- Email: [email protected]
Assignment of Mouse Cardiac Two-Pore Background K+Channel Gene (Kcnk4) to the Proximal Region of Mouse Chromosome 5
BRIEF MAPPING REPORTS
Assignment of Mouse Cardiac TwoPore Background K1 Channel Gene (Kcnk4) to the Proximal Region of Mouse Chromosome 5 Akikazu Fujita, Yoshiyuki Horio, Neal G. Copeland,* Debra J. Gilbert,* Nancy A. Jenkins,* and Yoshihisa Kurachi1 Department of Pharmacology II, Faculty of Medicine and Graduate School of Medicine, Osaka University, 2-2 Yamada-oka, Suita, Osaka 565-0871, Japan; and *Mammalian Genetics Laboratory, ABL-Basic Research Program, National Cancer Institute-Frederick Cancer Research and Development Center, Frederick, Maryland 21702
183
sis using progeny derived from matings of [(C57BL/6J 3 Mus spretus)F1 3 C57BL/6J] mice. This interspecific backcross mapping panel has been typed for over 2600 loci that are well distributed among all the autosomes as well as the X chromosome (2). C57BL/6J and M. spretus DNAs were digested with several enzymes and analyzed by Southern blot hybridization for informative restriction fragment length variant (RFLV) using a 0.6-kb SfiI/XbaI fragment of mouse cTBAK-1 cDNA as a probe. The probe detected BglII fragments of 19.0 and 15.0 kb (C57BL/6J) and of 10.5 kb (M. spretus). The presence or absence of the M. spretus-specific 10.5-kb BglII fragment was followed in backcross mice. Results: Kcnk4 mapped to the proximal region of mouse chromosome 5 (see Fig. 1). The recombination frequencies (expressed as genetic distances in centimorgans 6 the stan-
Received July 22, 1998; accepted September 11, 1998
Functional gene description: Potassium (K1) channels can be structurally classified into three major groups that contain two, four, and six putative transmembrane (TM) regions. The K1 channels of six TM regions with a single pore region include voltage-dependent K1 channels and calciumactivated K1 channels, while those of two TM regions with a single pore region are the inwardly rectifying K1 channels. Recently, K1 channels of four TM regions with two pore regions have been cloned (3–5). We have cloned cTBAK (cardiac two-pore background K1 channel)-1/TASK from a mouse heart cDNA library (4). cTBAK-1 (Kcnk4) consists of 409 amino acid residues and is predominantly expressed in the heart. The nucleotide and deduced amino acid sequences of cTBAK-1 showed about 30 – 40 and 22–25% identities with those of other 4TM K1 channels, respectively (4). cTBAK-1 is a K1-selective channel with a small conductance (16 pS) and is inhibited by the decrease of extracellular pH (3, 4; and unpublished observation). Name of clone: The clone used was cTBAK-1 (GenBank Accession No. AB008537) (4). Description of clone: The probe used in mapping Kcnk4 was a 0.6-kb SfiI/XbaI fragment of mouse cTBAK-1 cDNA. Method used to validate gene identity: The probe used has been sequenced. Flanking markers: En2, Il6, and Adra2c were used to position Kcnk4. Methods of mapping: The mouse chromosomal location of (Kcnk4) was determined by interspecific backcross analyThis research was supported in part by the National Cancer Institute, DHHS, under contract with ABL and also by grants from The Ministry of Education, Science, Sports and Culture of Japan, “Research for the Future” Program of The Japan Society for Promotion of Science (96L00302), the Human Frontier Science Program (RG0158/1997-B), and Yamanouchi Foundation for Research on Metabolic Disorders. 1 To whom correspondence should be addressed. Telephone: 1816-879-3510. Fax: 181-6-879-3519. E-mail: [email protected]. osaka-u.ac.jp.
GENOMICS 54, 183–184 (1998) ARTICLE NO. GE985573
0888-7543/98 $25.00 Copyright © 1998 by Academic Press All rights of reproduction in any form reserved.
