GENOMICS

6,545-547

(1990)

SHORT COMMUNICATION Assignment of the Structural Gene for Argininosuccinate Synthetase to Proximal Mouse Chromosome 2 MARIAN

J. JACKsON,*st

*Howard

LINDA

Hughes Medical

C. SURH,t-#

Institute Baylor

June 2, 1989;

revised

Press,

October

L. &AumT*st

of Biochemistry,

17, 1989

preparation). In the human there are at least 14 dispersed pseudogeneswhich map to many different sites in the genome in addition to the expressed locus which has been mapped to 9q34-qter (Carritt and Povey, 1979; Su et al., 1984). The human argininosuccinate synthetase cDNA has been used to detect argininosuccinate synthetase-related sequences in the mouse (Nakamura et al., 1985). Analysis of Chinese hamstermouse somatic cell hybrids led to the assignment of the structural locus to mouse chromosome 2. As with the human case, many pseudogenes were present in the mouse genome. Argininosuccinate synthetase-related sequences were also mapped to mouse chromosomes7 and 10. In the current study we have developed linkage markers for the mouse argininosuccinate synthetase expressed locus in order to map the locus more precisely using recombinant inbred strains (Bailey, 1971; Taylor, 1978). Genomic DNAs from seven recombinant inbred (RI) progenitor strains were studied using cloned sequences from the mouse argininosuccinate synthetase structural gene. Probe fragments that were free of repeated sequences and that were composed of predominantly intron sequenceswere used in order to minimize crosshybridization to pseudogenes.Three different two-allele restriction fragment length polymorphism systems were detected using probe fragments derived from genomic clones (data not shown). For each of these three polymorphisms, SWR/J mice had a unique fragment compared to strains AKR/J, C57BL/6J, BALB/cByJ, DBA/ 25, C3H/HeJ, and C57L/J. The latter group of strains all had the same fragment. The strain distribution pattern for one such polymorphism among seven SWXL RI strains was identical to that of the fifth component of complement (Hc), which has been mapped to proximal mouse chromosome 2 (data not shown). Since the genomic fragments tested failed to identify polymorphisms that were informative in any other RI

In order to develop linkage markers for the murine argininosuccinate synthetase locus (Ass-l), we have searched for restriction fragment length polymorphisms in the mouse genome using cloned sequences from the mouse argininosuccinate synthetase structural gene. Five restriction fragment length polymorphisms were found among the recombinant inbred progenitor strains AKR/J, BALB/cByJ, C3H/HeJ, C6’7BL/6J, C67L/J, DBA/BJ, and SWR/J. Of these, four polymorphisms were found to distinguish the SWR/J strain from the other six strains, which all had the same fragment. The fifth polymorphism revealed differences among the progenitor strains for recombinant inbred strain sets AKXL, BXD, and SWXL. The strain distribution pattern for this polymorphism indicated close linkage of Ass-l to Hc (the tlfth component of complement) on proximal mouse chromosome 2 with a recombination fraction of 0.016 and a 95% confidence interval of 0.003 to 0.054. These data place Ass-l in a syntenic group with the genes Hc, Abl, Fpgs, and Ak-1 whose linkage has been conserved between human chromosome 9q and mouse chromosome 2. Academic

AND ARTHUR

and tlnstitute for Molecular Genetics and *Department College of Medicine, Houston, Texas 77030

Received

Q 1990

E. O’BRIEN,*‘t’$

WILLIAM

Inc.

Argininosuccinate synthetase catalyzes the condensation of citrulline and aspartate to form argininosuccinate. This enzyme is important in the biosynthesis of arginine and serves as one of the enzymes of the urea cycle in mammals. We have been interested for some time in the regulation and structure of the argininosuccinate synthetase locus in humans and in mice. Argininosuccinate synthetase is subject to developmental, hormonal, nutritional, and tissue-specific regulation (reviewed in Jackson et al., 1986). Cloned sequences from the structural gene and the cDNA are available for both human (Freytag et al., 1984; Su et al., 1981) and mouse (L. C. Surh et al., manuscript in 545

All

Copyright 0 1990 righta of reproduction

oE!afJ-7643/90 $3.00 by Academic Press, Inc. in any form reserved.

