Mammalian Genome 3:112-118, 1992
9 Springer-VerlagNew York Inc. 1992
Characterization of a new hybrid mink-mouse clone panel: chromosomal and regional assignments of the GLO, ACY, NP, CKBB, ADH2, and ME1 loci in mink (Mustela vison) Svetlana D. Pack, Vladimir M. Bedanov, Olga V. Sokolova, Natalia S. Zhdanova, Natalia M. Matveeva, and Oleg L. Serov Laboratory of Developmental Genetics, Institute of Cytology and Genetics, Academy of Sciences of the USSR, Siberian Branch, 630090, Novosibirsk-90, Russia Received August 26, 1991; accepted December 2, 1991
Abstract. To expand the mink map, we established a new panel consisting of 23 mink-mouse clones. On the basis of statistical criteria (Wijnen et al. 1977; Burgerhout 1978), we developed a computer program for choice of clones of the panel. Assignments of the following mink genes were achieved with the use of the hybrid panel: glyoxalase (GLO), Chromosome (Chr) 1; acetyl acylase (ACY), Chr 5; creatine phosphokinase B (CKBB), Chr 10; alcohol dehydrogenase-2 (subunit B) (ADH2), Chr 8. Using a series of clones carrying rearrangements involving mink Chr 1 and 8, we assigned the gene for ME1 to the short arm of Chr 1 and that for ADH2 to Chr 8, in the region 8p12-p24. Mapping results confirm the ones we previously obtained with a mink-Chinese hamster panel. However, by means of an improved electrophoretic technique, we revised the localization of the gene for purine nucleoside phosphorylase (NP), which has been thought to be on mink Chr 2. It is reassigned to mink Chr 10.
Introduction The genetic map of mink now includes more than 60 biochemical markers, of which 16 were assigned by regional localization (Serov and Pack 1990). Furthermore, two linkage groups located on Chrs 7 and 9, respectively, were identified (Serov et al. 1987; Yermolaev et al. 1989). In most of these localizations we used a panel of American mink-Chinese hamster clones (Rubtsov et al. 1981a; Serov et al. 1987). To expand the mink map, we set up a new minkmouse hybrid clone panel. We thereby hoped to confirm and revise our previous gene assignments made Offprint requests to: S.D. Pack
with the use of the mink-Chinese hamster clones. A characterization of the new mink-mouse clone panel, its efficiency, and prospects as a mapping tool are presented. By means of the new panel, we determined the chromosomal localization of the genes for ACY, CKBB, GLO, and ADH2 and also checked the accuracy of our previous assignments of ten loci based on the mink-Chinese hamster panel. The revised localization of the mink NP gene is now on Chr 10; it had been previously localized to Chr 2; the regional localization for the ME1 gene is on the short arm of Chr 1 and that for the ADH2 gene is on Chr 8, in the region 8p12-p24.
Materials and methods
Cell lines and cultivation conditions Somatic cell hybrids obtained by fusion of mouse cells L M T K (series LM) or A9 (series AB) with mink bone marrow cells were used (Pack et al. 1989). All the hybrid clones were cultivated in selective HAT medium (13.6 ~g/ml hypoxanthine; 0.4 ~g/ml aminopterin; 12.5 p~g/ml thymidine; and 7.5 I~g/ml glycine (Littlefield 1964) supplemented with 10% calf fetal serum.
Cytogenetic analysis of mink-mouse clones Preparation and G-banding of the metaphase chromosomes were as described (Pack et al. 1989). To produce prometaphase chromosomes, ethidium bromide (5-8 ~g/ml) (Ikeuchi 1984) was added to the culture 2 h before fixation. During the last 10 min, colcemid (0.07 p~g/ml) was added, followed by hypotonic treatment with 0.56% KC1 and fixation in 3:1 methanol/acetic acid. The fixative was renewed four times; the cells were carefully resuspended each time the fixative was changed. The first cytogenetic analysis of the hybrid clones was based on examination of 15-25 metaphase plates. A clone was discarded when a chromosome rearrangement was detected in 5-10 metaphase
S.D. Pack et al.: N e w hybrid m i n k - m o u s e clone panel
113
Table 1. List of isozyme markers examined in mink-mouse hybrids.
