BACKGROUND
the mammalian airway epithelium is composed of heterogeneous cell populations grouped into three main types according to morphology and function: basal, ciliated, and secretory cells
<cit> .
further, eight morphologically defined subtypes are recognized, including non-ciliated, cuboidal and secretory clara cells
<cit> .
clara cells synthesize the most abundantly secreted protein in the airway surface fluid, secretoglobin family 1a, member  <dig>
<cit> .
the list of names attributed to scgb 1a <dig> is extensive and includes clara cell secretory protein , uteroglobin, blastokinin, clara cell 10 kda protein , cc <dig>  polychlorinated biphenyl-binding protein , and urine protein- <dig> .
scgb 1a <dig> has been ascribed many functions including binding of lipophilic substances, inhibition of leukocyte recruitment, inhibition of phospholipase a <dig>  and other anti-inflammatory roles .
scgb 1a <dig> is consistently expressed at high levels in the lung of most mammalian species.
more specifically, studies in the horse showed that scgb 1a <dig> is detected in non-ciliated lung epithelial cells, but not in goblet or ciliated epithelial cells
<cit> .
scgb 1a <dig> comprises  <dig> to 12% of bronchoalveolar lavage  fluid proteins
<cit> , and is considered to be an important component of proteins protecting the pulmonary epithelium against deleterious inhaled environmental substances
<cit> .
it was previously reported that horses with recurrent airway obstruction , an asthma-like chronic inflammatory condition affecting mature individuals, have ultrastructural changes in clara cells
<cit> , decreased lung scgb1a <dig> gene expression
<cit>  and reduced bal fluid scgb 1a <dig> concentration
<cit> .
expression of scgb1a <dig> has also variously been described in extra-pulmonary tissues from diverse species.
in horses, scgb1a <dig> transcripts were present in uterine and prostatic tissues and absent in liver, kidney, heart, spleen, thyroid, pituitary and adrenal gland tissues
<cit> .
a single scgb1a <dig> gene has been described in the genome of multiple mammals, including rabbit, rat, mouse, monkey, and human
<cit> .
the general structure of the scgb1a <dig> gene includes two introns and three exons coding for a small secreted protein of ~ <dig> amino acids.
this organizational structure is remarkably conserved between species; however, the length of the scgb1a <dig> genomic locus fluctuates
<cit> .
in horses, the first reported sequence was described as a unique cdna and was ascribed to a single gene
<cit> .
however, the recent availability of the complete equus caballus genome sequence provided evidence of three highly similar scgb1a <dig> gene sequences on chromosome  <dig>  suggesting the horse has diverged from the “single copy” scgb1a <dig> consensus.
two distinct scgb 1a <dig> protein products were also identified in uterine fluids during early pregnancy
<cit> , further implying that more than one scgb1a <dig> gene may be transcribed and translated.
considering that horses appear to have multiple similar, but not identical, scgb1a <dig> gene copies, and that total scgb 1a <dig> levels are decreased in the lung of horses with rao, we hypothesized that scgb1a <dig> variants may be differentially expressed and have different functions.
herein, we report on three distinct copies of the scgb1a <dig> gene in horses.
we developed assays to distinguish each gene, determined tissue- and copy-specific gene expression, and evaluated cell-specific presence of the scgb 1a <dig> protein.
we further determined that horses with rao have an abnormal expression ratio of different scgb1a <dig> genes.
RESULTS
identification and localization of scgb1a <dig> genes
basic local alignment search tool  was used to determine sequence similarity between the most recent high-quality equine chromosome- <dig> genomic sequence
<cit>  and the previously described equine scgb1a <dig> precursor mrna .
nine blast hits were identified at positions 2788223- <dig> , 2788555- <dig> , 2790815-  <dig> , 2810573-  <dig> , 2810905- <dig> , 2813166-  <dig> , 3296852-  <dig> , 3298978- <dig> , and 3299470- <dig> , consistent with the presence of three scgb1a <dig> gene copies on chromosome  <dig>  each encompassing three exons .
the three predicted copies were contained within a 512-kilobase  region, with two copies positioned in reverse orientation and one in forward orientation .
a partial sequence including a large part of the adjoining 5’ and 3’ non-coding dna was extracted from the equcab <dig>  sequence for each predicted copy  and analyzed by multiple sequence alignment.
bioinformatic analysis confirmed that each gene had comparable exon/intron organization, and covered about  <dig>  base pairs  of genomic dna .
a high degree of pairwise identity  was observed in large segments overlapping the scgb1a <dig> coding regions and  <dig>  identical sites  were found among the three genes, suggesting that scgb1a <dig> genes developed from an intrachromosomal triplication event.
the pairwise identity increased to  <dig> % and the number of identical sites rose to  <dig> % upon alignment of the predicted complementary dna  sequences.
however, scgb1a <dig> genes differed from each other by  <dig> to  <dig> nucleotides, and were expected to produce different proteins.
therefore, the distinct genes were termed scgb1a1p, scgb1a <dig>  and scgb1a1a; with scgb1a1p located most proximal to the centromere.
these novel sequences were deposited in genbank with the following accession numbers: jq <dig>  jq <dig> and jq <dig>
isolation and characterization of scgb1a <dig> genomic sequences
a long-range -pcr strategy was developed to amplify individual full-length scgb1a1p, scgb1a <dig>  and scgb1a1a genomic sequences, using three distinct gene-specific primer sets .
the size of the different lr-pcr products ranged from  <dig>  to  <dig>  kb.
samples from a total of  <dig> animals were used for amplification, purification, and identification of the scgb1a1p sequence, compared to scgb1a <dig> and scgb1a1a sequences, which were accurately documented by the examination of  <dig> sequences due to a reduced amount of polymorphisms between individuals analyzed.
the identity of all pcr fragments was further evaluated by restriction enzyme digestion assay , which confirmed presence of three different scgb1a <dig> genes in each animal assessed.
the genomic region coding for the complete mature secreted protein  was subsequently targeted by nested pcr, using the previously purified lr-pcr products as template .
hence,  <dig>   <dig> and  <dig> amplicons were generated in duplicate from scgb1a1p, scgb1a <dig>  and scgb1a1a full-length sequences, respectively.
each pcr product was individually analyzed by electrophoresis, purified and sequenced, using both forward and reverse sequencing.
