Characterization of a second human clathrin heavy chain polypeptide gene (CLH-22) from chromosome 22q11
Characterization of a second human clathrin heavy chain polypeptide gene ( CLH-22 ) from chromosome 22q11Darek Kedra1, Myriam Peyrard1, Ingegerd Fransson1, John E. Collins2, Ian Dunham2, Bruce A. Roe3 and Jan P. Dumanski1,*
1Department of Molecular Medicine, Clinical Genetics Unit, Karolinska Hospital, Building L-6, S-17176 Stockholm, Sweden, 2The Sanger Centre, Hinxton Hall, Hinxton, Cambridge CB10 1RQ, UK and 3Department of Chemistry and Biochemistry, University of Oklahoma, 620 Parrington Oval, Norman, OK 73019-0370, USA
Received February 7, 1996; Revised and Accepted February 28, 1996GenBank accession nos. X95486-X95488
We report cloning and characterization of the second human clathrin heavy chain polypeptide gene (CLH-22) localized to chromosome 22q11. Hence H. sapiens is the first species for which two clathrin heavy chain genes have been reported. We provide 5470 bp cDNA sequence covering the entire open reading frame of the CLH-22 gene. The predicted polypeptide is composed of 1640 amino acids. Its 6 kb transcript is expressed in all of 16 tested human tissues, suggesting it is a housekeeping gene. Skeletal muscle, testis and heart show significantly higher expression levels. Compared to the previously characterized human clathrin heavy chain gene localized on chromosome 17 (CLH-17), CLH-22 shows different transcript size and expression profile in human tissues. Northern analysis of CLH-22 suggests that several alternatively spliced transcripts exist. A presumably single, 171 bp long alternatively spliced exon has been characterized. Amino acid sequence comparison between CLH-22 and CLH-17 shows an overall identity and similarity of 84.7 and 91.1%, respectively. At the nucleic acid level, identity between open reading frames of both genes is 74.3%. Sequence comparison with previously cloned genes in other species suggests that counterparts of the CLH-17 gene have been cloned in B. taurus and R. norvegicus, whereas presumptive mammalian homologues of the CLH-22 gene are yet to be characterized. Our Northern and Southern blot analyses of meningiomas clearly suggest the CLH-22 gene may be involved in the tumor development and can be considered as a candidate for a tumor suppressor.
Clathrin-coated vesicles are dynamic intracellular organelles involved in endocytosis. Two main transport pathways mediated via clathrin-coated vesicles are recognized. One directs vesicles from the plasma membrane to the lysosomes and the second transports vesicles from the trans-Golgi network to the lysosomes or to the plasma membrane. Clathrin is a major protein component of these structures and its role is to promote formation of clathrin-coated pits and clathrin-coated vesicles (for review refs 1 ,2 ). Two types of clathrin light chain genes have been described (3 -5 ). To date, only single clathrin heavy chain genes were cloned in R. norvegicus (6 ), S. cerevisiae (7 ), H. sapiens (8 ,9 ), D. discoideum (10 ), D. melanogaster (11 ), and B. taurus (12 ).
We report the cloning and characterization of the second human clathrin heavy chain gene (CLH-22) from chromosome 22q11. Also, in this issue of Human Molecular Genetics (pp. 617-624) another group reports cloning of apparently the same gene (13 ). Here we extend the finding of Sirotkin et al. (13 ) by showing that the gene is alternatively spliced and highly expressed in skeletal muscle, testis and heart. In addition, our Northern and Southern blot analysis of meningiomas clearly suggests that this gene may be involved in the tumorigenesis and can be considered as a candidate for a tumor suppressor.