Isozyme
Chromosomal localization
Conditions of electrophoretic analysis
ME1 PP PEPA IDH1 SOD1 LDHA LDHB NP MDH1 PEPC G6PD ESD GLO ACY PEPS PEPD APRT UMPH2 ADH2
malate dehydrogenase-1 (NADP dependent) inorganic pyrophosphatase peptidase A isocitrate dehydrogenase- 1 superoxide dismutase-1 lactate dehydrogenase A lactate dehydrogenase B purine nucleoside phosphorylase malate dehydrogenase-1 (NAD dependent) peptidase C glucose-6-phosphate dehydrogenase esterase D glyoxalase acetyl acylase peptidase S peptidase D adenine phosphoribosyltransferase uridine monophosphate phosphohydrolase-2 alcohol dehydrogenase B
1 2 4 4 5 7 9 10 11 13 X 8 1 5 6 7 7 8 8
Rubtsov et al. 1981a, b, 1982
CKBB
creatine phosphokinase B
10
PGP PSP
phosphoglycolate phosphatase phosphoserine phosphatase
14 14
plates; the second analysis of the clones for setting up the panel was based on examination of 30 metaphase or prometaphase spreads.
Analysis of isozyme markers The list of isozyme markers examined in hybrid cells, as well as conditions of analysis, are given in Table 1.
" " " " " Gradov et al. 1985 Parr et al. 1977 Mullakandov et al. 1986 " " Nesterova et al. 1987 Wilson et al. 1986 Holmes 1979; Parr et al. 1977 Chern et al. 1980; Povey et al. 1979 Baker and Hopkinson 1978 Koch et al. 1983
much more than five clones showing discordant segregation, which made up 22% of the total clone number, the highest value being 87% when 20 clones were discordant (Table 2). The efficiency of the panel was estimated by localization of the genes for ME1, PP, PEPA, PEPC, IDH1, SOD1, LDHA, LDHB, G6PD, MDH1, NP, and
Results and discussion
To establish the clone panel, we used 82 clones of LM and 19 clones of AB series. All the clones were of independent origin. The results of our previous cytogenetic and biochemical analyses demonstrated that the only segregating chromosomes were mink (Pack et al. 1989). Chromosome rearrangements of mink chromosomes were detected in 21 of 78 hybrid clones (27%) by cytogenetic analysis, and these were excluded from further analysis. After the first step, characterization of chromosome constitution, 52 clones that seemed promising for setting up the panel were chosen. A computer program based on the criteria of Wijnen and colleagues (1977) and Burgerhout (1978) guided our choice of the clones. As a result, a panel consisting of 23 hybrid clones was established (Fig. 1). The condition to be met in the choice was not less than five clones with any chromosome pair segregating discordantly. Estimation of the efficiency of an established clone panel can be based on the determination of the chromosomal localization of the relevant genes. This is feasible when a marker and a chromosome segregate concordantly in the majority of the clones in a panel, and, in the ideal situation, there would be no clones with a detectable chromosome and an absent relevant gene. For most chromosome pairs, the panel contained
Fig. 1. Distribution of mink c h r o m o s o m e s in the panel of hybrid m i n k - m o u s e clones. Designations for mink c h r o m o s o m e : filled square, presence; open square, absence. Left, designations of individual clones.
