Mammalian Genome 3: 286-289, 1992
elIOllle 9 Springer-VerlagNew York Inc. 1992
The gene for proliferating cell nuclear antigen Chromosome 2
(Pcna) maps to mouse
Cathy Abbott, 1 Alison Pilz, 1 Heather Moseley, 2 and Jo Peters 2 1Department of Genetics and Biometry, University College London, Wolfson House, 4 Stephenson Way, London N W l 2HE, UK; 2MRC Radiobiology Unit, Chilton, Didcot, Oxon O X l l ORD, U K Received November 25, 1991; accepted February 20, 1992
Abstract. The structural gene for proliferating cell nu-
clear antigen (Pcna) has been mapped to mouse Chromosome (Chr) 2 by use of a PCR-based assay. With somatic cell hybrids, Pcna was mapped between the T(2;4)13H and T(2;4)ISn breakpoints. An interspecific backcross was employed to map Pcna 1.9 +-- 1.3 cM distal to II-lb. This was confirmed by mapping Pcna in the BXH recombinant inbred (RI) strains; no recombinants were seen between II-la and Pcna. In addition, a PCNA-related sequence (Pcna-rsl) was mapped to Chr 19 in the BXH RI strains.
Introduction
Proliferating cell nuclear antigen (PCNA) is involved in cell replication; PCNA expression occurs in cells at the G1/S boundary of the cell cycle and increases during S phase (Almendral et al. 1987). Inhibition of PCNA expression with antisense oligonucleotides in actively dividing cells has been shown to cause the cells to quiesce (Jaskulski et al. 1988). The highest level of expression of PCNA in the mouse is seen in tissues such as thymus, testis, and spleen, which are undergoing active cell division. Expression is also high, perhaps surprisingly, in brain, but is low in heart, pancreas, and liver (Yamaguchi et al. 1991). PCNA is an auxiliary protein of DNA polymerase ~ and is inducible by growth signals such as insulin and epidermal growth factor (Bravo and McDonald-Bravo 1984). The genes for human, rat, and mouse PCNA have recently been cloned. The 5' flanking region of the Drosophila melanogaster PCNA gene has been shown to contain sequences to which homeodomain proteins bind (Yamaguchi et al. 1990); likewise, the 5' regions of the mouse and human PCNA genes contain the oc-
Offprint requests to: C. Abbott
tamer-like sequence to which the OCT transcription factor (which contains a homeodomain) is predicted to bind. It is, therefore, possible that PCNA has a role in morphogenesis (Yamaguchi et al. 1991). The structural gene for PCNA in humans has been mapped to Chr 20 by use of human/rodent somatic cell hybrids (Ku et al. 1989). It was subsequently localized to Chr 20p13 by in situ hybridization (Webb et al. 1990). The p r e s e n c e of at least three possible pseudogenes of PCNA has been reported. Ku et al. (1989) detected pseudogenes on the X Chr and Chr 6p12 ~ pter, and Webb et al. (1990) detected hybridization of their probe to 1lp15.5 and Xpl 1.4 as well as to the presumed structural locus. The mouse Pcna gene has not been mapped. Two processed pseudogenes have been detected (Yamaguchi et al. 1991). One of these, + PCNA-II, was found to be present only in certain inbred strains of laboratory mice and was tentatively mapped to Chr 17, on the basis of the strain distribution pattern of the gene in the CXB RI strains. We have now mapped the structural gene, Pcna, in the mouse by use of a length polymorphism that is detectable with the polymerase chain reaction (PCR). Materials and methods The backcross used has been described previously (Pilz et al. 1992). The Acra typings were also reported in that paper. The mouse/ hamster somatic cell hybrids used were EBS-18AZ, the kind gift of Dr. Peter Lalley, and the Sn and 13H mouse/hamster somatic cell hybrids (Pilz et al. 1991). PCR was carried out with the primers described in Table 1 (Pcna and Pcna-rsl) or the II-lb primers described by Love and co-workers (1990). PCR was carried out on a Hybaid Thermal Reactor with 500 ng DNA, 50 pmoles each primer, 200 ~M each dNTP, 1 • PCR buffer (Promega), and 1 unit Taq polymerase (Promega) in a total volume of 100 t~1. Cycling conditions for Pcna were an initial incubation at 95~ for 5 min followed by 35 cycles of 94~ 15 s, 55~ 1 min, and 72~ 1 min 30 s. Pcna-rsl cycling conditions and II-lb cycling conditions were an initial incubation at 95~ for 5 min, followed by 30 cycles of 94~ 15 s, 55~ 30 s, and 72~ 30 s.
