cDNA-RDA of genes expressed in fetal and adult lungs identifies factors important in development and function

Paul Cooper, Beatrice Mueck, Shida Yousefi, Suzanne Potter, and Gabor Jarai

Novartis Horsham Research Centre, Molecular and Cell Biology Unit, Horsham, RH13 5AB, United Kingdom


    ABSTRACT
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

The identification of genetic factors important in lung development and function will help in understanding the underlying molecular mechanisms of respiratory disease. Representational difference analysis of cDNA (cDNA-RDA) is a PCR-based subtractive enrichment procedure for the isolation of differentially expressed genes. We performed cDNA-RDA and isolated genes expressed more abundantly in fetal and adult lungs. Fifty-four clones potentially representing genes with higher transcript levels in the fetal lung were sequenced. Sequence similarity searches indicated that these clones included 12 known genes, a discoidin-like domain-containing gene, six expressed sequence tags (ESTs), and one novel sequence. Fifty-six clones potentially representing genes expressed more abundantly in the adult lung were also cloned and sequenced. Of these, 16 known human genes were represented along with two sequences significantly similar to known mouse genes and two novel sequences. Several of these known genes are implicated in stress response and lung protection. Thus cDNA-RDA was successfully used to isolate known and novel differentially expressed genes, which putatively play an important role in human lung development.

complementary deoxyribonucleic acid; representational difference analysis; discoidin-like domain; stress response genes; intelectin gene; von Ebner minor salivary gland gene


    INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

THE IDENTIFICATION OF GENES in which expression is correlated with defined developmental, physiological, or pathological processes can provide valuable insight into the molecular mechanisms of these processes. Recently, such studies have become an important area in biomedical research, and increasing efforts are expected to focus on categorizing the expression pattern of transcripts and linking these changes to altering cellular behavior, ultimately contributing to the determination of the respective function of these genes.

The functional elucidation of factors involved in fetal and postnatal development of the lung and their role in the functioning of the adult lung is an important and formidable undertaking. During pre- and postnatal development, the lung develops into one of the most complex organs of the body and is characterized by over 40 different cell types (10). These cells, the exact function of which in many instances is still unknown, play a role in a wide range of processes including mechanisms of gas exchange or host defense. The cellular and morphological complexity and the unique function of this organ in the neonate and the adult make understanding of the mechanisms that govern its function difficult. The identification of genetic factors involved in these processes may help elucidate the underlying mechanisms at the molecular level. Furthermore, the identification of genes involved in pre- and postnatal lung development will not only lead to a better understanding of lung biology but may also provide genetic factors potentially involved in the progression of respiratory diseases. Lung tissue appears to undergo repeated cycles of repair and remodeling in a number of inflammatory and degenerative respiratory diseases such as asthma, chronic obstructive pulmonary disease, and acute respiratory distress syndrome (20, 21, 37, 55), and some of the mechanisms involved in these processes may be the same or similar to the ones that are active during lung development. Indeed, it has been suggested that repair after injury to the adult lung engages some of the same factors that regulate lung development (36).

Representational difference analysis (RDA) is a PCR-based subtractive enrichment procedure. Originally developed for the identification of differences between complex genomes, it has now been adapted to enable the isolation of genes with an altered expression between various tissues or cell samples (cDNA-RDA) (23). This technique offers several advantages over other approaches including the isolation of few false positives and the fact that unwanted difference products can be competitively eliminated, and genes producing rare transcripts that may not be represented in the currently available databases are also detectable. Recently, modifications have been implemented in the cDNA-RDA protocol that allow the use of small amounts (<300 ng) of starting mRNA, making this technique suitable when only limited amounts of tissue are available (12, 43, 64). RDA, therefore, provides a valuable tool for genetic comparison because it is sensitive enough to isolate genes even if they are expressed in only a small percentage of cells in a complex tissue (23). Taking these advantages into account, we used cDNA-RDA to identify genes differentially expressed between adult and fetal lungs.