The gene was cloned using the so called `software-based exon-trapping' approach based on the availability of genomic sequence combined with use of exon prediction computer programs. In the course of transcriptional characterization of several regions on human chromosome 22, we selected numerous cosmid clones for large scale genomic sequencing. Among these, cosmid E75F1 mapping to the vicinity of the telomere of 22q was partially sequenced. At present, it consists of 13 sequence contigs ranging from 1 to 11 kb spanning over 48 kbp. Partial sequence of E75F1 was used for exon prediction (using the grail program) (14 ), as well as for database searches. One of the contigs, number 464 (1116 bp, Fig. 1 , A2), which could not be joined or related to the rest of the sequencing project, produced one 147 bp presumptive exon, scored as `excellent' and with high similarity at the protein level to members of clathrin heavy chain (CLH) polypeptides (data not shown). Further FastA searches in the EMBL/GenBank database using contig 464, filtered by the PYTHIA repetitive DNA sequence server, showed 74.4% identity with rat CLH (accession no. J03583), 72.7% with randomly sequenced human cDNA corresponding to CLH from chromosome 17 (accession no. D21260) and 72.5% identity with partial human CLH (accession no. X55878), respectively. A PCR-derived probe, called 75L (374 bp), encompassing the presumptive 147 bp exon was generated and used for screening of a human foetal brain cDNA library (Fig. 1 , A2). One clone was found after screening of ~600 000 phage clones (termed cfb4, Fig. 1 B). The DNA sequence of its 4.5 kbp insert was determined using a combined shot-gun and primer walking strategy. Inserts of 2 shot-gun sequencing subclones, one from the 5' and one from the 3' end of cfb4 were used as probes for further full-length cDNA screening. Two skeletal muscle cDNA libraries (adult and foetal) were chosen for these experiments, based on results from Northern blot analysis using clone cfb4 (Fig. 2 and below). Our cDNA screening results are summarized in Figure 1 B. Twenty-four cDNA clones were isolated and sequenced at least using vector-derived primers on both sides flanking the inserts. A 5470 bp of cDNA sequence covering the entire open reading frame of the gene, capable of encoding 1640 amino acids was obtained. Our sequence encompasses the whole 3' untranslated part of the transcript and ends with a polyA tail. Based on the results from Northern blot analysis (Fig. 3 and below), the estimated CLH-22 transcript size is 6 kb. Thus, ~500-600 bp of the 5' untranslated part can be estimated to be missing. This cDNA has been sequenced at least once on both strands throughout its 5470 bp. The cDNA sequencing project consists of 270 independent gel readings generated on the ABI (Applied Biosystems, Inc.) 373A sequencer, and reached an average redundancy of over 10 gel characters per consensus character.
We have earlier reported the [beta]-adaptin gene (BAM22) localized in 22q12 as a candidate for meningioma tumor suppressor. This gene has been cloned from a small homozygous tumor deletion and lost its expression in 12% of sporadic meningiomas studied on Northern blots (16 ). Furthermore, results from detailed deletion mapping of chromosome 22 in tumors failed to identify any single minimal overlapping region deleted in tumors, pointing to whole 22q as a candidate area harboring putative tumor suppressor gene(s) (17 ,18 ). Considering that CLH-22 may be a component in the same pathways of intracellular protein transport and signal transduction, as the BAM22 gene, we analysed expression of CLH-22 in 46 sporadic meningiomas on Northern blots prepared with total cellular RNA isolated from the tumors. Expression of CLH-22 could only be detected in nine cases (Fig. 4 ). Thirty-seven tumors did not reveal any detectable transcript, suggesting loss of gene expression in 80% of the tumors. Interestingly, 37 meningiomas without the CLH-22 gene expression belonged to both main categories of tumors, with and without large deletions on one copy of chromosome 22. Among the 37 tumors with loss of CLH-22 gene expression, five cases did not display any deletions of chromosome 22, while three out of nine tumors with CLH-22 gene expression were without any detectable loss of heterozygosity on chromosome 22. The amount and quality of RNA present on the Northern blots used in the above described experiments were tested with several control genes, prior to and after the experiments with CLH-22: the Ewing's sarcoma gene (EWS) (19 ); the anonymous gene pK1.3 from 22q12 (16 ); the [beta]'-adaptin gene (BAM22) (16 ); the glyceraldehyde-3-phosphate dehydrogenase gene, (GAPDH) (16 ); and the retinoblastoma gene (RB1) (20 ).
