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Isolation, in silico characterization and chromosomal localization of a group of cDNAs from ciliated epithelial cells after in vitro ciliogenesis
Genome Biologyvolume 2, Article number: research0026.1 (2001)
Immotile cilia syndrome (ICS) or primary ciliary dyskinesia (PCD) is an autosomal recessive disorder in humans in which the beating of cilia and sperm flagella is impaired. Ciliated epithelial cell linings are present in many tissues. To understand ciliary assembly and motility, it is important to isolate those genes involved in the process.
Total RNA was isolated from cultured ciliated nasal epithelial cells after in vitro ciliogenesis and expressed sequenced tags (ESTs) were generated. The functions and locations of 63 of these ESTs were derived by BLAST from two public databases. These ESTs are grouped into various classes. One group has high homology not only with the mitochondrial genome but also with one or more chromosomal DNAs, suggesting that very similar genes, or genes with very similar domains, are expressed from both mitochondrial and nuclear DNA. A second class comprises genes with complete homology with part of a known gene, suggesting that they are the same genes. A third group has partial homology with domains of known genes. A fourth group, constituting 33% of the ESTs characterized, has no significant homology with any gene or EST in the database.
We have shown that sufficient information about the location of ESTs could be derived electronically from the recently completed human genome sequences. This strategy of EST localization should be significantly useful for mapping and identification of new genes in the forthcoming human genome sequences with the vast number of ESTs in the dbEST database.
Immotile cilia syndrome (ICS) or primary ciliary dyskinesia (PCD) is a human autosomal recessive disorder with a frequency of 1 in 20,000. Patients with PCD have recurrent respiratory tract infections, bronchiectasis and often male sterility. About 50% of patients have situs inversus and hence a Kartagener syndrome. These patients show abnormalities in the beating of cilia in ciliated epithelial cells and of flagella of spermatozoa. Electron microscopic ultrastructural study of cilia and spermatozoa of patients show that this disease is extremely heterogenous [1,2]. Ciliated epithelial cell linings are present in the upper airways of the respiratory tract, sinuses, middle ear, efferent duct of testis, Fallopian tubes, brain and spinal cord. Embryonic heart contains nodal cilia that produce a directional movement and it has been shown in mice that failure of the movement of these nodal cilia causes breakdown of left-right asymmetry . Cilia and flagella are complex structures and ciliary assembly alone requires more than 250 different proteins . Upper airway epithelial cells are also important for studying cystic fibrosis and asthma, and are often cultured in vitro for drug testing for asthma and related diseases. The identification of genes expressed in these cells may be helpful in characterizing genes involved in such diseases.
Upper airway epithelial cells have not been used previously for isolation of ESTs. We cultured ciliated epithelial cells starting from a patient's nasal biopsy, and after in vitro degeneration and regeneration of cilia, total RNA was isolated from these cells. A catalog of the function and chromosomal location of the expressed sequence tags (ESTs) generated from the RNA was deduced by BLAST searches of the public databases (GenBank, normal and HTGS). This implies that comprehensive information about gene functions and chromosomal locations of ESTs could be derived from these databases.
Results and discussion
We have isolated a group of ESTs from ciliated epithelial cells after in vitro ciliogenesis starting from a patient's nasal biopsy. The probable functional significance of these ESTs and their chromosomal locations are derived from published databases. For homology searches, two databases were considered. The first was a normal database which gives the identity of the sequence with respect to the other transcribed sequences from all organisms. The second was a 'high throughput genome sequences' (HTGS) database, which was used to determine the genomic clones that are homologous to these transcribed sequences. According to the known position of the sequenced clone, ESTs are placed in between the two closest markers in the chromosome (see, for example Table 1). These transcribed short sequences are divided into four subgroups according to their homology with the database.
ESTs with homology with mitochondrial DNA
A number of the nasal epithelial ESTs show very high homology with sequences from the human mitochondrial genome (Table 1), implying that these sequences are derived from mitochondrial DNA. It is surprising, however, that most of these ESTs are not only homologous with the mitochondrial genome but also with chromosomal DNA, and that the same region and extent of homology with mitochondrial and genomic DNA is observed in many cases. Moreover, an individual EST can have very high homology with a HTGS genomic clone from more than one chromosome (see 34-18, 36-62, 5R22 and 36-100 in Table 1). ESTs 34-18 and 9694, for example, have homology with the mitochondrial urf4 gene and with the same genomic clone in chromosome 5. Multiple alignment of ESTs 34-18 and 9694 suggests that these are different sequences and from different regions of the genomic clone.