Here we describe the identification of transcripts expressed more abundantly in fetal lung tissue, several of which are represented by uncharacterized expressed sequence tags (ESTs): one is a novel sequence and one may represent a new discoidin-like domain (DLD)-containing gene. Known genes, several previously shown to be more actively transcribed in the developing fetus (6, 8, 35, 39, 41, 42, 63), were also identified, serving to confirm the success of the experiment. Genes isolated as having increased expression in the adult lung included those involved in stress response and postnatal water balance, two homologs of mouse genes, and two entirely novel sequences. Identification of these transcripts provides genetic factors involved in fetal lung development and adult lung function.


    MATERIALS AND METHODS
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

mRNA and cDNA synthesis. Adult and fetal lung mRNA samples were purchased from Clontech. Total RNA was isolated by a modified guanidium thiocyanate method and subjected to two rounds of oligo(dT)-cellulose selection to obtain poly(A)+ RNA. Adult lung mRNA was derived from a pool of five tissue specimens from Caucasian men and women who had died suddenly at ages 14-40 yr and whose lung tissue was not diseased. The fetal lung mRNA sample was obtained from nine male and female 20- to 25-wk-old Caucasian fetuses who did not have diseased lung tissue and were the results of spontaneous abortions. cDNA was synthesized from 300 ng of mRNA with the Promega Universal Riboclone cDNA Synthesis System and the oligo(dT) primer for first-strand synthesis. Double-stranded cDNA was extracted once with an equal volume of phenol-chloroform-isoamyl alcohol and precipitated with a 0.5 volume of 7.5 M ammonium acetate and 2.5 volumes of ethanol. After centrifugation, the resultant pellet was washed with 70% ethanol and resuspended in 16 µl of sterile water.

Generation of subtracted libraries by cDNA-RDA. RDA for cDNA was performed essentially as described by O'Neill and Sinclair (43), with fast-performance liquid chromatography-purified primers. Briefly, double-stranded cDNA was digested with Dpn II and ligated to R-adaptors (23, 43) (a 24 mer annealed to a 12 mer). Amplicons for both "tester" and "driver" were generated with Expand Long Template PCR System and Expand Buffer 1 (Boehringer Mannheim). Typically, five 100-µl PCRs for each tester and 10 for each driver were performed. Dpn II digestion was used to remove the R-adaptors from both driver and tester amplicons followed by ligation of J-adaptors (23, 43) to the tester. Subtractive hybridizations were performed in 5-µl reactions at 67°C for 20 h in a GeneAmp 2400 (Perkin-Elmer). To generate difference product 1 (DP1), 250 ng of tester cDNA were mixed with 25 µg of driver cDNA at a ratio of 100:1. DP1 cDNA was digested with Dpn II to remove J-adaptors before ligation of N-adaptors (23, 43). To generate difference product 2 (DP2), 31.25 ng of tester were mixed with 25 µg of driver cDNA at a ratio of 800:1. Digested and excess adaptors were removed by washing the cDNA on Microcon 30 filters (Amicon) as recommended by O'Neill and Sinclair (43).

Cloning of difference products. The subtracted library was fractionated by agarose gel electrophoresis. Fractions in 1.5% low-melting-point agarose gels were digested with Agarase (Boehringer Mannheim) at 42°C overnight, washed three times with sterile water, and concentrated with Microcon 30 filters (Amicon). Before ligation, the subtracted PCR cDNA mix was incubated for 20 min with additional dATP and Taq DNA polymerase (Boehringer Mannheim) to ensure that most of the cDNA fragments contained "A overhangs." Approximately 10 ng of cDNA were ligated to 25 ng of pCR2.1 vector (TA Cloning Kit, Invitrogen), and the ligation was introduced into 50 µl of One Shot competent cells (Invitrogen). The libraries were plated onto agar plates containing 50 µg/ml of carbenicillin, 100 µM isopropyl-beta -D-thiogalactopyranoside, and 50 µg/ml of X-Gal. The plates were incubated at 37°C overnight and then briefly at 4°C to allow the blue and/or white staining to be clearly distinguishable. The plasmids were purified from 3-ml cultures of the white colonies with the Wizard Plus Minipreps DNA Purification System from Promega.

