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Human Outer Dense Fiber Gene, ODF2, Localizes to Chromosome 9Q34

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Human Outer Dense Fiber Gene, ODF2, Localizes to Chromosome 9Q34 Cytogenet Cell Genet 83:221–223 (1998) Human outer dense fiber gene, ODF2, localizes to chromosome 9q34 X. Shao,a S. Murthy,a,b D.J. Demetricka,b and F.A. van der Hoorna a Department of Biochemistry and Molecular Biology, and b Department of Pathology, University of Calgary, Calgary, Alberta (Canada) Abstract. We have isolated the human homolog of the rat sperm axoneme. We compared homology and genomic struc- Odf2 gene. In rat, Odf2, the 84-kDa major outer dense fiber ture to rat and mouse Odf2 genes. Using fluorescence in situ protein, interacts strongly and specifically with Odf1, the 27- hybridization, we mapped the human Odf2 gene (ODF2) to kDa major outer dense fiber protein. The interaction is me- chromosome 9q34. diated by leucine zippers during ODF assembly along the The mammalian spermatozoon contains characteristic cy- ODF components. Until recently only one ODF gene, ODF1, toskeletal structures associated with the central axoneme: nine which encodes the major 27-kDa ODF protein, had been outer dense fibers (ODF) extend taperingly throughout the cloned (van der Hoorn et al., 1990; Burfeind and Hoyer-Fen- midpiece and terminate in the principal piece while the fibrous der, 1991; Morales et al., 1994). Human ODF1 localizes to sheath (FS) replaces two of the ODF and surrounds the remain- chromosome 8q22 (Gastmann et al., 1993). ing ODF in the principal piece (Fawcett, 1975). The polypep- Recently, we cloned several testis-specific proteins which tide composition of ODF in rats has been known to contain six interact strongly and specifically with Odf1 by using the N- major polypeptides as well as several minor polypeptides, terminus of Odf1, which includes the leucine zipper motif many of which are highly insoluble (Olson and Sammons, (Shao and van der Hoorn, 1996), as a bait in a yeast genetic 1980; Vera et al., 1984; Oko and Clermont, 1988). These pro- screen (Shao et al., 1997). One of the novel genes characterized teins are produced exclusively in spermatids and assembled in encodes the major 84-kDa ODF protein, Odf2 (previously a proximal-to-distal manner along the axoneme (Oko and Cler- called Odf84) (Shao et al., 1997). We determined that a second mont, 1989). The structural integrity of ODF as well as the FS novel gene, SPAG4, encoding a spermatid-specific protein, is believed to be associated with sperm motility and male fertil- which also interacts strongly with Odf1 (Shao et al., manuscript ity. However, little is known about the relationship between the in preparation), localizes to human chromosome 20q11.2 (Tar- genetic defects of the ODF genes and abnormal ODF morpho- nasky et al., 1998). In the present study, we isolated a human genesis underlying human male infertility. This is largely due to genomic clone of Odf2 and used it as a probe to map human the uncharacterized biochemical properties of the individual ODF2 to chromosome 9q34 by fluorescence in situ hybridiza- tion (FISH). Supported by grants to F.A.v.d.H. from the Medical Research Council of Canada and Materials and methods to D.J.D. from the Canadian Breast Cancer Research Initiative. X.S. was sup- ported by a studentship from the Alberta Heritage Foundation for Medical Isolation of human ODF2 Research. D.J.D. is a Medical Research Council of Canada Clinician-scientist. To isolate human genomic clones of ODF2, the rat Odf2 cDNA was used Received 9 October 1998; manuscript accepted 18 November 1998. as a probe to screen a human genomic library (Stratagene), of which the DNA Request reprints from Dr. Frans A. van der Hoorn, Department of Biochemistry and was prepared from Caucasian male placenta and cloned into the XhoI site of Molecular Biology, University of Calgary Health Sciences Center, lambda FIX_II vector under stringent conditions. Insert sizes range from 9 to 3330 Hospital Drive N.W., Calgary, Alberta, T2N 4NI (Canada); 22 kb. Approximately 1 × 106 pfu were screened and three positive genomic telephone: 403-220-3323; fax: 403-283-8727; e-mail: [email protected] clones were isolated using standard techniques (Sambrook et al., 1989). The E-mail [email protected] © 1997 S. Karger AG, Basel Accessible online at: ABC Fax + 41 61 306 12 34 0301–0171/98/0834–0221$17.50/0 http://BioMedNet.com/karger http://www.karger.com Fig. 1. Nucleotide sequence of a human ODF2 exon. The nucleotide sequence of one exon of human ODF2 is compared to the corresponding rat Odf2 cDNA sequence. The 5) and 3) exon-intron boundaries are indicated. genomic DNA was isolated from these clones individually and characterized by restriction mapping and Southern blot hybridization and selected frag- ments that hybridized to rat Odf2 cDNA probes were sequenced to define exon-intron boundaries. A Fluorescence in situ hybridization FISH was performed using previously established methods on metho- trexate-thymidine synchronized, phytohemagglutinin