It is possible that families of very similar genes (or of genes with similar domains) are expressed from mitochondrial and nuclear genomic DNA or that a massive amount of domain fusion has occurred between mitochondrial and nuclear genes. Only one chimeric urf4 cDNA (fused mitochondrial and nuclear DNA) has previously been recovered experimentally from a viral integration, over a decade ago . On the basis of the high homology of each EST with both mitochondrial and nuclear DNA, we suggest that nuclear and mitochondrial domain fusion is not an isolated phenomenon, but is rather common. This remains to be rigorously investigated.
ESTs with complete homology with known genes
A second group of ESTs are completely homologous with known genes in the human genome (Table 2). We assume that these are either the same gene as their genomic counterpart or a gene containing the same domain. From this evidence it is interesting to note that a number of important genes whose functions are known are also expressed in ciliated epithelial cells, although the significance of this expression is unknown. Although further rigorous experiments are needed to characterize these genes in ciliated epithelium, the probable functions of some important genes are discussed below.
Cytohesin (EST 14-49) is involved in signal transduction pathways and regulates cell adhesion . Expression of this gene may play an important role in the adhesion of epithelial cells during the expansion of the cell layer.
Cyclophilin C (EST 24-51). In response to endotoxin, mice deficient in cyclophilin-associated protein overproduce interleukin-12 and interferon-gamma systemically and tumor necrosis factor-alpha locally. These are proinflammatory molecules that also promote helper T-cell responses . The role of this gene in ciliated epithelial cells remains elusive, however, and an important concern for further investigation.
Epidermal growth factor receptor (EGFR) kinase (EST 6034). EGFR is an important ligand-binding protein and its level is elevated in many tumors . The EGFR gene is a potential oncogene and expression of EGFR kinase may be required during formation of epidermis by epithelial cells.
The Drosophila Staufen genes (EST 6092) are RNA-binding proteins important for RNA transport and localization in the oocyte and neurons in Drosophila. The motor protein dynein (ddlc1) in conjunction with Staufen and Swallow acts as an adaptor for transporting bicoid RNA along microtubules to their minus ends at the anterior pole of the oocyte [9,10]. As dynein genes are highly expressed in ciliated epithelial cells  a potential interaction of dynein with the human Staufen homolog could be deduced.
Decay-accelerating factor (DAF, CD55) (EST 9661) protects host cells from the activation of autologous complement on their surfaces. It functions to disable the C3 convertases, the central amplification enzymes of the complement cascade . Expression of this gene in nasal epithelial cells could be explained by the need for protection against antigen-induced complement activation.
ESTs with partial homology to domains of known genes
A number of ESTs (Table 3) are partially homologous (that is in part of the EST sequence) to domains of known genes and could be of interest. However, further investigation of complete cDNAs and their functions may reveal the true identity of these genes. A few ESTs which are partly homologous with the domains of important genes are discussed below.
EST 24-17 (H3 pseudogene). A stretch of 21 base pairs (bp) of this EST has homology with the pseudogene of histone H3 but not with the normal histone H3 gene. Histone H3 is an important housekeeping protein involved in chromatin packing . It is possible that another H3 RNA is transcribed, which may be different from both the normal H3 gene and the pseudogene.
EST 36-5 (retinoic acid responder). As retinoic acid plays an important role in in vitro ciliogenesis, expression of the retinoic acid responder domain is not unexpected in ciliated epithelial cells, where it may modulate a number of dynein heavy-chain genes during ciliogenesis .
EST 9010 (Attractin precursor). The protein attractin is secreted by activated T cells and has also been detected in the central nervous system . It is suggested to be involved in immunity, obesity and pigmentation . The mouse mahogany mutation is caused by a mutation in the attractin gene . A portion (192 bp out of 194 bp) of EST 9010 has a very high similarity or identity with the attractin gene, suggesting that this EST is derived from a gene containing an attractin precursor domain. Expression of such a gene in ciliated epithelial cells is of unknown significance.
EST 906 (MDM2). MDM2 is an oncogene and the MDM2 oncoprotein binds to the p53 protein, inhibiting p53's function as a transcription factor and inducing its degradation. An MDM2-p53 autoregulatory feedback loop regulates the function of the p53 tumor suppressor gene . The significance of the expression of MDM2 or of a gene carrying an MDM2 domain in ciliated epithelial cells remains to be investigated.
EST 907 (Mkp5 dual-specificity phosphatase). Mkp5 is a member of the mitogen-activated kinase (MAP kinase) family (10 genes in total) and has an important role in apoptosis, tumor progression and immune responses . MAP kinases contain a docking motif that increases the efficiency of the reaction . Part of EST 907 (52 bp out of 391 bp) has high similarity with the docking motif, possibly implying that another member of the MAP family could exist.