DNA sequencing and analysis. Cycle sequencing of 300 ng of plasmid DNA was performed on an automated ABI310 sequencer (Perkin-Elmer) with M13 reverse and forward primers according to the manufacturer's instructions. Sequence similarity searches were performed with the BLAST algorithm (3), and sequence alignments were done with the GCG software package (Wisconsin Package version 9.1). Multiple sequence alignments and phylogenetic tree analysis were performed with the MegAlign software of DNASTAR (LaserGene).

Northern and Southern blot analyses. For Northern blot analyses, a master mix of mRNA from adult or fetal lung was combined with RNA loading buffer (Sigma), and aliquots were loaded for the generation of eight identical 300-ng and two 1-µg blots. Adult and fetal lung mRNAs were electrophoresed in 1% agarose-formaldehyde denaturing gels for 2 h. mRNA was then transferred to Hybond N membrane (Amersham) by capillary transfer with 20× saline-sodium citrate (SSC) and ultraviolet cross-linked to the filter. Southern blots were prepared by electrophoresing a 500-ng amplicon cDNA in 1.5% agarose gels and blotting with capillary transfer. Clone inserts for hybridization analysis were isolated from 1% low-melting-point agarose gels after digestion with EcoR I. Probes were synthesized by random priming 25 ng of DNA in the presence of [alpha -32P]dATP (Amersham) with the Strip-EZ DNA Random Primed Stripable DNA Probe Synthesis and Removal Kit (Ambion). Hybridizations were performed overnight in ExpressHyb hybridization solution (Clontech) at 65°C. Northern blots were washed in 2× SSC-0.05% SDS for 20 min at room temperature followed by two 20-min washes in 0.1× SSC-0.1% SDS at 50°C. For Southern blots, three consecutive 15-min washes were performed at 65°C in solutions containing 0.5× SSC-0.1% SDS followed by 0.2× SSC-0.1% SDS and finally 0.1× SSC-0.1% SDS. Filters were exposed to phosphorimager plates for between 2 h and 5 days and visualized by a STORM 840 PhosphorImager (Molecular Dynamics). A representative Northern blot was hybridized with a glyceraldehyde-3-phosphate dehydrogenase probe (Ambion) to control for loading. Individual blots were stripped and reprobed no more than three times to ensure that no significant reduction of signal intensity occurs.


    RESULTS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

cDNA-RDA with adult and fetal lung mRNAs. cDNA-RDA was performed with the fetal and adult mRNA samples as described in Generation of subtracted libraries by cDNA-RDA. Significantly, both samples were derived from pools of tissue specimens obtained from a mixture of male and female nondiseased Caucasian individuals and hence were representative of normal lung tissue at these respective stages of development. To isolate the genes more actively transcribed in the embryonic lung, we used cDNA from the fetal lung as the tester and cDNA from the adult lung as the driver. Conversely, to identify genes preferentially expressed in adult lung, we used adult lung cDNA as the tester and fetal lung cDNA as the driver. Two rounds of cDNA-RDA were used to generate DP2 in both instances because this has previously been shown to be sufficient to remove unwanted background and to enrich for differentially expressed fragments (43). To confirm that the experiments had been successful in enriching the sequences expressed at a higher abundance in the tester populations, we hybridized each DP2 to Northern blots containing mRNAs from adult and fetal lungs (Fig. 1). More intense hybridization signals were observed in the expected lanes, indicating that the subtractive hybridizations had been successful.


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Fig. 1.   Northern blot analysis of adult (B) and fetal (A) lung difference product 2 (DP2). Northern blots were generated with denaturing agarose gels containing 300 ng/lane of adult and fetal mRNAs and hybridized with radiolabeled DP2 probes as indicated. Positions of RNA molecular-weight markers fragments are indicated. See text for discussion.

Sequence analysis of subtraction products. Difference products for both subtractions were characterized by DNA sequence analysis. After purification by agarose gel electrophoresis, difference products (DP2) was cloned into the pCR2.1 vector and sequenced. Fifty-four clones were isolated from fetal lung DP2 and 56 clones from adult lung DP2. Analysis of these sequences by comparison with the sequences present in the available public databases indicated that 21 different sequences were represented by the 54 fetal lung DP2 clones. Of these 21 sequences, 13 were identical or similar to known genes, 6 were identified as human or mouse ESTs, 1 contained an Alu-repetitive element along with novel sequence, and 1 did not show any similarity to any known sequence (Table 1).

