file 11: Table S4).
Tammar ELP expression is up-regulated at parturition and
is mammary-specific
Northern analysis showed that tammar ELP was up-regu-
lated at parturition, consistent with brushtail possum ELP
[28] (Figure 7A). ELP transcripts were detected in the tam-
mar mammary gland from~day 17 of pregnancy onwards,
throughout early lactation (Phase 2A) until~day 87 of lac-
tation. ELP was then down-regulated to minimal levels for
the remainder of lactation. This was consistent with a pre-
vious study of late Phase 2A/Phase 2B mammary tissues,
but the precise timing of ELP gene induction was not
investigated[13,20,21].Neither
expressed in the virgin mammary gland and both genes
were down-regulated postpartum in the non-sucked glands
(Figure 7A), as in the brushtail possum [28].
LGB expression peaked in the mammary gland during
Phase 3, consistent with [68].
Although cDNA microarray analysis of the tammar mam-
mary gland (Figure 7B; Additional file 12: Table S5) was
based upon comparative expression levels rather than actual
transcript levels, the data was consistent with quantitative
analysis of the Northern blot (data not shown) and micro-
array data reported by [69]. Lastly, Northern analysis of
assorted tammar tissue samples indicated that expression of
ELP, norLGBwas
Opossum
Chr. 1
(501.25 to
501.67Mb)
Dog
Chr. 24
(35.64 to
35.89 kb)
Cow
Chr. 13
(74.48 to
75.20 kb)
Human
Chr. 7
p13-p15.1
Human
Chr. 20
q12-q13.12
Mouse
Chr. 2H3
Chicken
Chr. 20 (-)
(5.08 to
4.93 Mb)
Zebrafish
Chr. 23
(12.27 to
12.72 Mb)
SPINT3
WFDC8
LOC
611069
CE10
LOC100
130157
RPL5P2
WFDC6
SYS1-DBNDD2
Gm14317
HSPD
1P21
PIGTWFDC2
TP53TG5SYS1
DBNDD2
PigT
Tp53tg5
Sys1
Dbndd2
Spint3
Spinlw1
Wfdc6a
Wfdc2
PIGT
CTI
TP53TG5
SYS1
DBNDD2WFDC2
PIGT
CTI
TP53TG5
SYS1
DBNDD2WFDC2
LOC
611096
SPINLW1
SPINLW1
PIGTWFDC2ELPAEBP1 POLD2MYL7
TP53TG5
SYS1
DBNDD2
GCK YKT6
AEBP1 POLD2 MYL7 GCKYKT6
~602 kbp
PIGT
TP53TG5
DBNDD2
SYS1
LOC419
182
SRC BLCAPEMILIN3 MANBAL GHRH
PIGT
snx21
haus8 wfdc2
si:zfos-
452g4.1
sycp2
phactr3a
ppplr3da
LOC100
128997
CTI
Cti
Figure 4 Chromosomal location of the ELP/CTI gene in different species. The ELP/CTI gene was located within a syntenic block on opossum
Chr. 1 (~501.34 Mb), human Chr. 20q12-13.12, mouse Chr. 2 H3, dog chr. 24 (~35.7 Mb) and cow Chr. 13 (~74.5 Mb) [49,55]. However, ELP/CTI was
reduced to a pseudogene in the human and mouse (red arrow, white diagonal stripes) and was absent in the chicken and zebrafish. The ELP/CTI
gene was located on the reverse strand and was generally flanked by one, or both of the single-copy genes PIGT and WFDC2. The region
upstream of PIGT was conserved in mammals and the chicken and included the SYS1 [Golgi-localized integral membrane protein homolog (S.
cerevisiae)], TP53TG5 (TP53-target gene 5 protein), and DBNDD2 [dysbindin (dystrobrevin binding protein 1) domain containing 2] genes. However, a
chromosomal breakpoint was located downstream from the eutherian WFDC2 gene. Opossum chromosome 1 contained the AEBP1 (Adipocyte
enhancer binding protein 1), POLD2 [polymerase (DNA directed), delta 2, regulatory subunit 50 kDa], MYL7 (myosin, light chain 7, regulatory) and YKT6
[YKT6 v-SNARE homolog (S. cerevisiae)] genes and was orthologous to human chromosome 7p13-p15.1. In contrast, the eutherian chromosomes
contained a number of genes which encoded Kunitz and/or WAP domains. These included SPINT3, SPINLW1, WFDC8 and WFDC6, which were
likely to have arisen by gene and domain duplications [62]. Notably, there was an insert of ~602 kb between bovine CTI and WFDC2. Arrows
indicate the arrangement and orientation of genes and are not drawn to scale.
