An intronic insertion in KPL2 results in aberrant splicing and causes the immotile short-tail sperm defect in the pig.
ABSTRACT The immotile short-tail sperm defect is an autosomal recessive disease within the Finnish Yorkshire pig population. This disease specifically affects the axoneme structure of sperm flagella, whereas cilia in other tissues appear unaffected. Recently, the disease locus was mapped to a 3-cM region on porcine chromosome 16. To facilitate identification of candidate genes, we constructed a porcine-human comparative map, which anchored the disease locus to a region on human chromosome 5p13.2 containing eight annotated genes. Sequence analysis of a candidate gene KPL2 revealed the presence of an inserted retrotransposon within an intron. The insertion affects splicing of the KPL2 transcript in two ways; it either causes skipping of the upstream exon, or causes the inclusion of an intronic sequence as well as part of the insertion in the transcript. Both changes alter the reading frame leading to premature termination of translation. Further work revealed that the aberrantly spliced exon is expressed predominantly in testicular tissue, which explains the tissue-specificity of the immotile short-tail sperm defect. These findings show that the KPL2 gene is important for correct axoneme development and provide insight into abnormal sperm development and infertility disorders.
- SourceAvailable from: hmg.oxfordjournals.org[show abstract] [hide abstract]
ABSTRACT: Few autosomal recessive disorders display the degree of pleiotropism and genetic heterogeneity found in Bardet-Biedl syndrome (BBS), a genetic disorder characterized primarily by retinal dystrophy, obesity, polydactyly, cognitive impairment and gonadal and renal dysgenesis. This relatively rare condition has been reported frequently, but we have only recently begun to appreciate the genetic complexities that give rise to this constellation of clinical findings. During the last 12 months, the first three of at least six BBS genes have been identified, providing us for the first time with the ability to formulate hypotheses regarding the molecular etiology of the disorder. Here we review the key elements of the phenotype and discuss the significance of the discovery of the first three BBS genes on the effort to identify the cellular causes of this syndrome.Human Molecular Genetics 11/2001; 10(20):2293-9. · 7.69 Impact Factor
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ABSTRACT: Four mutants of Chlamydomonas reinhardtii representing independent gene loci have been shown to lack totally (pf-18, pf-19, and pf-15) or nearly totally (pf-20) the central microtubular pair complex in isolated axonemal preparations. Analysis of 35S-labeled axonemal proteins, using two methods of electrophoresis, reveals that all four mutants lack or are markedly deficient in 18 polypeptides, ranging in molecular weight from 360,000 to 20,000, that are regularly present in wild-type axonemes. Analyses of axonemal proteins labeled by cellular growth on 32P-labeled medium indicates that a subset of 8 of the 18 polypeptides are phosphorylated. Mutant and wild-type axonemes and flagella have been analyzed for their content of tubulin subunits using a high resolution two-dimensional electrophoresis system combined with agarose gel overlays containing either anti-alpha or anti-beta tubulin sera prepared from Chlamydomonas tubulins. The immunoprecipitates identify two major alpha tubulins, a major beta tubulin, and a minor component which is also precipitated by the anti-beta serum. None of these tubulins shows a specific defect in mutant axonemes, nor do the tubulin polypeptides show altered two-dimensional map positions in the mutant flagella. The 18 polypeptides provide a useful signature for identifying other mutants affecting the central-pair microtubular complex. Such mutants could be useful in defining the structural or functional role of these polypeptides in the central microtubules. Efforts to obtain additional central-pair mutants based on the motility phenotype of the four mutants analyzed here have yielded mutants which are allelic to three of the four mutants.The Journal of Cell Biology 11/1981; 91(1):69-76. · 10.82 Impact Factor