                              
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Table 1.   Summary of fetal lung sequences cloned by cDNA-RDA

Sequence analysis of the 56 clones isolated from the adult lung DP2 indicated that 20 different genes were represented. Of these 20 sequences, 15 were identical to known genes, 2 were similar to mouse sequences, 1 was similar to a Mir-repetitive element, and 2 did not show any similarity to any known sequence (Table 2).

                              
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Table 2.   Summary of sequences identified by cDNA-RDA present in adult lung DP2

Expression analysis of individual transcripts. Because methods based on subtractive enrichment procedures have been shown to yield false positives, the expression pattern of individual clones in adult versus fetal lungs was further verified by Northern blot analyses. The cDNA inserts were isolated from the sequenced clones and used as hybridization probes on blots containing fetal and adult lung mRNAs (Figs. 2 and 3). A representative Northern blot was hybridized with a glyceraldehyde-3-phosphate dehydrogenase probe as a control for equal loading (see MATERIALS AND METHODS). Tables 1 and 2 summarize the results of these hybridization experiments. Multiple transcripts were detected for the hepatoma-derived growth factor gene (Fig. 2) and also for the gene represented by adult DP2 clone 17.2 (Fig. 3). Of the 35 genes in which expression was tested, 16 showed no detectable expression on Northern blots (Tables 1 and 2). This most probably indicates the ability of the RDA technique to identify differentially expressed transcripts present only at low abundance. In summary, of the 19 transcripts detected, 17 clearly showed higher expression as expected, i.e., in the mRNA sample that had been used as a tester in the original subtraction experiment (not all hybridization results are shown). For two clones, those representing glucosidase II (Table 1) and aldehyde dehydrogenase (Table 2), no differential expression could be demonstrated between the two mRNA samples and hence may represent experimental artifacts (results not shown). Using the data for the transcripts that were detected by Northern blot analysis indicates that ~90% of the genes identified by the cDNA-RDA experiments were indeed differentially expressed.


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Fig. 2.   Northern analysis of individual fetal lung clones. Inserts from individual clones isolated from fetal lung DP2 were used as hybridization probes on Northern blots containing adult and fetal lung mRNAs as indicated. Loading of Northern blots was corrected for by hybridizing a glyceraldehyde-3-phosphate dehydrogenase (GAPDH) probe to a representative blot (see MATERIALS AND METHODS). See text for discussion.



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Fig. 3.   Northern analysis of individual adult lung clones. Inserts from individual clones derived from adult lung DP2 were used as hybridization probes on Northern blots containing adult and fetal lung mRNAs as indicated. Loading of Northern blots was corrected for by hybridizing a GAPDH probe to a representative blot (see MATERIALS AND METHODS). HSP70 and HSP40, 40- and 70-kDa heat shock protein, respectively; SLPI, secretory leukocyte protease inhibitor; AQP1, aquaporin-1. See text for discussion.

Furthermore, to obtain evidence that the transcripts that were not detected on Northern blots are also preferentially expressed in one or the other tissue, we performed the following Southern ("virtual Northern") analysis. Inserts from four clones were isolated and used as probes on Southern blots that were prepared with the same unsubtracted representations that were used as the tester or driver during the enrichment procedures (Fig. 4). Consistent with Northern analysis for the genes detected, virtual Northern analysis indicated that the sequences examined were more abundant in the tester population (fetal lung) compared with those in the driver population (adult lung). These data most probably indicate the detection limit of the Northern blot analysis while showing the advantages of PCR-based subtraction approaches for the isolation of low-abundance differentially expressed transcripts that could only be detected on more sensitive virtual Northern blots containing amplified cDNA populations.


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Fig. 4.   Virtual Northern hybridizations with individual clones isolated from fetal lung DP2. Five hundred nanograms of tester and driver representations were run in each lane. Probes used are indicated. See text for discussion.

