- Email: [email protected]
Characterisation of the human plunc gene, a gene product with an upper airways and nasopharyngeal restricted expression pattern
Biochimica et Biophysica Acta 1493 (2000) 363^367
www.elsevier.com/locate/bba
Short sequence-paper
Characterisation of the human plunc gene, a gene product with an upper airways and nasopharyngeal restricted expression pattern Colin D. Bingle, Lynne Bingle * Respiratory Cell and Molecular Biology, Division of Molecular and Genetic Medicine, University of She¤eld Medical School, M128, Royal Hallamshire Hospital, Glossop Road, She¤eld S10 2RX, UK Received 23 May 2000 ; received in revised form 25 July 2000; accepted 28 July 2000
Abstract Here we report the cloning and characterization of the human homologue of plunc, a murine gene expressed specifically in the upper airways and nasopharyngeal regions. The human plunc cDNA codes for a leucine-rich protein of 256 amino acids which is 72% identical to the murine protein. RNA blot analysis suggests that expression of plunc is restricted to the trachea, upper airway, nasopharyngeal epithelium and salivary gland. The human plunc gene contains nine exons and is localised to chromosome 20q11.2. The unique expression pattern of the human plunc suggest that it may prove a useful model gene with which to study the regulatory mechanisms which direct expression of genes specifically to the upper airways. ß 2000 Elsevier Science B.V. All rights reserved. Keywords : Gene expression ; Airway; Trachea; Nasopharyngeal; Pulmonary epithelium
The pulmonary epithelium extending from the trachea to the alveoli is derived from an outpocketing of the foregut endoderm into the mesenchyme of the fetal thorax. Proliferation and di¡erentiation of this epithelium, mediated in part by mesenchymal interactions, results in regional di¡erentiation and the formation of a large number of di¡erentiated cell types which provide the varied functions associated with the mature pulmonary system [1,2]. A number of genes are expressed in a cell-type restricted manner in di¡erent regions within the pulmonary system. Perhaps the best studied of these are genes whose expression is restricted to the bronchial and alveolar regions of the lung, for example the surfactant associated proteins, SP-A, SP-B, SP-C and SP-D and the Clara cell secretory protein (CCSP). The regulatory regions of these genes have been extensively studied and the transcriptional mechanisms which restrict their expression is partially understood [1,3]. The promoters of these genes have proved useful in targeting expression of proteins to the lower airways. Less well studied are genes whose expression is limited to the upper airways and the nasopharyngeal regions. Expression of SP-A, SP-D and CCSP has been
* Corresponding author. Fax: +44-114-272-1104; E-mail : c.d.bingle@she¤eld.ac.uk
shown to occur within the trachea [4^7] but as these genes are also expressed in the lower regions of the lungs they do not represent true upper airway restricted gene products. The expression pro¢le of gene products recently identi¢ed as being expressed within the nasopharyngeal region of the airways has still to be fully examined [8^10]. Consequently, information on genomic regions which direct expression of genes to these regions of the airways are limited [11]. The identi¢cation and characterization of such gene products may help us to gain insights into the mechanisms which govern expression of genes within the upper airways and may provide us with tools to target gene expression to these regions of the airways. Recently, a novel gene, plunc (palate, lung and nasal epithelium clone) was identi¢ed during a study into genes speci¢cally expressed during palate closure in the developing mouse [12]. Plunc has a unique expression pro¢le, being expressed around the developing palate, in nasal septum and nasal conchae, as well as in the trachea and main stem bronchi [12]. The function of plunc is unknown but the amino acid sequence is most closely similar to secretory proteins produced by the salivary glands and glandular epithelium of the trachea, the von Ebner minor salivary protein (also known as LCN1) and the parotid secretory protein [13^15]. Consistent with this sequence similarity plunc contains an N terminal region similar to that of a signal peptide, suggesting that plunc may be
0167-4781 / 00 / $ ^ see front matter ß 2000 Elsevier Science B.V. All rights reserved. PII: S 0 1 6 7 - 4 7 8 1 ( 0 0 ) 0 0 1 9 6 - 2
BBAEXP 91463 20-9-00
364