287
C. Abbott et al.: P c n a maps to mouse Chr 2 Table 1. Sequences of PCR primers used to map (a) Pcna and (b) Pcna-rsl. (a) (b)
Pcna: sense: antisense Pcna-rsl : sense: antisense:
HYBRID
13H 10-1
5' GGA AGG CAA AGG AAG ATG TTC 3' 5' AGG GAC AGT AAA CCA GTC TTC 3'
13H 3-1
Sn 3 - I
Abl
Abl
5' GAA CAG GAG TAC AGC TGT GTA 3' 5' CAG GCT CAT TCA TCT CTA TGG 3'
Aliquots (10 ~1) o f the Pcna-rsl and II-lb PCR products were analyzed by electrophoresis in a 2% agarose gel that was stained with ethidium bromide and viewed under U V light. PCR products for Pcna were digested in 20 Ixl volumes with 1 ixl HinfI at 37~ for 1 h and 1 Ixl TaqI at 65~ for 1 h. A 15-t-tl aliquot o f digested PCR product was then run on a 4% Nusieve 3:1 agarose gel, stained with ethidium bromide, and viewed under UV light. DNAs from the B X H RI strains were obtained from The Jackson Laboratory (Bar Harbor, Me.).
N-lb
Li-lb
Ad@
pcna
-
"}"
q"
[]MMU2
Results
The fourth intron of the mouse Pcna gene contains the sequence (T)10 C(T)6 AAG(T)4 (Yamaguchi et al. 1991). The gene contains no obvious microsatellite sequences, but mononucleotide repeats have been shown frequently to be polymorphic (Aitman et al. 1991). PCR primers were, therefore, designed to flank this region (Table la) and were used to map the Pcna gene in somatic cell hybrids and by linkage analysis. These primers, which correspond to sequence within intron 4, are specific for the structural gene Pcna, since both p s e u d o g e n e s of Pcna are p r o c e s s e d pseudogenes (Yamaguchi et al. 1991). The primers amplify mouse but not hamster DNA and are thus suitable for analyzing Pcna in mouse/hamster somatic cell hybrids. As there is extensive conserved synteny between human Chr 20 and mouse Chr 2 (reviewed in Davisson et al. 1991), we first amplified DNA from a mouse/hamster somatic cell hybrid (EBS-18AZ) that contains Chr 2 as its only genetic material from the mouse. A clear band of 562 bp was seen with the Pcna primers in hybrid DNA but not hamster, showing that the Pcna gene is on mouse Chr 2. DNA from somatic cell hybrids containing chromosomes carrying reciprocal translocations of mouse Chrs 2 and 4 [T(2;4)ISn and T(2;4)13H; Pilz et al. 1991) was then analyzed. Hybrid 13H 3-1, which contains only that part of Chr 2 distal to the T(2;4)13H breakpoint, and hybrid Sn 3-I, which contains the part of Chr 2 proximal to the T(2;4) 1Sn breakpoint, were both positive for Pcna. Hybrid 13H 10-1, which contains Chr 2 proximal to the T(2;4) 13H breakpoint, was negative for Pcna (data not shown; Fig. 1). The Pcna gene must, therefore, map between the T(2;4) 13H and T(2;4) 1Sn breakpoints. Having established that Pcna maps to central Chr 2, a more precise localization was achieved by mapping the gene by linkage analysis in an interspecific backcross. The intron primers were again used to search for variation in restriction fragment length between Mus spretus and laboratory mice. From the sequence of Pcna intron 4 from C57BL/6J (Yamaguchi et al. 1991), it is predicted that amplification with the Pcna primers will yield a band of 562 bp, which after
m
MMU4
Fig. 1. S c h e m a i c representation of Pcna m ~ p i n g in somatic cell hybrids.
digestion with HinfI and TaqI will yield bands of 245 bp and 215 bp as well as two smaller bands. This was indeed found to be the case, and similar results were found for DBA/2J and Mus spretus (Fig. 2). However, when DNA from C3H/HeJ and the laboratory stock AN (derived from C3H) was amplified and digested, the visible digestion products were 245 bp and 190 bp (Fig. 2). Since this difference in size between C3Hbased strains and C57BL/6J, DBA/2J, and Mus spretus could be revealed by digestion with other restriction enzymes, the difference seems to be due to a length variation in the region containing the sequence (T)m C(T) 6 AAG(T)4. Undigested bands from AN and Mus spretus appeared to be of slightly different length; however, it was difficult to resolve this difference clearly on agarose gels. This variation was used to map the Pcna gene by linkage analysis in an interspecific backcross of (AN x M. spretus) F~ females backcrossed to AN males, and in the BXH RI strains. A typical gel is shown in Fig. 1, and the results of analysis of the backcross are shown
Fig. 2. Agarose gel electrophoresis o f HinfI/TaqI-digested PCR products of P c n a in backcross offspring.