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ABSTRACT: Sperm motility is generated by a highly organized, microtubule-based structure, called the axoneme, which is constructed from approximately 250 proteins. Recent studies have revealed the molecular structures and functions of a number of axonemal components, including the motor molecules, the dyneins, and regulatory substructures, such as radial spoke, central pair, and other accessory structures. The force for flagellar movement is exerted by the sliding of outer-doublet microtubules driven by the molecular motors, the dyneins. Dynein activity is regulated by the radial spoke/central pair apparatus through protein phosphorylation, resulting in flagellar bend propagation. Prior to fertilization, sperm exhibit dramatic motility changes, such as initiation and activation of motility and chemotaxis toward the egg. These changes are triggered by changes in the extracellular ionic environment and substances released from the female reproductive tract or egg. After reception of these extracellular signals by specific ion channels or receptors in the sperm cells, intracellular signals are switched on through tyrosine protein phosphorylation, Ca2+, and cyclic nucleotide-dependent pathways. All these signaling molecules are closely arranged in each sperm flagellum, leading to efficient activation of motility.ZOOLOGICAL SCIENCE 10/2003; 20(9):1043-56. · 1.08 Impact Factor
An intronic insertion in KPL2 results in aberrant
splicing and causes the immotile short-tail sperm
defect in the pig
Anu Sironen*†, Bo Thomsen‡, Magnus Andersson§, Virpi Ahola¶, and Johanna Vilkki*
*MTT Agrifood Research Finland, Animal Production Research, Animal Breeding, FIN-31600, Jokioinen, Finland;‡Department of Genetics and
Biotechnology, Danish Institute of Agricultural Sciences, P.O. Box 50, DK-8830 Tjele, Denmark;§Department of Clinical Veterinary Sciences,
Saari Unit, Faculty of Veterinary Medicine, University of Helsinki, FIN-04920, Saarentaus, Finland; and¶MTT Agrifood Research Finland,
Food Research, FIN-31600, Jokioinen, Finland
Edited by Ryuzo Yanagimachi, University of Hawaii, Honolulu, HI, and approved February 3, 2006 (received for review July 25, 2005)
The immotile short-tail sperm defect is an autosomal recessive
disease within the Finnish Yorkshire pig population. This disease
specifically affects the axoneme structure of sperm flagella,
whereas cilia in other tissues appear unaffected. Recently, the
disease locus was mapped to a 3-cM region on porcine chromo-
some 16. To facilitate identification of candidate genes, we con-
structed a porcine-human comparative map, which anchored the
disease locus to a region on human chromosome 5p13.2 containing
eight annotated genes. Sequence analysis of a candidate gene
KPL2 revealed the presence of an inserted retrotransposon within
an intron. The insertion affects splicing of the KPL2 transcript in
in the transcript. Both changes alter the reading frame leading to
premature termination of translation. Further work revealed that
the aberrantly spliced exon is expressed predominantly in testic-
ular tissue, which explains the tissue-specificity of the immotile
short-tail sperm defect. These findings show that the KPL2 gene is
important for correct axoneme development and provide insight
into abnormal sperm development and infertility disorders.
cilia ? retrotransposon ? spermatogenesis
perception, and development. The biogenesis and maintenance
of cilia depend on the intraflagellar transport system, which is
required for the assembly and elongation of cilia by transporting
ciliary precursors to their site of incorporation (1). The internal
cytoskeletal structure of cilia, flagella, basal bodies, and cent-
rioles, called the axoneme, is highly conserved among eukaryotic
cells and consists of ?250 polypeptides (2, 3). The axoneme
structure of most motile cilia and flagella consists of nine outer
doublet microtubules surrounding a central pair of singlet mi-
crotubules. Projecting from the doublet microtubules are an
inner and an outer row of dyneins, which are ATP-dependent
motor proteins. Neighboring peripheral doublet microtubules
are linked to each other by the elastic protein nexin, which are
also connected to the inner singlets by radial spokes. Axonemal
bending, which provides the force for cilia movement, is gener-
ated by transient interactions of dyneins and doublet microtu-
bules that cause sliding between pairs of outer microtubules. The
central pair and radial spokes complex selectively interact with
subsets of dynein arms to regulate the sliding movements of
microtubules (4, 5). Furthermore, primary cilia are usually
immotile and contain a ‘‘9 ? 0’’ axoneme that lacks the central
pair singlets, the radial spokes, and the dynein complex. Primary
cilia are ubiquitous organelles in most vertebrates (6).
Mutations in proteins that function in basal bodies, in-
traflagellar transport system machinery, axonemes, ciliary ma-
trix, and ciliary membrane can lead to cilia related diseases in the
human such as polycystic kidney disease, retinal dystrophy,
ilia and flagella play important roles in many physiological
processes, including cellular and fluid movement, sensory
neurosensory impairment, Bardet-Biedl syndrome, or primary
ciliary dyskinesia (PCD) (7–10). PCD is a genetically heteroge-
neous group of disorders with axonemal abnormalities affecting
one in 16,000 individuals (11). PCD is characterized by the
complete absence of or occurrence of defective cilia and flagella.