stimulated, normal pe- ripheral blood lymphocytes (Demetrick, 1995). Suppression for 30 min with a mixture of sonicated human DNA (Sigma) and cot-1 DNA (Gibco/BRL) was required to reduce the background. The stained slides were counter- stained with DAPI and actinomycin D (for a DA-DAPI banding pattern) and B were mounted in antifade medium and visualized utilizing a Zeiss Axioplan 2 microscope. Approximately 30 metaphase spreads were examined for Fig. 2. Localization of ODF2 on human metaphase chromosomes. probe location. Images of representative mitoses were captured using a (A) Metaphase spread with specific dual-chromatid staining of a Cy3-labeled cooled CCD camera (Photometrics PXL1400). Digital alignment of the genomic ODF2 probe (red) to 9q34 on DAPI/AD (white-blue) stained nor- images from each fluor was done after registration calibration through a tri- mal human chromosomes. (B) Several enlarged pairs of chromosome 9 from ple bandpass filter (FITC/Texas Red/DAPI) to minimize registration error, different metaphases showing consistent localization of the ODF2 probe utilizing commercial software (Electronic Photography v 1.3, Biological (red) to normal DAPI/AD stained human chromosomes. Detection Inc., Pittsburgh PA). Results and discussion Human genomic clones were isolated from a lambda FIX_II clearly show localization of the probe to 9q34. At least one spe- human genomic library using rat Odf2 cDNA as a probe. cific probe signal was present in more than 90% of the mitoses Genomic DNA prepared from each positive clone was ana- examined. Approximately 80% of the spreads showed labeling lyzed by restriction mapping and Southern blot hybridization. of two chromatids of a single chromosome and more than half One of these clones, called hgÏ1, was further analyzed. A 1.3-kb of these showed specific labeling of both chromatids of both PstI fragment of hgÏ1 DNA, which can be hybridized to rat chromosomes. More than 90% of these signals were localized to Odf2 cDNA, was subcloned and sequenced to determine the a single band, 9q34. exon-intron boundaries. Figure 1 shows the sequence of the The location of ODF2 on human chromosome 9q34, to exon and exon-intron boundaries in comparison to the corre- which area a number of genes have been mapped, is not linked sponding rat Odf2 cDNA sequence. The human and rat Odf2 to any known testis-specific gene. Human ODF1 has been exons are 90% identical at the nucleotide level and the amino localized to chromosome 8q22 (Gastmann et al., 1993), we acid sequence encoded by this exon is 100% identical. The 5) recently mapped SPAG4, encoding an Odf1 interacting protein and 3) splice junction sequences of the human ODF2 clone (Shao et al., manuscript in preparation), to human chromo- match 5) and 3) splice site consensus sequences. Interestingly, some 20q11.2 (Tarnasky et al., 1998) and acrosin, an acrosomal the genomic organization of this exon is identical to that of the protease, was localized to 22q13→qter (Vazquez-Levin et al., mouse gene (Shao and van der Hoorn, unpublished data), indi- 1992). It appears that genes which function in spermiogenesis cating evolutionary conservation of the genomic organization are dispersed throughout the genome. Odf2, which has two leu- of this exon between mouse, rat and human. cine zippers, interacts strongly and specifically with Odf1 via To determine the chromosome location of ODF2, a 20-kb its upstream leucine zipper during ODF assembly in elongating insert of human genomic clone hgÏ1 was labeled with digoxige- spermatids, and the second leucine zipper may interact with nin-dUTP and hybridized to synchronized human lymphocyte other ODF or sperm tail structural proteins (Shao et al., 1997). metaphase spreads as detailed above (Fig. 2). These results Thus Odf2 protein is probably a crucial player in the organiza- 222 Cytogenet Cell Genet 83:221–223 (1998) tion of ODF morphogenesis. Interestingly, we recently demon- and Spag4 proteins will allow us to investigate and diagnose strated that Odf2 protein is also detectable in the sperm con- human male infertility syndromes, which are a consequence of necting piece (Schalles et al., 1998), a structure derived from genetic defects in the ODF1, SPAG4 and ODF2 genes. The the centriole which is involved in aster formation after fertiliza- phenotype associated with such genetic defects likely includes tion of the egg (Long et al., 1997). Our observations are directly abnormal sperm tail function and/or motility, as well as inabili- relevant to the investigation of human male infertility. Male ty to fertilize eggs. infertility contributes to 50% of couples unable to conceive children: sperm structural abnormalities are a large fraction of these cases, but so far cytogenetic analyses of infertile males can Acknowledgements only detect gross chromosome abnormalities (Yoshida et al., We thank the Faculty of Medicine, University of Calgary for an Estab- 1997). Therefore, the chromosomal localization of ODF2 in lishment Grant for the FISH equipment.
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