C.D. Bingle, L. Bingle / Biochimica et Biophysica Acta 1493 (2000) 363^367
secreted [12]. Despite its unknown function, the expression pattern of plunc suggests that this gene may be a suitable model with which to study the regulatory mechanisms which govern expression of genes in the tracheal and nasopharyngeal epithelium. With this end point in mind we set out to isolate and characterise the human plunc gene. By screening the human EST database with the full length mouse plunc cDNA sequence we identi¢ed a number of homologous clones. To determine the complete sequence of the human plunc homologue we sequenced two of these clones. The human plunc cDNA is 1020 bp in length and contains a single open reading frame, which
Fig. 2. Human plunc is closely related to its murine counterpart but is missing a N-terminal region containing PLPL repeats. The derived amino acid sequence of human plunc (H) is aligned with that of the mouse protein (M) and the partial sequence of the rat (R) and bovine (B) sequences. Identical amino acids are indicated by (3) and (.) indicate where gaps are inserted for maximum alignment. The position of the G(L/P/Q)(P/L)LPL repeats in the mouse sequence are indicated by underlining.
Fig. 1. cDNA and derived amino acid sequence of human plunc. The complete nucleotide and derived amino acid sequence of human plunc was obtained by sequencing EST N23239. The 5P-most nine nucleotides were obtained from the sequence of AA315985. Numbers to the left represent the nucleotides whilst those at the right represent the amino acids. The start and stop codons are shown in italics and the relative position of the intron/exon boundaries (see below) are shown by the arrows above the sequence. The human plunc cDNA has the accession number AF172993.
encodes for a leucine rich (24.6%) protein of 256 amino acids (Fig. 1). Comparison of the human sequence with the mouse homologue, which is 278 amino acids long, shows that overall there is a s 72% sequence identity (Fig. 2). Further analysis of sequences deposited with in the EST databases identi¢ed the existence of cDNAs corresponding to partial clones from rat and bovine sources. Translation and alignment of these partial clones suggests that plunc from these species will also prove to be highly related to the human and murine protein sequences (Fig. 2). Throughout the human and murine sequences there are regions which are completely conserved. Interestingly, there is one region towards the N-terminal which is completely absent from the human protein when compared to the mouse sequence. This portion of the mouse protein contains three of the four G(L/P/Q)(P/L)LPL repeat regions which are unique to mouse plunc [12]. The remaining repeat is also incomplete as the invariant L at position 4 is replaced in the human protein with a V, suggesting that these repeated regions may not represent functionally signi¢cant motifs within the plunc protein, although their true role requires analysis of the proteins themselves. To determine the expression pattern of human plunc we initially probed a poly(A) dot blot with the plunc cDNA. Signi¢cant hybridization was seen in the sample from adult trachea and to a lesser extent in the sample from adult lung (Fig. 3A). Prolonged exposure of the blot failed to show expression of plunc RNA in any other tissues on the blot. This observation is consistent with the expression pattern seen with mouse plunc [12] and suggests that human plunc may be a marker for upper airway epithelium. Total RNA extracted from nasal septal epithelium, was
BBAEXP 91463 20-9-00
C.D. Bingle, L. Bingle / Biochimica et Biophysica Acta 1493 (2000) 363^367
365
Fig. 3. Expression of human plunc is restricted to the trachea, lung and nasopharyngeal epithelium and cellular derivatives. (A) A commercial multiple tissue poly(A) dot blot was hybridized with a random primed plunc cDNA probe. Expression of plunc is highest in the sample from trachea (arrow) and low-level expression was also found in the sample of adult lung (arrow). (B) plunc mRNA was also expressed in total RNA isolated from normal nasal septum epithelium (lane 1), salivary gland (lane 2) and trcheobronchial epithelial cells (lane 4) but not in liver (lane 3). (C) Plunc expression in NCI-H647 cells (upper) is reduced by PMA (lane 2) and interferon-Q (lane 5) treatment compared to control (lane 1). Levels of plunc RNA were not signi¢cantly altered by treatment with retinoic acid (lane 3), interleukin-1L (lane 4), tumour necrosis factor K (lane 6), dexamethasone (lane 7) or lipopolysaccharide (lane 8).