Structural defects have been observed in several axoneme
components, including outer and inner dynein arms, radial
spokes, nexin links, and microtubules. Thus far, only mutations
in genes DNAI1, DNAH5, and DNAH11 encoding for proteins of
the outer dynein arms have been identified (11–13). The com-
mon clinical manifestations of PCD are situs inversus, bronchi-
ectasis, chronic sinusitis, and male sterility.
The first case of the immotile short-tail sperm (ISTS) defect
in pigs was detected in Finnish Yorkshire (Large White) boars
in 1987 and, to date, 82 boars are known to be affected. The ISTS
phenotype is characterized as lowered sperm counts, short
sperm tails, and axonemal abnormalities. Electron microscopic
examination of flagella cross-sections has revealed that typically
one or both of the central microtubules are missing, and often
there are less than nine doublets and the subunits of the doublets
are broken apart. However, dynein arms appear normal in
cross-sections. Approximately 5% of spermatozoa of affected
boars have flagella of normal length, but none are motile (14).
The disorder appears to be specific to sperm tail development,
because no effects on the structure of cilia in the respiratory or
female reproductive tract have been observed (14). The ISTS
defect provides an ideal opportunity to analyze the function of
a gene affecting cilia and sperm tail development. Homozygosity
mapping and haplotype analysis has located the ISTS associated
gene to porcine chromosome 16 within a 3-cM region proximal
to SW419 (15). In the present study, we fine-mapped the
causative mutation to an interval corresponding to 1.158 kbp on
human chromosome 5 containing eight annotated genes. We
show that, in one of these genes, KPL2, a retrotransposon within
in testicular tissue. These findings are consistent with earlier
studies reporting that KPL2 is expressed predominantly in
ciliated tissues and at specific stages of sperm cell development
in the rat (16).
Fine Mapping. Fine mapping was initiated by isolating porcine
BAC clones containing the markers SW2411, SW419, and S0006.
Conflict of interest statement: No conflicts declared.
This paper was submitted directly (Track II) to the PNAS office.
for the Sus scrofa KPL2 partial genomic sequence).
†To whom correspondence should be addressed. E-mail: email@example.com.
© 2006 by The National Academy of Sciences of the USA
March 28, 2006 ?
vol. 103 ?
End sequences of BAC clones were used to construct a partial
contig of the disease-associated region by chromosome walking
(data not presented). BLASTN searches with BAC-end sequences
from the contig against the human NCBI database located the
chromosome 5p13.2. Porcine ESTs corresponding to genes in
this region were sequenced and SNPs were found in the genes
AMACR and RAI14. A recombination in two affected boars
between the ISTS defect and RAI14 reduced the disease-
associated region to 1,158 kbp in the human map, which harbors
eight annotated genes (RAI14, FLJ25439, RAD1, BRIX,
LOC134218, AGXT2, PRLR, and FLJ23577). Intriguingly, the
seminiferous tubules, and is therefore an attractive candidate
using the isolated BAC-clones as a template showed that the
KPL2 gene is indeed located in close proximity to the disease-
linked marker SW419. Expression of KPL2 in porcine testis was
confirmed by PCR amplification of testicular cDNA with KPL2-
specific primers. The cDNA of KPL2 was sequenced from a
normal and an affected boar. This analysis identified 10 SNPs
and also revealed that exon 30 was absent in the KPL2 transcript
of the affected boar. Furthermore, genotyping of additional
animals showed that all 10 SNPs were homozygous in several
normal boars as well as in affected boars, excluding the possi-
bility that these SNPs are disease-causing mutations. Mutation-
associated exon skipping is known to be the underlying cause for
an increasing number of diseases (17, 18), which prompted us to
investigate the mechanism leading to the absence of exon 30 in
affected animals in greater detail.
Mutation Detection. The KPL2 intron 29 (3,305 bp) and the first
1,500 bp of intron 30 were sequenced by using genomic DNA of
a normal and an affected boar. Three SNPs were found, all of
which were heterozygous in affected boars. During this sequenc-
ing, some primer pairs designed to amplify the beginning of
intron 30 were found not to produce a product using DNA from
possible insertions or deletions by positioning overlapping
primer pairs in intron 30 (Fig. 1A). Most primer pairs resulted
in identical PCR fragments in normal and affected boars.
However, certain primer combinations generated a product only
in normal animals. The inability to generate a PCR fragment
suggested the presence of a large insertion in intron 30 in
affected animals. To corroborate this, we performed a Southern
blot analysis using a probe that spans the junction between exon
30 and intron 30 (Fig. 1B). The data indicate that a large segment
of ?9,000 bp has been inserted into intron 30 in affected boars.
