SHORT COMMUNICATION Assignment of the Human Homologue of the Mouse t-Complex Gene TCTE3 to Human Chromosome 6q27 G. A. RAPPOLD,“,’ J. TROWSDALE,t AND P. LICHTER# */nstitut
fiir Humangenetik, University of Heidelberg, Heidelberg, Germany; tlmperial Cancer Research fund, Lincoln’s London WC 2A 3PX, Great Britain; and SDeutsches Krebsforschungszentrum, Heidelberg, Germany Received
October
28, 1991;
A gene with male germ cell-specific expression from the t-complex on mouse chromosome 17, TCTE3 (probe 117~; symbol D17Leh117c), has been previously cloned and characterized (8). The significance of the testis-specific expression of TCTE3 being present in greatest abundance in later spermatogenic stages is further enhanced by its genetic mapping to an -~-CM subregion in the middle part of the t-complex called In(17)3, which has been shown to contain the gene for the distorter/ sterility gene Ted-3 (1, 6). The homologues of several t-complex genes have by now been mapped in human, and all that have been mapped to date are located on human chromosome 6 (9). To determine the chromosome location of human sequences related to TCTE3 and to further define the syntenic relationship existing between the t-complex genes and their human counterparts, somatic cell hybrid mapwere carried out. Using ping and in situ hybridization the mouse 117~ gene sequence as a probe, a filter with genomic DNA samples from total mouse and total human DNAs and somatic cell hybrid lines carrying portions of chromosome 6 was hybridized (see Fig. 1). Under normal stringency conditions, 117~ hybridized to human DNA (lane 1) and to DNA from cell line MCP-6, which carries a portion of chromosome 6 (6p21.3-qter), but not to DNA from cell line 56-47, which carries the at the Institut Neuenheimer
February
24, 1992
6p21.1-pter portion of human chromosome 6. These results indicate that human sequences related to TCTES do indeed map to human chromosome 6, specifically within the 6pl2-qter region. To determine this location more precisely, a human X clone was isolated from a genomic library constructed in XEMBLS (a), using the mouse cDNA 117~ as a probe. This X clone, termed h117, was labeled with biotin by nick-translation and hybridized to metaphase spreads of human chromosomes under conditions that suppress signals from repetitive DNA sequences as previously described (4). Detection via FITC-conjugated avidin revealed a highly specific hybridization signal in the telomerit region of chromosome 6q27 (see Fig. 2). Of 35 metaphases evaluated, 31 showed the signal on both
The gene TCTE3 from the mouse t-complex region is expressed specifically in testicular germ cells. It maps in the central subregion of the t-complex on mouse chromosome 17 containing loci involved in transmission ratio distortion and male sterility. In this study, somatic cell hybrid lines have been used to map the human homologue, TCTE3, to the long arm of chromosome 6. CISS hybridization with the human X clone h117 refined this chromosome assignment to the very distal position of chromosome 6q27, thus providing further evidence that loci from the t-complex of mouse chromosome 17 can map to opposite arms of human chromosome 6. 0 1992 Academic Press, Inc.
1 TO whom correspondence should be addressed Humangenetik der Universitlt Heidelberg, Im 328, 6900 Heidelberg, Germany.
revised
Inn Fields,
fur Feld
1337
-23.1
-4.4
-2.3 -2.0 FIG. 1. 117~ restriction patterns observed in DNA prepared from somatic cell hybrid lines. (1) Total human DNA, (2) total mouse DNA, (3) MCP-6, (4) 56-47. Both MCP-6 (3) and 56-47 (7) contain portions of human chromosome 6. DNA samples were cleaved with EcoRI, electrophoresed in an 0.8% agarose gel, transferred to a nylon membrane, and hybridized to the 0.7-kb 32P-labeled 117~ probe (8). The filter was washed in 6X SSC at 65°C and exposed for 7 days. An -8-kb EcoRI fragment could not be mapped on the hybrids because of a mouse band at a similar position. The size of the human-specific EcoRI fragment present on lanes 1 and 3 is -3.5 kb (indicated by an arrow). GENOMICS
13,
1337-1339 (19%) 0888-7543/92 $5.00
Copyright Q 1992 by Academic Press, Inc. All rights of reproduction in any form reserved.