also shown to hybridize strongly with the plunc probe (Fig. 3B, lane 1), whereas no expression was seen in total RNA samples isolated from human peripheral lung samples (results not shown). Hybridization in the human lung poly(A) RNA sample on the dot blot may have arisen from tissue from the bronchiolar and/or tracheal regions being present in the sample. In addition we also observed that plunc was highly expressed in tracheobronchial epithelial cells (Fig. 3B, lane 4), derived from the proximal portions of the airways [16] as well as in RNA from salivary gland (Fig. 3B, lane 2). Taken together these results appear to show that plunc may be restricted to the tracheal epithelium and nasopharyngeal regions. However, at present we have no information as to which cell types within the airways express plunc in situ. In the mouse, plunc expression appears to be localized throughout the epithelial cells of the trachea and main bronchi [12]. As an initial way to gain insights into which cell types might express plunc, we searched for plunc mRNA in human lung derived cell lines. Amongst s 20 lines tested we identi¢ed one, the NCI-H647 adeno-squamous carcinoma cell
line, that was positive for plunc mRNA (Fig. 3C). Little is known about NCI-H647 cells. They do not express distal lung epithelial markers, for example surfactant apoproteins (results not shown), but do express the antiproteinase SKALP/ela¢n, which is expressed throughout the airway. We were unable to show that these same cells expressed the recently described nasopharyngeal epithelium restricted gene, NESG1 [8] or human LCN1 [14] (results not shown). When these cells were treated with a variety of in£ammatory mediators we were able to show that interferon-Q and phorbol myristate acetate clearly reduced plunc mRNA expression (Fig. 3C), whereas treatment with a variety of other mediators had either a lesser or had no e¡ect on plunc expression. To add further weight to our hypothesis that plunc is an upper airway and nasal epithelium restricted gene, all of the ESTs corresponding to human plunc contained in the database are derived either from lung or olfactory epithelium. Other genes suggested to be markers for these epithelia [8^10] have some of their corresponding ESTs derived from other tissues. We cloned the human plunc gene by three, overlapping, genomic
BBAEXP 91463 20-9-00
366
C.D. Bingle, L. Bingle / Biochimica et Biophysica Acta 1493 (2000) 363^367
Fig. 4. The structure of the human plunc gene. The complete human plunc gene was isolated by genome walking as described. The genomic sequence has the accession number AF214562. (A) A schematic representation of the structure shows the position of the exons, and the arrows indicate the extent of the individual genomic walks. Representative restriction sites are indicated (E, EcoRI; B, BglII; P, PstI). (B) The sequence of the intron/exon boundaries and the size of the introns and exons. The divergent sequences arising from alternative splicing of exon 9 are indicated as exons 9, 9a and 9b. The derived sequence for the EST originally identi¢ed is arbitrarily called exon 9.