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ELP and LGB was mammary gland-specific (Figure 8), un-
like the ubiquitously expressed cystatin C (CST3) gene
(data not shown).
Discussion
ELP was originally thought to be a marsupial-specific gene
[19]. However, we have shown that the marsupial ELP and
eutherian CTI genes evolved from a common therian an-
cestral gene (Figure 9). Mammalian ELP/CTI was generally
flanked by one or both of the single copy PIGT and
WFDC2 genes in a region that was syntenic to that of
other mammals. The conserved genomic structure of 3
exons and 2 introns and homologous transposable element
fragments confirmed that ELP and CTI were true ortholo-
gues. CTI was also identified as the putative ancestral gene
of the ruminant-specific PTI, STI and TKDP1-5 genes.
Based upon current genome sequencing and assemblies,
ELP/CTI was not found in birds, fish, reptiles, nor amphi-
bians, suggesting the gene was present in the therian
ancestor before the divergence of marsupials and euther-
ians at least 130 million years ago [1,2,70].
Mammalian ELP/CTI and the evolution of bovine PTI, STI
and the TKDPs
The Kunitz-type inhibitor domain has been duplicated
many times throughout evolutionary history [38]. This
was no more evident than for the region of bovine
chromosome 13 on which CTI and the 7 CTI-like genes
were located. The PTI, STI and TKDP1-5 genes were
specific to the order Cetartiodactyla, sub-order Rumi-
nantia [50,51,63,72], strong evidence they evolved from
CTI after the divergence of the Ruminantia ~25-35 MYA
[1]. The CTI, PTI and STI genes had a similar 3-exon
structure and conserved regions within both coding and
non-coding segments. The PTI and STI genes and pro-
teins were homologous and almost certainly arose by
gene duplication [73]. However, the TKDP1-5 genes
had one or more additional exons inserted between
Dog
Cow
Elephant
Horse
Mouse
Human
Opossum
WFDC2
35,682 35,684 35,686 35,688 35,690 35,692 35,694 35,696 kbp
gene
exon UTRCNS
Dog
Cow
Elephant
Horse
Mouse
Human
Opossum
100%
10%
100%
10%
100%
100%
10%
10%
100%
10%
100%
10%
100%
10%
100%
10%
100%
100%
10%
10%
100%
10%
100%
10%
kbp 35,698 35,700 35,702 35,704 35,706 35,708 35,710 35,712
~602 kbp
PIGTELP/CTI
Figure 5 VISTA plot of pairwise alignments for selected mammals in the region containing the PIGT, ELP/CTI and WFDC2 genes.
Sequence homology within the PIGT-ELP/CTI-WFDC2 region of the dog, cow, elephant, horse, human, mouse and opossum genomes was
determined with mVISTA [64]. The dog sequence was used as the reference sequence (horizontal axis, dog chromosome 24 numbering). Grey
horizontal arrows indicate gene location and direction of transcription. Blue rectangles indicate coding exons and untranslated regions (UTRs) of
the gene are depicted by light green rectangles. Exon 1 of canine WFDC2 was missing (gap in the current assembly) from the dog genome and is
indicated by a blue rectangle with diagonal white stripes. The right axis indicates the percentage identity within a 100 bp window for each
pairwise comparison, ranging from 10% to 100%. Regions sharing greater than 25% identity are shaded and the black horizontal line indicates
70% identity. The region containing the Kunitz domain-encoding ELP/CTI exon 2 was conserved in the cow, horse, mouse and opossum, but was
absent in the elephant and human CTI genes (red boxes).
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the signal- and pro-peptide-encoding and Kunitz do-
main-encoding exons (equivalent to intron 1 of CTI,
PTI and STI) resulting in an expansion to 4 (TKDP5),
6 (TKDP2, 3 and 4) and 12 exons (TKDP1) [50,51,72].
These added exons encode tripartite N-domains which
had no similarity to database sequences or motifs and
evolved recently due to the “exonization” of an intron
within an active MER retrotransposon and its subse-
quent duplication [50,63]. These elements have been
associated with genetic rearrangements and deletions
[74]. This may explain the excision of CTI exons 2
(Kunitz domain) and 3 (C terminus) for the elephant
and primates, based upon current genome sequencing
and assemblies.