1338
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COMMUNICATION
FIG. 2. Mapping of X clone h117 (insert size 15 kb) by in situ hybridization to human metaphase chromosomes. Biotinylated probe DNA was hybridized to metaphase chromosomes from stimulated lymphocytes of a normal male individual under conditions as described previously (4). The hybridized probe was detected via avidin-conjugated FITC, and chromosomes were banded with DAPI. (A) Specific hybridization signals in the telomeric region of chromosome 6q. (B) The same metaphase section as in A depicting the two chromosome 6 homologues delineated by cohybridization with digoxigenin-labeled DNA of library pBS6 followed by detection with anti-digoxigenin antibody conjugated to rhodamine. (C) Signal on a DAPI-banded elongated chromosome 6 showing the localization of clone h117 to 6q27. Note that band 6q27 is negatively “stained” by DAPI. Images of FITC-, DAPI-, and rhodamine were taken separately by using a cooled charged coupled device camera system (Photometrics) and overlaid electronically using the software NIH image 1.33. A and C are overlays of DAPI and FITC images, and B is an overlay of a DAPII and rhodamine image. FITC signals are depicted by arrows.
chromosome homologues and no other signals were found. Chromosome identification and banding assignment were confirmed by cohybridization with DNA library pBS6 (kindly provided by Joe Gray, San Francisco), derived from sorted human chromosome 6 (see Fig. 2B), and Alu-enriched human DNA to achieve an R-banding-like AL-banding (data not shown) as described previously (4, 5). The localization on the distal end of the long arm of chromosome 6 places the human homologue of TCTE3 in close vicinity to human homologues of t-complex genes such as Tcp-1, Sod-2, and the human TCPlO gene family (homologous to Tcp-10 in mouse 17) (see reviews of Spence et al. (9) and Artzt et al. (1)). In contrast, the human homologues of the MHC and a number of closely linked mouse loci (including Pim-1, Glo-1, and Pgk-2) have been mapped to the short arm of chromosome 6 (6pll-~21) (see Spence et al. (9)). This suggests a syntenic relationship between the proximal and middle portions (In(17)2-In(17)3) of the t-complex of mouse chromosome 17 and 6q and between the distal portion of the t-complex and 6p (1). It is now clear that the major features of the t-complex, e.g., male-specific transmission distortion and crossover suppression, cannot be generalized to man. Therefore, mouse t-complex as a functional, genetic unit most probably does not have a human equivalent. However, the homology of human chromosomes 6p and 6q (separated by nonhomologous DNA) with mouse chromosome 17 is well established and human homologues of genes associated with t-complex developmental lethal
and sterility mutations are likely to exist. An analysis of the segregation of chromosome 6 alleles with familial cases of male infertility may therefore reveal a subset of male sterilities in humans that could be the result of mutations in these testis-expressed genes. ACKNOWLEDGMENTS We thank Denise Barlow and Lisa Stubbs for careful reading of the manuscript. This work was supported by grants from the Deutsche Forschungsgemeinschaft and by the Verein zur Fiirderung der Krebsforschung in Deutschland.
REFERENCES 1.
2.
3.
4.
5.
Artzt, K., Barlow, D., Dove, W., Fischer Lindahl, K., Klein, J., Lyon, M. F., and Silver, L. M. (1991). Maps of mouse chromosome 17: First report. Mammalian Genome 1: 5-29. Frischauf, A-M. (1987). Construction and characterization of a genomic library in X. In “Methods in Enzymology” (S. L. Berger and A. R. Kimmel, Eds.), Vol. 152, pp. 190-199. Academic Press, San Diego. Goodfellow, P. N., Banting, G., Trowsdale, J., Chambers, S., and Solomon, E. (1982). Introduction of a human X-6 translocation chromosome into a mouse teratocarcinoma: Investigation of control of HLA-A, B, C expression. Proc. Natl. Acad. Sci. USA 79: 1190-1194. Lichter, P., Tang, C-J. C., Call, K., Hermanson, G., Evans, G. A., Housman, D., and Ward, D. C. (1990). High-resolution mapping of human chromosome 11 by in situ hybridization with cosmid clones. Science 2: 64-69. Lichter, P., Boyle, A. L., Cremer, T., and Ward, D. C. (1991). Analysis of genes and chromosomes by non-isotopic in situ hybridization. Genet. Anal. Technol. Appl. 8: 24-35.
SHORT 6. 7.
COMMUNICATION
1339
Lyon, M. F. (1986). Male sterility of the mouse t-complex is due to homozygosity of the distorter genes. Cell 44: 357-363. Nagarajan, L., Lonie, E., Tsujimoto, Y., Ar-Rushdi, A., Huebner, K., and Croce, C. M. (1986). Localization of the human pim on-
8.
Rappold, G. A., Stubbs, L., Labeit, S., Crkvenjakow, R. B., and Lehrach, H. (1987). Identification of a testis-specific gene from the mouse t-complex next to a CpG-rich island. EMBO J. 6: 1975-1980.
cogene (PIM) tions in acute
9.
Spence,
2560.
to a region leukemias.
of chromosome
6 involved Sci.
Proc. Natl. Acad.
in transloca-
USA 83: 2556-
M. A., Spurr,
N. K., and Field,
the committee on the genetic constitution togenet. Cell Genet. 51: 149-165.