FIG. 1. Kcnk4 maps in the proximal region of mouse chromosome 5. Kcnk4 was placed on mouse chromosome 5 by interspecific backcross analysis. The descriptions of the probes and RFLV for the loci linked to Kcnk4 have been described (1). The segregation patterns of Kcnk4 and flanking genes in 179 backcross mice that were typed for all loci are shown at the top of the figure. For individual pairs of loci, more than 179 animals were typed (see below). Each column represents the chromosome identified in the backcross progeny that was inherited from the (C57BL/6J 3 M. spretus)F1 parent. The shaded boxes represent the presence of a C57BL/6J allele, and white boxes represent the presence of a M. spretus allele. The number of offspring inheriting each type of chromosome is listed at the bottom of each column. A partial chromosome 5 linkage map showing the location of Kcnk4 in relation to linked genes is shown at the bottom of the figure. The number of recombinant N2 animals over the total number of N2 animals typed plus the recombination frequency 6 the standard error is shown for each pair of loci to the left of the chromosome map. The positions of loci in human chromosomes, where known, are shown to the right.
184
BRIEF MAPPING REPORTS
dard error) are centromere–En2–2.5 6 1.1–Il6– 0.5 6 0.5– Kcnk4–1.7 6 1.0 –Adra2c. Additional comments: We have compared our interspecific map of chromosome 5 with a composite mouse linkage map that reports the map locations of many uncloned mouse mutations (provided from the Mouse Genome Database, a computerized database maintained at The Jackson Laboratory, Bar Harbor, ME). Kcnk4 mapped in a region of the composite map that lacks mouse mutations with a phenotype that might be expected for an alteration in this locus (data not shown). Homologies: The proximal region of mouse chromosome 5 shares regions of homology with human chromosomes 7p, 2p, 18p, and 4p, suggesting that the human homologue of Kcnk4 will map to one of these human chromosomes. References 1.
Chang, N.-C., Jenkins, N. A., Gilbert, D. J., Copeland, N. G., Chang, Y.-H., Chen, W.-M., and Chang, A. C. (1994). Assignment of two alpha 2 adrenoceptor subtype genes to murine chromosomes. Neurosci. Lett. 167: 105–108. 2. Copeland, N. G., and Jenkins, N. A. (1991). Development and applications of a molecular genetic linkage map of the mouse genome. Trends Genet. 7: 113–118. 3. Duprat, F., Lesage, F., Fink, M., Reyes, R., Heurteaux, C., and Lazdunski, M. (1997). TASK, a human background K1 channel to sense external pH variations near physiological pH. EMBO J. 16: 5464 –5471. 4.
Kim, D., Fujita, A., Horio, Y., and Kurachi, Y. (1998). Cloning and functional expression of a novel cardiac two-pore background K1 channel (cTBAK-1). Circ. Res. 82: 513–518.
5.
Lesage, F., Guillemare, E., Fink, M., Duprat, F., Lazdunski, M., Romey, G., and Barhanin, J. (1996). TWIK-1, a ubiquitous human weakly inward rectifying K1 channel with a novel structure. EMBO J. 15: 1004 –1011.
Assignment of the Human Gene for the Sarcomeric M-Band Protein Myomesin (MYOM1) to 18p11.31–p11.32 Ernst J. M. Speel,*,1 Peter F. M. van der Ven,† Jozefa C. M. Albrechts,* Frans C. S. Ramaekers,* Dieter O. Fu¨rst,† and Anton H. N. Hopman* *Department of Molecular Cell Biology and Genetics, University Maastricht, 6200 MD Maastricht, The Netherlands; and †Department of Cell Biology, University of Potsdam, D-14471 Potsdam, Germany
is responsible for the stable and highly ordered arrangement of thick and thin filaments (1). Together with its associated proteins, the giant protein titin (also called connectin) interconnects the major structures of sarcomeres: the M-bands and Z-discs. Apart from titin, only a few structural proteins, localized within the M-band, have been identified, including skelemin, M-protein, and myomesin. Myomesin has been shown to bind strongly to titin, and this binding occurs in the conspicuous globular head portion of the titin molecules that localizes to the M-band (3, 7). Since myomesin is found in all striated muscle fiber types, it has been assumed to constitute the principal titin-anchoring protein in the M-band. Because it also interacts with myosin (3, 7), myomesin might play an important role in the assembly and stabilization of myofibrils. DNA source and its description: The cDNA of myomesin was isolated and previously characterized from human muscles [(7); EMBL/GenBank Accession No. X69090]. The