Finally, long-range PCR was used to amplify a 9-kbp fragment,
verifying the presence of a large insertion in intron 30 (Fig. 1B).
Sequencing of the fragment is currently ongoing. Preliminary
sequence information was used to search GenBank, which
indicated that one end of the sequence was homologous to a
porcine genomic sequence containing the PERV-A retrovirus
(GenBank accession no. AY160111, identities 542?544), and to
the 5? genomic sequence of a porcine endogenous retrovirus
clone PERV-A (GenBank accession no. AJ304824, identities
387?387). The sequence of the other end of the insertion showed
86% identity over ?900 bp to LINE-1 elements, which are
abundant retrotransposons in mammals (19). These data indi-
cate that a retrotransposition event has disrupted the intron,
although the exact structure of the retrotransposon remains
To generate a PCR fragment (KPL2i) diagnostic of the
presence of the insertion, we designed a reverse primer within
the insertion and used this primer in combination with a forward
primer within exon 30 to genotype normal, carrier, and ISTS-
affected pigs. Another fragment (KPL2n) amplified with a
forward primer at the end of exon 30, and a reverse primer in the
intron 30 downstream of the insertion site was used as a marker
for unaffected chromosomes. This PCR-based assay showed that
the insertion was homozygous only in ISTS affected boars and
heterozygous in carrier pigs, whereas no product for KPL2i was
observed in samples of normal individuals from the Yorkshire
(n ? 10), Duroc, Hampshire, and Landrace breeds (four indi-
viduals from each breed). Thus, these data show that the
presence of the 9,000-bp insertion in intron 30 is associated with
the ISTS defect.
Characterization of Aberrant Splice Products. Subsequently, KPL2
transcript splicing in various tissues was characterized. Semi-
quantitative PCR amplification of cDNA across base pairs
3978–4466 (KPL2e29–36) produces a fragment of 487 bp in
normal boars and a fragment of 257 bp in ISTS affected boars.
A less abundant fragment of 998 bp was also detected in two
affected boars (Fig. 2A). Sequencing of these fragments showed
that exon 30 (230 bp) was missing in the shorter fragment, and
that exon 30 was present in the longer fragment, but only in
combination with part of the intron 30 (59 bp preceding the
insertion) and the beginning of the insertion (452 bp). Impor-
tantly, the reading frame in both of these aberrantly spliced
Identification of the insertion site in individuals affected with ISTS by PCR
analysis. Primer pairs on both sides of the insertion resulted in similar PCR
fragments for normal (Nor) and affected boars (Aff), whereas primer pairs
over the insertion site only produced a PCR product for normal boars. (B) A
schematic presentation of the genomic region containing the insertion in
intron 30 of the porcine KPL2 gene. Positioning of the EcoRI restriction sites
is shown in Lower Left, and long-range PCR in Lower Right. The data are
affected boars. Long-range PCR allowed a more precise estimate of the
insertion size of ?9,000 bp.
Characterization of an insertion in intron 30 of the KPL2 gene. (A)
Sironen et al.
March 28, 2006 ?
vol. 103 ?
no. 13 ?
transcripts is disrupted, generating premature stop codons, and
as a result truncation of the encoded protein (Fig. 3). This
analysis also revealed that expression of KPL2e29–36 was tissue-
specific, being expressed in the testis, and at a lower level in the
trachea, but not in any of the other tissues examined. Likewise,
another fragment containing exons 40, 41, and 43 (KPL2e40–43,
base pairs 5191–5389) was also expressed specifically in the testis
and trachea. In contrast, a fragment from KPL2 exons 7–8
(KPL2e7–8, base pairs 956-1081) was found to be expressed in
all tissues analyzed, but at lower levels in the lung, liver, and
kidney, for both normal and affected boars.