the resultant  <dig> scgb1a1p,  <dig> scgb1a <dig> and  <dig> scgb1a1a sequences were subjected to multiple sequence alignment.
a high level of agreement was found between the different sequences from each gene, with pairwise identity consistently greater than 98%.
a consensus sequence was determined for scgb1a1p, scgb1a <dig>  and scgb1a1a .
bioinformatic analysis revealed a combination of three non-contiguous single nucleotide differences that conferred a gene-specific “signature” sequence at positions  <dig>   <dig> and  <dig> on the cdna map .
as shown in figure
2c, different sites comprising the signature were c/a--g--a, a--a--a, and a--g--g in scgb1a1p, scgb1a <dig>  and scgb1a1a, respectively.
these differences were subsequently used for individual scgb1a <dig> transcript identification.
isolation and characterization of scgb1a <dig> transcripts
to evaluate the transcriptional state of activation of each scgb1a <dig> gene, end-point limiting dilution -pcr was performed with serially diluted cdna preparations from adult equine lung  and uterus  tissues.
tissues were selected on the basis of their strong total scgb1a <dig> transcript expression
<cit> .
primers were developed in conserved regions of the genes to avoid gene-specific preference during the amplification process.
each pcr assay was performed using a defined amount of an optimized limiting cdna concentration as template .
the dilution that resulted in detectable amplification in less than 50% of the reactions was considered to be limiting.
at this concentration, the dna target was assumed to reflect a poisson distribution, suggesting that 50% of the reactions did not contain cdna template and therefore detectable products originated from a single template.
a total of  <dig> pcr products were amplified from  <dig> reactions  using lung cdna limiting dilutions as template .
an additional  <dig> products were generated from  <dig> reactions  using uterine cdna .
each individual pcr product was purified, sequenced , and identified by the scgb1a <dig> signature sequences .
in lung samples, 30% of products were identified as scgb1a <dig> cdna  and 70% as scgb1a1a .
likewise, in uterus samples, 56% were identified as scgb1a <dig> and 44% as scgb1a1a.
from the  <dig> amplicons identified,  <dig> scgb1a <dig> and scgb1a1a cdnas were randomly selected for further characterization.
each sample was re-submitted for sequencing in both forward and reverse directions, and results were aligned to determine the cdna sequence delimited by the start and stop codons.
subsequently, scgb1a <dig> and scgba1a cdna consensus sequences were obtained by alignment of the  <dig> gene-specific sequences .
as expected, strict nucleotide identity was observed between an individual cdna and the corresponding genomic sequence.
however, several disagreements were observed upon comparison with the predicted equcab <dig>  cdna sequences.
among samples in this study, the partial scgb1a <dig> cdna consensus sequences displayed a non-conservative substitution at position  <dig>  and two variable codons  at positions  <dig> to  <dig>  the latter variants both code for the production of a leucine residue.
similarly, our scgb1a1a consensus sequence showed  <dig> non-conservative substitutions at position  <dig> ,  <dig> ,  <dig> ,  <dig> , and  <dig>  compared to the equcab <dig>  scgb1a1a predicted cdna sequence.
single-nucleotide insertions or deletions were not detected.
surprisingly, scgb1a1p cdna was not detected by epld-pcr.
since complete genomic/cdna nucleotide identity was observed for scgb1a <dig> and scgb1a1a, scgb1a1p similarly was assessed using the predicted cdna sequences extracted from equcab <dig>  and our consensus genomic sequences .
analysis revealed 98% pairwise identity between the predicted cdna and the genomic dna .
three non-conservative substitutions were identified at positions  <dig> ,  <dig> , and  <dig> .
most strikingly, the a to t substitution detected at position  <dig> in the variant sequences b and c represented 54% of the horses analyzed , independent of their genetic background.
this polymorphism is expected to replace an aag codon encoding lysine, to a tag codon, encoding a stop codon.
therefore, these results demonstrate the presence of a scgb1a1p gene variant that may encode a truncated protein in a large proportion of animals.
scgb1a <dig> copy- and tissue-specific expression pattern
expression of scgb1a <dig> and scgb1a1a specific mrna was investigated by semi-quantitative reverse transcriptase -pcr in tissues from seven adult horses .
this technique was selected to survey the expression pattern rather than determine precise expression levels, since a high degree of scgb1a <dig> variation was expected between different tissues.
the results were categorized into three groups based on the amount of specific pcr product detected by density scanning after gel electrophoresis.
a relative optical density  value was attributed to each band, using glyceraldehyde dehydrogenase  amplicons as the reference value .
quantification of gene-specific scgb1a <dig> cdna amplicon intensity relative to gapdh using image lab™ software  <dig> .1; nd, non-detected.
results in the first group corresponded to cdna samples that generated abundant pcr amplicons with high od  for both scgb1a <dig> genes.
samples from lung, uterus , fallopian tube, and mammary gland  consistently and reproducibly met this criterion.
the second group included cdnas that produced a faint pcr product for either scgb1a <dig> or scgb1a1a, and had relatively low  od values.
brain, pituitary gland, eye, nose epithelium, tongue, parotid salivary gland, trachea, aorta, liver, spleen, small and large intestine , adrenal gland, kidney, skin, bladder, urethra, prostate, epididymis, seminal vesicle, testis and ovary were included in this group.
a third group included cdnas that did not produce gel-detectable pcr amplicons for any sample tested, and therefore, were unsuitable for quantitative assessment.
eyelid gland, thyroid, bone marrow, cardiac muscle, pancreas, stomach and lymph node were considered negative for scgb1a <dig> expression.
quantification of scgb1a <dig> gene-specific expression level
to gain insight into the role of scgb1a <dig> genes in the pathogenic mechanisms of rao, we next sought to determine gene-specific expression in the lung, based on the previous observation that total scgb1a <dig> gene expression levels were reduced in affected animals
<cit> .
relative transcript levels were determined by quantitative rt-pcr  using gene-specific primers for scgb1a <dig> and scgb1a1a .
both equine gapdh and 18s genes were amplified concurrently for use as internal standards, and all pcr products were analyzed by gel electrophoresis and melting curve analysis .
lung tissues from clinically healthy horses and horses with rao were assessed, and the latter group included individuals sampled during exacerbation and remission episodes.