Lack of conservation of the ELP/CTI putative P1reactive
site residue
All putative ELP/CTI peptides were predicted to be secreted
and shared a conserved single 51 amino acid Kunitz do-
main. The conserved location of the 6 cysteine residues
which form three disulphide bonds suggested ELP/CTI
would, like bovine CTI [75] and PTI [46] form a globular
protein. However, neither the identity, physiochemical prop-
erties of the ELP/CTI P1reactive site residue, the trypsin
interaction site, nor the N- and C-terminus of the proteins
were conserved. The P1“warhead” residue plays an essential
role in the interaction of a Kunitz inhibitor domain with a
serine protease and a P1mutation may alter the protease
specificity of the Kunitz domain to a particular substrate
and the reaction kinetics [48,76]. Kunitz inhibitors with a
basic residue, K (Cetartiodactyla) or R (Carnivora) at P1
generally inhibit trypsin or trypsin-like serine endopepti-
dases such as chymotrypsin, pepsin, plasmin and kallikrein
in vitro (e.g. bovine CTI and PTI) [31,38,77]. However,
Kunitz domains with smaller, uncharged residues at P1,
such as serine, generally inhibit elastase-like proteases (eg.
neutrophil elastase) [43,47,76]. In contrast, Kunitz domains
with an acidic, negatively-charged P1residue (e.g. TKDP2)
exhibit minimal antiprotease activity in vitro [72]. Compari-
son of BPTI Kunitz domains suggested that the marsupial
ELP P1amino acids were quite rare [43,49,55]. Furthermore,
the absence of purifying selection within the putative ELP/
CTI trypsin interaction site and the lack of conservation of
P1residues provides intriguing questions as to the role(s) of
the marsupial ELP and eutherian CTI proteins in vivo.
Not all Kunitz domains act as protease inhibitors [43].
As mentioned previously, snake and spider venoms con-
tain proteins with Kunitz domains [40]. Some domains in-
hibit trypsin or chymotrypsin via P1, whilst others lack
anti-protease activity but have neurotoxic effects by acting
as potassium channel blockers [41]. Peigneur and collea-
gues [78] recently reported a sea anemone Kunitz domain
protein, APEKTx1 (Anthopleura elegantissima potassium
channel toxin 1) which had dual functions. It exhibited
both trypsin-inhibitor activity and selectively blocked the
Kv1.1 type of voltage-gated potassium channels. Further-
more, not all Kunitz protease inhibitors act via the P1
Tammar
Possum
Koala
Dunnart FT
Dunnart SF
Opossum
Dog
Panda
Cat
Dolphin
Cow
Pig
STI
PTI
TKDP2
TKDP1
TKDP3
TKDP4
TKDP5
74
61
84
100
100
100
100
100
91
35
40
68
89
100
100
82
100
ELP/CTI
Bovine PTI,
STI and
the TKDPs
Figure 6 A phylogenetic tree of ELP/CTI and the CTI-like bovine
PTI, STI and TKDP1, 2, 3, 4 and 5 family. The evolutionary
relationship between the protein-coding regions of the marsupial ELP,
eutherian CTI and bovine TKDP1-5, PTI and STI transcripts was
determined by maximum likelihood analysis using a molecular clock
assumption. The bovine SLPI transcript was used as an outgroup (data
not shown). Two main groups were formed: 1. mammalian ELP/CTI and
2. bovine CTI, PTI and the TKDPs. Numbers at branch points indicate
confidence levels as determined by bootstrap values (100 replicates).
Phylogenetic trees were produced with Phylip software version 3.69.
Transcripts were aligned with MUSCLE and boostrapped values
generated with SEQBOOT. Maximum likelihood trees were generated
with DNAMLK using a transition/transversion ratio of 1.34, a Gamma
distribution shape of 1.39 with 5 Hidden Markov Model categories,
global rearrangements and with a randomised input order jumbled
once. The protein-coding regions of the following transcripts were used
in the analysis: ELP/CTI, tammar [GenBank: JN191338], fat-tailed dunnart
[GenBank: JN191339], stripe-faced dunnart [GenBank: AC186006], koala
[GenBank: JN191337] opossum [GenBank: JN191340], brushtail possum,
cow [GenBank: JN191341], dog [GenBank: JN191342], cat [GenBank:
BK008083], pig [Ensembl: F1SD34_PIG (ENSSSCT00000008098)], Giant
panda [GenBank: BK008084], and Common bottlenose dolphin
[GenBank: BK008086], and the following bovine transcripts: PTI
[GenBank: NM_001001554], STI [GenBank: NM_205786], TKDP1 [GenBank:
NM_205776], TKDP2 [GenBank: NM_001012683], TKDP3 [GenBank:
XM_584746], TKDP4 [GenBank: NM_205775], and TKDP5 [GenBank:
XM_614808] and SLPI [GenBank: NM_001098865].
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residue. The tick anticoagulant peptide (TAP) inhibits Fac-
tor X, Factor Xa and thrombin but the reactive site is
located towards the N-terminus of the protein, rather than
at the P1residue of the Kunitz domain [79].