KPL2 Expression Levels in Various Tissues. Testis tissue of three
normal and three ISTS-affected boars were analyzed for relative
expression of KPL2e7–8 and KPL2e29–30 using quantitative
PCR (qPCR). Expression of KPL2 was also determined in
samples of lung, trachea, and liver of three normal and three
ISTS-affected boars. In normal boars, the expression of the
fragment KPL2e7–8 was 2.3-, 6-, and 12-fold lower in the
trachea, lung, and liver, respectively, relative to the expression in
the testis (Fig. 4A). In addition, affected boars showed a down-
possibly caused by nonsense-mediated RNA decay of the mu-
tated transcript, whereas the trachea, lung, and liver appeared
less affected (Fig. 4A). In normal boars, the expression of
KPL2e29–30 fragment was highest in the testis and ?4.3-fold
lower in the trachea, whereas no transcription was detected in
the liver or lung. In affected boars, the expression is decreased
15-fold in the testis and ?3-fold in the trachea (Fig. 4B),
compared with normal individuals. The qPCR data were sup-
ported by Northern blot analysis, confirming that KPL2 is
transcribed predominantly in the testis and that the expression
pattern is markedly altered in ISTS boars (Fig. 2B). Taken
together, these data show that the KPL2 gene is differentially
expressed in healthy animals. Thus, the region coding for the
testis and trachea (tissues with motile cilia), whereas the region
coding for the N-terminal part is expressed in all of the tissues
analyzed (including the lung, liver, and kidney). Lung tissue
consists of bronchioles and alveoli, but only bronchioles contain
motile cilia. The lower number of motile cilia in lung tissue
relative to the testis and trachea would account for the lack of
expression of the KPL2e29–30 fragment in lung tissue samples.
Furthermore, the intronic 9,000-bp insertion in affected animals
generates a C-terminally truncated version of the protein in the
testis and trachea, and also leads to a decreased level of KPL2
expression of different parts of the KPL2 gene in different tissues. RT-PCR was
used to analyze the fragments KPL2e7–8 (exons 7–8), KPL2e29–36 (exons
affected boars, and in different tissues of a normal boar. The KPL2e29–36
fragment is mainly expressed in the testis and at lower levels in the trachea.
shorter fragment (257 bp) is depleted of exon 30, and the longer fragment
(998 bp) includes exon 30 together with part of intron 30 and the beginning
of the insertion, as verified by sequencing. KPL2e40–43 is also only expressed
in the testis and trachea. The KPL2e7–8 fragment was expressed in all tissues
is moderate in normal testis and extremely low in the lung, liver, and trachea.
Furthermore, KPL2 expression is reduced in ISTS affected boars. Expression of
?-actin was used as a control.
Aberrant splicing of exon 30 in affected boars. (A) Analysis of
31 of the KPL2 gene in normal and ISTS affected boars. Both skipping of exon 30 in ISTS affected individuals (ISTS1) and partial inclusion of intron 30 and the
insertion (ISTS2) disrupt the reading frame and produce several translation stop codons (gray shading), leading to premature termination of translation.
Aberrant splice products of ISTS-affected boars. The DNA and protein sequences are shown for the end of exon 29, exon 30, and the beginning of exon
www.pnas.org?cgi?doi?10.1073?pnas.0506318103Sironen et al.
Sequence Alignments. The porcine KPL2 nucleotide sequence
from the present study (GenBank accession no. DQ119847) was
translated into an amino acid sequence and aligned with the
available full-length protein sequences from the NCBI database
for the human KPL2 isoform 1 (GenBank accession no.
NP?079143) and the rat KPL2 (GenBank accession no.
NP?072142) using CLUSTALW (20). The pairwise sequence align-
ment scores were: human–pig 80%, human–rat 73%, and pig–rat
70%. Protein sequence lengths were 1,822, 1,812, and 1,744 aa,
for the human, pig, and rat, respectively.
The NCBI LocusLink was used to identify exon?intron bound-
whereas the rat gene includes 40 exons spanning 170,943 bp, with
exon numbering for the rat starting at exon 2 in the human gene.