288
C. Abbott et at.: P c n a m a p s to m o u s e Chr 2
in Fig. 3. Pcna was found to map 18.6 +- 3.9 cM distal to Acra and 1.9 - 1.3 cM distal to II-lb. The most likely gene order is Acra-18.6 +- 3.9 cM-II-lb-l.9 +1.3 cM-Pcna since with this order there are no double recombinants. This result is consistent with that obtained with the BXH RI strains (shown in Table 2a), which shows that in the 12 strains tested, no recombinants have been seen with II-la, a gene that maps very close to II-lb (reviewed in Siracusa and Abbott 1991). Yamaguchi and colleagues (1991) described the presence of two processed pseudogenes of Pcna, one of which, referred to as qJ PCNA II, is present in only certain inbred laboratory strains of mice. Since this pseudogene, for which we suggest the gene name Pcna-rsl, is absent in C3H/HeJ, AN, and Mus spretus, is could not be mapped in the same backcross as Pcna. Pcna-rsl is, however, present in C57BL/6J. We therefore designed for PCR primers (Table 1) that amplify Pcna-rsl, but not Pcna, to give a 220-bp band. These primers were used to map the gene in the BXH RI strains. The strain distribution pattern (SDP) obtained is shown in Table 2b. Yamaguchi and co-workers (1991) also mapped the gene in the CXB RI strains, obtaining SDPs in lines D, E, G, H, I, J, and K of CBBCBBB respectively, identical to that of Ly-l. Yamaguchi and colleagues suggested that Pcna-rsl maps to Chr 17, based on the SDP. However, when the resuits from both sets of RI lines are combined, the SDPs for Pcna-rsl and Ly-1 are identical in 19 RI lines, and thus the most likely map position for Pcna-rsl is the proximal region of Chr 19.
Discussion
The PCR-based assay for Pcna-rsl provides an easily scorable polymorphic locus on Chr 19, with the proviso that the pseudogene is present in one of the strains used in the backcross and absent in the other, to which the F 1 animals are backcrossed. So far, the
Aora 9 1 4 9 II-lb 9 Pcna B I D I B I I B I B No. of animals
1
2
2
2
51 30 1
[]
AN allele
9
Mus staretus allele
9
Not tested
1
1] 6
Fig. 3. Analysis of b a c k c r o s s progeny for Acra, II-lb, and Pcna. A total of 106 mice were analyzed for Acra, 105 for l l - l b , and 109 for Pcna. R e c o m b i n a t i o n frequencies found were 19/102 b e t w e e n Acra and II-lb, 2/105 b e t w e e n l l - l b and Pcna, and 21/106 b e t w e e n Ac r a and Pcna.
Table 2. SDPs of (a) Pcna in the BXH RI lines, together with the tl-la SDP (D'Eustachio et al. 1987) and (b) Pcna-rsl in the BXH RI lines, together with the Ly-1 SDP (Hogarth et al. 1988). (a) BXH line
2
3
4
6
7
8
9
10
11
12
14
19
Ilila Pcna
]] B
B B
H H
H H
B B
H H
B B
H H
B B
H H
H H
B B
(b) BXH line
2
3
4
6
7
8
9
10
11
12
14
19
H H
H H
H H
B B
B B
H H
H H
B B
B B
B B
H H
H H
Ly-1 Pcna-rsl
PCR assay for the Pcna polymorphism can be used only to map Pcna in backcrosses involving C3H/HeJ or stocks (such as AN) derived from C3H, as has been described. However, it is possible that by re-designing the PCR primers so that they more closely flank the microsatellite-like sequence in intron 4, followed by subjection of the PCR products to high-resolution polyacrylamide gel electrophoresis, length variation of the PCR product could be detected in other strains of mice. The mapping of Pcna to mouse Chr 2 extends the known region of homology between mouse Chr 2 and human Chr 20. To date, 10 loci on lISA 20 that have been firmly mapped in the mouse have been found to reside on MMU 2 (Davisson et al. 1991). The positioning of Pcna on the consensus map of MMU 2 supports the localization in the human to 20p13, since Pcna maps to the same region of MMU 2 as Prn and Itp, the human homologs of both of which map to HSA 20p. A number of phenotypic mutants map to the same region of MMU 2 as Pcna (reviewed in Siracusa and Abbott 1991). These include welhaarig (we; Falconer 1954), mahogany (mg; Lane and Green 1960), and tight-skin (Tsk; Green et al. 1976). Pcna should provide a useful additional molecular marker for mapping studies aimed at cloning these mutants. Other genes that code for proteins involved in control of the cell cycle have also recently been mapped (Sakaguchi et al. 1991). Cyclin genes and sequences homologous to cyclin genes have been mapped to mouse Chrs 3, 4, 6, 10, and 15 by cross-hybridization with two cloned probes for human CDC2. These data, together with the mapping of Pcna to MMU 2, show the genes involved in cell cycle control to be scattered throughout the genome. Disruption of these genes in the mouse by homologous recombination could lead to valuable insights into the control of cell replication and, potentially, the basic mechanisms involved in carcinogenesis. A c k n o w l e d g m e n t . W e are very grateful to Dr. Ben Taylor for his
help in analyzing the data from the B X H RI strains.