deduced amino acid sequence predicted unique Nterminal domains (approximately 110 residues), followed by repeating immunoglobulin-like and fibronectin type IIIlike motifs. Together with titin, M-protein, skelemin, Cprotein, and H-protein, myomesin was ascribed to the group of intracellular proteins of the immunoglobulin superfamily (1). DNA probes used: Two oligonucleotides specific for the 39 UTR of the myomesin cDNA (59-GGTCAGAGGGCAGACGAATGTG-39 and 59-GGGGAAGAGGACAGACGTGAA-39) were used for dot-blot analysis of a somatic cell hybrid panel, and a partial myomesin cDNA probe representing the 59 situated 3457 bp (7) was used for fluorescence in situ hybridization (FISH) analysis. Flanking markers used: Three cosmid probes with known mapping sites, i.e., SCW0403E (18p11.1–p11.21), SCW0202F (18p11.21–p11.31), and SCW0203D (18p11.31– p11.32) (2), were used for double-target FISH analysis. Methods of mapping: For chromosomal assignment of the human myomesin gene, denatured DNA samples (10 mg) from the NIGMS human/rodent somatic cell hybrid mapping panel 2 (Human Genetic Mutant Cell Repository, Coriell Institute, Camden, NJ) were spotted on nitrocellulose, together with the parental DNAs, using a home-made dot-blot apparatus. One hundred picograms of human myomesin and M-protein cDNAs cloned in pBluescript KS(1) (7) served as positive and negative controls, respec-
Received May 8, 1998; accepted July 27, 1998
Functional gene description: In the sarcomeres of cross-striated muscles, a complex cytoskeletal framework The authors thank Dr. A. Geurts van Kessel (University of Nijmegen, Nijmegen, The Netherlands) for donation of somatic cell hybrid DNA; Dr. T. Imamura (National Institute of Genetics, Mishima, Japan) for providing the 18p-specific cosmid probes; Dr. L. Brecevic
GENOMICS 54, 184 –186 ARTICLE NO. GE985503
(1998)
0888-7543/98 $25.00 Copyright © 1998 by Academic Press All rights of reproduction in any form reserved.
for screening the Cytogenetics Database, and N. Wey for photographic and computer-assisted reproductions (University of Zu¨rich, Zu¨rich, Switzerland). This work was supported in part by the Deutsche Forschungsgemeinschaft (D.O.F.) and the Netherlands Organization for Scientific Research (N.W.O.). 1 To whom correspondence and reprint requests should be addressed at present address: Department of Pathology, University of Zu¨rich, Schmelzbergstrasse 12, CH-8091 Zu¨rich, Switzerland. Telephone: 141-1-2553030: Fax: 141-1-2554551. E-mail: ernst-jan.speel@ pty.usz.ch.
Assignment of Mouse Cardiac TwoPore Background K1 Channel Gene (Kcnk4) to the Proximal Region of Mouse Chromosome 5 Akikazu Fujita, Yoshiyuki Horio, Neal G. Copeland,* Debra J. Gilbert,* Nancy A. Jenkins,* and Yoshihisa Kurachi1 Department of Pharmacology II, Faculty of Medicine and Graduate School of Medicine, Osaka University, 2-2 Yamada-oka, Suita, Osaka 565-0871, Japan; and *Mammalian Genetics Laboratory, ABL-Basic Research Program, National Cancer Institute-Frederick Cancer Research and Development Center, Frederick, Maryland 21702
183
sis using progeny derived from matings of [(C57BL/6J 3 Mus spretus)F1 3 C57BL/6J] mice. This interspecific backcross mapping panel has been typed for over 2600 loci that are well distributed among all the autosomes as well as the X chromosome (2). C57BL/6J and M. spretus DNAs were digested with several enzymes and analyzed by Southern blot hybridization for informative restriction fragment length variant (RFLV) using a 0.6-kb SfiI/XbaI fragment of mouse cTBAK-1 cDNA as a probe. The probe detected BglII fragments of 19.0 and 15.0 kb (C57BL/6J) and of 10.5 kb (M. spretus). The presence or absence of the M. spretus-specific 10.5-kb BglII fragment was followed in backcross mice. Results: Kcnk4 mapped to the proximal region of mouse chromosome 5 (see Fig. 1). The recombination frequencies (expressed as genetic distances in centimorgans 6 the stan-
Received July 22, 1998; accepted September 11, 1998