walks using the polymerase chain reaction (PCR)-based Genome Walker system. A comparison of the genomic clones with that of the cDNA sequence revealed that the human plunc gene consists of nine exons and covers approximately 7.3 kb (Fig. 4A). The ¢rst (and shortest) exon and ninth exon are non-coding. A putative TATAA box is located within 40 bp 5P of the end of exon 1. The exons range in size from 40 to 174 bp and the introns between 1642 and 159 bp (Fig. 4B). The longest intron separates the ¢rst and second exons. The fact that the intron/exon boundaries do not directly correspond to the divergent region of the human and mouse protein sequences suggests that the di¡erence is not due to alternative splicing of an exon. In support of this we were unable to identify any alternative splicing of coding region of plunc in NCI-H647 RNA by reverse transcriptase^PCR using multiple primer pairs (results not shown). However, amongst the ESTs present within the database, we were able to characterise three distinct cDNA populations, which di¡ered in their 3PUTRs around the junction of exons 8 and 9. In addition to the sequence contained within the clone that we have described (Fig. 1), the two cDNAs use additional 3P-splice acceptor sites which add either 41 bp (exon 9b) or 55 bp (exon 9a) to the sequence of exon 9. Both of these acceptor sites conform to the expected consensus and it remains to be determined how these additional regions of sequence
may function in the regulation of plunc mRNA turnover and stability. One of the ESTs within the database (Z24371) corresponds to the microsatellite marker D20S195. This marker, which corresponds to a portion of exon 2, the whole of intron 2, exon 3 and a portion of intron 3, has been localised to chromosome 20q11.2 in a newly identi¢ed Graves disease locus [17]. Linkage of human plunc to chromosome 20 was unequivocally con¢rmed by the existence of partial genomic clones in the chromosome 20 sequencing project database (http://www.sanger.ac.uk/ HGP/Chr20/). No human conditions associated with defects in the tissues in which we have shown plunc to be expressed have been mapped to this region of the genome. In summary, we have cloned and characterized the human plunc gene and shown that, as is the case with the murine homologue, its expression is limited to the upper airways and nasopharyngeal regions, including the trachea and nasal epithelium. Using this genomic sequence information it should prove possible to identify and study the regulatory regions which confer this unique expression pattern to the gene. We wish to thank Beth LeClair for discussions concerning the role of plunc, Yaron Tomer for suggesting we look at the Chromosome 20 sequencing web pages, Scott Randell for providing the human tracheobronchial epithelial
BBAEXP 91463 20-9-00
C.D. Bingle, L. Bingle / Biochimica et Biophysica Acta 1493 (2000) 363^367
cells and Rob Read for providing the human nasal septum samples. This work was funded by the Wellcome Trust. References [1] B.P. Hackett, C.D. Bingle, J.D. Gitlin, Annu. Rev. Physiol. 58 (1996) 51^71. [2] B.L. Hogan, J.M. Yingling, Curr. Opin. Genet. Dev. 8 (1998) 481^ 486. [3] J.A. Whitsett, S.W. Glasser, Biochim. Biophys. Acta 1408 (1998) 303^311. [4] J.J. Coalson, V. Winter, F. Yang, Anat. Rec. 250 (1998) 300^315. [5] K.L. Goss, A.R. Kumar, J.M. Snyder, Am. J. Respir. Cell Mol. Biol. 19 (1998) 613^621. [6] A. Khoor, M.E. Gray, G. Singh, M.T. Stahlman, J. Histochem. Cytochem. 44 (1996) 1429^1438. [7] C.J. Wong, J. Akiyama, L. Allen, S. Hawgood, Pediatr. Res. 39 (1996) 930^937.