114
S.D. P a c k et al.: N e w hybrid m i n k - m o u s e clone panel
Table 2. Discordancy percentage in a mink-mouse clone panel for paired comparisons of mink chromosomes. Chr B ChrA
1
1
0
2 3 4 5 6 7 8 9 10 11 12 13 14 X
57 43 39 43 30 35 47 30 56 39 26 30 35 56
2
3
4
5
6
7
8
9
10
11
12
13
14
0 30 52 83 39 47 43 70 52 52 56 52 56 43
0 30 61 26 43 22 56 39 47 52 47 35 65
0 47 47 47 35 43 52 43 47 43 39 52
0 70 43 47 30 30 56 43 30 61 47
0 43 47 52 56 39 35 47 43 56
0 30 30 30 47 43 39 43 56
0 35 35 61 70 52 39 87
0 52 52 39 43 56 61
0 61 56 43 39 52
0 22 61 56 52
0 39 52 30
0 47 35
65
ESD, which we have previously mapped by means of mink-Chinese hamster hybrids (Serov et al. 1987) As Fig. 2 shows, complete concordant segregation was observed for 9 of the 12 isozyme markers; MEI, Chr 1; PP, Chr 2; PEPA and IDHI, Chr 4; SOD1, Chr 5; ESD, Chr 8; LDHB, Chr 9; MDH1, Chr 11; and G6PD, Chr X. A low discordancy level of 4% (a single discordant clone of the studied 23) was observed in two instances: LDHA, Chr 7; PEPC, Chr 13. The complete concordant segregation of the NP gene with Chr 10 (Fig. 2) deserves comment. This gene is located on Chr 2 according to previous data (Rubtsov et al. 1982). However, in further analysis of a series of subclones of a mink-mouse hepatoma hy-
X
0
0
brid clone carrying a rearrangement of Chr 2, we revealed discordant segregation of NP with Chr 2, as well as that of NP with the other markers of this chromosome (Serov et al. 1987). In analysis of the subclones of another mink-mouse hepatoma clone, we observed apparent cosegregation of NP with PP, hexokinase-1 (HK1), and glutamate-oxaloacetate transaminase-1 (GOT1). The cytogenetic analysis in both series of the subclones was, regretfully, incomplete (Serov et al. 1987). Thus, in the previous study we could not explain adequately the discordancy between NP and Chr 2 and its enzyme markers (Serov et al. 1987). Figure 3A compares the electrophoretic NP pat-
Fig. 2. A synopsis of c h r o m o s o m a l localizations. T w e n t y - t w o ge ne s l o c a l i z e d w i t h t he use of 23 m i n k - m o u s e hybri d clones. Left, clone designations; top, ge ne s y m b o l s ; bottom, c h r o m o s o m e n u m b e r ; filled s qua re , p r e s e n c e of c h r o m o s o m e and ma rke r; open square, a b s e n c e of c h r o m o s o m e and m a r k e r ; hatched square, d i s c o r d a n t clones: marker+/chromosome - (+) and marker-/ c h r o m o s o m e + ( - ).
S.D.
Pack
et al.:
New
Fig. 3.
Electrophoretic
ADH2
(D) in parent
ster
(1); channel hybrid
clone
hybrid
clone
2, mouse;
mink
ACY;
ACY.
(C) Channel
mouse mouse
hybrid
channel for mink for mink
channel
2, mink;
ADH2; ADH2.
for
mink
negative
channels
for
hybrid
2, mink;
for mink
channels
CKBB; CKBB. ( D )
negative
for
for mink
3,5,6, mink-
channel
4,
channel
1, m o u s e ;
hybrid
4,5, mink-mouse
a mink1, m i n k ;
positive
mink
ham-
4, a mink-
clone
clone
3,6, mink-mouse
channels
channel
clones
hybrid
bearing Chr 10) and LM48 (30% of cells bearing it) were lower than in the remaining cells positive for mink NP (the percentage of cells with Chr 10 varied from 80% to 123% in these clones). Two discordant mink-Chinese hamster clones, L26 (NP +/Chr 2 -) and F9B (NP -/Chr 2 + ), are noteworthy (Rubtsov et al. 1982; Serov et al. 1987) in that mink Chr 10 is present in the former and absent in the latter. The efficiency of the mink-mouse panel was tested with the use of 11 markers (ME1, PP, PEPA, PEPC, IDH1, SOD1, LDHA, LDHB, G6PD, MDH1, and ESD) with known chromosomal localization. Of the 253 tested marker-chromosome combinations, just 2 segregated discordantly. This less than 1% discordancy frequency testifies to the efficiency of the panel. The revised localization of the NP gene on Chr 10, with reliance on the new panel, appears to be more correct than the previous localization on Chr 2 with the use of the mink-Chinese hamster panel. The more recent analyses of NP in a series of mink-Chinese hamster clones confirmed that NP is, indeed, located on Chr 10 (Table 3). The new panel was utilized for determining the chromosomal localization of the genes for ACY, GLO, CKBB, and ADH2. As seen in Fig. 2, the expression of mink GLO is concordant with the presence of mink Chr 1 and its marker ME1 in all the 23 clones of the mouse-mink panel. It may be inferred that the GLO gene is located on mink Chr 1. The distribution for ACY in the cells of mouse, mink, and several mouse-mink clones is shown in Fig. 3B. From the data of Fig. 2 it follows that there is a 100% concordancy segregation of the mink ACY gene with Chr 5 and its marker, SOD1. Analysis of mink ACY in the mink-Chinese hamster clone panel also provided evidence for the assignment of the AC Y gene to mink Chr 5. The electrophoretic patterns of CKBB from mink, mouse, and hybrid clone cells are shown in Fig. 3C. CKBB activity was not detected in the L M T K - and A9 cells under the conditions used, as in the case reported by Povey and co-workers (1979). For this reason, the clones negative for mink CKBB show no
(C), and
1, C h i n e s e
NP; a n d c h a n n e l 5 , NP. ( B ) C h a n n e l
hybrid
channel
positive
115
ACY(B), CKBB
3, mouse;
3, mink-mouse
1, m o u s e ;
clone
(A),
(A) Channel
4, mink-mouse
clones
panel
cells.