We report here molecular cloning of a novel, human clathrin heavy chain gene, which we localized to chromosome 22q11. The fact that two clathrin heavy chain genes exist at all, is an interesting and unexpected finding, and H. sapiens is so far the first species in which this has been shown. Clathrin heavy and light chain polypeptides together form three-armed structures called triskelions. It was believed that clathrin heavy chain polypeptides are uniform components of all clathrin coated vesicles (1 ,2 ). This unexpected diversity at the level of clathrin heavy chains raises the question as to what degree these two polypeptides are functionally interchangeable. Comparison of the amino acid sequences from CLH-17 and CLH-22 (Fig. 2 ), shows that their overall amino acid similarity is high. It has previously been shown that two clathrin light chain genes exist in several species (3 -5 ). It may be hypothesized that two different clathrin heavy chains participate in formation of distinct clathrin triskelions and which have partially or completely different functions in the intracellular receptor sorting and transport. Our findings of alternatively spliced forms of the CLH-22 gene are also new, as no alternative splicing has been shown to date for any other clathrin heavy chain genes. It will be important to characterize the entire genomic exon-intron structure of the gene, in parallel with a more detailed delineation of different alternatively spliced forms of CLH-22 in many tissues. Assuming that these splice variants reflect differences in protein function, they may be informative in helping to further understand the role of different domains of the protein. The strong variation in expression levels of the CLH-22 gene in human tissues is also surprising, especially when viewed in context of clearly uniform expression of the CLH-17 gene in the same set of human tissues studied on the same Northern blots (Fig. 3 ). The high expression level in testis is unlikely to be unspecific, as no elevated levels of the CLH-17 gene expression was noted in the same tissue (Fig. 3 ). Disruptions of clathrin heavy chain genes have been carried out in for instance D. melanogaster, and suggest that the clathrin heavy chain gene is essential in this organism. While most mutations of this gene in D. melanogaster are lethal, one allele has been described, which although not lethal, caused sterility in males (11 ). The CLH-22 gene may therefore be important in an as yet not fully characterized process which is important for normal function of testis.
Clathrin triskelions interact with adaptor complexes, which is one of the main events during formation of the clathrin coated pits and vesicles. Two different adaptor complexes have been described; trans Golgi network adaptors and plasma membrane adaptors. Both adaptors are heterotetrameric protein complexes composed of [gamma]- plus [beta]'- and [alpha]- plus [beta]-adaptins, respectively, as well as two additional smaller proteins. Because [beta]- and [beta]'-adaptins are the most homologous components of the adaptors it has been proposed that their main function is to interact with clathrin triskelions (2 ). It has also been shown that monomeric [beta]-adaptins promote clathrin assembly in vitro (21 ). Therefore, a documented functional link exists between clathrin and [beta]-adaptins. We have previously characterized a human [beta]'-adaptin gene from chromosome 22q12 (BAM22) and shown that this gene may be viewed as a candidate tumor suppressor gene involved in the development of meningioma (16 ). In this context it should be emphasized that the CHL-22 gene also shows that it may be important in the tumor development and it is located within the region heterozygously deleted in up to 65% of meningiomas (18 ). Our results call therefore for a full-scale screening of this gene for point mutations in meningiomas and in other malignancies which show specific deletions of chromosome 22. This analysis should be performed from tumor DNA as the starting material, since we have observed a frequent loss of the CLH-22 gene expression in meningiomas. This in turn, will necessitate characterization of the exon-intron borders for the entire CLH-22 gene. The large scale genomic sequencing of the CLH-22 gene region is well advanced (Z. Wang, G. Zhang, F. Chen, B. A. Roe, B. Emanuel and M. Budarf, personal communication). Our results from the CLH-22 gene expression on Northern blots with meningiomas also indicate that the CLH-22 promoter region should be studied for mutations affecting the elements controlling gene expression and a possible change of its methylation status.
The following human cDNA libraries from Stratagene were used: fetal brain, polyT+ random primed, #936206; adult muscle, polyT primed, #937209; fetal muscle, polyT+ random primed, #836201. Approximately 600-800 000 plaques were plated for each screen (16 ). In vivo excision of the cDNA clones were performed according to the recommendations of the supplier. Filters were hybridized and washed using stringent conditions (0.1* SSC, 0.1% SDS, 65oC) (22 ). Northern blot analysis was performed as previously described (16 ,17 ). Two PCR-derived probes were designed for the CLH-17 gene, based on the sequence deposited in the databases (accession no. D21260) and amplified from the human spleen and placenta cDNA libraries using the following two pairs of primers: C17AU, TAA GAT GGA AGG AAA TGC; C17AL, TTA GCC CTT AGG TAC ACA; and C17BU, TGC TGA GAA ATG TAA TGC, C17BL, TCA GGA TCG TAG CAG TTG.
Cosmid DNA was prepared using Qiagen columns (QIAGEN, Inc.). Plasmid DNA was mini-prepared through alkaline lysis (22 ). Plasmid inserts DNA and cosmid DNA was mechanically sheared using a nebulizer (IPI Medical Products, USA no. 4207) (23 ). DNA fragments between 800-1500 bp were shot-gun cloned into pUC18 vector (Pharmacia). We used fluorescent end-labeled universal primers, or Taq Dye Deoxy Terminators (ABI). The sequences were assembled using the Staden program package (24 ). Gaps in the shot-gun sequences were closed by primer walking strategy. Putative exonic sequences were predicted using the grail program (14 ). Database searches and sequence comparisons were done using FastA and gap programs from Wisconsin Package, Genetics Computer Group, WI, USA. Repetitive sequences were filtered out using Pythia server (25 ).