367
[8] L.F. Fung, P.W. Yuen, W. Wei, H.S. Kwan, S.W. Tsao, Int. J. Oncol. 13 (1998) 85^89. [9] Z. Li, K. Yao, Y. Cao, Gene 237 (1999) 235^240. [10] M.I. Merkulova, S.G. Andreeva, T.M. Shuvaeva, S.V. Novoselov, I.V. Peshenko, M.F. Bystrova, V.I. Novoselov, E.E. Fesenko, V.M. Lipkin, FEBS Lett. 450 (1999) 126^130. [11] A. Medvedev, N.A. Saunders, H. Matsuura, A. Chistokhina, A.M. Jetten, J. Biol. Chem. 274 (1999) 3887^3896. [12] W.M. Weston, E.E. LeClair, W. Trzyna, K.M. McHugh, P. Nugent, C.M. La¡erty, L. Ma, R.S. Tuan, R.M. Greene, J. Biol. Chem. 274 (1999) 13698^13703. [13] H.O. Madsen, J.P. Hjorth, Nucleic Acids Res. 13 (1985) 1^13. [14] B. Redl, P. Wojnar, H. Ellemunter, H. Feichtinger, Lab. Invest. 78 (1998) 1121^1129. [15] L. Mirels, W.D. Ball, J. Biol. Chem. 267 (1992) 2679^2687. [16] S.H. Bernacki, A.L. Nelson, L. Abdullah, J.K. Sheehan, A. Harris, D.C. William, S.H. Randell, Am. J. Respir. Cell Mol. Biol. 20 (1999) 595^604. [17] Y. Tomer, G. Barbesino, D.A. Greenberg, E. Concepcion, T.F. Davies, Am. J. Hum. Genet. 63 (1998) 1749^1756.
BBAEXP 91463 20-9-00
www.elsevier.com/locate/bba
Short sequence-paper
Characterisation of the human plunc gene, a gene product with an upper airways and nasopharyngeal restricted expression pattern Colin D. Bingle, Lynne Bingle * Respiratory Cell and Molecular Biology, Division of Molecular and Genetic Medicine, University of She¤eld Medical School, M128, Royal Hallamshire Hospital, Glossop Road, She¤eld S10 2RX, UK Received 23 May 2000 ; received in revised form 25 July 2000; accepted 28 July 2000
Abstract Here we report the cloning and characterization of the human homologue of plunc, a murine gene expressed specifically in the upper airways and nasopharyngeal regions. The human plunc cDNA codes for a leucine-rich protein of 256 amino acids which is 72% identical to the murine protein. RNA blot analysis suggests that expression of plunc is restricted to the trachea, upper airway, nasopharyngeal epithelium and salivary gland. The human plunc gene contains nine exons and is localised to chromosome 20q11.2. The unique expression pattern of the human plunc suggest that it may prove a useful model gene with which to study the regulatory mechanisms which direct expression of genes specifically to the upper airways. ß 2000 Elsevier Science B.V. All rights reserved. Keywords : Gene expression ; Airway; Trachea; Nasopharyngeal; Pulmonary epithelium
The pulmonary epithelium extending from the trachea to the alveoli is derived from an outpocketing of the foregut endoderm into the mesenchyme of the fetal thorax. Proliferation and di¡erentiation of this epithelium, mediated in part by mesenchymal interactions, results in regional di¡erentiation and the formation of a large number of di¡erentiated cell types which provide the varied functions associated with the mature pulmonary system [1,2]. A number of genes are expressed in a cell-type restricted manner in di¡erent regions within the pulmonary system. Perhaps the best studied of these are genes whose expression is restricted to the bronchial and alveolar regions of the lung, for example the surfactant associated proteins, SP-A, SP-B, SP-C and SP-D and the Clara cell secretory protein (CCSP). The regulatory regions of these genes have been extensively studied and the transcriptional mechanisms which restrict their expression is partially understood [1,3]. The promoters of these genes have proved useful in targeting expression of proteins to the lower airways. Less well studied are genes whose expression is limited to the upper airways and the nasopharyngeal regions. Expression of SP-A, SP-D and CCSP has been
* Corresponding author. Fax: +44-114-272-1104; E-mail : c.d.bingle@she¤eld.ac.uk