546

SHORT

COMMUNICATION

TABLE Recombinant

Inbred

Strain

Distribution

Patterns AKXL

Locus

5

Ass-l

AALLAAALLALLAAALAA AALLAAALLALLAAALAL

HC

1

6

2

7

5

8

6

9

8

12

13

14

16

1 at the Ass-l and Hc Loci

of Polymorphisms

strain

17 19

21

SWXL 24

25

28

29

37

38

4

L s LSSSLSS

cl

9

11

12

13

14

15

16

BXD

strain

18

19

20

21

22

7

23

24

25

27

strain

12

14

15

16

17

s

s

L

s

s

28

29

30

31

32

Ass-l HC

Note. Data for Ass-l were obtained by hybridization of EcoRI digests of DNA from p3C-eb2 as described in the text. Data for Hc are from D’Eustachio et al. (3). Boxed loci. A, AKR/J; B, C57BL/6J; D, DBA/ZJ; L, C57L/J; S, SWR/J.

strain set, we searched for additional polymorphisms using the mouse cDNA for argininosuccinate synthetase. Two different EcoRI polymorphisms were found using the cDNA probe pTZASM12. Like the polymorphisms detected with the intron probes, one polymorphism with pTZASM12 was found only in the SWR/ J parental strain (Fig. 1, bands Al and A2). Analysis of the SWXL RI strains showed that this additional marker had the same strain distribution pattern as that of the intron probes and Hc (data not shown). The second polymorphic system detected with the cDNA was more informative and revealed differences among the progenitor strains for RI sets AKXL, BXD, and SWXL (Fig. 1, bands Bl and B2). The strain distribution pattern for this restriction fragment length polymorphism is shown in Table 1. Comparison to the strain distribution pattern for Hc (D’Eustachio et al, 1986) showed three recombination events among 51 recombinant inbred strains characterized for both Ass1 and Hc. The pooled data yield a recombination fraction of 0.016 with a 95% confidence interval of 0.003 to 0.054 (Taylor, 1978; Silver, 1985). In order to confirm that the second EcoRI polymorphism was indeed in the structural gene rather than in a pseudogene, we searched for the appropriate EcoRI fragment in our genomic clones which had been isolated from a BALB/ c genomic library. A 3.5-kb EcoRI fragment was identified in a genomic clone. Subclone p3C-eb2, which was free of repeated sequences,was isolated from this EcoRI fragment. Filters containing EcoRI digests of the recombinant inbred strain DNAs were rehybridized using this probe. This genomic fragment identified the 3.5and 7.2-kb polymorphic fragments detected as the second cDNA polymorphism and gave the same strain distribution pattern as that shown in Table 1. We have demonstrated close linkage, in recombinant inbred strains, of the structural gene for argininosuc-

recombinant inbred strains with both pTZASM12 and areas indicate a recombination event between the two

cinate synthetase (Ass-l) to the fifth component of complement (Hc). Hc is an anchor locus on proximal mouse chromosome 2. The human argininosuccinate synthetase expressed locus has been mapped to 9q34-

MW 9.4 -

6.6 -

4.4 82

2.3 FIG. 1. EcoRI fragment length polymorphisms detected with the mouse cDNA for argininosuccinate synthetase. Genomic DNA from recombinant inbred progenitor strains was digested with EcoFZI, fractionated on 0.6% agarose gels run at 20 V for 3 days to allow resolution of higher molecular weight fragments, and transferred to Nytran membranes. The filter was hybridized to 32P-labeled pTZASM12. Bands Al and A2 correspond to a two-allele polymorphism which distinguishes SWR/J mice from the other six strains examined. Bands Bl and B2 correspond to a second two-allele polymorphism.

SHORT

547

COMMUNICATION

qter (Carritt and Povey, 1979; Su et al., 1984). Recently, the fifth component of complement has been mapped in humans to 9q (Jeremiah et al., 1987; Lemons et al., 1987). Several other markers mapped to human chromosome 9q have been assigned to proximal mouse 2. Murine adenylate kinase-1 (A&l) and folylpolyglutamate synthetase (Fpgs) genes were localized to ZcenCl using microcell hybrids (Fournier and Moran, 1983) and the mouse Abl proto-oncogene was mapped by in situ hybridization to 2B (Threadgill and Womack, 1988). Thus, it seems that a syntenic group containing the genes Hc, Ass-l, Abl, Fpgs, and Ak-1 has been conserved between human chromosome 9 and proximal mouse chromosome 2. It is interesting to note that for each probe used, at least one restriction fragment length polymorphism that distinguished the SWR/J strain from the other strains examined was detected. These findings suggest that the proximal region of mouse chromosome 2 present in SWR/J mice may be of more genetically distant origin than the other chromosomes studied.