ELP/CTI – a conserved N-glycosylation site predicted
within the Kunitz domain
All ELP/CTI proteins shared a putative conserved N-glyco
sylation site within the Kunitz domain at asparagine-42 (as-
ELP
RNA
PREGNANCY
⎨
Phase 1
LACTATION
⎨
LGB
⎧⎧
10P17P
18P
21P 26P
20L
70L
76L 80L87L
127L130L
163L
151L 180L
240L260L
13P15P 16P
20P
22P 25P
VIRGIN
1L2L 2L
2L NS 3L NS4L NS
10L15L
40L
168L
Phase 2A
Phase 2BPhase 3
⎧⎧
⎪⎪
A
Lactation stage (days)
Phase 1
Day 2
Day 260
Day 35 Day 210Day 158
Average gene expression
0
1.0
2.0
3.0
4.0
5.0
6.0
7.0
8.0
204060 80 100120140 160 180 200220 240 2600 -20
280
Phase 2APhase 2B Phase 3Virgin gland
Day 95
9.0
Day 25
fetus
B
ELP LGB GAPDH
Figure 7 ELP expression in the tammar mammary gland throughout the reproductive cycle. A. Northern analysis of total RNA (10 μg)
extracted from the mammary glands of tammar wallabies during pregnancy (P, Phase 1) and lactation (L, Phase 2A, Phase 2B and Phase 3), from
non-sucked (NS) glands and from a virgin female (~220 days of age). Tammar ELP expression was undetected in the virgin gland, minimal during
pregnancy (Phase 1) and then induced at parturition and expressed during early lactation (Phase 2A). ELP was down-regulated at mid-lactation
(Phase 2B), consistent with [13,20,21]. ELP transcripts were not detected in Phase 3. ELP expression also declined postpartum in non-sucked glands.
Tammar LGB was used as a positive control for lactation and exhibited a similar expression pattern to ELP, but with LGB expression increased (but
not significantly so) during Phases 2B and 3, as reported previously [13,68,69]. Ribosomal RNA bands indicate RNA integrity and loading. B.
Microarray analysis of the tammar mammary gland [ArrayExpress: E-MTAB-1057] supported the quantitative analysis of Northern blot (data not
shown) and microarray data reported by [69]. Expression of the ELP and LGB milk protein genes and the housekeeping gene GAPDH
(glyceraldehyde 3-phosphate dehydrogenase) is depicted as average normalised raw intensity based upon the expression n=3, 7 and 2 clones on
each microarray respectively±SEM (Additional file 12: Table S5). Whilst ELP (red) and LGB (blue) expression differed during the reproductive cycle,
GAPDH (green) expression was constant.
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paragine-40 for koala ELP), consistent with the site identi-
fied for bovine CTI in vitro [58]. The proportion of sugars
attached to glycosylated bovine CTI, possum ELP and tam-
mar ELP varies, 25-40% [58,80], 60% [25] and ~47-55%
[20,21,26], respectively. However, as the N-glycosylation site
occurs at the base of the pear-shaped protein and at the op-
posite end to the P1site, it is unlikely to affect protease-
binding activity [58]. Unlike bovine CTI, the Kunitz
domains of neither bovine PTI, STI, nor for the placenta-
specific TKDPs are predicted to be N-glycosylated. In fact,
very few Kunitz domains are N-glycosylated, or predicted to
be so [43,49,55]. The exceptions are SPINT4, SPINLW1,
the first Kunitz domains of bikunin and hepatocyte growth
factor activator inhibitor, the second domain of tissue factor
pathway inhibitor 1, as well as selected sea anemone pep-
tides. The precise effect of N-glycosylation is uncertain, but
it may enhance protein hydrophilicity and solubility, reduce
proteolysis, influence cell surface signalling and adhesion
Muscle (Psoas)
Testes
Epididymis
Virgin
Abdominal fat
Heart (Atrium)
Heart (Ventricle)
Ovary
Pouch skin
24P
76L
150L
260L
Adrenal gland
Spleen
Liver
Lung
Pituitary gland
Ltmph node
Kidney
Salivary gland
Pancreas
Brain
Small intestines
Hind gut
Mammary gland
⎨⎧
ELP
LGB
RNA
⎧
Figure 8 Tammar ELP expression was specific to the mammary gland. Northern analysis of total RNA (10 μg) extracted from assorted
tammar tissues indicated that both ELP and LGB expression were specific to the mammary gland. Ribosomal RNA bands indicate RNA integrity
and loading.