shown to occur within the trachea [4^7] but as these genes are also expressed in the lower regions of the lungs they do not represent true upper airway restricted gene products. The expression pro¢le of gene products recently identi¢ed as being expressed within the nasopharyngeal region of the airways has still to be fully examined [8^10]. Consequently, information on genomic regions which direct expression of genes to these regions of the airways are limited [11]. The identi¢cation and characterization of such gene products may help us to gain insights into the mechanisms which govern expression of genes within the upper airways and may provide us with tools to target gene expression to these regions of the airways. Recently, a novel gene, plunc (palate, lung and nasal epithelium clone) was identi¢ed during a study into genes speci¢cally expressed during palate closure in the developing mouse [12]. Plunc has a unique expression pro¢le, being expressed around the developing palate, in nasal septum and nasal conchae, as well as in the trachea and main stem bronchi [12]. The function of plunc is unknown but the amino acid sequence is most closely similar to secretory proteins produced by the salivary glands and glandular epithelium of the trachea, the von Ebner minor salivary protein (also known as LCN1) and the parotid secretory protein [13^15]. Consistent with this sequence similarity plunc contains an N terminal region similar to that of a signal peptide, suggesting that plunc may be
0167-4781 / 00 / $ ^ see front matter ß 2000 Elsevier Science B.V. All rights reserved. PII: S 0 1 6 7 - 4 7 8 1 ( 0 0 ) 0 0 1 9 6 - 2
BBAEXP 91463 20-9-00
364
C.D. Bingle, L. Bingle / Biochimica et Biophysica Acta 1493 (2000) 363^367
secreted [12]. Despite its unknown function, the expression pattern of plunc suggests that this gene may be a suitable model with which to study the regulatory mechanisms which govern expression of genes in the tracheal and nasopharyngeal epithelium. With this end point in mind we set out to isolate and characterise the human plunc gene. By screening the human EST database with the full length mouse plunc cDNA sequence we identi¢ed a number of homologous clones. To determine the complete sequence of the human plunc homologue we sequenced two of these clones. The human plunc cDNA is 1020 bp in length and contains a single open reading frame, which
Fig. 2. Human plunc is closely related to its murine counterpart but is missing a N-terminal region containing PLPL repeats. The derived amino acid sequence of human plunc (H) is aligned with that of the mouse protein (M) and the partial sequence of the rat (R) and bovine (B) sequences. Identical amino acids are indicated by (3) and (.) indicate where gaps are inserted for maximum alignment. The position of the G(L/P/Q)(P/L)LPL repeats in the mouse sequence are indicated by underlining.
Fig. 1. cDNA and derived amino acid sequence of human plunc. The complete nucleotide and derived amino acid sequence of human plunc was obtained by sequencing EST N23239. The 5P-most nine nucleotides were obtained from the sequence of AA315985. Numbers to the left represent the nucleotides whilst those at the right represent the amino acids. The start and stop codons are shown in italics and the relative position of the intron/exon boundaries (see below) are shown by the arrows above the sequence. The human plunc cDNA has the accession number AF172993.
encodes for a leucine rich (24.6%) protein of 256 amino acids (Fig. 1). Comparison of the human sequence with the mouse homologue, which is 278 amino acids long, shows that overall there is a s 72% sequence identity (Fig. 2). Further analysis of sequences deposited with in the EST databases identi¢ed the existence of cDNAs corresponding to partial clones from rat and bovine sources. Translation and alignment of these partial clones suggests that plunc from these species will also prove to be highly related to the human and murine protein sequences (Fig. 2). Throughout the human and murine sequences there are regions which are completely conserved. Interestingly, there is one region towards the N-terminal which is completely absent from the human protein when compared to the mouse sequence. This portion of the mouse protein contains three of the four G(L/P/Q)(P/L)LPL repeat regions which are unique to mouse plunc [12]. The remaining repeat is also incomplete as the invariant L at position 4 is replaced in the human protein with a V, suggesting that these repeated regions may not represent functionally signi¢cant motifs within the plunc protein, although their true role requires analysis of the proteins themselves. To determine the expression pattern of human plunc we initially probed a poly(A) dot blot with the plunc cDNA. Signi¢cant hybridization was seen in the sample from adult trachea and to a lesser extent in the sample from adult lung (Fig. 3A). Prolonged exposure of the blot failed to show expression of plunc RNA in any other tissues on the blot. This observation is consistent with the expression pattern seen with mouse plunc [12] and suggests that human plunc may be a marker for upper airway epithelium. Total RNA extracted from nasal septal epithelium, was