Sequence analysis of fetal lung clone 35 and adult lung clones 17.2 and 19.2. BLAST searches of the public database with the sequence of clone 35 derived from fetal lung DP2 (Table 1) indicated that it showed similarity to several sequences known to contain DLDs. Significant similarity was identified between our sequence and the DLD containing the protein carboxypeptidase X2 (CPX-2) (68), aortic carboxypeptidase-like protein (31), coagulation factors (24, 25, 58), milk fat globule membrane proteins (53), endothelial cell protein del-1 (19), and neuropilins (18, 29, 56). Bestfit (GCG program) comparison with a conceptual-translated amino acid sequence from clone 35 with that of the most similar protein, mouse CPX-2, indicated that they were 67% similar across the DLD region (results not shown). Figure 5 shows multiple sequence alignment and phylogenetic tree analysis of clone 35 with that of its nearest neighbors.


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Fig. 5.   A: multiple sequence alignment (Clustal) for discoidin-like domain-containing proteins aligned with conceptual translated sequence from clone 35. Mouse sequence of carboxypeptidase X2 (GenBank accession no. AF017639) and human (GenBank accession no. AF053944) and mouse (GenBank accession no. AF053943) sequences of aortic carboxypeptidase-like proteins (ACLPs) are shown. Residues are shown blocked (solid boxes) if they are present in 2 or more sequences. B: phylogenetic relationship between sequences. Length of each pair of branches represents distances between sequence pairs, with units at bottom indicating no. of substitution events. See text for discussion.

Searches of the public databases with the sequences from two adult lung DP2-derived clones, clones 17.2 and 19.2 (Table 2), found no similarity with any known human genes. However, each displayed a high degree of similarity to recently identified mouse transcripts. The insert in clone 19.2 is 81.5% similar to the mouse intelectin gene transcript (30) over a 375-bp-long region, and the conceptual translation of the human cDNA sequence aligned with the mouse protein sequence, indicating that the sequences were 88.8% similar over a region of 107 amino acids (Fig. 6, A and B). The insert in clone 17.2 shows 72.2% similarity over a region of 283 bp to the gene for the von Ebner minor salivary gland protein (Fig. 6C); however, due to the quality of the sequence deposited in the public database, we were unable to meaningfully align the human amino acid sequence with that of the mouse. Figure 6 shows Bestfit (GCG) alignments of these newly cloned human genes with the mouse sequences. These alignments indicate that we have most probably isolated cDNA fragments from the human homologs of both mouse genes.


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Fig. 6.   Sequence alignments for 2 RDA clones that show similarity to mouse genes. A: represesentational difference analysis (RDA) clone 19.2 (top) aligned with nucleotide sequence for Mus musculus gene for intelectin (GenBank accession no. AB016496; bottom). B: conceptual amino acid translation from nucleotide sequence of clone 19.2 (top) aligned with that of Mus musculus intelectin gene product (GenBank accession no. 3357909; bottom). C: nucleotide sequence from RDA clone 17.2 (top) aligned with Mus musculus von Ebner minor salivary gland gene sequence (GenBank accession no. U46068; bottom). Alignments were made using Bestfit program (GCG package). Percent similarities and identities over intervals shown are given. See text for discussion.


    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

The determination of the expression patterns of genes and correlation of this with the changes in cellular phenotype will provide insights into the genetic mechanisms involved in various physiological or pathological processes. The biophysical basis of lung function and biochemical characterization of lung development indicate that both processes are extremely complex and involve the interactions of multiple gene products. Aberrant expression or mutations of these genes may lead to lung function abnormalities and predisposition to disease. RDA provides a valuable tool for the determination of expression changes and is also sensitive enough to isolate genes present in only a small fraction of cells in a complex tissue (12). We have therefore used cDNA-RDA to identify and clone several genes, among them some as yet unknown, that are differentially expressed between adult and fetal lung tissues.

Isolation of the elastin gene from the fetal lung DP2 (Table 1) confirms the success of the subtraction experiment because this gene as well as the genes for other structural proteins have been shown to be expressed more actively in the fetal lung. Elastin is a critical component of the lung interstitium, providing the property of recoil to the vascular, conducting airway and terminal air space compartments of the lung (6, 46). In pathological circumstances such as emphysema or pulmonary fibrosis, elastin gene expression may be reactivated; however, normal elastic fiber assembly does not appear to occur (46). Similarly, collagens have also been reported to be actively synthesized in embryonic and early postnatal development of the lung in mice and humans; however, specific references to alpha 1(III) procollagen expression in this respect have not yet been made (8, 35).