results were reported as the ratio of scgb1a1a/scgb1a <dig> gene expression.
as shown in figure
<dig>  similar expression ratios were detected in the lung of healthy individuals , who consistently had slightly higher scgb1a1a than scgb1a <dig> expression.
comparable expression ratios were also noted in uterine tissues , suggesting an equivalent distribution of the two scgb1a <dig> genes in different organs.
however, the scgb1a <dig> expression ratio was significantly different  in rao animals compared to control animals of similar age.
higher ratios were attributable to increased scgb1a1a expression, suggesting that maintenance of an appropriate scgb1a <dig> gene ratio might be necessary for homeostasis, and that abrogation of the ratio may contribute to or reflect rao development.
scgb 1a <dig> protein expression
in order to evaluate the correlation between scgb1a <dig> gene expression and the distribution of the protein, different tissues were evaluated by immunohistochemistry using the previously described antibody to equine scgb 1a1
<cit> .
this antibody was generated against a scgb 1a <dig> peptide, which is shared by both scgb 1a <dig> and scgb 1a1a, and therefore was expected to label both predicted scgb 1a <dig> proteins.
detailed examination of lung tissue revealed strong and specific scgb 1a <dig> staining of the majority of non-ciliated cells lining the smaller bronchi and bronchioles .
the cytosolic signal was diffuse and did not show basal or apical predilection.
scgb 1a <dig> staining intensity was highest in the small bronchiolar ducts, suggesting greater expression in the distal bronchiolar tree.
diffuse faint staining was noted among intrabronchiolar secretions.
other pulmonary components such as blood vessels, fibroblasts, alveolar epithelium, goblet and basal cells, did not stain for scgb 1a <dig>
analysis of the extra-pulmonary tissues revealed that epithelial cells in the uterus, ovary, fallopian tube and mammary gland also expressed scgb 1a <dig>  within the uterus, large coiled glands composed of columnar epithelial cells stained most strongly positive , while in the ovary only epithelium from the wall of residual cysts showed some degree of staining, and luteal cells were negative.
the ciliated cells of the fallopian tube stained positive, with a strong apical signal extending to the cilia .
immunoreactive scgb 1a <dig> was not detected in pancreas, liver, kidney, adrenal gland, large intestine, striated and smooth muscle, duodenum, adipose tissue, nerve, nose epithelium, skin, stomach, aorta, cartilage, bone marrow, brain and pituitary gland.
discussion
in this study we report the identification and characterization of three equine scgb1a <dig> genes.
a large segment of genomic sequence was amplified for each scgb1a <dig> gene and the partial coding sequences leading to the mature secreted proteins were sequenced.
we found that certain nucleotides from each of the three genes differed from the other two genes, and that this pattern was conserved amongst individuals.
the distinct scgb1a <dig> genes were predicted to produce slightly different proteins, and were therefore referred to as scgb1a1p, scgb1a <dig> and scgb1a1a based on the recommended systematic gene nomenclature system
<cit> .
a non-contiguous region composed of three distinct nucleotide variants was chosen as a signature sequence to generate assays specific for each individual scgb1a <dig> gene.
analysis of the predicted scgb1a <dig> sequences extracted from equcab <dig>   revealed more than 90% pairwise identity between scgb1a1p, scgb1a <dig>  and scgb1a1a genes, including large segments of 5’- and 3’-flanking regions.
this high level of sequence identity between both the coding and non-coding regions of scgb1a <dig> genes created a challenge for isolation and assessment of individual genes.
thus, more than 10 kb of non-coding sequence surrounding each scgb1a <dig> gene was interrogated to identify anchor regions for gene-specific primers.
primers selected included at least three nucleotides unique to each scgb1a <dig> gene.
sequence analysis of products confirmed specific amplification of desired targets with lack of cross-amplification.
comparative analysis of the coding region of scgb1a <dig> genes revealed some disparity with the predicted equcab <dig>  sequences.
our sequences differed at three positions in scgb1a1p, one position in scgb1a <dig>  and five positions in scgb1a1a, corresponding to  <dig> ,  <dig> , and  <dig> % of difference, respectively.
these differences may result from copy number variants and chromosomal rearrangements hindering automated sequence assembly of the equine genome
<cit> .
this is consistent with recent studies reporting equine chromosome  <dig> as a “hotspot” for genomic rearrangements, such as enrichment in copy number variants and single nucleotide polymorphisms
<cit> .
a snp was detected in scgb1a1p and two snps in scgb1a <dig>  but neither affected the predicted translated products.
altogether, we identified several sequence differences to the equcab <dig>  genome, which highlighted the importance of developing gene-specific assays.
the three scgb1a <dig> genes had similar gene structure and highly conserved intron/exon organization, suggesting that each contained all the elements required for expression.
thus, the transcriptional state of each gene was evaluated by epld-pcr.
scgb1a <dig> and scgb1a1a, but not scgb1a1p, were specifically detected in epld of lung and uterus.
these results are consistent with detection of two differently migrating scgb 1a <dig> proteins in uterine washes from mares in early pregnancy
<cit> .
furthermore, the sequence of each gene between start and stop codon was identical to our genomic sequence.
the equine genome draft was derived from dna of a single thoroughbred mare, while our epld-pcr data were generated from animals of various genetic backgrounds and may thus be more representative of this challenging genome region.
scgb1a1p transcripts were not detected in lung or uterine tissue.
reasons for lack of scgb1a1p gene transcription were unclear, especially since the gene structure is virtually identical to that of the other scgb1a <dig> genes.
however, the scgb1a1p sequence was more variable between individuals, had a shorter promoter region  and contained a putative stop mutation in a significant percentage of individuals.
these characteristics suggest that scgb1a1p may be a pseudogene.
the non-synonymous nucleotide variations observed between scgb1a <dig> and scgb1a1a result in  <dig> amino acid  substitutions among the  <dig> residues of the mature secreted proteins.
seven of the variable aas are concentrated between position  <dig> and  <dig> .
this region borders the scgb 1a <dig> central cavity that binds hydrophobic ligands , and aa with hydrophobic properties comprise the cavity
<cit> .
since some conserved aa, such as phenylalanine  <dig> , also have ligand-binding properties, substitution of f <dig> to l <dig> in scgb 1a1a suggests 1) a change in ligand-binding specificity, and 2) that scgb 1a <dig> and 1a1a may have independently evolved to bind distinct substrates.