BBAEXP 91463 20-9-00
C.D. Bingle, L. Bingle / Biochimica et Biophysica Acta 1493 (2000) 363^367
365
Fig. 3. Expression of human plunc is restricted to the trachea, lung and nasopharyngeal epithelium and cellular derivatives. (A) A commercial multiple tissue poly(A) dot blot was hybridized with a random primed plunc cDNA probe. Expression of plunc is highest in the sample from trachea (arrow) and low-level expression was also found in the sample of adult lung (arrow). (B) plunc mRNA was also expressed in total RNA isolated from normal nasal septum epithelium (lane 1), salivary gland (lane 2) and trcheobronchial epithelial cells (lane 4) but not in liver (lane 3). (C) Plunc expression in NCI-H647 cells (upper) is reduced by PMA (lane 2) and interferon-Q (lane 5) treatment compared to control (lane 1). Levels of plunc RNA were not signi¢cantly altered by treatment with retinoic acid (lane 3), interleukin-1L (lane 4), tumour necrosis factor K (lane 6), dexamethasone (lane 7) or lipopolysaccharide (lane 8).
also shown to hybridize strongly with the plunc probe (Fig. 3B, lane 1), whereas no expression was seen in total RNA samples isolated from human peripheral lung samples (results not shown). Hybridization in the human lung poly(A) RNA sample on the dot blot may have arisen from tissue from the bronchiolar and/or tracheal regions being present in the sample. In addition we also observed that plunc was highly expressed in tracheobronchial epithelial cells (Fig. 3B, lane 4), derived from the proximal portions of the airways [16] as well as in RNA from salivary gland (Fig. 3B, lane 2). Taken together these results appear to show that plunc may be restricted to the tracheal epithelium and nasopharyngeal regions. However, at present we have no information as to which cell types within the airways express plunc in situ. In the mouse, plunc expression appears to be localized throughout the epithelial cells of the trachea and main bronchi [12]. As an initial way to gain insights into which cell types might express plunc, we searched for plunc mRNA in human lung derived cell lines. Amongst s 20 lines tested we identi¢ed one, the NCI-H647 adeno-squamous carcinoma cell
line, that was positive for plunc mRNA (Fig. 3C). Little is known about NCI-H647 cells. They do not express distal lung epithelial markers, for example surfactant apoproteins (results not shown), but do express the antiproteinase SKALP/ela¢n, which is expressed throughout the airway. We were unable to show that these same cells expressed the recently described nasopharyngeal epithelium restricted gene, NESG1 [8] or human LCN1 [14] (results not shown). When these cells were treated with a variety of in£ammatory mediators we were able to show that interferon-Q and phorbol myristate acetate clearly reduced plunc mRNA expression (Fig. 3C), whereas treatment with a variety of other mediators had either a lesser or had no e¡ect on plunc expression. To add further weight to our hypothesis that plunc is an upper airway and nasal epithelium restricted gene, all of the ESTs corresponding to human plunc contained in the database are derived either from lung or olfactory epithelium. Other genes suggested to be markers for these epithelia [8^10] have some of their corresponding ESTs derived from other tissues. We cloned the human plunc gene by three, overlapping, genomic
BBAEXP 91463 20-9-00
366
C.D. Bingle, L. Bingle / Biochimica et Biophysica Acta 1493 (2000) 363^367
Fig. 4. The structure of the human plunc gene. The complete human plunc gene was isolated by genome walking as described. The genomic sequence has the accession number AF214562. (A) A schematic representation of the structure shows the position of the exons, and the arrows indicate the extent of the individual genomic walks. Representative restriction sites are indicated (E, EcoRI; B, BglII; P, PstI). (B) The sequence of the intron/exon boundaries and the size of the introns and exons. The divergent sequences arising from alternative splicing of exon 9 are indicated as exons 9, 9a and 9b. The derived sequence for the EST originally identi¢ed is arbitrarily called exon 9.