Million years ago (MYA)
CENOZOICPALEOZOICMESOZOIC
Tertiary
JurassicTriassic Cretaceous
PermianCarboniferous
0 65
146
250 208 360
290
Eutherians
Cetartiodactyla
(cow, pig, dolphin)
Marsupials
(tammar wallaby,
dunnarts, possum,
koala, opossum)
Amniotes
166
MYA
148
MYA
Viviparity
Placentation
Nutritive Lactation
Homeothermy
Prolonged gestation
315
MYA
Monotremes
Mammalian ancestor
Birds
Reptiles
Oviparity and lactation
Carnivora
(cat, dog)
ELP
CTI
ELP/
CTI
X
X
?
?
?
ELP/
CTI
Prolonged and
complex lactation
Figure 9 Evolution of the ELP/CTI gene in therians. Tree depicting the relationship between the amniotes: birds, reptiles, monotremes,
marsupials and eutherians [1,3,70,71] and the distribution of the ELP/CTI gene. The divergence times used are based upon the analysis by Bininda-
Emonds and colleagues [1]. Extant species which have a functional ELP/CTI gene are indicated by green tree branches. Extant species in which the
ELP/CTI gene has not been detected are indicated by a red cross. Lineages on the tree for which the presence or absence of the ELP/CTI gene
remains inconclusive are indicated by a red question mark. Based upon current analyses, the functional ELP/CTI gene evolved at least 130 million
years ago (MYA) and has been retained by extant marsupials and the Laurasiatherian orders Cetartiodactyla and Carnivora. Whether the ELP/CTI
gene is present in monotremes is unknown.
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and affect protein folding, turnover and quality control [81-
83]. Furthermore, oligosaccharides may act as soluble re-
ceptor analogues for bacterial and viral pathogens, prevent-
ing them from attaching to the wall of the intestines,
thereby stopping their passage through the gastrointestinal
and urinary tracts of the young [84,85].
The lack of conservation of the ELP/CTI N- and
C-terminus was intriguing, particularly the positive
Darwinian selection (p<0.05) acting upon the coil-
like marsupial ELP N-terminus. In contrast, the eu-
therian CTI N-terminus tended towards neutral se-
lection. The N- and C-termini of proteins have been
associated with sub-cellular targeting, protein-protein
and protein-lipid interactions and macromolecular
complex formation [86]. The marsupial- and euther-
ian-specific homology of the mature ELP/CTI N-
terminus suggested these regions may have different
activities. However, the lack of conservation of the
ELP/CTI C-terminus suggested these areas may have
species-specific effects. Interestingly, the conservation
of the TGA codon used by the tammar, koala, pig,
dolphin and cow for all species but the cat (CGA)
suggested it was the ancestral ELP/CTI stop codon,
with more recent mutations producing a shortened
ELP/CTI C-terminus in some species. Furthermore, a
conserved marsupial-specific region within the 3'
UTR may regulate ELP gene transcription.
ELP/CTI is expressed and secreted in milk during the
early lactation/colostrogenesis period only [this study,
[20,21,25-28,31,36,37]]. Furthermore, all mammalian neo-
nates have an innate immune system but an immature
adaptive immune system and a gut which is yet to undergo
maturation or ‘closure’ and is therefore permeable to
macromolecules [16,29,87-89]. For the calf, gut maturation
occurs 24–36 hr pp [16], whereas for the tammar, this
process does not occur until ~200 days pp [87]. Therefore,
maternal milk immunoglobulins such as IgG can be pas-
sively transferred via colostrum and Phase 2A/2B milk to
the gut of the young calf and tammar, respectively, where
they are absorbed by the intestines and enter the circulatory
system [16,89]. Hence ELP/CTI may enhance the survival
of the young by preventing the proteolytic degradation of
maternal immunoglobulins [31], or by protecting the young
against pathogens [25]. Although sequence comparisons
predict the ELP/CTI peptides are likely to inhibit serine
endopeptidases, their true function(s) will only be deter-
mined through in vitro and/or in vivo studies.
The importance of local control mechanisms in the regu-
lation of the tammar mammary glands and ELP were high-
lighted in this study. Whilst ELP expression proceeds in the
sucked gland, the gene is down-regulated and milk produc-
tion ceases in the non-sucked glands, as for the possum
[28]. However, this partitioning of mammary glands and
lactation does not occur in eutherians [6]. Marsupial ELP/
eutherian CTI expression was specific to the mammary
gland and lactation (Figure 8), unlike the genes that most
likely evolved from bovine CTI. PTI and STI are produced
in mast cells, which have a protective role and are distribu-
ted throughout the body to tissues such as the duodenum,
pancreas, lung, pituitary gland, spleen and chondrocytes
[90]. In contrast, the five bovine TKDPs are differentially
expressed in trophoblast cells of the ruminant placenta only
during the peri-implantation period, suggesting they have
an important role in the maintenance of the conceptus and
pregnancy [51,63,72]. Hence, the bovine PTI, STI and
TKDP1-5 genes have undergone positive (adaptive) selec-
tion, changes in tissue-specific expression and function
compared to the putative CTI ancestral gene, consistent
with gene duplication and neofunctionalisation [91,92].