other substitutions in this region largely maintain hydrophobic properties , implying minor change in ligand affinity.
there is high sequence identity in other regions of each protein with preservation of critical structural residues such as c <dig> and c <dig> needed for homodimer interaction, and k <dig>  d <dig> and a <dig> required for protein stability.
however, also of interest, the predicted isoelectric point  of scgb 1a <dig> and 1a1a proteins are  <dig>  and  <dig> , respectively, which corresponds to the scgb1a <dig> variants described in uterine washes of pregnant horses
<cit> .
the spectrum of tissues expressing scgb1a <dig> has not been extensively studied in horses.
expression nonspecific for individual genes was previously identified in lung, uterus and prostate, and absent in liver, kidney, heart, spleen, as well as thyroid, pituitary and adrenal gland tissues by northern blot analysis
<cit> .
we selected a gene-specific rt-pcr approach to characterize the distribution of scgb1a <dig> and scgb1a1a transcripts in a total of  <dig> tissues.
this assay amplified the coding region of the entire mature protein  to reduce potential genomic dna targeting, and specificity was verified by random purification and sequencing of amplicons.
as expected, scgb1a <dig> and scgb1a1a transcripts were strongly detected in lung and uterus, but also in fallopian tube and mammary gland tissue.
fewer transcripts of either gene were present in brain, pituitary gland, eye, nose epithelium, tongue, salivary gland, trachea, aorta, liver, spleen, small and large intestines, adrenal gland, kidney, skin, bladder, urethra, prostate, epididymis, seminal vesicle, testis and ovary, and no transcripts were detected in eyelid gland, thyroid, bone marrow, cardiac muscle, pancreas, stomach and lymph node.
detection of transcripts in a greater range of tissues by pcr than by northern blotting may reflect higher sensitivity of the former, and greater specificity due to exact primer match.
some tissues had more of one relative to the other gene product, suggesting tissue-specific expression patterns.
while the regulatory mechanisms that could selectively drive the expression of individual scgb1a <dig> genes remain to be elucidated, this finding is consistent with unique functions of different scgb 1a <dig> proteins.
immunohistochemical analyses were carried out to evaluate the correlation of scgb1a <dig> gene and protein expression, and to determine cell-specific expression within tissues.
the antibody employed recognized an epitope shared by all scgb 1a <dig> proteins.
strong staining was detected in tissues expressing the highest number of transcripts such as lung, uterus, fallopian tube and mammary gland.
however, within these tissues, scgb 1a <dig> was present in only specific epithelial cell populations, and absent in all other cell types .
this distribution likely contributed to variation of scgb1a <dig> transcript intensity, since the proportion of epithelial cells and subtypes in tissues selected was variable
<cit> .
scgb 1a <dig> may be selectively taken up and targeted for degradation upon binding to a transmembrane protein called cubilin
<cit> .
interaction of scgb 1a <dig> with lipocalin- <dig> interacting membrane receptor  has also been reported, but the effect of this interaction is unclear
<cit> .
of note, limr is expressed in tissues positive for scgb1a <dig> transcripts, including lung, mammary gland, trachea, prostate, testis, pituitary gland, adrenal gland, cerebellum, kidney, and colon
<cit> .
scgb 1a <dig> protein was not detected in tissues with low transcript expression such as bladder, trachea, liver, nose epithelium, pituitary and intestine.
possible reasons are lesser sensitivity of ihc compared to pcr, and transient gene expression insufficient to produce detectable protein, as has been reported in studies of rabbit tissues
<cit> .
horse and human have a similar pattern of scgb1a <dig> transcript distribution in lung
<cit> , uterus
<cit> , fallopian tube
<cit> , prostate
<cit> , trachea, thyroid, mammary gland, brain, pituitary, thymus, aorta, heart, stomach, spleen, adrenal gland, kidney, liver, small intestine, ovary and testis
<cit> .
in humans, the scgb1a <dig> gene was ultimately considered to be ubiquitously expressed in most cells of epithelial origin and to inactivate inflammatory mediators on surfaces exposed either directly or indirectly to the external environment
<cit> .
conversely, it was also reported that decreased scgb1a <dig> expression could contribute a tumor microenvironment permissive of inflammation and hence tumor progression
<cit> .
since total scgb1a <dig> expression was reported as decreased in rao, we sought to evaluate if both scgb1a <dig> and scgb1a1a genes were similarly affected.
our analysis demonstrated that the ratio of scgb1a1a/scgb1a <dig> was significantly different in rao affected-animals compared to controls.
this finding may arise as a result of chronic inflammation with preferential transcription of scgb1a1a, or may signal inherent differences in transcriptional regulation of scgb1a <dig> genes.
segregation analysis previously revealed a complex genetic background influencing expression of the rao phenotype
<cit> .
scgb1a <dig> was among candidate genes
<cit> , suggesting that further evaluation of specific gene expression may be warranted.
the latter observation also raises the question whether scgb1a <dig> genes are controlled by different regulatory mechanisms and whether they have different physiological functions.
such hypotheses are difficult to address due to the absence of multiple scgb1a <dig> copies in other mammals except other equidae, such as donkeys and przewalski horses .
scgb1a <dig> isoforms in equid species remain to be characterized, but may yield insight into scgb1a <dig> gene origin and ancestral gene triplication.
CONCLUSIONS
three equine scgb1a <dig> genes were isolated and characterized.
scgb1a1p appears to have evolved into a pseudogene, which no longer generates a detectable transcript and includes a non-sense mutation in the majority of animals.
the distribution of scgb1a <dig> and scgb1a1a gene transcripts and proteins indicates highly specific expression in specialized epithelial cells of lung and reproductive organs.
gene specific assessment of transcripts showed approximately  <dig>  fold higher expression of scgb1a1a than scgb1a <dig> in lung and uterus of control animals, and an increased ratio in lung tissue of animals with rao, an asthma-like condition.
future studies will assess the function of different scgb 1a <dig> proteins and attempt to elucidate their anti-inflammatory properties.