ACKNOWLEDGMENTS We thank Dr. Benjamin Taylor for analysis of the recombinant inbred strain distribution pattern data and Drs. Leroy Hood and John Weis for provision of mouse genomic libraries. This work was supported by USPHS Grants RR05425-26 and GM 27593. L.C.S. is supported by a research fellowship from the Medical Research Council of Canada.

3.

D’EUSTACHIO, P., KF~ISTENSEN, T., WETSEL, R. A., RIBLET, R., TAYLOR, B. A., AND TACK, B. F. (1986). Chromosomal location of the genes encoding complement components C5 and factor H in the mouse. J. Zmmwwl. 137: 3990-3995.

4.

FOURNIER, R. E. K., AND MORAN, R. G. (1983). Complementation mapping in microcell hybrids: Localization of l$gs and Ak-1 on MLLS musculus chromosome 2. Somat. Cell Genet. 9: 69-M.

5.

FREYTAG, S. O., BEAUDET, A. L., BOCK, H. G. O., AND O’BRIEN, W. E. (1984). Molecular structure of the human argininosuccinate synthetase gene: Occurrence of alternative mRNA splicing. Mol. Cell. Biol. 4: 1978-1984.

6.

JACKSON, Mammalian 464.

7.

JEREMIAH, S. J., WEST, L. F., DAVIS, M. B., POVEY, S., CARRITI, B., AND FEY, G. (1987). Assignment of human complement component C5 to chromosome 9. Cytogenet. Cell Genet. 46: 634.

8.

LEMONS, R. S., LE BEAU, R. A. (1987). Chromosomal fifth component of human 46: 647.

9.

NAKAMURA, D., POPP, R., EICHER, E., AND LALLEY, P. A. (1985). Comparison of the argininosuccinate synthetase gene family in mouse and man. Cytogenet. Cell Genet. 40: 710. SILVER, J. (1985). Confidence Iimits for estimates of gene linkage based on analysis of recombinant inbred strains. J. Hered. 76: 436-440.

10.

M. J., BFAUDET, A. L., AND O’BRIEN, W. E. (1986). urea cycle enzymes. Annu. Reu. Genet. 20: 431-

M. M., TACK, B. F., AND WETSEL, mapping of the gene encoding the complement. Cytogenet. Cell Genet.

11.

Su, T.-S., BOCK, H.-G. O., O’BRIEN, W. E., AND BEAUDET, A. L. (1981). Cloning of cDNA for arginmosuccinate synthetase mRNA and study of enzyme overproduction in a human cell line. J. Biol. Chem. 266: 11826-11831.

12.

Su, T.-S., NUSSBAUM, R. L., AIRHART, S., LEDBETTER, D. H., MOHANDAS, T., O’BRIEN, W. E., AND BEAUDET, A. L. (1984). Human chromosomal assignments for 14 argininosuccinate synthetase pseudogenes: Cloned DNAs as reagents for cytogenetic analysis. Amer. J. Hum. Genet. 36: 954-964. TAYLOR, B. A. (1978). Recombinant inbred strains: Use in gene mapping. In “Origins of Inbred Mice” (H. C. Morse III, Ed.), pp. 423-438, Academic Press, New York. THREADGILL, D. W., AND WOMACK, J. E. (1988). Regional localization of mouse Abl and Mos proto-oncogenes by in situ hybridization. Genomics 3: 82-86.

REFERENCES 1.

BAILEY, D. W. (1971). Recombinant-inbred strains: An aid to finding identity, linkage, and function of histocompatibiity and other genes. Transplnntution 11: 325-327.

13.

2.

CAR-, B., AND POVJZY, S. (1979). Regional assignments of the loci AK,, ACONs and ASS on human chromosome 9. Cytogenet. Cell Genet. 23: 171-181.

14.

Assignment of the structural gene for argininosuccinate synthetase to proximal mouse chromosome 2.

In order to develop linkage markers for the murine argininosuccinate synthetase locus (Ass-1), we have searched for restriction fragment length polymo...
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