Functional gene description: Potassium (K1) channels can be structurally classified into three major groups that contain two, four, and six putative transmembrane (TM) regions. The K1 channels of six TM regions with a single pore region include voltage-dependent K1 channels and calciumactivated K1 channels, while those of two TM regions with a single pore region are the inwardly rectifying K1 channels. Recently, K1 channels of four TM regions with two pore regions have been cloned (3–5). We have cloned cTBAK (cardiac two-pore background K1 channel)-1/TASK from a mouse heart cDNA library (4). cTBAK-1 (Kcnk4) consists of 409 amino acid residues and is predominantly expressed in the heart. The nucleotide and deduced amino acid sequences of cTBAK-1 showed about 30 – 40 and 22–25% identities with those of other 4TM K1 channels, respectively (4). cTBAK-1 is a K1-selective channel with a small conductance (16 pS) and is inhibited by the decrease of extracellular pH (3, 4; and unpublished observation). Name of clone: The clone used was cTBAK-1 (GenBank Accession No. AB008537) (4). Description of clone: The probe used in mapping Kcnk4 was a 0.6-kb SfiI/XbaI fragment of mouse cTBAK-1 cDNA. Method used to validate gene identity: The probe used has been sequenced. Flanking markers: En2, Il6, and Adra2c were used to position Kcnk4. Methods of mapping: The mouse chromosomal location of (Kcnk4) was determined by interspecific backcross analyThis research was supported in part by the National Cancer Institute, DHHS, under contract with ABL and also by grants from The Ministry of Education, Science, Sports and Culture of Japan, “Research for the Future” Program of The Japan Society for Promotion of Science (96L00302), the Human Frontier Science Program (RG0158/1997-B), and Yamanouchi Foundation for Research on Metabolic Disorders. 1 To whom correspondence should be addressed. Telephone: 1816-879-3510. Fax: 181-6-879-3519. E-mail: [email protected]. osaka-u.ac.jp.
GENOMICS 54, 183–184 (1998) ARTICLE NO. GE985573
0888-7543/98 $25.00 Copyright © 1998 by Academic Press All rights of reproduction in any form reserved.
FIG. 1. Kcnk4 maps in the proximal region of mouse chromosome 5. Kcnk4 was placed on mouse chromosome 5 by interspecific backcross analysis. The descriptions of the probes and RFLV for the loci linked to Kcnk4 have been described (1). The segregation patterns of Kcnk4 and flanking genes in 179 backcross mice that were typed for all loci are shown at the top of the figure. For individual pairs of loci, more than 179 animals were typed (see below). Each column represents the chromosome identified in the backcross progeny that was inherited from the (C57BL/6J 3 M. spretus)F1 parent. The shaded boxes represent the presence of a C57BL/6J allele, and white boxes represent the presence of a M. spretus allele. The number of offspring inheriting each type of chromosome is listed at the bottom of each column. A partial chromosome 5 linkage map showing the location of Kcnk4 in relation to linked genes is shown at the bottom of the figure. The number of recombinant N2 animals over the total number of N2 animals typed plus the recombination frequency 6 the standard error is shown for each pair of loci to the left of the chromosome map. The positions of loci in human chromosomes, where known, are shown to the right.
184
BRIEF MAPPING REPORTS
dard error) are centromere–En2–2.5 6 1.1–Il6– 0.5 6 0.5– Kcnk4–1.7 6 1.0 –Adra2c. Additional comments: We have compared our interspecific map of chromosome 5 with a composite mouse linkage map that reports the map locations of many uncloned mouse mutations (provided from the Mouse Genome Database, a computerized database maintained at The Jackson Laboratory, Bar Harbor, ME). Kcnk4 mapped in a region of the composite map that lacks mouse mutations with a phenotype that might be expected for an alteration in this locus (data not shown). Homologies: The proximal region of mouse chromosome 5 shares regions of homology with human chromosomes 7p, 2p, 18p, and 4p, suggesting that the human homologue of Kcnk4 will map to one of these human chromosomes. References 1.
Chang, N.-C., Jenkins, N. A., Gilbert, D. J., Copeland, N. G., Chang, Y.-H., Chen, W.-M., and Chang, A. C. (1994). Assignment of two alpha 2 adrenoceptor subtype genes to murine chromosomes. Neurosci. Lett. 167: 105–108. 2. Copeland, N. G., and Jenkins, N. A. (1991). Development and applications of a molecular genetic linkage map of the mouse genome. Trends Genet. 7: 113–118. 3. Duprat, F., Lesage, F., Fink, M., Reyes, R., Heurteaux, C., and Lazdunski, M. (1997). TASK, a human background K1 channel to sense external pH variations near physiological pH. EMBO J. 16: 5464 –5471. 4.
Kim, D., Fujita, A., Horio, Y., and Kurachi, Y. (1998). Cloning and functional expression of a novel cardiac two-pore background K1 channel (cTBAK-1). Circ. Res. 82: 513–518.