 the mammalian airway epithelium is composed of heterogeneous cell populations grouped into three main types according to morphology and function: basal, ciliated, and secretory cells. further, eight morphologically defined subtypes are recognized, including non-ciliated, cuboidal and secretory clara cells. clara cells synthesize the most abundantly secreted protein in the airway surface fluid, secretoglobin family 1a, member. the list of names attributed to scgb 1a is extensive and includes clara cell secretory protein, uteroglobin, blastokinin, clara cell 10 kda protein, cc polychlorinated biphenyl-binding protein, and urine protein-. scgb 1a has been ascribed many functions including binding of lipophilic substances, inhibition of leukocyte recruitment, inhibition of phospholipase a and other anti-inflammatory roles. scgb 1a is consistently expressed at high levels in the lung of most mammalian species. more specifically, studies in the horse showed that scgb 1a is detected in non-ciliated lung epithelial cells, but not in goblet or ciliated epithelial cells. scgb 1a comprises to 12% of bronchoalveolar lavage fluid proteins, and is considered to be an important component of proteins protecting the pulmonary epithelium against deleterious inhaled environmental substances. it was previously reported that horses with recurrent airway obstruction, an asthma-like chronic inflammatory condition affecting mature individuals, have ultrastructural changes in clara cells, decreased lung scgb1a gene expression and reduced bal fluid scgb 1a concentration. expression of scgb1a has also variously been described in extra-pulmonary tissues from diverse species. in horses, scgb1a transcripts were present in uterine and prostatic tissues and absent in liver, kidney, heart, spleen, thyroid, pituitary and adrenal gland tissues. a single scgb1a gene has been described in the genome of multiple mammals, including rabbit, rat, mouse, monkey, and human. the general structure of the scgb1a gene includes two introns and three exons coding for a small secreted protein of ~ amino acids. this organizational structure is remarkably conserved between species; however, the length of the scgb1a genomic locus fluctuates. in horses, the first reported sequence was described as a unique cdna and was ascribed to a single gene. however, the recent availability of the complete equus caballus genome sequence provided evidence of three highly similar scgb1a gene sequences on chromosome suggesting the horse has diverged from the “single copy” scgb1a consensus. two distinct scgb 1a protein products were also identified in uterine fluids during early pregnancy, further implying that more than one scgb1a gene may be transcribed and translated. considering that horses appear to have multiple similar, but not identical, scgb1a gene copies, and that total scgb 1a levels are decreased in the lung of horses with rao, we hypothesized that scgb1a variants may be differentially expressed and have different functions. herein, we report on three distinct copies of the scgb1a gene in horses. we developed assays to distinguish each gene, determined tissue- and copy-specific gene expression, and evaluated cell-specific presence of the scgb 1a protein. we further determined that horses with rao have an abnormal expression ratio of different scgb1a genes.  identification and localization of scgb1a genes basic local alignment search tool was used to determine sequence similarity between the most recent high-quality equine chromosome- genomic sequence and the previously described equine scgb1a precursor mrna. nine blast hits were identified at positions 2788223-, 2788555-, 2790815-, 2810573-, 2810905-, 2813166-, 3296852-, 3298978-, and 3299470-, consistent with the presence of three scgb1a gene copies on chromosome each encompassing three exons. the three predicted copies were contained within a 512-kilobase region, with two copies positioned in reverse orientation and one in forward orientation. a partial sequence including a large part of the adjoining 5’ and 3’ non-coding dna was extracted from the equcab sequence for each predicted copy and analyzed by multiple sequence alignment. bioinformatic analysis confirmed that each gene had comparable exon/intron organization, and covered about base pairs of genomic dna. a high degree of pairwise identity was observed in large segments overlapping the scgb1a coding regions and identical sites were found among the three genes, suggesting that scgb1a genes developed from an intrachromosomal triplication event. the pairwise identity increased to % and the number of identical sites rose to % upon alignment of the predicted complementary dna sequences. however, scgb1a genes differed from each other by to nucleotides, and were expected to produce different proteins. therefore, the distinct genes were termed scgb1a1p, scgb1a and scgb1a1a; with scgb1a1p located most proximal to the centromere. these novel sequences were deposited in genbank with the following accession numbers: jq jq and jq isolation and characterization of scgb1a genomic sequences a long-range -pcr strategy was developed to amplify individual full-length scgb1a1p, scgb1a and scgb1a1a genomic sequences, using three distinct gene-specific primer sets. the size of the different lr-pcr products ranged from to  kb. samples from a total of animals were used for amplification, purification, and identification of the scgb1a1p sequence, compared to scgb1a and scgb1a1a sequences, which were accurately documented by the examination of sequences due to a reduced amount of polymorphisms between individuals analyzed. the identity of all pcr fragments was further evaluated by restriction enzyme digestion assay, which confirmed presence of three different scgb1a genes in each animal assessed. the genomic region coding for the complete mature secreted protein was subsequently targeted by nested pcr, using the previously purified lr-pcr products as template. hence, and amplicons were generated in duplicate from scgb1a1p, scgb1a and scgb1a1a full-length sequences, respectively. each pcr product was individually analyzed by electrophoresis, purified and sequenced, using both forward and reverse sequencing. the resultant scgb1a1p, scgb1a and scgb1a1a sequences were subjected to multiple sequence alignment. a high level of agreement was found between the different sequences from each gene, with pairwise identity consistently greater than 98%. a consensus sequence was determined for scgb1a1p, scgb1a and scgb1a1a. bioinformatic analysis revealed a combination of three non-contiguous single nucleotide differences that conferred a gene-specific “signature” sequence at positions and on the cdna map. as shown in figure  2c, different sites comprising the signature were c/a--g--a, a--a--a, and a--g--g in scgb1a1p, scgb1a and scgb1a1a, respectively. these differences were subsequently used for individual scgb1a transcript identification. isolation and characterization of scgb1a transcripts to evaluate the transcriptional state of activation of each scgb1a gene, end-point limiting dilution -pcr was performed with serially diluted cdna preparations from adult equine lung and uterus tissues. tissues were selected on the basis of their strong total scgb1a transcript expression. primers were developed in conserved regions of the genes to avoid gene-specific preference during the amplification process. each pcr assay was performed using a defined amount of an optimized limiting cdna concentration