We would like to thank Dr Thomas Sejersen for the human foetal skeletal muscle cDNA library and Dr Hans Mehlin for valuable help in computer-assisted DNA sequence analysis. This study was supported by the Swedish Cancer Society, the Swedish Medical Research Council, the Cancer Society in Stockholm, the National Center for Human Genome Research, the Magnus Bergvall Foundation, and the Markus Borgström's Fond.
ABI, Applied Biosystems Inc.; BAM22, [beta]'-adaptin gene from 22q12; CLH-17, clathrin heavy chain polypeptide gene from human chromosome 17; CLH-22, clathrin heavy chain polypeptide gene from human chromosome 22.
1 Kreis, T.E. (1992) Regulation of vesicular and tubular membrane traffic of the Golgi complex by coat proteins. Curr. Opinion Cell Biol., 4, 609-615.MEDLINE Abstract
2 Robinson, M.S. (1994) The role of clathrin, adaptors and dynamin in endocytosis. Curr. Opinion Cell Biol., 6, 538-544.MEDLINE Abstract
3 Kirchhausen, T., Scarmato, P., Harrison, S.C., Monroe, J.J., Chow, E.P., Mattaliano, R.J., Ramachandran, K.L., Smart, J.E., Ahn, A.H. and Brosius, J. (1987) Clathrin light chains LCA and LCB are similar, polymorphic, and share repeated heptad motifs. Science, 236, 320-324.MEDLINE Abstract
4 Jackson, A.P., Seow, H.F., Holmes, N., Drickamer, K. and Parham, P. (1987) Clathrin light chains contain brain-specific insertion sequences and a region of homology with intermediate filaments. Nature, 326, 154-159.MEDLINE Abstract
5 Jackson, A.P. and Parham, P. (1988) Structure of human clathrin light chains. Conservation of light chain polymorphism in three mammalian species. J. Biol. Chem., 263, 16688-16695.MEDLINE Abstract
6 Kirchhausen, T., Harrison, S.C., Chow, E.P., Mattaliano, R.J., Ramachandran, K.L., Smart, J. and Brosius, J. (1987) Clathrin heavy chain: molecular cloning and complete primary structure. Proc. Natl Acad. Sci. USA, 84, 8805-8809.MEDLINE Abstract
7 Lemmon, S.K., Pellicena-Palle, A., Conley, K. and Freund, C.L. (1991) Sequence of the clathrin heavy chain from Saccharomyces cerevisiae and requirement of the COOH terminus for clathrin function. J. Cell. Biol., 112, 65-80.MEDLINE Abstract
8 Dodge, G.R., Kovalszky, I., McBride, W., Yi, H.F., Chu, M.L., Saitta, B., Stokes, D.G. and Iozzo, R.V. (1991) Human clathrin heavy chain (CTLC): partial molecular cloning, expression and mapping of the gene to human chromosome 17q11-qter. Genomics, 11, 174-178.MEDLINE Abstract
9 Nomura, N., Miyajima, N., Sazuka, T., Tanaka, A., Kawarabayashi, Y., Sato, S., Nagase, T., Seki, N., Ishikawa, K. and Tabata, S. (1994) Prediction of the coding sequences of unidentified human genes. I. The coding sequences of 40 new genes (KIAA0001-KIAA0040) deduced by analysis of randomly sampled cDNA clones from human immature myeloid cell line KG-1. DNA Res, 1, 27-35.MEDLINE Abstract
10 O'Halloran, T.J. and Anderson, R.G. (1992) Characterization of the clathrin heavy chain from Dictyostelium discoideum. DNA Cell Biol., 11, 321-30.MEDLINE Abstract
11 Bazinet, C., Katzen, A.L., Morgan, M., Mahowald, A.P. and Lemmon, S.K. (1993) The Drosophila clathrin heavy chain gene: clathrin function is essential in a multicellular organism. Genetics, 134, 1119-1134.MEDLINE Abstract
12 Liu, S.H., Wong, M.L., Craik, C.S. and Brodsky, F.M. (1995) Regulation of clathrin assembly and trimerization defined using recombinant triskelion hubs. Cell, 83, 257-267.MEDLINE Abstract