for mink
negative
clone
ofNP
channel
positive
channel
hybrid
hybrid
2, mink;
mouse
mink-mouse
patterns and
mouse channel
hybrid
mink-
positive
clones
negative
terns yielded by mink, mouse, Chinese hamster, and several mink-mouse hybrids. Clearly, electrophoresis separates mink and mouse NP much better than mink and Chinese hamster NP. In this way, we obtained assurance that NP segregates concordantly with mink Chr 10, and not with mink Chr 2 (Fig. 2). We have further analyzed eight additional mink-mouse clones not included in the established mink-mouse clone panel. Here again, the concordancy results are consistent with the assignment of NP to mink Chr I0. There was another line of evidence for the association of the NP gene and mink NP activity. The evidence came from the relation between the activity level of mink NP and number of cells carrying Chr 10 in the clones. Thus, judging by visual estimation, the activity levels of mink NP in clones LM7 (23% of cells
T a b l e 3. S e g r e g a t i o n o f the m i n k
NP, ADH2, ACY1, a n d
Mink marker
mink chromosomes
Chromosome
in t h e m i n k - C h i n e s e
hamster cell hybrid clones. NT means not tested.
of mink
Clone
NP
ADH2
ACY
1
2
3
4
5
6
7
8
9
10
II
12
13
14
X
F3M F12B-1 FD9M KO2-1 L22-1 KOI-I D7BI D12M D13M K12-1 L15-1 L25-1 R14-1 D3M DllB
+ + + + + + + + + + NT
+ + + + + + + . NT
+ + + + + . +
+ + + + + + + + . .
+ + + -
+ + . + + + + +
+ +
+ + . + + +
+ -
+ -
+ + + + + + _ + + +
+ _ + +
+ + _ + + + _ + _ -
+ + + + + + + + + ~ _ _ _ +
+ _ + _ + + + . + + + + + .
_ _ + _ _ + + _
_ + + + + _ + +
+ _ + + + + _ _
+ _ + + +
_ + _ + _
+ + + + _ + _ _ + + + _ _ _ +
+ + + + + + + + + + + + + + +
.
. .
.
. .
+ .
.
. .
.
. + + + + + +
.
. .
.
. + .
.
-
.
.
.
.
+ + _ _ _ .
.
.
116
S . D . P a c k et al.: N e w h y b r i d m i n k - m o u s e
F i g . 4. E l e c t r o p h o r e t i c A D H p a t t e r n s in d i f f e r e n t m i n k t i s s u e s : s t o m a c h (1,4), k i d n e y (2,5), a n d M V c e l l s (3,6). 1 , 2 , 3 - - s t a i n i n g w i t h the use of butanol as a substrate; 4,5,6---staining with the use of e t h a n o l as a s u b s t r a t e . ADH1, ADH2, a n d A D H 3 a r e A D H i s o z y m e s having presumably subunit compositions AA, BB, and CC, respect i v e l y . It is s e e n t h a t M V cells c o n t a i n ADH2 i s o z y m e .