as template. the dilution that resulted in detectable amplification in less than 50% of the reactions was considered to be limiting. at this concentration, the dna target was assumed to reflect a poisson distribution, suggesting that 50% of the reactions did not contain cdna template and therefore detectable products originated from a single template. a total of pcr products were amplified from reactions using lung cdna limiting dilutions as template. an additional products were generated from reactions using uterine cdna. each individual pcr product was purified, sequenced, and identified by the scgb1a signature sequences. in lung samples, 30% of products were identified as scgb1a cdna and 70% as scgb1a1a. likewise, in uterus samples, 56% were identified as scgb1a and 44% as scgb1a1a. from the amplicons identified, scgb1a and scgb1a1a cdnas were randomly selected for further characterization. each sample was re-submitted for sequencing in both forward and reverse directions, and results were aligned to determine the cdna sequence delimited by the start and stop codons. subsequently, scgb1a and scgba1a cdna consensus sequences were obtained by alignment of the gene-specific sequences. as expected, strict nucleotide identity was observed between an individual cdna and the corresponding genomic sequence. however, several disagreements were observed upon comparison with the predicted equcab cdna sequences. among samples in this study, the partial scgb1a cdna consensus sequences displayed a non-conservative substitution at position and two variable codons at positions to the latter variants both code for the production of a leucine residue. similarly, our scgb1a1a consensus sequence showed non-conservative substitutions at position,,,, and compared to the equcab scgb1a1a predicted cdna sequence. single-nucleotide insertions or deletions were not detected. surprisingly, scgb1a1p cdna was not detected by epld-pcr. since complete genomic/cdna nucleotide identity was observed for scgb1a and scgb1a1a, scgb1a1p similarly was assessed using the predicted cdna sequences extracted from equcab and our consensus genomic sequences. analysis revealed 98% pairwise identity between the predicted cdna and the genomic dna. three non-conservative substitutions were identified at positions,, and. most strikingly, the a to t substitution detected at position in the variant sequences b and c represented 54% of the horses analyzed, independent of their genetic background. this polymorphism is expected to replace an aag codon encoding lysine, to a tag codon, encoding a stop codon. therefore, these results demonstrate the presence of a scgb1a1p gene variant that may encode a truncated protein in a large proportion of animals. scgb1a copy- and tissue-specific expression pattern expression of scgb1a and scgb1a1a specific mrna was investigated by semi-quantitative reverse transcriptase -pcr in tissues from seven adult horses. this technique was selected to survey the expression pattern rather than determine precise expression levels, since a high degree of scgb1a variation was expected between different tissues. the results were categorized into three groups based on the amount of specific pcr product detected by density scanning after gel electrophoresis. a relative optical density value was attributed to each band, using glyceraldehyde dehydrogenase amplicons as the reference value. quantification of gene-specific scgb1a cdna amplicon intensity relative to gapdh using image lab™ software.1; nd, non-detected. results in the first group corresponded to cdna samples that generated abundant pcr amplicons with high od for both scgb1a genes. samples from lung, uterus, fallopian tube, and mammary gland consistently and reproducibly met this criterion. the second group included cdnas that produced a faint pcr product for either scgb1a or scgb1a1a, and had relatively low od values. brain, pituitary gland, eye, nose epithelium, tongue, parotid salivary gland, trachea, aorta, liver, spleen, small and large intestine, adrenal gland, kidney, skin, bladder, urethra, prostate, epididymis, seminal vesicle, testis and ovary were included in this group. a third group included cdnas that did not produce gel-detectable pcr amplicons for any sample tested, and therefore, were unsuitable for quantitative assessment. eyelid gland, thyroid, bone marrow, cardiac muscle, pancreas, stomach and lymph node were considered negative for scgb1a expression. quantification of scgb1a gene-specific expression level to gain insight into the role of scgb1a genes in the pathogenic mechanisms of rao, we next sought to determine gene-specific expression in the lung, based on the previous observation that total scgb1a gene expression levels were reduced in affected animals. relative transcript levels were determined by quantitative rt-pcr using gene-specific primers for scgb1a and scgb1a1a. both equine gapdh and 18s genes were amplified concurrently for use as internal standards, and all pcr products were analyzed by gel electrophoresis and melting curve analysis. lung tissues from clinically healthy horses and horses with rao were assessed, and the latter group included individuals sampled during exacerbation and remission episodes. results were reported as the ratio of scgb1a1a/scgb1a gene expression. as shown in figure  similar expression ratios were detected in the lung of healthy individuals, who consistently had slightly higher scgb1a1a than scgb1a expression. comparable expression ratios were also noted in uterine tissues, suggesting an equivalent distribution of the two scgb1a genes in different organs. however, the scgb1a expression ratio was significantly different in rao animals compared to control animals of similar age. higher ratios were attributable to increased scgb1a1a expression, suggesting that maintenance of an appropriate scgb1a gene ratio might be necessary for homeostasis, and that abrogation of the ratio may contribute to or reflect rao development. scgb 1a protein expression in order to evaluate the correlation between scgb1a gene expression and the distribution of the protein, different tissues were evaluated by immunohistochemistry using the previously described antibody to equine scgb 1a1. this antibody was generated against a scgb 1a peptide, which is shared by both scgb 1a and scgb 1a1a, and therefore was expected to label both predicted scgb 1a proteins. detailed examination of lung tissue revealed strong and specific scgb 1a staining of the majority of non-ciliated cells lining the smaller bronchi and bronchioles. the cytosolic signal was diffuse and did not show basal or apical predilection. scgb 1a staining intensity was highest in the small bronchiolar ducts, suggesting greater expression in the distal bronchiolar tree. diffuse faint staining was noted among intrabronchiolar secretions. other pulmonary components such as blood vessels, fibroblasts, alveolar epithelium, goblet and basal cells, did not stain for scgb 1a analysis of the extra-pulmonary tissues revealed that epithelial cells in the uterus, ovary, fallopian tube and mammary gland also expressed scgb 1a within the uterus, large coiled glands composed of columnar epithelial cells stained most strongly positive, while in the ovary only epithelium from the wall of residual cysts showed some degree of staining, and luteal cells were negative. the ciliated cells of the fallopian tube stained positive, with a strong apical signal extending to the cilia. immunoreactive scgb 1a was not