Exon numbering differs between species, such that the exon
skipped in ISTS boars corresponds to exon 29 in the rat and exon
30 in the human. No published protein sequence contains all
exons in the human, and exon differences are known to exist
between the human and rat. Full-length protein sequences for
the rat, human (isoform 1), and pig (testis cDNA), which are
used for the alignment reported in Fig. 5, which is published as
supporting information on the PNAS web site, lack human exons
A complete protein–protein alignment between the human,
rat, and pig showed that human exons 4 and 19 were missing in
the rat sequence, and residues 1204–1226 (four imperfect copies
of the sequence QAKKEKE) in exon 27 were absent in the
human and pig sequence compared with that of the rat. The
KPL2 protein sequence contained several highly conserved
exons (Fig. 5), including the porcine exon 30. A BLAST search
with exon 30 also resulted in partial KPL2 sequences for the dog,
monkey, mouse, and chicken. The protein sequence for exon 30
was highly conserved across all mammalian species, with an
alignment score varying mostly between 81 and 96%, being 89%
between the human and pig, but only 53% between the chicken
We present strong evidence that the presence of a retrotrans-
poson in the KPL2 gene causes the infertility phenotype of ISTS
boars. The data demonstrate that processing of the transcript
two abnormal splice products. Thus, the majority of the KPL2
transcripts in affected boars lacked exon 30, whereas a minor
fraction retained exon 30, but also included intronic as well as
retroelement sequences. Notably, the reading frames of both of
these abnormally spliced transcripts are disrupted, generating
premature translation stop codons, which truncate the protein at
residues 1403 and 1487, respectively, from a total of 1,812 amino
acids. The result is an entirely different and shorter C terminus,
which is most likely the cause for the loss of function. Further-
more, the amount of KPL2 transcripts were reduced in affected
testicular tissue, possibly as a result of mRNA degradation by
nonsense-mediated decay of transcripts containing premature
In the rat, KPL2 is expressed in tissues containing cilia-like
structures such as the lung, trachea, testis, brain, and at lower
in the heart or liver, suggesting a role for this gene in ciliogenesis.
Consistent with this suggestion, KPL2 expression is closely
correlated with ciliated cell differentiation in cultures of primary
tracheal epithelial cells. Likewise, the spatio-temporal expres-
sion pattern in the seminiferous tubules at specific stages during
sperm cell development supports KPL2 as having a central role
in the differentiation of axoneme-containing cells (16). The
present study confirmed the expression pattern reported for rats
with a high level expression in the testis, followed by an
intermediate level in the trachea, and much lower expression in
the lung, kidney, and liver. This suggests that the function of
KPL2 is not confined to motile cilia with a 9 ? 2 axoneme
structure, but that it may also play a role in immotile primary
cilia. Within this context, it is important to note that several
posttranscriptional regulatory pathways operate in combination
with gene expression levels to determine the proteomic profile
of cells. One mechanism is alternative splicing, which can
generate a range of protein isoforms by the inclusion or skipping
of exons, often in a cell-type or developmental stage-specific
manner. In the human, two isoforms of KPL2 have been
identified (GenBank accession nos. NP?079143 and NP?653323),
and several sequences with varying exon content from different
tissues have been deposited in GenBank. Our results indicate
that, in the pig, the primary KPL2 transcript undergoes tissue-
specific splicing to include exon 30 and presumably some other
3?end exons only in the testis and trachea. The intronic insertion
of translation, which explains the sperm tail defects in ISTS
boars. However, no respiratory dysfunction has been observed in
no apparent effect on the axonemal structure (data not shown).
It is unclear whether this is related to the 4- to 5-fold higher
expression level in the testis relative to the trachea, which may
indicate a more crucial role of at least one variant of KPL2 in
sperm tail development than in cilia differentiation. Alterna-
tively, the phenotype could be dependent interactions of KPL2
with other proteins, which are only expressed in the testis.
in the trachea, and would not be detected in affected boars,
which are usually slaughtered at a young age once infertility has
been diagnosed. The clinical features of patients suffering form
PCD also vary significantly (21). Typically males with immotile
spermatozoa also have defective cilia in other tissues. However,
cases have been reported where patients have immotile sperm,
yet the structure and motility of other examined cilia are normal,
ISTS affected boars relative to normal testis expression (100%). (A) The qPCR
analysis of an mRNA fragment spanning KPL2 exons 7 and 8. (B) The qPCR
analysis of an mRNA fragment spanning KPL2 exons 29 and 30. Amplification
by qPCR was performed in triplicate on 50-ng cDNA samples of testicular,
tracheal, lung, and liver tissues from normal and ISTS-affected boars. Mean
of three cDNA samples).
Expression of different KPL2 exons in various tissues of normal and
Sironen et al.
March 28, 2006 ?
vol. 103 ?
no. 13 ?
or in patients with no history of respiratory tract disorders
Comparative sequence alignment of KPL2 showed a high
degree of cross-species conservation. Based on the human KPL2
annotation (GenBank accession no. NP?079143), we performed
sequence scans against protein domain databases to predict
functional domains. This revealed the presence of a domain of
unknown function called DUF1042 in the N terminus, which
classifies KPL2 together with other proteins implicated in fla-
gella function such as the human SPATA4 protein (spermato-
genesis associate 4, GenBank accession no. NP?653245), the
mouse sperm flagella protein Spef1 (GenBank accession no.