CKBB activity, while those positive for it displayed CKBB activity of the mink type (Fig. 3C). The results of segregation analysis of mink chromosomes and CKBB in the hybrid clone cells are summarized in Fig. 2. CKBB segregated concordantly with mink Chr I0, as well as its MDH1 marker in all 23 hybrid clone cells. Based on the obtained results, the CKBB gene may be assigned to mink Chr 10. The ADH patterns were obtained from mink tissues and MV cells. When butanol was used as substrate, three forms of ADH are revealed: one anodal B (stomach), two cathodal A (liver, kidneys), and C, with low activity (stomach) (Fig. 4). When the substrate was ethanol, no ADHB activity was detectable (Fig. 4). This is the feature distinguishing the B from the A and C forms of ADH after incubation with ethanol. The electrophoretic patterns of ADH2 from mink, mouse, and a number of hybrid clone cells are presented in Fig. 3D; mink ADH2 activity is detectable, whereas the activity of mouse ADH2 is undetectable in L M T K - and A9 cells. Mink ADH2 is clearly seen in the hybrid clones positive for it, and mink ADH2 is missing in those negative for it. Figure 2 shows how the activity of mink ADH2 is distributed in the hybrid clone cells. As also evident from Fig. 2, in 96% of cases the presence (or absence) of mink ADH2 in the hybrid mink-mouse clones is concordant with the presence (or absence) of mink Chr
clone panel
8. It may be important that, in all the clones positive for mink ADH2, the activities of UMPH2 and ESD, the other markers of mink Chr 8, were also detected. Furthermore, hybrid clone LM6, which contained in the background of mouse chromosomes a single mink Chr 8, was also positive for ADH2 (Table 4). Analysis of ten more clones not included in the panel and of the mink-Chinese hamster clone panel (Table 3) persuasively confirmed concordancy between ADH2 and Chr 8. Thus, taken together, the results demonstrate the localization of the ADH2 on mink Chr 8. In our efforts to localize the ADH2 gene to mink Chr 8, we analyzed transformant clones obtained by transfer of mink genes to mouse cells (Sukoyan et al. 1984) and also mouse-mink clones in which different rearrangements involving mink Chr 8 were detected (Pack et al. 1989). The translocation t(1;8) (lqterlcen::8cen-8pter) identified in clone LM24 involved the apparently intact long arm of Chr 1 and the short one of Chr 8 (Fig. 5). The breakpoints are located in the centromeric regions of the corresponding chromosomes. LM55 contained translocation t(1;8)(lqterlq21::8q24-p26) (Fig. 5), which involved translocation o f the long arm of Chr 1 to Chr 8, whose breakpoint was on segment 8q24. Both clones, LM24 and LM55, showed activity of ADH2 (Table 4). The results of segregation analysis of the enzyme markers of Chr 1 and 8 and their fragments are summarized in Table 4. Chromosomal localization of the TKI, GALK (galactokinase), ALDC (aldolase C), and ESD genes was determined with the use of minkChinese hamster hybrid clones (Gradov et al. 1985), and that of the UMPH-2 and ADH2 genes with the panel of mink-mouse clones (Pack et al. 1989; this communication). From the data for the segregation of the markers and fragments of mink Chr 8 (Table 4) it follows that ADH2 activity correlates with the retention of the integrity of a region 8p24-p12 of the short arm of Chr 8. Fragment 8pter-pl2 was present in clone STT13-1 cells expressing mink A D H 2 . Clones STT16-8 and STT16-3, which contained respectively regions p25-cen and p24-q24 of mink Chr 8, as well as STT13-1, showed expression of ADH2. This suggested that the ADH2 gene is localized in the 8p24-p12 region of mink Chr 8. The genes in the short arm of Chr 8 may b e o r d e r e d as c e n - ( A D H 2 , T K 1 ) - G A L K (ALDC,HOX-2)-UMPH2 (Fig. 6). We further regionally localized the gene for ME1, which resides on mink Chr 1. The clones used for this
Table 4. Cytogenetic and biochemical characterization of hybrid and transformed mouse-mink clones containing different fragments of mink Chrs 1 and 8. Enzyme markers of mink Clone
Chr 8 and its fragments
TK1
GALK
ALDC
UMPH-2
ESD
ADH2
ME1
LM6 STT13-1 a STT16-8 a STT16-3 a LM24 LM55
intact pter--pl2 p25--cen p24---q24 t(1;8)(lqter-lcen::8cen-8pter) t(1 ;8)(lqter-I q21 ::8q24-8p26)
+ + + + + +
0 + + . 0 0
+ + -
+ + -
+ -
+ -
-
+ + + + 4 +
0 0 0 0 -
Presence ( + ) or absence ( - ) of marker; (0) not examined. a For detailed cytogenetic characterization of the clones, see Gradov et al. 1985.