detected in pancreas, liver, kidney, adrenal gland, large intestine, striated and smooth muscle, duodenum, adipose tissue, nerve, nose epithelium, skin, stomach, aorta, cartilage, bone marrow, brain and pituitary gland. discussion in this study we report the identification and characterization of three equine scgb1a genes. a large segment of genomic sequence was amplified for each scgb1a gene and the partial coding sequences leading to the mature secreted proteins were sequenced. we found that certain nucleotides from each of the three genes differed from the other two genes, and that this pattern was conserved amongst individuals. the distinct scgb1a genes were predicted to produce slightly different proteins, and were therefore referred to as scgb1a1p, scgb1a and scgb1a1a based on the recommended systematic gene nomenclature system. a non-contiguous region composed of three distinct nucleotide variants was chosen as a signature sequence to generate assays specific for each individual scgb1a gene. analysis of the predicted scgb1a sequences extracted from equcab revealed more than 90% pairwise identity between scgb1a1p, scgb1a and scgb1a1a genes, including large segments of 5’- and 3’-flanking regions. this high level of sequence identity between both the coding and non-coding regions of scgb1a genes created a challenge for isolation and assessment of individual genes. thus, more than 10 kb of non-coding sequence surrounding each scgb1a gene was interrogated to identify anchor regions for gene-specific primers. primers selected included at least three nucleotides unique to each scgb1a gene. sequence analysis of products confirmed specific amplification of desired targets with lack of cross-amplification. comparative analysis of the coding region of scgb1a genes revealed some disparity with the predicted equcab sequences. our sequences differed at three positions in scgb1a1p, one position in scgb1a and five positions in scgb1a1a, corresponding to,, and % of difference, respectively. these differences may result from copy number variants and chromosomal rearrangements hindering automated sequence assembly of the equine genome. this is consistent with recent studies reporting equine chromosome as a “hotspot” for genomic rearrangements, such as enrichment in copy number variants and single nucleotide polymorphisms. a snp was detected in scgb1a1p and two snps in scgb1a but neither affected the predicted translated products. altogether, we identified several sequence differences to the equcab genome, which highlighted the importance of developing gene-specific assays. the three scgb1a genes had similar gene structure and highly conserved intron/exon organization, suggesting that each contained all the elements required for expression. thus, the transcriptional state of each gene was evaluated by epld-pcr. scgb1a and scgb1a1a, but not scgb1a1p, were specifically detected in epld of lung and uterus. these results are consistent with detection of two differently migrating scgb 1a proteins in uterine washes from mares in early pregnancy. furthermore, the sequence of each gene between start and stop codon was identical to our genomic sequence. the equine genome draft was derived from dna of a single thoroughbred mare, while our epld-pcr data were generated from animals of various genetic backgrounds and may thus be more representative of this challenging genome region. scgb1a1p transcripts were not detected in lung or uterine tissue. reasons for lack of scgb1a1p gene transcription were unclear, especially since the gene structure is virtually identical to that of the other scgb1a genes. however, the scgb1a1p sequence was more variable between individuals, had a shorter promoter region and contained a putative stop mutation in a significant percentage of individuals. these characteristics suggest that scgb1a1p may be a pseudogene. the non-synonymous nucleotide variations observed between scgb1a and scgb1a1a result in amino acid substitutions among the residues of the mature secreted proteins. seven of the variable aas are concentrated between position and. this region borders the scgb 1a central cavity that binds hydrophobic ligands, and aa with hydrophobic properties comprise the cavity. since some conserved aa, such as phenylalanine, also have ligand-binding properties, substitution of f to l in scgb 1a1a suggests 1) a change in ligand-binding specificity, and 2) that scgb 1a and 1a1a may have independently evolved to bind distinct substrates. other substitutions in this region largely maintain hydrophobic properties, implying minor change in ligand affinity. there is high sequence identity in other regions of each protein with preservation of critical structural residues such as c and c needed for homodimer interaction, and k d and a required for protein stability. however, also of interest, the predicted isoelectric point of scgb 1a and 1a1a proteins are and, respectively, which corresponds to the scgb1a variants described in uterine washes of pregnant horses. the spectrum of tissues expressing scgb1a has not been extensively studied in horses. expression nonspecific for individual genes was previously identified in lung, uterus and prostate, and absent in liver, kidney, heart, spleen, as well as thyroid, pituitary and adrenal gland tissues by northern blot analysis. we selected a gene-specific rt-pcr approach to characterize the distribution of scgb1a and scgb1a1a transcripts in a total of tissues. this assay amplified the coding region of the entire mature protein to reduce potential genomic dna targeting, and specificity was verified by random purification and sequencing of amplicons. as expected, scgb1a and scgb1a1a transcripts were strongly detected in lung and uterus, but also in fallopian tube and mammary gland tissue. fewer transcripts of either gene were present in brain, pituitary gland, eye, nose epithelium, tongue, salivary gland, trachea, aorta, liver, spleen, small and large intestines, adrenal gland, kidney, skin, bladder, urethra, prostate, epididymis, seminal vesicle, testis and ovary, and no transcripts were detected in eyelid gland, thyroid, bone marrow, cardiac muscle, pancreas, stomach and lymph node. detection of transcripts in a greater range of tissues by pcr than by northern blotting may reflect higher sensitivity of the former, and greater specificity due to exact primer match. some tissues had more of one relative to the other gene product, suggesting tissue-specific expression patterns. while the regulatory mechanisms that could selectively drive the expression of individual scgb1a genes remain to be elucidated, this finding is consistent with unique functions of different scgb 1a proteins. immunohistochemical analyses were carried out to evaluate the correlation of scgb1a gene and protein expression, and to determine cell-specific expression within tissues. the antibody employed recognized an epitope shared by all scgb 1a proteins. strong staining was detected in tissues expressing the highest number of transcripts such as lung, uterus, fallopian tube and mammary gland. however, within these tissues, scgb 1a was present in only specific epithelial cell populations, and absent in all other cell types. this distribution likely contributed to variation of scgb1a transcript intensity, since the proportion of epithelial cells and subtypes in tissues selected was variable. scgb 1a may be selectively taken up and targeted for degradation upon binding to a transmembrane protein called cubilin. interaction