References Aitman, T., Hearne, C., McAleer, M., and Todd, J.: M o n o n u c l e otide repeats are an a b u n d a n t source of length variants in m o u s e genomic D N A . M a m m a l i a n G e n o m e 1: 206-210, 1991. Almendral, J., H u e b s c h , D., Blundell, P., M c D o n a l d - B r a v o , H . , and Bravo, R.: Cloning and s e q u e n c e of the h u m a n nuclear pro-
C. Abbott et al.: Pcna maps to mouse Chr 2 tein cyclin: homology with DNA-binding proteins. Proc Natl Acad Sci USA 84: 1575-1579, 1987.
Bravo, R. and McDonald-Bravo, H.: Induction of the nuclear protein 'cyclin' in quiescent mouse 3T3 cells stimulated by serum and growth factors. Correlation with DNA synthesis. EMBO J 3: 3177-3181, 1984. Davisson, M.T., Lalley, P.A., Peters, J., Doolittle, D.P., Hillyard, A.L., and Searle, A.G.: Report of the comparative committee for human, mouse and other rodents (HGM 11). Cytogenet Cell Genet 58: 1152-1189, 1991. D'Eustachio, P., Jadidi, S., Fuhlbrigge, R., Gray, P., and Chaplin, D.: Interleukin-1 a and 13genes: linkage on chromosome 2 in the mouse. Immunogenetics 26: 339-343, 1987. Falconer, D.: Linkage in the mouse: the sex-linked genes and 'rough'. Z Indukt Abstammungs-Vererbungsl 86: 263-268, 1954. Green, M., Sweet, H., and Bunker, L.: Tight-skin, a new mutation of the mouse causing excessive growth of connective tissue and skeleton. Am J Pathol 82: 493-512, 1976. Hogarth, P., Houlden, B., Latham, S., Cherry, M., Taylor, B., and McKenzie, I.: The mouse Ly-12.1 specificity: genetic and biochemical relationship to Ly-1. Immunogeneties 27: 383-387, 1988. Jaskulski, D., deRiel, J., Mercer, W., Calabretta, B., and Baserga, R.: Inhibition of cellular proliferation by antisehse oligodeoxynucleotides to PCNA Cyclin. Science 240: 1544-1546, 1988. Ku, D., Travali, S., Calabretta, B., Huebner, K., and Baserga, R.: H u m a n gene for proliferating cell n u c l e a r antigen has pseudogenes and localizes to chromosome 20. Somatic Cell Mol Genet 15: 297-307, 1989. Lane, P. and Green, M.: Mahogany, a recessive colour mutation in linkage Group V of the mouse. J Hered 51: 228-230, 1960. Love, J., Knight, A., McAleer, M., and Todd, J.: Towards con-
289 struction of a high resolution map of the mouse genome using PCR-analysed microsatellites. Nucleic Acids Res. 18: 4123--4130, 1990. Pilz, A., Fox, M., Povey, S., and Abbott, C.: A panel of somatic cell hybrids with translocation breakpoints on mouse chromosome 2 and their characterization by PCR. Mammalian Genome 1 (Suppl): $517, 1991. Pilz, A., Moseley, H., Peters, J. and Abbott, C.: Comparative mapping of mouse chromosome 2 and human chromosome 9q: the genes for gelsorin and dopamine 13 hydroxylase map to mouse chromosome 2. Genomics 12: 715-719, 1992. Sakaguchi, A., Martinez, L., Lalley, P., Sylvia, V., Han, E., Killary, A., and Ghosh Choudhury, G.: Chromosome mapping in mouse of genes that regulate the eukaryotic cell cycle. Cytogenet Cell Genet 58: 2138, 1991. Siracusa, L. and Abbott, C.: Mouse Chromosome 2. Mammalian Genome: I: S15-$41, 1991. Webb, G., Parsons, P., and Chenevix-Trench, G.: Localization of the gene for human proliferating cell nuclear antigen/cyclin by in situ hybridization. Hum Genet 86: 84-86, 1990. Yamaguchi, M., Nishida, Y., Moriuchi, T., Hirose, F., Hui, C-C., Suzuki, Y., and Matsukage, A.: Drosophila proliferating cell nuclear antigen (cyclin) gene: structure, expression during development, and specific binding of homeodomain proteins to its 5' flanking region. Mol Cell Biol 10: 872-879, 1990. Yamaguchi, M., Hayashi, Y.Y., Hirose, F., Matsuoka, S., Moriuchi, T., Shiroishi, T., Moriwaki, K., and Matsukage, A.: Molecular cloning and structural analysis of mouse gene and pseudogenes for proliferating cell nuclear antigen. Nucleic Acids Res 19: 2403-2410, 1991.