DLDs have been previously identified in the carboxypeptidases of CPX-2 and aortic carboxypeptidase-like protein (31, 68), coagulation factors V and VIII (24, 25, 58), milk fat globule membrane proteins (53), endothelial cell protein del-1 (19), and the A5/neuropilin protein (18, 29, 56). Discoidin is a lectin produced by the slime mold Dictyostelium discoideum and is thought to facilitate cellular aggregation and migration (51). It has been suggested that DLDs may be important for cell-cell recognition or for cell migration mediated through homotypic and heterotypic interactions (31). Sequence comparison suggests that we may have isolated a novel member of the DLD containing proteins. Its expression in the fetal lung might suggest that it plays a role in cellular fate determination. However, further studies are needed to determine its function.

Hepatoma-derived growth factor (Table 1, Fig. 2) is an endothelial and fibroblast mitogen previously shown to be important in embryonic kidney development (39, 41). Our results suggest that it may also play a role in the developing lung and, therefore, may be a more general developmental factor. gamma 2-Adaptin has also previously been shown to have higher expression in the fetal compared with the adult lung (Table 1, Fig. 2) (45). The gene product is involved in the intracellular transport of ligand-receptor complexes and appears to be ubiquitously expressed in most adult and fetal tissues. However, no explanation has so far been suggested for its weak expression in the adult lung (45).

Isolation of the globin gene from fetal lung DP2 (Table 1) most probably highlights the difficulties in obtaining pure tissue samples because the most likely reason for its isolation is contamination of the lung tissue by erythrocytes. These cells are known to have a highly upregulated expression of embryonic globin genes (42, 65). Isolation of the red blood cell anion exchanger gene from the fetal lung DP2 may be due to similar reasons (50).

Because 90% of the genes detected by Northern analysis were differentially expressed, this indicated that the DP2 libraries we had generated were of high quality and indeed subtracted. Virtual Northern hybridization also showed that the clones that were not detected by conventional Northern blotting were present at higher concentrations in the tester compared with those in the driver representation (Fig. 4). In our laboratory, results from other systems have shown that all genes determined to be differentially expressed by virtual Northern analysis were differentially expressed when analyzed by Northern blotting or semiquantitative RT-PCR (data not shown). Hence this evidence suggests that the four genes analyzed on virtual Northern blots are most probably more abundant in fetal RNA but were of too low abundance to be detected by conventional Northern analysis. Similarity searches of the dbEST database with sequences from clones 16, 13, and 38 (Table 1) resulted in matches mainly with EST sequences derived from human or mouse fetal cDNA libraries, providing indirect evidence for these genes having higher expression in fetal lung tissue. Analysis of the expression pattern of these genes in the developing embryo can help determine whether they are important for development in general or are indeed specific for fetal lung generation.

The identification of genes that are more abundantly expressed in the adult compared with the fetal lung will help determine the factors important in adult lung function, protection, and development. Several of the genes identified as having a higher transcript level in the adult lung have been previously linked to postnatal lung biology. PRELP, a 55-kDa matrix protein, has originally been described in adult articular cartilage. Expression analysis in human chondrocytes indicated that message levels increased from the fetus to early adulthood but then decreased with age. Analysis of noncartilaginous tissue revealed that the adult lung had the highest expression levels of all the tissues studied (4). The exact function of the PRELP protein is as yet unknown; however, sequence similarities have been identified with several connective tissue proteins (4, 16).

Aquaporin-1 (AQP-1) is the first characterized member of a protein family that forms water-specific channels involved in reabsorption, osmoregulation, and secretion (Table 2) (2, 5). Studies of fetal rat lung have demonstrated that AQP-1 expression first appears late in gestation, increases before birth, and is sustained at high levels in adult animals. It has been suggested that AQP-1 plays a role in perinatal lung water clearance and also performs a similar function in later life (27). Water balance in the lung is also controlled via Na+ and Cl- transport. The lung amiloride-sensitive Na+ channel, which is involved in this control, is composed of alpha -, beta -, and gamma -subunits (61, 63). Although only the expression of the alpha -subunit has been shown to be increased between the fetal and adult stages, transcription of all three subunits is thought to be regulated by similar mechanisms (7, 48, 60). Because we have shown that the beta -subunit has a higher expression in the adult lung (Table 2), it appears probable that all three subunits are similarly temporally expressed.