of scgb 1a with lipocalin- interacting membrane receptor has also been reported, but the effect of this interaction is unclear. of note, limr is expressed in tissues positive for scgb1a transcripts, including lung, mammary gland, trachea, prostate, testis, pituitary gland, adrenal gland, cerebellum, kidney, and colon. scgb 1a protein was not detected in tissues with low transcript expression such as bladder, trachea, liver, nose epithelium, pituitary and intestine. possible reasons are lesser sensitivity of ihc compared to pcr, and transient gene expression insufficient to produce detectable protein, as has been reported in studies of rabbit tissues. horse and human have a similar pattern of scgb1a transcript distribution in lung, uterus, fallopian tube, prostate, trachea, thyroid, mammary gland, brain, pituitary, thymus, aorta, heart, stomach, spleen, adrenal gland, kidney, liver, small intestine, ovary and testis. in humans, the scgb1a gene was ultimately considered to be ubiquitously expressed in most cells of epithelial origin and to inactivate inflammatory mediators on surfaces exposed either directly or indirectly to the external environment. conversely, it was also reported that decreased scgb1a expression could contribute a tumor microenvironment permissive of inflammation and hence tumor progression. since total scgb1a expression was reported as decreased in rao, we sought to evaluate if both scgb1a and scgb1a1a genes were similarly affected. our analysis demonstrated that the ratio of scgb1a1a/scgb1a was significantly different in rao affected-animals compared to controls. this finding may arise as a result of chronic inflammation with preferential transcription of scgb1a1a, or may signal inherent differences in transcriptional regulation of scgb1a genes. segregation analysis previously revealed a complex genetic background influencing expression of the rao phenotype. scgb1a was among candidate genes, suggesting that further evaluation of specific gene expression may be warranted. the latter observation also raises the question whether scgb1a genes are controlled by different regulatory mechanisms and whether they have different physiological functions. such hypotheses are difficult to address due to the absence of multiple scgb1a copies in other mammals except other equidae, such as donkeys and przewalski horses. scgb1a isoforms in equid species remain to be characterized, but may yield insight into scgb1a gene origin and ancestral gene triplication.  three equine scgb1a genes were isolated and characterized. scgb1a1p appears to have evolved into a pseudogene, which no longer generates a detectable transcript and includes a non-sense mutation in the majority of animals. the distribution of scgb1a and scgb1a1a gene transcripts and proteins indicates highly specific expression in specialized epithelial cells of lung and reproductive organs. gene specific assessment of transcripts showed approximately fold higher expression of scgb1a1a than scgb1a in lung and uterus of control animals, and an increased ratio in lung tissue of animals with rao, an asthma-like condition. future studies will assess the function of different scgb 1a proteins and attempt to elucidate their anti-inflammatory properties. 
bioinformatic analysis showed that scgb1a genes differ from each other by to nucleotides, and that they code for different proteins. airway fluid and lung tissue of horses with recurrent airway obstruction, a chronic inflammatory lung disease affecting mature horses similar to environmentally induced asthma of humans, have reduced total scgb 1a concentration. analysis of scgb1a and scgb1a1a sequences by endpoint-limiting dilution pcr identified a consistent difference affecting 3 bp within exon which served as a gene-specific “signature”. however, analysis of the equine genome suggested that horses might have multiple scgb1a gene copies. secretoglobin 1a, also called clara cell secretory protein, is the most abundantly secreted protein of the airway. cc10clara cellclara cell secretory proteinend-point limiting dilution pcrhorseimmunohistochemistrylong-range pcrrecurrent airway obstructionuteroglobin non-ciliated lung epithelial cells produce scgb 1a during inhalation of noxious substances to counter airway inflammation. assessment of gene- and organ-specific expression by semiquantitative rt-pcr of tissues showed strong expression of scgb1a and scgb1a1a in lung, uterus, fallopian tube and mammary gland, which correlated with detection of scgb 1a protein by immunohistochemistry. significantly altered expression of the ratio of scgb1a1a to scgb1a was detected in rao-affected animals compared to controls, suggesting different roles for scgb 1a and scgb 1a1a in this inflammatory condition. we identified three scgb1a gene copies on equine chromosome contained within a 512-kilobase region. the scgb1a1p gene had most inter-individual variability and contained a non-sense mutation in many animals, suggesting that scgb1a1p has evolved into a pseudogene.
 secretoglobin 1a, also called clara cell secretory protein, is the most abundantly secreted protein of the airway. the scgb1a gene has been characterized in mammals as a single copy in the genome. however, analysis of the equine genome suggested that horses might have multiple scgb1a gene copies. non-ciliated lung epithelial cells produce scgb 1a during inhalation of noxious substances to counter airway inflammation. airway fluid and lung tissue of horses with recurrent airway obstruction, a chronic inflammatory lung disease affecting mature horses similar to environmentally induced asthma of humans, have reduced total scgb 1a concentration. herein, we investigated whether horses have distinct expressed scgb1a genes; whether the transcripts are differentially expressed in tissues and in inflammatory lung disease; and whether there is cell specific protein expression in tissues.  we identified three scgb1a gene copies on equine chromosome contained within a 512-kilobase region. bioinformatic analysis showed that scgb1a genes differ from each other by to nucleotides, and that they code for different proteins. transcripts were detected for scgb1a and scgb1a1a, but not for scgb1a1p. the scgb1a1p gene had most inter-individual variability and contained a non-sense mutation in many animals, suggesting that scgb1a1p has evolved into a pseudogene. analysis of scgb1a and scgb1a1a sequences by endpoint-limiting dilution pcr identified a consistent difference affecting 3 bp within exon which served as a gene-specific “signature”. assessment of gene- and organ-specific expression by semiquantitative rt-pcr of tissues showed strong expression of scgb1a and scgb1a1a in lung, uterus, fallopian tube and mammary gland, which correlated with detection of scgb 1a protein by immunohistochemistry. significantly altered expression of the ratio of scgb1a1a to scgb1a was detected in rao-affected animals compared to controls, suggesting different roles for scgb 1a and scgb 1a1a in this inflammatory condition.  this is the first report of three scgb1a genes in a mammal. the two expressed genes code for proteins predicted to differ in function. alterations in the gene expression ratio in rao suggest cell and tissue specific regulation and functions. these findings may be important for understanding of lung and reproductive conditions. cc10clara cellclara cell secretory proteinend-point limiting dilution pcrhorseimmunohistochemistrylong-range pcrrecurrent airway obstructionuteroglobin 