The location of the CTI gene in a rapidly evolving region
of the eutherian chromosome [51,62] may explain the con-
version of CTI into a putative pseudogene in Afrotheria
(elephant), Xenarthra (sloth, armadillo), Euarchontoglires
(humans, primates, rodents) and in selected Laurasiather-
ians such as the horse and flying fox.
This region included many additional genes with Kunitz
and WAP 4-DSC domains [62], unlike for marsupials. It is
possible that the role of CTI is fulfilled by one of these
genes and hence the loss of the CTI gene is tolerated. Alter-
natively, CTI function may have become non-essential due
to physiological changes in selected species. Notably, milk
protein gene loss is not common amongst mammals, as
genes involved in milk production are generally under nega-
tive selection [93]. However, the conservation of the ELP/
CTI gene in marsupials and Laurasiatherian orders Carniv-
ora (dog, cat, dolphin, panda) and Cetartiodactyla (cow, pig)
suggests ELP/CTI has an important role in these species.
Conclusions
Marsupial ELP and eutherian CTI evolved from a common
ancestral gene and encode a milk protein with a single
BPTI-Kunitz serine protease inhibitor domain. Although
CTI was identified as the putative ancestral gene of PTI,
STI and the placenta-specific trophoblast TKDP1-5 gene
family, the origin of the ELP/CTI gene is inconclusive. ELP/
CTI expression in the postpartum mammary gland is brief
(~24-48 hrs) in eutherians but prolonged in the tammar
and other marsupials (up to 100 days). However, this period
correlates with the provision of milk to an immuno-incom-
petent young, suggesting ELP/CTI may play a vital role in
immune protection of the young at this time.
Methods
Animals
Tammar wallabies (Macropus eugenii) were provided
from two different marsupial colonies: VIAS (Victorian
Institute of Animal Science), DPI (Department of Primary
Industries), Attwood, Victoria and The University of
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Melbourne, Victoria. Animals were kept in open grassy
yards with ad libitum access to food, water and shelter,
using standard animal husbandry conditions in accord-
ance with the National Health and Medical Research
Council guidelines [94]. All experiments were approved
by the Animal Experimentation Ethics Committees of the
Department of Primary Industries and The University of
Melbourne.
Tissues
Tissues (salivary gland, adrenal gland, pituitary gland,
lymph node, spleen, liver, kidney, lung, pancreas, brain,
small intestines, hind gut, muscle, heart, ovaries) were col-
lected from adult female tammars (n=2). Mammary
glands were also collected from adult females at different
stages of pregnancy and lactation (n=60). Mammary
glands from virgin females were collected from tammar
pouch young (~220 days of age, n=3). Testes and epididy-
mides were collected from adult tammar males (n=2).
Tissue samples derived from ear-tagging of a population of
koalas (Phascolarctos cinereus) located on French Island,
Victoria, were donated by Dr. Kath Handasyde and Dr.
Emily Hynes from the Department of Zoology, The Uni-
versity of Melbourne. Total RNA extracted from a grey
short-tailed opossum (Monodelphis domestica) mammary
gland from day 15 of lactation (early-lactation) was pro-
vided by Dr Denijal Topcic (The University of Melbourne)
from animals provided by Professor Norman Saunders
(The University of Melbourne). Dr Peter Frappell (Latrobe
University) provided fat-tailed dunnart mammary gland
tissue from day 37 of lactation (Phase 2) and liver tissue.
Dr Amelia Brennan (The University of Melbourne) pro-
vided total RNA isolated from the mammary gland of a
late-pregnant (~8 months) Holstein-Friesian cow. A small
quantity of dog colostrum (~20 μL) from a late-pregnant
(~2 weeks prepartum) Labrador in its first pregnancy was
also kindly donated by Cate Pooley (The University of
Melbourne). All samples were snap frozen in liquid nitro-
gen and stored at −80°C until use, with the exception of
the koala ear punches, which were stored at 4°C.
RNA extraction and northern analysis
Total RNA was extracted from tissues using the Qiagen
RNeasy Midi Kit (Qiagen) and from cells isolated from
colostrum using RNAWIZ (Ambion). RNA extracted
from cells shed into milk during the lactation process
provides a good representation of gene expression in the
mammary gland [95] and therefore eliminates the need
for destructive tissue sampling. RNA was electrophor-
esed through a 1% agarose, low-formaldehyde (1.1%) gel
with 1X MOPS [3(N-Morpholino) Propane Sulfonic
Acid] buffer at 4°C and then transferred to Zeta-Probe
GT Blotting Membrane (BioRad) in 20X SSC (3.0 M
sodium chloride, 0.3 M trisodium citrate, pH 7.0)
overnight.