elIOllle 9 Springer-VerlagNew York Inc. 1992
The gene for proliferating cell nuclear antigen Chromosome 2
(Pcna) maps to mouse
Cathy Abbott, 1 Alison Pilz, 1 Heather Moseley, 2 and Jo Peters 2 1Department of Genetics and Biometry, University College London, Wolfson House, 4 Stephenson Way, London N W l 2HE, UK; 2MRC Radiobiology Unit, Chilton, Didcot, Oxon O X l l ORD, U K Received November 25, 1991; accepted February 20, 1992
Abstract. The structural gene for proliferating cell nu-
clear antigen (Pcna) has been mapped to mouse Chromosome (Chr) 2 by use of a PCR-based assay. With somatic cell hybrids, Pcna was mapped between the T(2;4)13H and T(2;4)ISn breakpoints. An interspecific backcross was employed to map Pcna 1.9 +-- 1.3 cM distal to II-lb. This was confirmed by mapping Pcna in the BXH recombinant inbred (RI) strains; no recombinants were seen between II-la and Pcna. In addition, a PCNA-related sequence (Pcna-rsl) was mapped to Chr 19 in the BXH RI strains.
Introduction
Proliferating cell nuclear antigen (PCNA) is involved in cell replication; PCNA expression occurs in cells at the G1/S boundary of the cell cycle and increases during S phase (Almendral et al. 1987). Inhibition of PCNA expression with antisense oligonucleotides in actively dividing cells has been shown to cause the cells to quiesce (Jaskulski et al. 1988). The highest level of expression of PCNA in the mouse is seen in tissues such as thymus, testis, and spleen, which are undergoing active cell division. Expression is also high, perhaps surprisingly, in brain, but is low in heart, pancreas, and liver (Yamaguchi et al. 1991). PCNA is an auxiliary protein of DNA polymerase ~ and is inducible by growth signals such as insulin and epidermal growth factor (Bravo and McDonald-Bravo 1984). The genes for human, rat, and mouse PCNA have recently been cloned. The 5' flanking region of the Drosophila melanogaster PCNA gene has been shown to contain sequences to which homeodomain proteins bind (Yamaguchi et al. 1990); likewise, the 5' regions of the mouse and human PCNA genes contain the oc-
Offprint requests to: C. Abbott
tamer-like sequence to which the OCT transcription factor (which contains a homeodomain) is predicted to bind. It is, therefore, possible that PCNA has a role in morphogenesis (Yamaguchi et al. 1991). The structural gene for PCNA in humans has been mapped to Chr 20 by use of human/rodent somatic cell hybrids (Ku et al. 1989). It was subsequently localized to Chr 20p13 by in situ hybridization (Webb et al. 1990). The p r e s e n c e of at least three possible pseudogenes of PCNA has been reported. Ku et al. (1989) detected pseudogenes on the X Chr and Chr 6p12 ~ pter, and Webb et al. (1990) detected hybridization of their probe to 1lp15.5 and Xpl 1.4 as well as to the presumed structural locus. The mouse Pcna gene has not been mapped. Two processed pseudogenes have been detected (Yamaguchi et al. 1991). One of these, + PCNA-II, was found to be present only in certain inbred strains of laboratory mice and was tentatively mapped to Chr 17, on the basis of the strain distribution pattern of the gene in the CXB RI strains. We have now mapped the structural gene, Pcna, in the mouse by use of a length polymorphism that is detectable with the polymerase chain reaction (PCR). Materials and methods The backcross used has been described previously (Pilz et al. 1992). The Acra typings were also reported in that paper. The mouse/ hamster somatic cell hybrids used were EBS-18AZ, the kind gift of Dr. Peter Lalley, and the Sn and 13H mouse/hamster somatic cell hybrids (Pilz et al. 1991). PCR was carried out with the primers described in Table 1 (Pcna and Pcna-rsl) or the II-lb primers described by Love and co-workers (1990). PCR was carried out on a Hybaid Thermal Reactor with 500 ng DNA, 50 pmoles each primer, 200 ~M each dNTP, 1 • PCR buffer (Promega), and 1 unit Taq polymerase (Promega) in a total volume of 100 t~1. Cycling conditions for Pcna were an initial incubation at 95~ for 5 min followed by 35 cycles of 94~ 15 s, 55~ 1 min, and 72~ 1 min 30 s. Pcna-rsl cycling conditions and II-lb cycling conditions were an initial incubation at 95~ for 5 min, followed by 30 cycles of 94~ 15 s, 55~ 30 s, and 72~ 30 s.