L. L. (1989).
Report
on
on chromosome 6. Cy-
fiir Humangenetik, University of Heidelberg, Heidelberg, Germany; tlmperial Cancer Research fund, Lincoln’s London WC 2A 3PX, Great Britain; and SDeutsches Krebsforschungszentrum, Heidelberg, Germany Received
October
28, 1991;
A gene with male germ cell-specific expression from the t-complex on mouse chromosome 17, TCTE3 (probe 117~; symbol D17Leh117c), has been previously cloned and characterized (8). The significance of the testis-specific expression of TCTE3 being present in greatest abundance in later spermatogenic stages is further enhanced by its genetic mapping to an -~-CM subregion in the middle part of the t-complex called In(17)3, which has been shown to contain the gene for the distorter/ sterility gene Ted-3 (1, 6). The homologues of several t-complex genes have by now been mapped in human, and all that have been mapped to date are located on human chromosome 6 (9). To determine the chromosome location of human sequences related to TCTE3 and to further define the syntenic relationship existing between the t-complex genes and their human counterparts, somatic cell hybrid mapwere carried out. Using ping and in situ hybridization the mouse 117~ gene sequence as a probe, a filter with genomic DNA samples from total mouse and total human DNAs and somatic cell hybrid lines carrying portions of chromosome 6 was hybridized (see Fig. 1). Under normal stringency conditions, 117~ hybridized to human DNA (lane 1) and to DNA from cell line MCP-6, which carries a portion of chromosome 6 (6p21.3-qter), but not to DNA from cell line 56-47, which carries the at the Institut Neuenheimer
February
24, 1992
6p21.1-pter portion of human chromosome 6. These results indicate that human sequences related to TCTES do indeed map to human chromosome 6, specifically within the 6pl2-qter region. To determine this location more precisely, a human X clone was isolated from a genomic library constructed in XEMBLS (a), using the mouse cDNA 117~ as a probe. This X clone, termed h117, was labeled with biotin by nick-translation and hybridized to metaphase spreads of human chromosomes under conditions that suppress signals from repetitive DNA sequences as previously described (4). Detection via FITC-conjugated avidin revealed a highly specific hybridization signal in the telomerit region of chromosome 6q27 (see Fig. 2). Of 35 metaphases evaluated, 31 showed the signal on both
The gene TCTE3 from the mouse t-complex region is expressed specifically in testicular germ cells. It maps in the central subregion of the t-complex on mouse chromosome 17 containing loci involved in transmission ratio distortion and male sterility. In this study, somatic cell hybrid lines have been used to map the human homologue, TCTE3, to the long arm of chromosome 6. CISS hybridization with the human X clone h117 refined this chromosome assignment to the very distal position of chromosome 6q27, thus providing further evidence that loci from the t-complex of mouse chromosome 17 can map to opposite arms of human chromosome 6. 0 1992 Academic Press, Inc.
1 TO whom correspondence should be addressed Humangenetik der Universitlt Heidelberg, Im 328, 6900 Heidelberg, Germany.
revised
Inn Fields,
fur Feld
1337
-23.1
-4.4
-2.3 -2.0 FIG. 1. 117~ restriction patterns observed in DNA prepared from somatic cell hybrid lines. (1) Total human DNA, (2) total mouse DNA, (3) MCP-6, (4) 56-47. Both MCP-6 (3) and 56-47 (7) contain portions of human chromosome 6. DNA samples were cleaved with EcoRI, electrophoresed in an 0.8% agarose gel, transferred to a nylon membrane, and hybridized to the 0.7-kb 32P-labeled 117~ probe (8). The filter was washed in 6X SSC at 65°C and exposed for 7 days. An -8-kb EcoRI fragment could not be mapped on the hybrids because of a mouse band at a similar position. The size of the human-specific EcoRI fragment present on lanes 1 and 3 is -3.5 kb (indicated by an arrow). GENOMICS
13,
1337-1339 (19%) 0888-7543/92 $5.00
Copyright Q 1992 by Academic Press, Inc. All rights of reproduction in any form reserved.