Membranes were rinsed in 2X SSC, UV crosslinked at
1200 J (Stratagene UV Stratalinker1800) and hybridized
in 25 mL [30% deionised formamide, 5 X SSC, 50 mM
sodium acetate, herring sperm DNA (100 μg/μL), 5 mL
Denhart’s 50X stock solution, 0.1% SDS] with an [α-32P]
dCTP-labelled probe [DECAprime II Random Priming
DNA Labelling Kit (Ambion)] and incubated for ~16 hr
at 42°C. The tammar ELP, RsaI digested LGB (to detect
both LGB transcripts [96]) and CST3 probes were either
amplified by RT-RCR from tammar mammary gland
total RNA or sourced from clones in a tammar mam-
mary gland EST library held by the Cooperative
Research Centre for Innovative Dairy Products [19], with
plasmid DNA isolated and the cDNA insert amplified by
PCR. Membranes were washed (0.1X SSC, 0.1% SDS)
twice for 15 min at 60°C, wrapped in cling film, sealed
into plastic pockets and exposed to a General Purpose
Storage Phosphor screen and scanned on a Typhoon
8600 Scanner (Molecular Dynamics/GE Healthcare).
Membranes were stripped of probes by incubation with
boiling (100°C) 1X SSC, 0.1% SDS on a shaking platform
for two 15 min periods, then rinsed with RT 1X SSC,
0.1% SDS.
RT-PCR and cloning of ELP/CTI
cDNA was generated using Superscript III Reverse Tran-
scriptase (Invitrogen), oligo(dT)20 primer (50 μM;
Sigma-Proligo) and 5 μg of total RNA isolated from
mammary tissue or cells separated from milk. PCR was
performed using 2 μL (10%) of the first strand reaction,
the proof-reading Platinum Taq DNA Polymerase High
Fidelity (Invitrogen), plus the appropriate forward and
reverse primers and conditions to amplify ELP/CTI tran-
scripts (Table 3). PCR products were cloned into the
pGEM-TEasy VectorSystem
sequenced. Full protein-coding ELP/CTI transcripts were
cloned from total RNA extracted from the fat-tailed dun-
nart, cow and opossum mammary gland tissues and from
cells in canine colostrum.
I (Promega) and
Genomic DNA isolation and cloning
Genomic DNA was isolated from koala and fat-tailed
dunnart tissues as described [97]. The ELP/CTI genes
were amplified by PCR (Table 3) using Platinum Taq
DNA Polymerase and ~200 ng of genomic DNA tem-
plate, cloned into pGEM-T Easy and sequenced.
Isolation of the tammar ELP gene from a genomic library
A tammar genomic library (liver) in the E. coli phage
vector lambda EMBL3 T7/SP6 was screened with
tammar ELP cDNA and a positive clone isolated. The
clone was SalI digested and the ~14.7 kb genomic
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DNA fragment cloned into a modified pBeloBACII
plasmid vector. Digestion of pBeloBACII-14.7kbtELP
with SalI and HindIII yielded three fragments, 6.2 kb
SalI/HindIII, 5.2 kb HindIII/HindIII and 3.3 kb SalI/
HindIII. These fragments were sub-cloned into pBlue-
script SK and the latter two clones sequenced by the
Australian Research Genome Facility (Australia). The
remaining 6.2 kb was sequenced (Department of Path-
ology, The University of Melbourne), providing the
full sequence of the genomic clone (14.704 kb).
BLAST [98] searches of the NCBI Macropus eugenii
WGS (Whole Genome Shotgun) trace archives and
assembly of hits with CAP3 [99,100] produced a con-
tig of 54,363 bp which included ELP and the first 2
exons of WFDC2.
Fluorescence in situ hybridisation (FISH)
Metaphase spreads were prepared from the tammar and
FISH performed as described [101]. The 14.7 kb tammar
ELP genomic clone was used as a probe. Slides were
examined using a Zeiss Axioplan microscope and images
captured using the Spot Advance software package. Pic-
tures were processed with Confocal Assistant, Image J,
Adobe Illustrator and Adobe Photoshop. Chromosomal
location of ELP was verified by at least ten metaphase
spreads that had at least three or four signals out of a
maximum of four.