287
C. Abbott et al.: P c n a maps to mouse Chr 2 Table 1. Sequences of PCR primers used to map (a) Pcna and (b) Pcna-rsl. (a) (b)
Pcna: sense: antisense Pcna-rsl : sense: antisense:
HYBRID
13H 10-1
5' GGA AGG CAA AGG AAG ATG TTC 3' 5' AGG GAC AGT AAA CCA GTC TTC 3'
13H 3-1
Sn 3 - I
Abl
Abl
5' GAA CAG GAG TAC AGC TGT GTA 3' 5' CAG GCT CAT TCA TCT CTA TGG 3'
Aliquots (10 ~1) o f the Pcna-rsl and II-lb PCR products were analyzed by electrophoresis in a 2% agarose gel that was stained with ethidium bromide and viewed under U V light. PCR products for Pcna were digested in 20 Ixl volumes with 1 ixl HinfI at 37~ for 1 h and 1 Ixl TaqI at 65~ for 1 h. A 15-t-tl aliquot o f digested PCR product was then run on a 4% Nusieve 3:1 agarose gel, stained with ethidium bromide, and viewed under UV light. DNAs from the B X H RI strains were obtained from The Jackson Laboratory (Bar Harbor, Me.).
N-lb
Li-lb
Ad@
pcna
-
"}"
q"
[]MMU2
Results
The fourth intron of the mouse Pcna gene contains the sequence (T)10 C(T)6 AAG(T)4 (Yamaguchi et al. 1991). The gene contains no obvious microsatellite sequences, but mononucleotide repeats have been shown frequently to be polymorphic (Aitman et al. 1991). PCR primers were, therefore, designed to flank this region (Table la) and were used to map the Pcna gene in somatic cell hybrids and by linkage analysis. These primers, which correspond to sequence within intron 4, are specific for the structural gene Pcna, since both p s e u d o g e n e s of Pcna are p r o c e s s e d pseudogenes (Yamaguchi et al. 1991). The primers amplify mouse but not hamster DNA and are thus suitable for analyzing Pcna in mouse/hamster somatic cell hybrids. As there is extensive conserved synteny between human Chr 20 and mouse Chr 2 (reviewed in Davisson et al. 1991), we first amplified DNA from a mouse/hamster somatic cell hybrid (EBS-18AZ) that contains Chr 2 as its only genetic material from the mouse. A clear band of 562 bp was seen with the Pcna primers in hybrid DNA but not hamster, showing that the Pcna gene is on mouse Chr 2. DNA from somatic cell hybrids containing chromosomes carrying reciprocal translocations of mouse Chrs 2 and 4 [T(2;4)ISn and T(2;4)13H; Pilz et al. 1991) was then analyzed. Hybrid 13H 3-1, which contains only that part of Chr 2 distal to the T(2;4)13H breakpoint, and hybrid Sn 3-I, which contains the part of Chr 2 proximal to the T(2;4) 1Sn breakpoint, were both positive for Pcna. Hybrid 13H 10-1, which contains Chr 2 proximal to the T(2;4) 13H breakpoint, was negative for Pcna (data not shown; Fig. 1). The Pcna gene must, therefore, map between the T(2;4) 13H and T(2;4) 1Sn breakpoints. Having established that Pcna maps to central Chr 2, a more precise localization was achieved by mapping the gene by linkage analysis in an interspecific backcross. The intron primers were again used to search for variation in restriction fragment length between Mus spretus and laboratory mice. From the sequence of Pcna intron 4 from C57BL/6J (Yamaguchi et al. 1991), it is predicted that amplification with the Pcna primers will yield a band of 562 bp, which after
m
MMU4
Fig. 1. S c h e m a i c representation of Pcna m ~ p i n g in somatic cell hybrids.
digestion with HinfI and TaqI will yield bands of 245 bp and 215 bp as well as two smaller bands. This was indeed found to be the case, and similar results were found for DBA/2J and Mus spretus (Fig. 2). However, when DNA from C3H/HeJ and the laboratory stock AN (derived from C3H) was amplified and digested, the visible digestion products were 245 bp and 190 bp (Fig. 2). Since this difference in size between C3Hbased strains and C57BL/6J, DBA/2J, and Mus spretus could be revealed by digestion with other restriction enzymes, the difference seems to be due to a length variation in the region containing the sequence (T)m C(T) 6 AAG(T)4. Undigested bands from AN and Mus spretus appeared to be of slightly different length; however, it was difficult to resolve this difference clearly on agarose gels. This variation was used to map the Pcna gene by linkage analysis in an interspecific backcross of (AN x M. spretus) F~ females backcrossed to AN males, and in the BXH RI strains. A typical gel is shown in Fig. 1, and the results of analysis of the backcross are shown
Fig. 2. Agarose gel electrophoresis o f HinfI/TaqI-digested PCR products of P c n a in backcross offspring.