1338
SHORT
COMMUNICATION
FIG. 2. Mapping of X clone h117 (insert size 15 kb) by in situ hybridization to human metaphase chromosomes. Biotinylated probe DNA was hybridized to metaphase chromosomes from stimulated lymphocytes of a normal male individual under conditions as described previously (4). The hybridized probe was detected via avidin-conjugated FITC, and chromosomes were banded with DAPI. (A) Specific hybridization signals in the telomeric region of chromosome 6q. (B) The same metaphase section as in A depicting the two chromosome 6 homologues delineated by cohybridization with digoxigenin-labeled DNA of library pBS6 followed by detection with anti-digoxigenin antibody conjugated to rhodamine. (C) Signal on a DAPI-banded elongated chromosome 6 showing the localization of clone h117 to 6q27. Note that band 6q27 is negatively “stained” by DAPI. Images of FITC-, DAPI-, and rhodamine were taken separately by using a cooled charged coupled device camera system (Photometrics) and overlaid electronically using the software NIH image 1.33. A and C are overlays of DAPI and FITC images, and B is an overlay of a DAPII and rhodamine image. FITC signals are depicted by arrows.
chromosome homologues and no other signals were found. Chromosome identification and banding assignment were confirmed by cohybridization with DNA library pBS6 (kindly provided by Joe Gray, San Francisco), derived from sorted human chromosome 6 (see Fig. 2B), and Alu-enriched human DNA to achieve an R-banding-like AL-banding (data not shown) as described previously (4, 5). The localization on the distal end of the long arm of chromosome 6 places the human homologue of TCTE3 in close vicinity to human homologues of t-complex genes such as Tcp-1, Sod-2, and the human TCPlO gene family (homologous to Tcp-10 in mouse 17) (see reviews of Spence et al. (9) and Artzt et al. (1)). In contrast, the human homologues of the MHC and a number of closely linked mouse loci (including Pim-1, Glo-1, and Pgk-2) have been mapped to the short arm of chromosome 6 (6pll-~21) (see Spence et al. (9)). This suggests a syntenic relationship between the proximal and middle portions (In(17)2-In(17)3) of the t-complex of mouse chromosome 17 and 6q and between the distal portion of the t-complex and 6p (1). It is now clear that the major features of the t-complex, e.g., male-specific transmission distortion and crossover suppression, cannot be generalized to man. Therefore, mouse t-complex as a functional, genetic unit most probably does not have a human equivalent. However, the homology of human chromosomes 6p and 6q (separated by nonhomologous DNA) with mouse chromosome 17 is well established and human homologues of genes associated with t-complex developmental lethal
and sterility mutations are likely to exist. An analysis of the segregation of chromosome 6 alleles with familial cases of male infertility may therefore reveal a subset of male sterilities in humans that could be the result of mutations in these testis-expressed genes. ACKNOWLEDGMENTS We thank Denise Barlow and Lisa Stubbs for careful reading of the manuscript. This work was supported by grants from the Deutsche Forschungsgemeinschaft and by the Verein zur Fiirderung der Krebsforschung in Deutschland.
REFERENCES 1.
2.
3.
4.
5.
Artzt, K., Barlow, D., Dove, W., Fischer Lindahl, K., Klein, J., Lyon, M. F., and Silver, L. M. (1991). Maps of mouse chromosome 17: First report. Mammalian Genome 1: 5-29. Frischauf, A-M. (1987). Construction and characterization of a genomic library in X. In “Methods in Enzymology” (S. L. Berger and A. R. Kimmel, Eds.), Vol. 152, pp. 190-199. Academic Press, San Diego. Goodfellow, P. N., Banting, G., Trowsdale, J., Chambers, S., and Solomon, E. (1982). Introduction of a human X-6 translocation chromosome into a mouse teratocarcinoma: Investigation of control of HLA-A, B, C expression. Proc. Natl. Acad. Sci. USA 79: 1190-1194. Lichter, P., Tang, C-J. C., Call, K., Hermanson, G., Evans, G. A., Housman, D., and Ward, D. C. (1990). High-resolution mapping of human chromosome 11 by in situ hybridization with cosmid clones. Science 2: 64-69. Lichter, P., Boyle, A. L., Cremer, T., and Ward, D. C. (1991). Analysis of genes and chromosomes by non-isotopic in situ hybridization. Genet. Anal. Technol. Appl. 8: 24-35.
SHORT 6. 7.
COMMUNICATION
1339
Lyon, M. F. (1986). Male sterility of the mouse t-complex is due to homozygosity of the distorter genes. Cell 44: 357-363. Nagarajan, L., Lonie, E., Tsujimoto, Y., Ar-Rushdi, A., Huebner, K., and Croce, C. M. (1986). Localization of the human pim on-
8.
Rappold, G. A., Stubbs, L., Labeit, S., Crkvenjakow, R. B., and Lehrach, H. (1987). Identification of a testis-specific gene from the mouse t-complex next to a CpG-rich island. EMBO J. 6: 1975-1980.
cogene (PIM) tions in acute
9.
Spence,
2560.
to a region leukemias.
of chromosome
6 involved Sci.
Proc. Natl. Acad.
in transloca-
USA 83: 2556-
M. A., Spurr,
N. K., and Field,
the committee on the genetic constitution togenet. Cell Genet. 51: 149-165.
L. L. (1989).
Report
on
on chromosome 6. Cy-