EST 9667 (transcription regulator protein; HCNGP). This transcriptional regulator has been isolated from adrenal gland (C Jiang, J Shi, C Huang, S Ren, Y Li, J Zhou, Y Yu, S Xu, Y Wang, G Fu, et al., unpublished data; GenBank accession number AF119664). A part of EST 9667 (86 bp out of 87 bp) is highly similar to the HCNGP; expression of this gene in ciliated epithelial cells is of unknown significance.
EST C2s-3 (translocase). Protein translocation across the cytoplasmic membrane has not been studied extensively in mammalian cells. It is also not known how many genes are involved in this process . It is interesting that this EST shows homology (34 bp out of 38 bp) with a translocase gene and may carry out a translocase-like function.
EST 9640 (Sid3177). Sid3177 is a unique gene isolated from the mouse and assumed to be a part of the inactive progesterone receptor complex (N Seki, A Hattori, A Hayashi, S Kozuma, M Muramatsu, T Saito, unpublished data; GenBank accession number AB024935). The high homology of EST 9640 with a domain of Sid3177 suggests that a similar gene(s) is present in humans and is expressed in ciliated epithelial cells.
ESTs 3R1-32, 5r29, 5R5 (genomic scaffold-binding protein). Parts of these ESTs have high homology with the Drosophila scaffold-binding protein . Scaffold-binding proteins are important in the replication and segregation of chromosomes. However, human counterparts of the complete genes have yet to be isolated.
EST 9014 (asthmatic clone). Part of this EST has homology with a cDNA isolated from asthma patients and that appears to be expressed differentially in asthmatics (IC Kilty, PJ Vickers, unpublished data; GenBank accession number AF095856). Expression of such a gene in the upper airway epithelium is of important in the context of identifying genes responsible for asthma.
ESTs with no significant homology to sequences in the database
Twenty ESTs (33%) have no significant homology (Table 4) to sequences in normal databases. The identities of these genes are not known even after 2 million sequences have been accumulated in the dbEST database. BLAST searches against the HTGS database, however, reveals that most of them are highly homologous with known genomic clones. These ESTs have been mapped electronically and their chromosomal locations derived. In recent years, there has been an exponential rise in the number of sequences available in the public databases. Despite this, a high percentage of partial sequences of cDNAs (ESTs) submitted to the databases remain unrecognized (anonymous ESTs). This lack of similarity could be explained  in several ways. One explanation is that a different part of the transcript is present in GenBank; second, the transcript represents a novel gene not yet isolated; third, there is alternative splicing of the same gene in different species; fourth, inaccurate sequence data; and/or fifth, the sequence of the transcript has diverged to an extent that it is not recognized as an ortholog.
We have isolated a group of cDNAs that are expressed in ciliated epithelial cells in the upper airway of the human respiratory tract. These short cDNAs may be extremely helpful for isolating and characterizing the complete genes and for studying their expression pattern in the human body. We also noted that a number of ESTs are highly homologous to genes that are involved in cancers and immune reaction pathways. Expression of these genes in ciliated epithelial cells in the upper respiratory tract is of unknown significance. In addition, mapping these genes may be helpful for retrieving and characterizing complete genes. Subsequently, it may help in cloning those disease genes by the positional candidate gene approach. A number of the ESTs can be mapped electronically from the human genome sequence (HTGS database) and their probable function could be derived from the normal database. This shows that a large number of ESTs in the dbEST database could be mapped electronically by BLAST and a comprehensive EST map could be generated that may be helpful for characterizing a large number of genes in the human genome.
Materials and methods
Human epithelial cell culture
Human nasal epithelial cells were enzymatically dissociated from biopsies using a protease type XIV (Pronase) digestion overnight at 4°C. Pronase was inactivated by adding fetal calf serum (FCS) or NU-Serum (10% final concentration) and the cell suspension was washed three times in culture medium (Ham's F12- DME 1/1 with cholera toxin (10 ng/ml), streptomycin (50 μg/ml), penicillin (50 IU/ml) and 2% Ultroser G). The cell suspension was preplated on plastic for 1 h at 37°C to remove most of the contaminating fibroblasts [24,25]. Cells were plated in T75 tissue culture flasks on 0.2% collagen gel for monolayer culture and kept at 37°C at 5% CO2 atmosphere in a biological oxygen demand (BOD) incubator. Culture medium was changed three times a week. After three weeks of exponential growth the cultures reach confluence.