288
C. Abbott et at.: P c n a m a p s to m o u s e Chr 2
in Fig. 3. Pcna was found to map 18.6 +- 3.9 cM distal to Acra and 1.9 - 1.3 cM distal to II-lb. The most likely gene order is Acra-18.6 +- 3.9 cM-II-lb-l.9 +1.3 cM-Pcna since with this order there are no double recombinants. This result is consistent with that obtained with the BXH RI strains (shown in Table 2a), which shows that in the 12 strains tested, no recombinants have been seen with II-la, a gene that maps very close to II-lb (reviewed in Siracusa and Abbott 1991). Yamaguchi and colleagues (1991) described the presence of two processed pseudogenes of Pcna, one of which, referred to as qJ PCNA II, is present in only certain inbred laboratory strains of mice. Since this pseudogene, for which we suggest the gene name Pcna-rsl, is absent in C3H/HeJ, AN, and Mus spretus, is could not be mapped in the same backcross as Pcna. Pcna-rsl is, however, present in C57BL/6J. We therefore designed for PCR primers (Table 1) that amplify Pcna-rsl, but not Pcna, to give a 220-bp band. These primers were used to map the gene in the BXH RI strains. The strain distribution pattern (SDP) obtained is shown in Table 2b. Yamaguchi and co-workers (1991) also mapped the gene in the CXB RI strains, obtaining SDPs in lines D, E, G, H, I, J, and K of CBBCBBB respectively, identical to that of Ly-l. Yamaguchi and colleagues suggested that Pcna-rsl maps to Chr 17, based on the SDP. However, when the resuits from both sets of RI lines are combined, the SDPs for Pcna-rsl and Ly-1 are identical in 19 RI lines, and thus the most likely map position for Pcna-rsl is the proximal region of Chr 19.
Discussion
The PCR-based assay for Pcna-rsl provides an easily scorable polymorphic locus on Chr 19, with the proviso that the pseudogene is present in one of the strains used in the backcross and absent in the other, to which the F 1 animals are backcrossed. So far, the
Aora 9 1 4 9 II-lb 9 Pcna B I D I B I I B I B No. of animals
1
2
2
2
51 30 1
[]
AN allele
9
Mus staretus allele
9
Not tested
1
1] 6
Fig. 3. Analysis of b a c k c r o s s progeny for Acra, II-lb, and Pcna. A total of 106 mice were analyzed for Acra, 105 for l l - l b , and 109 for Pcna. R e c o m b i n a t i o n frequencies found were 19/102 b e t w e e n Acra and II-lb, 2/105 b e t w e e n l l - l b and Pcna, and 21/106 b e t w e e n Ac r a and Pcna.
Table 2. SDPs of (a) Pcna in the BXH RI lines, together with the tl-la SDP (D'Eustachio et al. 1987) and (b) Pcna-rsl in the BXH RI lines, together with the Ly-1 SDP (Hogarth et al. 1988). (a) BXH line
2
3
4
6
7
8
9
10
11
12
14
19
Ilila Pcna
]] B
B B
H H
H H
B B
H H
B B
H H
B B
H H
H H
B B
(b) BXH line
2
3
4
6
7
8
9
10
11
12
14
19
H H
H H
H H
B B
B B
H H
H H
B B
B B
B B
H H
H H
Ly-1 Pcna-rsl
PCR assay for the Pcna polymorphism can be used only to map Pcna in backcrosses involving C3H/HeJ or stocks (such as AN) derived from C3H, as has been described. However, it is possible that by re-designing the PCR primers so that they more closely flank the microsatellite-like sequence in intron 4, followed by subjection of the PCR products to high-resolution polyacrylamide gel electrophoresis, length variation of the PCR product could be detected in other strains of mice. The mapping of Pcna to mouse Chr 2 extends the known region of homology between mouse Chr 2 and human Chr 20. To date, 10 loci on lISA 20 that have been firmly mapped in the mouse have been found to reside on MMU 2 (Davisson et al. 1991). The positioning of Pcna on the consensus map of MMU 2 supports the localization in the human to 20p13, since Pcna maps to the same region of MMU 2 as Prn and Itp, the human homologs of both of which map to HSA 20p. A number of phenotypic mutants map to the same region of MMU 2 as Pcna (reviewed in Siracusa and Abbott 1991). These include welhaarig (we; Falconer 1954), mahogany (mg; Lane and Green 1960), and tight-skin (Tsk; Green et al. 1976). Pcna should provide a useful additional molecular marker for mapping studies aimed at cloning these mutants. Other genes that code for proteins involved in control of the cell cycle have also recently been mapped (Sakaguchi et al. 1991). Cyclin genes and sequences homologous to cyclin genes have been mapped to mouse Chrs 3, 4, 6, 10, and 15 by cross-hybridization with two cloned probes for human CDC2. These data, together with the mapping of Pcna to MMU 2, show the genes involved in cell cycle control to be scattered throughout the genome. Disruption of these genes in the mouse by homologous recombination could lead to valuable insights into the control of cell replication and, potentially, the basic mechanisms involved in carcinogenesis. A c k n o w l e d g m e n t . W e are very grateful to Dr. Ben Taylor for his
help in analyzing the data from the B X H RI strains.
