Isolation, in silico characterization and chromosomal localization of a group of cDNAs from ciliated epithelial cells after in vitro ciliogenesis

Abstract

Background

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.

Results

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.

Conclusions

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.

Background

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 [3]. Cilia and flagella are complex structures and ciliary assembly alone requires more than 250 different proteins [4]. 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.

Table 1.

Homology with mitochondrial DNA

Clone names	Homology to normal	Homologous	Homology to	Chromo-	Homologous	In between	Distance
	database (identity),	region, extent	human clone	some	region, extent	markers	from P-tel
	accession number	of similarity	(HTGS)		of similarity		(kb)
		(bp)*, identity	accession		(bp)*, identity
		(bp/bp)†	number		(bp/bp)†
34-18, 356 bp	L00016.1 Hs urf4	24-336, 313/313	AC021965	5	35-333, 271/299	D5S2400 &	124831
	gene, mitochondrial					SHGC-141614
			AC008670	5	24-145, 111/123	D5S2056 & AA007G12	82616
			AC012363	2	80-246, 136/137	stSG60109 &	30855
						RH120618
36-62, 100 bp	NC_001807.2 Hs	13-87, 74/76	AL160031	13	18-87, 70/71	D13S1223 & DD13s943	96955
	mitochondrion
			AC008670	5	30-77, 45/49	D5S2408 & EE8-Sp6	96746
5R22, 244 bp	NC_001807.2 Hs	19-224, 206/206	AC021965	5	19-217, 169/199	D5S2400 &	124831
	mitochondrion					SHGC-141614
			AC016571	5	13-269, 253/260	RH122707 & stSG3646	206852
36-105, 70 bp	NC_001807.2Hs	15-51, 31/36	NSH	NA	NA	NA	NA
	mitochondrion
24-16, 215 bp	NC_001807 Hs	1-196, 195/196	AC021965	5	1-196, 178/196	D5S2400 &	124831
	mitochondrion					SHGC-141614
			AC018806	5	1-167, 151/167	D5S409 & H119908	90813
5R16, 56 bp	NC_001807 Hs	20-37, 18/18	AC055801	2	19-35, 16/16	RH112539 &	110180
	mitochondrion					D2S2002
			AC010290	5	37-53,17/17	G-7042470 &	171125
						GDB:187625
			AP000686 1	11	37-53,17/17	RH79933 & DD11S3991	176404
9625, 135 bp	NC_001807.2 Hs	16-64, 48/49	AC068619	17	35-114, 66/80	NMF	124848
	mitochondrion
9639, 179 bp	NC_001807.2 Hs	13-169, 137/149	AC025380	X	55-140, 72/86	stSG9218 &	78184
	mitochondrion					HSSTS293
6085, 111 bp	NC_001807 Hs	31-100, 69/70	AL359496	1	31-100, 68/70	NMF	NA
	mitochondrion
36-16, 105 bp	NC_001807 Hs	24-82, 57/59	AC008670	5	27-82, 51/56	D5S2056 &	82616
	mitochondrion					A007G12
36-100, 342 bp	NC_001807 Hs	36-175,140/140	AC024033	12	36-175, 140/140	stSG62230 &	73482
	mitochondrion	172-320, 146/150			172-320, 146/150	RH47270
			AC010270	5	36-175,140/140	D5S2408 & EE8-Sp6	96746
					172-320, 146/150
36-34, 72 bp	NC_001807 Hs	18-55, 37/38	AC021914	11	21-52, 30/32	sTSG4656 &	26100
	mitochondrion					sts-N93614
36-36, 87 bp	AF134583 Hs	19-67, 49/49	AL049739	6	26-46, 21/21	S78653 &	39448
	mitochondrial					stSG46623
	DNA-like
9694, 150 bp	L00016.1 Hs urf4	19-132, 114/114	AC021965	5	19-121, 95/103	D5S2400 &	124831
	gene, mitochondrial					SHGC-141614
34-47, 103 bp	NC_001807.2 Hs	16-82, 67/67	AC022223	5	16-82, 67/67	RH12239 &	95113
	mitochondrion					WI-18379

^*Extent of similarity, the number corresponds to the starting and ending base pair in the respective homologous gene with the EST. †Identity, the number corresponds to the identical base pair of EST/homologous gene. NA, not applicable; Hs, Homo sapiens; Dm, Drosophila melanogaster; Dr, Drosophila radiodurans. NSH, no significant homology (identity less than 20 bases); NMF, no matches found.

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 [5]. 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.

Table 2.