Cells were then released from the collagen gel using 200 IU/ml collagenase type IV. Cell clusters, aggregates and cell sheets were washed three times in culture medium to eliminate collagenase and then placed in culture medium at 37°C on a gyratory shaker at 80 rpm to avoid attachment of the cells to the culture flask. During the first week, the medium (the same as used earlier) was changed every day. On the second day, the 2% Ultroser G was replaced by 10% NU-serum [24,25]. After 1 week stable aggregates, spheroids and vesicles were formed and showed no tendency to adhere to the culture flask; the culture was kept stationary for another few weeks. Generally, cilia appear in 2 weeks and cells were used to isolate total cellular RNA after the third or fourth week.
Primer design, RT-PCR amplification, cloning and sequencing of clones
We used degenerate primers for the reverse transcription and PCR amplification (RT-PCR). Primers are initially designed  to clone the dynein heavy-chain genes from ciliated epithelial cells and are taken from the P-loop region (ATP hydrolysis region) of dynein heavy-chain genes. Reverse transcription was done with primers 4, 6 and 3R. PCR amplifications were done in round-robin fashion with primers 1 and 4, 2 and 4, 3 and 4, 1 and 6, 2 and 6, 3 and 6, 1 and 3R, 2 and 3R, 3 and 3R, and so on. Primers  used in these studies were 1, 5'-TAY GGN TTY GAR TAY YTN GG-3'; 2, 5'-GTN CRR ACN CCN YTN ACN GA-3'; 3, 5'-ACN GGN AAR ACN GAR ACN AC-3'; 4, 5'-CCN GGR TTC ATN GTD ATR AA-3'; 6, 5'-CKN ARN CCR AAR TCR TAR TG-3', 3R, 5'-GTN GTY TCN GTY TTN CCN GT-3'.
Total RNA was isolated from the cultured cells by the method described in [26,27]. RT-PCR was carried out with GeneAmp RT-PCR Kit (Perkin Elmer). Each sample of RNA was routinely treated with DNase I for 6 h at 37°C to remove any genomic DNA contamination. Two micrograms of RNA were reverse transcribed by the downstream primer in 20 μl at 42°C and PCR amplified withthe addition of upstream primer in a 100 μl volume. In all cases, PCR conditions were: for denaturation, 94°C, 4 min; for amplification, 94°C for 1 min; 50°C for 1 min; 72°C for 1 min for 40 cycles; and for elongation, 72°C for 10 min. RNA without reverse transcriptase and water without RNA (plus reverse transcriptase) did not yield any product in any of the PCR reactions.
PCR products were cloned in PCR2.1 vector of the TA-cloning Kit (InVitrogen) and were subjected to blue/white selection. White colonies were checked by PCR for the presence of insert with the vector-specific primers (M13 forward and reverse). Approximately 400 clones were sequenced on an ABI377 Automated Fluorescence Sequencer (Perkin Elmer). Sequences were screened with BLAST for the identity of these clones. Along with the cloning of nine dynein heavy-chain genes , a number of non-dynein cDNAs were recovered which were studied in detail. From 400 clones, 63 were selected as unique by the following procedures. The dynein heavy-chain genes (82 clones) were ignored; only one sequence was selected when two or more clones containing similar sequences were obtained; very small sized clones (below 70 bases) were ignored. Only in one case was a 56 bp clone (5R16) selected, as this sequence was not obtained repeatedly.
Electronic mapping of ESTs and derivation of their probable function from database searching
ESTs were BLASTed against the normal database (in NCBI BLAST page normal database (GenBank) designated as 'nr') and highest similarities with the known genes were taken into account. In cases of homologies with more than one gene, only the gene with the highest homology (number of base pairs, highest similarity and identity) was taken as the homologous gene and functional characterization has been done on the basis of the function of the known gene.
EST sequences were also BLASTed against the HTGS databases and the accession number and chromosome number of the highest-similarity clone were noted. Each clone was searched by accession number in Locus Link  in 'GENEBANK MAP' and the clone carrying flanking markers in a particular chromosome was assigned in 'STS MAP'. The distance from P-tel of the chromosome was taken as the map position of the EST.
All ESTs are deposited in databases under the dbEST accession numbers 8451921 to 8451980 and 8452140 to 8452142 and the Genbank accession numbers BG673720 to BG673779 and BG687691 to BG687693.
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Locus Link. [http://www.ncbi.nlm.nih.gov/LocusLink/]
We thank Tom Willems and Sandra Deckker for helping with the cell culture. A.K.M. is supported by a postdoctoral fellowship by a grant from the European Union (BIOMED2). This work was supported by a grant from the European Union (Biomed2, BMH4 CT96-0302).
About this article
- Epidermal Growth Factor Receptor
- Epidermal Growth Factor Receptor Gene
- Primary Ciliary Dyskinesia
- Normal Database
- Nasal Epithelial Cell