References Aitman, T., Hearne, C., McAleer, M., and Todd, J.: M o n o n u c l e otide repeats are an a b u n d a n t source of length variants in m o u s e genomic D N A . M a m m a l i a n G e n o m e 1: 206-210, 1991. Almendral, J., H u e b s c h , D., Blundell, P., M c D o n a l d - B r a v o , H . , and Bravo, R.: Cloning and s e q u e n c e of the h u m a n nuclear pro-
C. Abbott et al.: Pcna maps to mouse Chr 2 tein cyclin: homology with DNA-binding proteins. Proc Natl Acad Sci USA 84: 1575-1579, 1987.
Bravo, R. and McDonald-Bravo, H.: Induction of the nuclear protein 'cyclin' in quiescent mouse 3T3 cells stimulated by serum and growth factors. Correlation with DNA synthesis. EMBO J 3: 3177-3181, 1984. Davisson, M.T., Lalley, P.A., Peters, J., Doolittle, D.P., Hillyard, A.L., and Searle, A.G.: Report of the comparative committee for human, mouse and other rodents (HGM 11). Cytogenet Cell Genet 58: 1152-1189, 1991. D'Eustachio, P., Jadidi, S., Fuhlbrigge, R., Gray, P., and Chaplin, D.: Interleukin-1 a and 13genes: linkage on chromosome 2 in the mouse. Immunogenetics 26: 339-343, 1987. Falconer, D.: Linkage in the mouse: the sex-linked genes and 'rough'. Z Indukt Abstammungs-Vererbungsl 86: 263-268, 1954. Green, M., Sweet, H., and Bunker, L.: Tight-skin, a new mutation of the mouse causing excessive growth of connective tissue and skeleton. Am J Pathol 82: 493-512, 1976. Hogarth, P., Houlden, B., Latham, S., Cherry, M., Taylor, B., and McKenzie, I.: The mouse Ly-12.1 specificity: genetic and biochemical relationship to Ly-1. Immunogeneties 27: 383-387, 1988. Jaskulski, D., deRiel, J., Mercer, W., Calabretta, B., and Baserga, R.: Inhibition of cellular proliferation by antisehse oligodeoxynucleotides to PCNA Cyclin. Science 240: 1544-1546, 1988. Ku, D., Travali, S., Calabretta, B., Huebner, K., and Baserga, R.: H u m a n gene for proliferating cell n u c l e a r antigen has pseudogenes and localizes to chromosome 20. Somatic Cell Mol Genet 15: 297-307, 1989. Lane, P. and Green, M.: Mahogany, a recessive colour mutation in linkage Group V of the mouse. J Hered 51: 228-230, 1960. Love, J., Knight, A., McAleer, M., and Todd, J.: Towards con-
289 struction of a high resolution map of the mouse genome using PCR-analysed microsatellites. Nucleic Acids Res. 18: 4123--4130, 1990. Pilz, A., Fox, M., Povey, S., and Abbott, C.: A panel of somatic cell hybrids with translocation breakpoints on mouse chromosome 2 and their characterization by PCR. Mammalian Genome 1 (Suppl): $517, 1991. Pilz, A., Moseley, H., Peters, J. and Abbott, C.: Comparative mapping of mouse chromosome 2 and human chromosome 9q: the genes for gelsorin and dopamine 13 hydroxylase map to mouse chromosome 2. Genomics 12: 715-719, 1992. Sakaguchi, A., Martinez, L., Lalley, P., Sylvia, V., Han, E., Killary, A., and Ghosh Choudhury, G.: Chromosome mapping in mouse of genes that regulate the eukaryotic cell cycle. Cytogenet Cell Genet 58: 2138, 1991. Siracusa, L. and Abbott, C.: Mouse Chromosome 2. Mammalian Genome: I: S15-$41, 1991. Webb, G., Parsons, P., and Chenevix-Trench, G.: Localization of the gene for human proliferating cell nuclear antigen/cyclin by in situ hybridization. Hum Genet 86: 84-86, 1990. Yamaguchi, M., Nishida, Y., Moriuchi, T., Hirose, F., Hui, C-C., Suzuki, Y., and Matsukage, A.: Drosophila proliferating cell nuclear antigen (cyclin) gene: structure, expression during development, and specific binding of homeodomain proteins to its 5' flanking region. Mol Cell Biol 10: 872-879, 1990. Yamaguchi, M., Hayashi, Y.Y., Hirose, F., Matsuoka, S., Moriuchi, T., Shiroishi, T., Moriwaki, K., and Matsukage, A.: Molecular cloning and structural analysis of mouse gene and pseudogenes for proliferating cell nuclear antigen. Nucleic Acids Res 19: 2403-2410, 1991.
