Mammalian Genome 8, 45--46 (1997).

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�9 Springer-Verlag New York Inc. 1997

Localization of Zap70, the gene for a T cell-specific protein tyrosine kinase, to mouse and rat chromosomes by fluorescence in situ hybridization and molecular genetic linkage analyses

T. Saito, 1 Y. Matsuda, 1 H. Ito, 1 N. Fusaki, z T. Hori, 1 T. Yamamoto 2

1Division of Genetics, National Institute of Radiological Sciences, 4-9-1 Anagawa, Inage-ku, Chiba 263, Japan 2Department of Oncology, Institute of Medical Science, Tokyo University, 4-6-1 Shirokanedai, Minato-ku, Tokyo 108, Japan

Received: 1 September 1995 / Accepted: 23 August 1996

Zap70, the gene for a non-SRC type protein tyrosine kinase that has recently been demonstrated to play an essential role in T cell development and function, was mapped to mouse Chromosome (Chr) 1B and rat Chr 9q22.1 by direct R-banding fluorescence in situ hybridization (FISH). The gene was localized also by genetic linkage analysis at 2.1 _+ 1.1 cM distal to D1Mit20 and 5.6 _ 1.9 cM proximal to D1Mitl8 on mouse Chr 1. The regions around Zap70 locus of the two species have conserved linkage homology to human Chr 2q, where the human ZAP70 has been localized.

The ZAP70 protein was first identified as a phosphotyrosine- containing protein tyrosine kinase 70 kilodalton that is recruited to the zeta chain of the T cell antigen receptor complex during T cell activation (Chart et al. 1991). It has been proposed that the ZAP70 protein participates in T cell activation (Chan et al. 1992; Kolanus et al. 1993; Weiss and Littman 1994). In 1994, several groups reported that mutations in ZAP70 are associated with an autosomal recessive form of human severe combined immunodeficiency dis-

Correspondence to: T. Yamamoto

ease (Arpaia et al. 1994; Chan et al. 1994; Elder et al. 1994). These studies indicated that absence of the ZAP70 protein results in an inability to mediate the signals of T cell receptor and produces a distinctive form of human severe combined immunodeficiency. Although detailed basic biochemical studies on the mechanism for the hereditary immunodeficiency are immature, animal model sys- tems, once established, should be very useful to develop the strat- egies for treatment of the patients by gene therapy, as well as to investigate T cell development and activation in vivo.

As the first step to establish experimental animal model sys- tems for ZAP70-defective diseases and to make an examination for the presence of correspondence to already known classical muta- tions, we performed comparative mapping of Zap70 on mouse and rat chromosomes by the FISH method (Matsuda et al. 1992), using a human ZAP70 cDNA fragment of 2.0 kb as a probe. The gene was mapped to mouse Chr 1B (Fig. l-a, b, and c ) and to rat chromosome 9q22.1 (Fig. l-d, e, and f). Both chromosomal re- gions have conserved linkage homology with each other and with human Chr 2q (Lyon and Kirby 1994; Seldin et al. 1993; Yamada

Fig. 1. Chromosomal localization of Zap70 to mouse (a, b, and c) and rat (d, e, and f) chromosomes by fluorescence in situ hybridization with a human ZAP70 cDNA fragment as a biotinylated probe. The cDNA frag- ment was prepared by PCR from a cDNA library of human peripheral blood lymphocytes. The PCR primers were 5'-GGACATCCACCTGTAC- GTCC-3' and 5'-GTFGTCTCCACACACAGCTG-3' derived from the 5' and 3' untranslated regions, respectively [Chan et al. 1992]. Arrows indi-

care the signals on mouse Chr 1B and on rat Chr 9q22.1. The metaphase spreads were photographed with Nikon B-2A (a, c, d, and f) and UV (b and e) filters. R- and G-banded patterns are shown in (a, c, d, and f) and (b and e), respectively. The chromosome diagram shown on the right represents rat Chromosome 9, indicating the position of the hybridization signal with a black arrowhead.

46 T. Saito et al.: Localization of ZapTO

o , . , e o �9 [] �9 [] �9 [] �9 [] z , , - , o �9 [] [] �9 �9 [] �9 []

�9 [] [] �9 [] �9 �9 [] �9 [] [] �9 [] �9 [] �9 66 58 1 2 2 6 4 3

Fig. 2. Results of DNA typing of backcross progeny. The backcross prog- eny were obtained by mating (C57BL/6 x Mus spretus) F 1 males and Mus spretus males. Genomic DNAs of the backcross progeny prepared in our laboratory were applied to DNA typing. The indicated loci were typed on 142 backcross progeny. Each column represents a chromosome haplotype composed of C57BL/6J (a black box) and Mus spretus (a white box) DNA sequences. Values at the bottom of each column are the number of animals inheriting the indicated haplotype. Amplification conditions were 94~ for 5 rain, 30 cycles of 94~ for 0.5 min, 55~ for 0.5 rain, and 72~ for 5 rain. The PCR products were analyzed by agarose gel electrophoresis.

Chromosome 1

Centromere ~ D I Mit20

2.1 ~. ZAP-70

5.6

D1Mit78

4.9 D 1Mit22

Telomere i

Fig. 3. Molecular genetic linkage map showing the chromosomal location of mouse Zap70. The order of loci typed in this study, are shown in a schematic form. Values to the left of the chromosome are estimated dis- tances in centimorgans.

et al. 1994). Our mapping data are in good accordance with both of the reported human and mouse chromosomal position of ZAP70/Zap70, which is reported to be in human Chr 2q (Chan et al. 1994; Ku et al. 1994) and mouse Chr 1A4-C1, respectively.

The location of the gene on mouse chromosome was also de- termied by genetic linkage analysis with DNA samples from a total number of 142 interspecific backcross mice between (C57BIJ6 x Mus spretus) F 1 females and M. spretus males. First, DNAs from the two parental mouse species, C57B1/6 and M. spretus, and from their F 1 were digested with various restriction endonucleases, and Southern blot hybridization analysis was performed with the hu- man ZAP70 cDNA probe to choose an appropriate restriction en- zyme to produce restriction fragment length polymorphisms (RFLPs) between the two species. KpnI gave clearly distinguish- able bands of 4.9 kb for C57BL/6 and 4.3 kb for M. spretus. Then,

the DNAs from 142 backcross animals were digested with KpnI and probed with the ZAP70 cDNA fragment to examine the seg- regation for RFLPs for each animal. Three simple sequence length polymorphism markers, DIMitl8, DIMit20, and DIMit22, were chosen for polymerase chain reaction (PCR) analysis based on the results of the cytogenetic mapping by FISH. Oligonucleotide prim- ers specific for these loci were prepared, and DNA typing by PCR was performed as described in the legend of Fig. 2. In all the 19 recombinants between DIMit20 and DiMit22, and gene order that minimized double recombination events was DIMit20, Zap70, DiMit18, DIMit22, from proximal to distal, with estimates of distance of 2.1 -+ 1.2 cM between DIMit20 and Zap70, 5.6 _+ 1.9 cM between Zap70 and DiMit18, and 4.9 _+ 1.8 cM between DIMit18 and DIMit22 (Fig. 3). No immunodeficient mutation or other already known classical mutation has been identified in these regions of mouse and rat chromosomes.

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