Complete homologies with known genes

Clone names	Homology to	Homologous	Homology to	Chromo-	Homologous	In between	Distance
	normal database	region, extent	human clone	some	region, extent	markers	from P-tel
	(identity),	of similarity	(HTGS)		of similarity		(kb)
	accession	(bp)*, identity	accession		(bp)*, identity
	number	(bp/bp)†	number		(bp/bp)†
14-49, 336 bp	NM_004228.2 Hs	22-317, 296/296	AC073131	19	110-225,116/116	D19S902 &	68477
	coiled/coil domain 2				223-317, 95/95	sTSg58178
	(cytohesin-2)
24-51, 234 bp	NM_000943.1 Hs	33-220, 186/186	AC012424,	5	99-220,121/122	RH101603 &	153095
	peptidylprolyl					RH103740
	isomerase C
	cyclophilin
3s-1, 121 bp	NM_006870.2 Hs	20-109, 90/90	Al132765,	20	20-109, 90/90	WI-22195 &	25762
	(actin depolymerizing					RH123144
	factor)
6034, 82 bp	NM_004447.1 Hs	18-66, 49/49	NSH	NA	NA	NA	NA
	epidermal growth
	factor receptor
	kinase
6086, 115 bp	M27024 Hs	19-115, 91/97	AL133223.3	14	19-115, 91/97	H14a433 &	119103
	heat shock protein					D14S305
			AC024731	11	21-117, 90/97	NMF	NA
6092, 71 bp	NM_004602.1 Hs	29-71, 41/43	AC068845	19	29-71, 41/43	NMF	NA
	staufen(STAU)
	(Dm RNA-binding
	protein)
9661, 257 bp	M31516.1 Hs	17-236, 218/220	AL355527	1	17-236, 218/220	NMF	NA
	decay-accelerating
	factor mRNA
968, 104 bp	AF203815 Hs	23-87, 63/65	AP000769	11	23-87, 63/65	NMF	NA
	alpha gene sequence

^*Extent of similarity, the number corresponds to the starting and ending base pair in the respective homologous gene with the EST. †Identity, the number corresponds to the identical base pair of EST/homologous gene. NA, not applicable; Hs, Homo sapiens; Dm, Drosophila melanogaster; Dr, Drosophila radiodurans. NSH, no significant homology (identity less than 20 bases); NMF, no matches found.

Cytohesin (EST 14-49) is involved in signal transduction pathways and regulates cell adhesion [6]. 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 [7]. 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 [8]. 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 [11] 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 [12]. 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.

Table 3.

Partial homologies with known genes

Clone	Homology with	Homologous	Homology with	Chromo-	Homologous	In between	Distance
names	normal database	region, extent	human clone	some	region, extent	marker	from
	(identity),	of similarity	(HTGS),		of similarity		P-tel
	accession	(bp)*, identity	accession		(bp)*, identity		(kb)
	number	(bp/bp)†	number		(bp/bp)†
24-17, 389 bp	AL137022 Histone	125-145, 21/21	NSH	NA	NA	NA	NA
	(H3) pseudogene
24-4, 323 bp	AF078904 Zeta	84-105 bp, 22/22	AC058816	6	1-135,121/122	stSN21216 and	7798
	globin gene					D6S1442
26-1, 138 bp	AE003672 Dm genome	56-78, 22/23	AC022237	15	1-20, 20/20	NMF	NA
	scaffold binding protein
3R1-2f, 429 bp	AE001826 Dr R1	6-32, 26/27	NSH	NA	NA	NA	NA
	megaplasmid MP1
36-5, 225 bp	NM_002888.1Hs	52-190, 136/139	AC025033	3	51-190,137/140	stSG53537 and	197037
	retinoic acid receptor					R95445
	responder
36-50, 104 bp	NM_014666.1| Hs	32-84, 48/53	AC026407	5	32-84, 48/53	D5S1853 and	123950
	KIAA0171 gene product					RH101108
36-98, 195 bp	AF068299	170-193, 24/25	AC069530	3	164-182 19/19	NMF	NA
	A. thaliana gamma
	glutamylcysteine
	synthetase gene
3R1-32, 182 bp	AE003819 Dm	102-121, 20/20	NSH	NA	NA	NA	NA
	genomic scaffold
5R29, 506 bp	AE003568 Dm	465-491,26/27	NSH	NA	NA	NA	NA
	genomic scaffold
5R5, 83 bp	AE003650 Dm	22-42, 21/21	AC027364	6	19-66, 48/48	RH112849 &	6789
	genomic scaffold					stSG47852
			AC024731	11	21-117, 90/97	NMF	NA
9010, 440 bp	AF218906.1 Hs	215-408,192/194	AC015847	17	215-408,192/194	NMF	NA
	attractin precursor
	(ATRN)
9014, 396 bp	AF095856 Hs	373-394, 22/22	AL133245.2	2	109-358,247/250	stSG60109 and	36134
	asthmatic clone 4 mRNA					RH120618
			AC010968	2	109-358,247/250	stSG22421 and	30222
						RH120618
905, 485 bp	X73004.1Hs EWS gene	173-415,237/239	AF121897	21	176-415,238/240	NMF	NA
906, 130 bp	AF144028.1Hs	19-72, 54/54	AC019009	14	19-67, 49/49	NMF	NA
	MDM2 gene
			AC022305	15	19-67, 49/49	NMF	NA
907, 391 bp	AB026436 Hs for	12-64, 52/53	AL049696.9	6	92-371,279/280	D6S1762 and	88638
	dual specificity					D6S1856
	phosphatase MKP5
9640, 271 bp	AB024935 Mus	106-200, 89/96	AC073620	12	13-249, 237/237	RH44840 and	9861
	musculus Sid3177 mRNA					RH83752
9646, 249 bp	NM_014928.1 Hs	17-124, 107/108	AC006207	12	17-235, 216/219	B568G1/T7 and	1911
	KIAA1046 protein					D12S2049
9667, 262 bp	AF119664 Hs	130-216, 86/87	AC019214	17	1-179, 177/179	D17S609 and	87186
	Transcriptional regulator					D17S1769
	protein HCNGP mRNA
c2s-3, 348 bp	AF207550 Hs	49-86, 34/38	AC019099	Y	4-286, 252/254	DYS215 and	21795
	protein translocase					DYS197