5.
Lesage, F., Guillemare, E., Fink, M., Duprat, F., Lazdunski, M., Romey, G., and Barhanin, J. (1996). TWIK-1, a ubiquitous human weakly inward rectifying K1 channel with a novel structure. EMBO J. 15: 1004 –1011.
Assignment of the Human Gene for the Sarcomeric M-Band Protein Myomesin (MYOM1) to 18p11.31–p11.32 Ernst J. M. Speel,*,1 Peter F. M. van der Ven,† Jozefa C. M. Albrechts,* Frans C. S. Ramaekers,* Dieter O. Fu¨rst,† and Anton H. N. Hopman* *Department of Molecular Cell Biology and Genetics, University Maastricht, 6200 MD Maastricht, The Netherlands; and †Department of Cell Biology, University of Potsdam, D-14471 Potsdam, Germany
is responsible for the stable and highly ordered arrangement of thick and thin filaments (1). Together with its associated proteins, the giant protein titin (also called connectin) interconnects the major structures of sarcomeres: the M-bands and Z-discs. Apart from titin, only a few structural proteins, localized within the M-band, have been identified, including skelemin, M-protein, and myomesin. Myomesin has been shown to bind strongly to titin, and this binding occurs in the conspicuous globular head portion of the titin molecules that localizes to the M-band (3, 7). Since myomesin is found in all striated muscle fiber types, it has been assumed to constitute the principal titin-anchoring protein in the M-band. Because it also interacts with myosin (3, 7), myomesin might play an important role in the assembly and stabilization of myofibrils. DNA source and its description: The cDNA of myomesin was isolated and previously characterized from human muscles [(7); EMBL/GenBank Accession No. X69090]. The deduced amino acid sequence predicted unique Nterminal domains (approximately 110 residues), followed by repeating immunoglobulin-like and fibronectin type IIIlike motifs. Together with titin, M-protein, skelemin, Cprotein, and H-protein, myomesin was ascribed to the group of intracellular proteins of the immunoglobulin superfamily (1). DNA probes used: Two oligonucleotides specific for the 39 UTR of the myomesin cDNA (59-GGTCAGAGGGCAGACGAATGTG-39 and 59-GGGGAAGAGGACAGACGTGAA-39) were used for dot-blot analysis of a somatic cell hybrid panel, and a partial myomesin cDNA probe representing the 59 situated 3457 bp (7) was used for fluorescence in situ hybridization (FISH) analysis. Flanking markers used: Three cosmid probes with known mapping sites, i.e., SCW0403E (18p11.1–p11.21), SCW0202F (18p11.21–p11.31), and SCW0203D (18p11.31– p11.32) (2), were used for double-target FISH analysis. Methods of mapping: For chromosomal assignment of the human myomesin gene, denatured DNA samples (10 mg) from the NIGMS human/rodent somatic cell hybrid mapping panel 2 (Human Genetic Mutant Cell Repository, Coriell Institute, Camden, NJ) were spotted on nitrocellulose, together with the parental DNAs, using a home-made dot-blot apparatus. One hundred picograms of human myomesin and M-protein cDNAs cloned in pBluescript KS(1) (7) served as positive and negative controls, respec-
Received May 8, 1998; accepted July 27, 1998
Functional gene description: In the sarcomeres of cross-striated muscles, a complex cytoskeletal framework The authors thank Dr. A. Geurts van Kessel (University of Nijmegen, Nijmegen, The Netherlands) for donation of somatic cell hybrid DNA; Dr. T. Imamura (National Institute of Genetics, Mishima, Japan) for providing the 18p-specific cosmid probes; Dr. L. Brecevic
GENOMICS 54, 184 –186 ARTICLE NO. GE985503
(1998)
0888-7543/98 $25.00 Copyright © 1998 by Academic Press All rights of reproduction in any form reserved.
for screening the Cytogenetics Database, and N. Wey for photographic and computer-assisted reproductions (University of Zu¨rich, Zu¨rich, Switzerland). This work was supported in part by the Deutsche Forschungsgemeinschaft (D.O.F.) and the Netherlands Organization for Scientific Research (N.W.O.). 1 To whom correspondence and reprint requests should be addressed at present address: Department of Pathology, University of Zu¨rich, Schmelzbergstrasse 12, CH-8091 Zu¨rich, Switzerland. Telephone: 141-1-2553030: Fax: 141-1-2554551. E-mail: ernst-jan.speel@ pty.usz.ch.