13 Sirotkin, H., Morrow, B., DasGupta, R., Goldberg, R., Patanjali, S.R., Shi, G., Cannizarro, L., Shprintzen, R., Weissman, S.M. and Kucherlapati, R. (1996) Isolation of a clathrin heavy chain gene with muscle-specific expession from the region commonly deleted in Velo-Cardio-Facial syndrome. Hum. Mol. Genet, 5, 617-624.MEDLINE Abstract
14 Uberbacher, E.C. and Mural, R.J. (1991) Locating protein-coding regions in human DNA sequences by a multiple sensor-neural networ approach. Proc. Natl Acad. Sci. USA, 88, 11261-11265.MEDLINE Abstract
15 Collins, J.E., Cole, C.G., Smink, L.J., Garrett, C.L., Leversha, M.A., Soderlund, C.A., Maslen, G.L., Everett, L.A., Rice, K.M., Coffey, A.J., Gregory, S., Gwilliam, R., Dunham, A., Davies, A.F., Hassock, S., Todd, C.M., Peyrard, M., Xie, Y.-G., Lehrach, H., Hulsebos, T.J.M., Weissenbach, J., Morrow, B., Kucherlapati, R.S., Wadey, R., Scambler, P.J., Kim, U.-J., Simon, M.I., Carter, N.P., Durbin, R., Dumanski, J.P., Bentley, D.R. and Dunham, I. (1995) A high resolution integrated yeast artificial chromosome clone map of human chromosome 22. Nature, 377, 367-379.MEDLINE Abstract
16 Peyrard, M., Fransson, I., Xie, Y.-G., Han, F.-Y., Ruttledge, M.H., Swahn, S., Collins, J.E., Dunham, I., Collins, V.P. and Dumanski, J.P. (1994) Characterization of a new member of the human [beta]-adaptin gene family from chromosome 22q12, a candidate meningioma gene. Hum. Mol. Genet., 3, 1393-1399.MEDLINE Abstract
17 Dumanski, J.P., Carlbom, E., Collins, V.P. and Nordenskjöld, M. (1987) Deletion mapping of a locus on human chromosome 22 involved in the oncogenesis of meningioma. Proc. Natl Acad. Sci. USA, 84, 9275-9279.MEDLINE Abstract
18 Ruttledge, M.H., Xie, Y.-G., Han, F.-Y., Peyrard, M., Collins, V.P., Nordenskjöld, M. and Dumanski, J.P. (1994) Deletions on chromosome 22 in sporadic meningioma. Genes, Chromosomes Cancer, 10, 122-130.MEDLINE Abstract
19 Delattre, O., Zuckman, J., Plougastel, B., Desmaze, C., Melot, T., Peter, M., Kovar, H., Joubert, I., de Jong, P., Rouleau, G., Aurias, A. and Thomas, G. (1992) Gene fusion with an ETS DNA-binding domain caused by chromosome translocation in human tumours. Nature, 359, 162-165.MEDLINE Abstract
20 Friend, S.H., Bernards, R., Rogelj, S., Weinberg, R.A., Rapaport, J.M., Albert, D.M. and Dryja, T.P. (1986) A human DNA segment with properties of the gene that predisposes to retinoblastoma and osteosarcoma. Nature, 323, 643-646.MEDLINE Abstract
21 Gallusser, A. and Kirchhausen, T. (1993) The [beta]1 and [beta]2 subunits of the AP complexes are the clathrin coat assembly components. EMBO J., 12, 5237-5244.MEDLINE Abstract
22 Sambrook, J., Fritsch, E.F. and Maniatis, T. (1989) Molecular cloning; a laboratory manual. Cold Spring Harbor, New York
23 Pan, H.-Q., Wang, Y.-P., Chissoe, S.L., Bodenteich, A., Wang, Z., Iyer, K., Clifton, S.W., Crabtree, J.S. and Roe, B.A. (1994) The complete nucleotide sequences of the SacBII Kan domain of the P1 pAD10-SacBII cloning vector and three cosmid cloning vectors: pTCF, svPHEP, and Lawrist16. GATA, 11, 181-186.
24 Staden, R. (1994) The Staden package. In Methods in molecular biology, A. M. Griffin and H. G. Griffin. Humana Press Inc: Totawa, NJ, pp. 9-170.MEDLINE Abstract
25 Milosavljevic, A. (1996) Handbook of Genome Analysis, Repeat Analysis, Chapter 13, Section 4.
*To whom correspondence should be addressed
This page is maintained by OUP admin. Last updated Thu Oct 31 15:23:49 GMT 1996. Part of the OUP Journals World Wide Web service.Copyright Oxford University Press, 1996