^*Extent of similarity, the starting and ending base pair in the respective homologous gene with the EST. †Identity, the number corresponds to the identical base pair of EST/homologous gene. number of base pair identical in EST with homologous gene. NA, not applicable. Hs, Homo sapiens; Dm, Drosophila melanogaster; Dr, Drosophila radiodurans. NSH, no significant homology (identity less than 20 bases); NMF, no matches found.

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 [13]. 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 [14].

EST 9010 (Attractin precursor). The protein attractin is secreted by activated T cells and has also been detected in the central nervous system [15]. It is suggested to be involved in immunity, obesity and pigmentation [16]. The mouse mahogany mutation is caused by a mutation in the attractin gene [17]. 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 [18]. 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 [19]. MAP kinases contain a docking motif that increases the efficiency of the reaction [20]. 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 [21]. 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 [22]. 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 [23] 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.

Table 4.

No significant homology in the normal database

Clone names	Homology to	Homologous	Homology to	Chromo-	Homologous	In between	Distance
	normal database	region, extent	human clone	some	region, extent	markers	from
	(identity),	of similarity (bp)*,	(HTGS)		of similarity (bp)*,		P-tel
	accession	identity (bp/bp)†	accession		identity (bp/bp)†		(kb)
	number		number
123-12, 324 bp	NSH	NA	NSH	NA	NA	NA	NA
24-16n, 401 bp	NSH	NA	AC015927	9	4-390, 378/395	NMF	NA
24-37, 170 bp	NSH	NA	NSH	NA	NA	NA	NA
26-16, 226 bp	NSH	NA	NSH	NA	NA	NA	NA
26-6, 280 bp	NSH	NA	NSH	NA	NA	NA	NA
26-6bis, 188 bp	NSH	NA	AC009086	16	30-146,114/117	NMF	NA
34-2, 175 bp	NSH	NA	NSH	NA	NA	NA	NA
36-39, 72 bp	NSH	NA	AC008670	5	30-77, 45/49	D5S2056 and 1007G12	82630
3R1-4F, 278 bp	NSH	NA	NSH	NA	NA	NA	NA
3s-16, 56 bp	NSH	NA	NSH	NA	NA	NA	NA
5r9, 310 bp	NSH	NA	AC009554	15	22-114, 91-93	WI-14756 and D15S553	65614
					113-268,155/156
6054, 285 bp	NSH	NA	NSH	NA	NA	NA	NA
904, 200 bp	NSH	NA	AC003072	22	18-124, 106/107	sts-M27288, stdJ2478f24	27298
9678, 156 bp	NSH	NA	AC009127	16	18-139,119/122	D16S2969 and D16s2943	68750
9682, 282 bp	NSH	NA	AC023885	5	1-179, 178/179	D5S1982 and RH118984	85497
9685, 420 bp	NSH	NA	AL359997	9	1-420, 415/420	stSU25414 and stSH67867	55819
3R1-6f, 328 bp	NSH	NA	NSH	NA	NA	NA	NA
14s-19,169 bp	NSH	NA	NSH	NA	NA	NA	NA
26-12,120 bp	NSH	NA	NSH	NA	NA	NA	NA
3R1-2f, 429 bp	NSH	NA	NSH	NA	NA	NA	NA
36-79, 164 bp	NSH	NA	NSH	NA	NA	NA	NA

^*Extent of similarity, the number corresponds to the starting and ending base pair in the respective homologous gene with the EST. ^†Identity, the number corresponds to the identical base pair of EST/homologous gene. NA, not applicable; Hs, Homo sapiens; Dm, Drosophila melanogaster; Dr, Drosophila radiodurans. NSH, no significant homology (identity less than 20 bases); NMF, no matches found.

Conclusions

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 [11] 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 [11] 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 [11], 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 [28] 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.

Accession numbers

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.