cDNA microarray analysis of tammar ELP gene expression
ELP gene expression in the tammar mammary gland
was investigated by analysing a microarray database
[69,102-104]producedfrom
microarray slides and total RNA collected from glands
at each phase of the lactation cycle [69,102-104]. Glass
microarray slides were printed by the Peter MacCallum
Cancer Centre Microarray Core Facility, Melbourne,
Australia and contained 10,368 tammar cDNA spots
which were derived from a commercially prepared (Life
Technologies, Rockville, MD, USA), normalised 15,001
tammar mammary gland EST (expressed sequence tag)
library. The library was prepared using tammar mam-
mary gland total RNA pooled from various time points
in pregnancy (P), lactation (L) and involution (I). These
included: day 26P, d55L, d87L, d130L, d180L, d220L,
d260L and d5I (tissue from a d45L female 5 days after
removal of the pouch young (RPY)) [19]. Gene expres-
sion changes in the tammar mammary gland during
the reproductive cycle were investigated by a large-
scale microarray experiment involving 36 comparisons
(72 slides including dye swaps, 144 channels in total)
[69,102-104].
Sixteen different time points were used in the experi-
ment: virgin female~300 days old (n=3), pregnancy
(Phase 1: d5P, d25P, d26P; n=1 per time point), lactation
(Phase 2A: d1L, d5L, d80L; Phase 2B: d130L, d168L,
d180L; Phase 3: d213L, d220L, d260L; n=1 per time
custom-madecDNA
Table 3 Primer sequences and conditions used to amplify ELP/CTI genes and transcripts
ELP/CTI gene/
transcript
FT dunnart
transcript
FT_ELP_R CCCAAAGTGCTGTTAATGCTTTATTGTAGC
Name Primer Sequence15' 3' PCR Product
Size (bp)
Primer Conditions
FT_ELP_F GTCAAGTGTTATCTACTGGCAGCACCATG488 94°C for 2 min; 35 cycles of 94°C for 30 sec;
59°C for 30 sec; 68°C for 1 min; 68°C for 10 min
Opossum transcript
mELP_NheI_F
GCTAGCAAGGTTTTCTCTCAGTGCCATC488 94°C for 2 min; 35 cycles of 94°C for 30 sec;
60°C for 30 sec; 68°C for 30 sec; 68°C for 10 min
mELP_BamHI_R
GGATCCTGTTAATGCTTTATTGTACCAG
Tammar transcript
tELP_NheI_F
GCTAGCAAGTGTAGTCTACCAGTGGCACC479 94°C for 2 min; 35 cycles of 94°C for 30 sec;
58°C for 30 sec; 68°C for 30 sec; 68°C for 10 min
tELP_BamHI_R
GGATCCTGTTAATGCTTTATTGTACCAG
Dog
transcript
Dog_ELP_Ex1_F GCCTAGAACATTCAGCTATTGGCACC 44994°C for 2 min; 35 cycles of 94°C for 30 sec;
55°C for 30 sec; 68°C for 1 min; 68°C for 10 min
Dog_ELP_Ex3_R TGAATGTTTTATTGACCTAGACCTGGAGG
Cow transcript
bELP_NheI_F
GCTAGCAACTCACAGCTCCTCACACCATG463 94°C for 2 min; 35 cycles of 94°C for 30 sec;
58°C for 30 sec; 68°C for 30 sec; 68°C for 10 min
bELP_BamHI_R
GGATCCGAACACTTTATTGACCCAGTCCTG
FT dunnart gene
FT_ELP_F GTCAAGTGTTATCTACTGGCAGCACCATG 4771 94°C for 2 min; 35 cycles of 94°C for 30 sec;
55°C for 30 sec; 68°C for 6 min; 68°C for 10 min
FT_ELP_R CCCAAAGTGCTGTTAATGCTTTATTGTAGC
Koala gene
tELP_Ex1_F GGTAGCAAGTGTAGTCTACCAGTGGCACC1428 94°C for 2 min; 35 cycles of 94°C for 30 sec;
52°C for 30 sec; 68°C for 4 min; 68°C for 10 min
tELP_BamHI_R
GGATCCTGTTAATGCTTTATTGTACCAG
Tammar gene
(6.2 kbpromoter)
T7 TAATACGACTCACTATAGGG6326 94°C for 2 min; 35 cycles of 94°C for 30 sec;
57°C for 30 sec; 68°C for 8 min; 68°C for 10 min
tELP_Prom_RGACTGATCAGACCAATATAAGCTT
Tammar gene
(7.9 kbpromoter)
T7 TAATACGACTCACTATAGGG804494°C for 2 min; 35 cycles of 94°C for 30 sec;
57°C for 30 sec; 68°C for 8 min; 68°C for 10 min
tELP_Ex1_R GAGGGCCAACGATGGTAAATTTCAT
1Restriction enzyme sites are indicated in bold, italicised text.
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