The stress response genes GADD34 and 70- and 40-kDa heat shock proteins (HSP70 and HSP40, respectively) were all found to be more abundantly expressed in the adult compared with the fetal lung (Table 2, Fig. 3). The upregulation of these genes may simply reflect the more stressful environment to which the adult lung is exposed. GADD34 transcription is known to be increased by a number of genotoxic agents including ionizing radiation, medium depletion, and contact inhibition (13) and has also been correlated with increased apoptosis (22). HSP70 is a member of a large family of conserved proteins induced by a variety of cell injuries including elevated temperature and exposure to reactive oxygen species and other inflammatory mediators (66). HSP70 has also been suggested to play a role in human lung diseases because increased expression in airway epithelium and macrophages has been observed in asthmatic and acute respiratory distress syndrome patients (26, 59). However, so far, a role for HSP40 in lung biology has not been shown, although it is known to physically interact with HSP70 (14, 54). Selective increased expression of stress proteins has also been suggested as a means to provide protection against acute lung injury in certain diseases (67). Secretory leukocyte protease inhibitor is thought to protect the airways from aberrant neutrophil proteinase activity. The identification of secretory leukocyte protease inhibitor in adult lung DP2 (Table 2, Fig. 3) also illustrates the protective nature of the genes required for adult lung function (28).

The alignment of sequences from clones 17.2 and 19.2 (Table 2) with that of the mouse von Ebner minor salivary gland and intelectin genes, respectively (Fig. 6), indicates that we have most probably isolated cDNA fragments from their respective human homologs. No information is available regarding the expression pattern or function of the protein encoded by the murine von Ebner minor salivary gland gene; however, information regarding the mouse intelectin gene was recently published (30). The intelectin gene was isolated from Paneth cells of the small intestine, which are proposed to be involved in host defense against microorganisms (30). Komiya et al. (30) have therefore suggested that intelectin may play a role in this defense. More work is needed to elucidate both of their roles in lung physiology. Sequences from clones 39.2 and 14.1 (Table 2) did not show similarity with any sequences in the public databases. These sequences are likely to represent two novel genes involved in adult lung function and need to be studied further.

cDNA-RDA is a powerful technique for the isolation of differentially expressed genes, but it also has limitations in that not all of the differentially expressed genes are necessarily enriched during the procedure. The lack of Dpn II restriction sites in the message and preferential amplification of some messages may result in the generation of <100% coverage of expressed genes in the representations. Hence such limitations may explain why genes known to be differentially expressed during lung development, such as surfactant proteins A and B (32, 49), have not been isolated. Although, large-scale cDNA sequencing provides an indispensable resource for gene discovery (1), we have isolated novel sequences not represented by any ESTs in the public databases. Not only does our data indicate the sensitivity and speed of this technique, but it also underlines its complementarity to other gene identification approaches.

The genes we have isolated provide information for the further characterization and understanding of the genetic nature of lung development and function. In the future, this information may help us devise new rational and gene therapeutic approaches able to ameliorate lung injury and augment lung repair in various respiratory diseases.


    ACKNOWLEDGEMENTS

We thank Michael O'Neill and Andrew Sinclair for providing the detailed protocol for cDNA-representational difference analysis from small amounts of starting mRNA and Mike Hubank and David Schatz for providing the original protocol.


    FOOTNOTES

The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. §1734 solely to indicate this fact.

Address for reprint requests and other correspondence: G. Jarai, Novartis Horsham Research Centre, Molecular and Cell Biology, Wimblehurst Road, Horsham, West Sussex RH13 5AB, UK (E-mail: gabor.jarai{at}pharma.novartis.com).

Received 13 May 1999; accepted in final form 27 September 1999.


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ABSTRACT
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MATERIALS AND METHODS
RESULTS
DISCUSSION
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