AY860964), and CPC1 (central pair complex 1, GenBank ac-
cession no. AAT40991) of the unicellular organism Chlamydo-
monas reinhardtii. In addition, the N terminus contains a calpo-
nin homology domain, indicating potential actin binding activity.
An adenylate kinase (ADK) domain and an ATP?GTP binding
site (P-loop) are located centrally, where a number of other
domains have been identified including two potential bipartite
nuclear localization signals and a calcium-binding EF-hand
motif, indicating that KPL2 activity is modulated by calcium. The
region containing the EF-hand is missing in ISTS boars, which
may, to some extent, explain the sperm-specific structural
changes (the positions, E values, and databases used are outlined
in Table 1, which is published as supporting information on the
PNAS web site). KPL2 and CPC1 share ?40% similarity and
both harbor several of the functional domains, including
DUF1042, ADK, and EF-hands, which strongly suggest that they
serve similar functions (25). Mutations in CPC1 disrupt the
assembly of the central pair microtubule-associated complex and
alter flagellar beat frequency (25). However, the KPL2 mutation
produces a complex and more severe phenotype with fully
disrupted central pair microtubules, as well as outer doublet
defects, which may suggest different or additional roles of KPL2
in the assembly of the axoneme. Furthermore, flagella in CPC1
mutants can still beat, whereas all sperm flagella are immotile in
ISTS boars, although ciliated cells other than spermatozoa are
motile, for example in the ductuli efferentes of the testis.
Mutagenesis studies in Chlamydomonas have revealed several
genes (e.g., pf18, pf19, pf20, pf6, pf16, and pf15) coding for the
central apparatus proteins that are essential for flagella motility,
Mutations in pf16 (spag6) and pf20 have also been shown to
affect spermatogenesis in the mouse, where spag6 interacts with
pf20. Mutated spag6 is known to cause infertility and truncated
flagella (28, 29). These gene products have been localized to the
central pair complex and are only expressed in the testis. These
findings support the hypothesis that the KPL2 protein may also
be part of, or interact with, the central pair complex. However,
the expression of KPL2 in tissues devoid of motile cilia, such as
the kidney and liver, suggests a wider role for at least one of the
putative KPL2 isoforms.
In conclusion, KPL2 appears to be expressed in all cilia
containing tissues, but presumably as different splice variants.
The isoform containing the exon 30 encoded domain appears to
axoneme primarily in spermatozoa. Localization of the KPL2
protein at the cellular level will elucidate the role of this gene in
sperm tail development, and further studies are required to
reveal the importance of different KPL2 variants in other tissues.
Mutations in various parts of KPL2 are likely to produce
different phenotypes, and thus the gene may be involved in
multiple types of ciliary defects. Analysis of the function of KPL2
may therefore provide a general insight into cilia malformations
and abnormal development.
Materials and Methods
Pig Genomic Library Screening and Comparative Mapping. For fine
mapping, BAC-clones (PigE BAC) from MCR geneservice
(www.geneservice.co.uk?home; ref. 30) were picked up by PCR
screening with markers SW2411, SW419, and S0006 located
within the disease-associated region on porcine chromosome 16.
The selected BAC clones were isolated with the Qiagen plasmid
midi kit protocol in accordance with the manufacturer’s recom-
mendations. The ends of extracted BAC clones were sequenced
and compared to the human sequence database (www.ncbi.
on the human map.
PCR Amplification and DNA Sequencing. PCR amplification using
BAC pools or pig genomic DNA as a template was performed
with Dynazyme DNA polymerase (Finnzymes) according to the
instructions from the supplier. Long-range PCR was performed
by using Dynazyme EXT polymerase (Finnzymes) or a Long
PCR Enzyme mix (Fermentas).
The PCR amplicons were purified by using ExoSAP-IT
(Amersham Pharmacia), whereas PCR fragments were se-
quenced in both directions with the same primers used in the
amplification procedures. The BAC-ends were sequenced with
universal primers T7 and SP6. Sequencing was performed on
MegaBace 500 capillary DNA sequencer (Amersham Phar-
macia) using DYEnamic ET Terminator kits with Thermo
Sequenase II DNA Polymerase (Amersham Pharmacia).