walks using the polymerase chain reaction (PCR)-based Genome Walker system. A comparison of the genomic clones with that of the cDNA sequence revealed that the human plunc gene consists of nine exons and covers approximately 7.3 kb (Fig. 4A). The ¢rst (and shortest) exon and ninth exon are non-coding. A putative TATAA box is located within 40 bp 5P of the end of exon 1. The exons range in size from 40 to 174 bp and the introns between 1642 and 159 bp (Fig. 4B). The longest intron separates the ¢rst and second exons. The fact that the intron/exon boundaries do not directly correspond to the divergent region of the human and mouse protein sequences suggests that the di¡erence is not due to alternative splicing of an exon. In support of this we were unable to identify any alternative splicing of coding region of plunc in NCI-H647 RNA by reverse transcriptase^PCR using multiple primer pairs (results not shown). However, amongst the ESTs present within the database, we were able to characterise three distinct cDNA populations, which di¡ered in their 3PUTRs around the junction of exons 8 and 9. In addition to the sequence contained within the clone that we have described (Fig. 1), the two cDNAs use additional 3P-splice acceptor sites which add either 41 bp (exon 9b) or 55 bp (exon 9a) to the sequence of exon 9. Both of these acceptor sites conform to the expected consensus and it remains to be determined how these additional regions of sequence
may function in the regulation of plunc mRNA turnover and stability. One of the ESTs within the database (Z24371) corresponds to the microsatellite marker D20S195. This marker, which corresponds to a portion of exon 2, the whole of intron 2, exon 3 and a portion of intron 3, has been localised to chromosome 20q11.2 in a newly identi¢ed Graves disease locus [17]. Linkage of human plunc to chromosome 20 was unequivocally con¢rmed by the existence of partial genomic clones in the chromosome 20 sequencing project database (http://www.sanger.ac.uk/ HGP/Chr20/). No human conditions associated with defects in the tissues in which we have shown plunc to be expressed have been mapped to this region of the genome. In summary, we have cloned and characterized the human plunc gene and shown that, as is the case with the murine homologue, its expression is limited to the upper airways and nasopharyngeal regions, including the trachea and nasal epithelium. Using this genomic sequence information it should prove possible to identify and study the regulatory regions which confer this unique expression pattern to the gene. We wish to thank Beth LeClair for discussions concerning the role of plunc, Yaron Tomer for suggesting we look at the Chromosome 20 sequencing web pages, Scott Randell for providing the human tracheobronchial epithelial
BBAEXP 91463 20-9-00
C.D. Bingle, L. Bingle / Biochimica et Biophysica Acta 1493 (2000) 363^367
cells and Rob Read for providing the human nasal septum samples. This work was funded by the Wellcome Trust. References [1] B.P. Hackett, C.D. Bingle, J.D. Gitlin, Annu. Rev. Physiol. 58 (1996) 51^71. [2] B.L. Hogan, J.M. Yingling, Curr. Opin. Genet. Dev. 8 (1998) 481^ 486. [3] J.A. Whitsett, S.W. Glasser, Biochim. Biophys. Acta 1408 (1998) 303^311. [4] J.J. Coalson, V. Winter, F. Yang, Anat. Rec. 250 (1998) 300^315. [5] K.L. Goss, A.R. Kumar, J.M. Snyder, Am. J. Respir. Cell Mol. Biol. 19 (1998) 613^621. [6] A. Khoor, M.E. Gray, G. Singh, M.T. Stahlman, J. Histochem. Cytochem. 44 (1996) 1429^1438. [7] C.J. Wong, J. Akiyama, L. Allen, S. Hawgood, Pediatr. Res. 39 (1996) 930^937.
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