Gene Expression. For analysis of candidate gene expression,
samples of testicular, liver, kidney, tracheal, and lung tissue from
normal and ISTS affected boars were collected and stored in
RNAlater buffer (Qiagen). Samples of testis, trachea, lung, and
liver were available from three affected and three normal boars,
but kidney samples were only available from one of the normal
boars. Total RNA purification was performed with RNeasy
Protect Mini and Midi kits (Qiagen). Total RNA was reverse
transcribed (RT-PCR) with random primers and an RNA PCR
kit (SuperScript, Invitrogen, and ImProm-II Reverse Transcrip-
tion System, Promega) according to the manufacturer’s instruc-
tions and amplified by using gene-specific primers. Control
reactions were performed with a ribosomal 18S RNA, and this
process was repeated using RNA isolated from three different
animals where possible. Fragments KPL2e7–8 (126 bp),
KPL2e29–36 (487 bp), and KPL2e40–43 (199 bp) were used to
examine the expression of various components of KPL2 in
5, and the primers used for PCR amplification are listed in Table
2, which is published as supporting information on the PNAS
Real-Time qPCR. qPCR was used to measure the relative RNA
transcript levels of KPL2 in the testis, trachea, lung, and liver.
Concentrations of cDNAs were measured and tissue cDNA
samples were diluted 1:100 before use. Two fragments from
different regions of KPL2 (KPL2e7–8 and KPL2e29–30) were
analyzed by using ribosomal 18S RNA as an internal reference
gene (a list of primers used is given in Table 2). The qPCR was
performed with an ABI 7000 Sequence Detection System in
96-well microtiter plates using Absolute qPCR SYBR Green
ROX Mix (VWR). Amplification by qPCR contained 12.5 ?l of
Absolute qPCR SYBR Green Mix, 50 ng of cDNA, and 70 nM
of each primer in a final volume of 25 ?l. Amplifications were
initiated with a 15-min enzyme activation at 95°C followed by 40
cycles of denaturation at 95°C for 15 s, primer annealing at 60°C
for 30 s, and extension at 72°C for 30 s. All samples were
amplified in triplicate, and the mean value was used for further
calculations. Each run comprised of the products of amplifica-
www.pnas.org?cgi?doi?10.1073?pnas.0506318103Sironen et al.
tion for three control and three test samples with two primer
pairs (reference and target gene) and a negative control also
analyzed in triplicate. A standard curve for each primer pair was
produced by serially diluting a control cDNA. Quantities of
specific mRNA in the sample were measured according to the
corresponding gene-specific standard curve. Raw data were
analyzed with the sequence detection software (Applied Biosys-
tems) and relative quantitation was performed within Microsoft
EXCEL applying the RELATIVE EXPRESSION SOFTWARE tool (31,
32). Ratios between the target and reference gene were calcu-
lated by using the mean of these measurements. Specificity of
RT-PCR products was determined by gel electrophoresis, which
KPL2e29–30, 388 bp; KPL2e7–8, 126 bp). In addition, a melting
curve analysis was performed allowing single product-specific
melting temperatures to be determined. No primer–dimer for-
mations were generated during the application of 40 real-time
PCR amplification cycles. Differences in amplification were
corrected by quantifying samples relative to the corresponding
standard curves using ribosomal 18S RNA as an internal refer-
Southern and Northern Blotting. Porcine genomic DNA was di-
gested with EcoRI, EcoRV, and BamHI, and separated by
electrophoresis on a 0.8% agarose gel in TBE buffer and
transferred onto positively charged Hybond-NX membranes
(Amersham Pharmacia). The membranes were hybridized with
the DNA Probe1 (1,177 bp, Table 1) labeled with EasyTides
[?-32P]dCTP, 250 ?Ci (PerkinElmer). Hybridization and wash-
the membranes were exposed to x-ray films.
and Midi kits (Qiagen). Poly(A)? RNA was isolated by using the
Dynabeads DIRECT mRNA kit (Dynal) following the manu-
facturer’s instructions. Each RNA sample was denatured by
boiling for 10 min and loaded onto a 1% agarose-formaldehyde
by using the NorthernMax kit (Ambion). The Probe2 (KPL2
exons 3–7, 756 bp, Table 2) was radioactively labeled with the
Nick Translation System (GIBCO?BRL) using [?-32P]dCTP
We thank Dr. O. Manninen for assistance with Southern blotting
analysis, Dr. P. Pakarinen for assistance with Northern blot analysis, and
A. Virta for sequencing analysis. This work was financially supported by
The Finnish Animal Breeding Association.
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no. 13 ?