.
. + +
.
S.D. Pack et al.: New hybrid mink-mouse clone panel
Fig. 5. Intact mink Chr 8 (1) and 1 (4) and translocations t(1;8) detected (3) in LM24 t(l ;8)(lqter-lcen::8cen-8pter) and (2) in LM55 t(1 ;8)(lqter-lq21::8q24-8p26). Numbers 2 and 3 on the chromosomes denote break-points in clones LM55 and LM24.
117
tical criteria (Wijnen et al. 1977; Burgerhout 1978). With the use of the mink-mouse hybrid clones, we localized ten new mink genes to individual chromosomes; of these ten, localization of four genes--GLO, ADH2, ACY, and CKBB is described here (Fig. 2), and that of six others was done in our previous report (Pack et al. 1989; Fig. 2). Chromosomal localizations for ACY, ADH2, and PGP were amply supported by analysis of the mink-Chinese hamster hybrid panel (Pack et al. 1989; Table 3). Circumstantial evidence was obtained allowing us to reassign the NP gene to mink Chr 10. Independent confirmation of assignments, which we have achieved with a mink-Chinese hamster panel for 11 genes, was obtained. This comparative approach is advantageous because, to our
purpose were LM24 and LM55 containing translocations made up of parts of Chr 1 and 8 (Table 4). LM24 contains the entire long arm of Chr 1, and LM55 the distal part of the long arm, qter-q21 (Fig. 5). No ME1 activity was detectable in the two clones (Table 4). This suggested the assignment of the ME1 gene to the short arm of mink Chr 1 (Fig. 7). Thus, to map mink chromosomes, we set up a panel of 23 hybrid mink-mouse clones fulfilling statis-
m
4.3
2
i
!
U]V[PH-2 ALDC,HOX-2
]
TKI
8,3
7.2
P 2
GALK
4.1 4 . 2 2.3
3
2.1
2.2
1.2
cx>o~
ADH-2
1
t
!
U~l
5
1.2
2 1.2
I
1
1.1
1 , 1 /~x,. 1.2
11 q
2.1 2.1 2.3
tl
2
1
2 , 2 2.1 2.3
2
3
i
4.1
,
4.2
!
1 i
4.3
i
5
.
I~D
Fig. 6. Subchromosomal localization of the gene for A D H 2 on the short arm of mink Chr 8.
3
Fig. 7. Subchromosomal localization of the gene for ME1 on mink Chr 1.
118
knowledge, there is no other strategy for directly checking the correctness of gene mapping. The segregation of each of the 22 markers to specific chromosomes provided us with a basis for estimation of the efficiency of the mink-mouse panel (Fig. 2). The number of hybrid clones tested for panel efficiency was 23. Of the 506 marker-chromosome combinations, only in 5 (less than 1%) was the segregation discordant, thereby yielding a 99% efficiency for the panel. This produces confidence in the mink-mouse panel as a tool in mink gene mapping. Acknowledgments. The authors are indebted to A. Fadeeva for translation of the paper from Russian into English and to I.B. Nosatova for technical assistance.
References Baker, R.F. and Hopkinson, D.A.: Genetic polymorphism of human phosphoglycolate phosphatase (PGP). Ann Hum Genet 42: 143151, 1978. Burgerhout, W.G.: A standard procedure for gene assignment by use of somatic cell hybrids. Cytogenet Cell Genet 22: 689--693, 1978. Chern, C.J., Tan, P., and Park, H.: Chromosomal mapping of human creatine kinase (brain type) using human-rodent somatic cell hybrids. Cytogenet Cell Genet 27: 232-237, 1980. Gradov, A.A., Pack, S.D., Sukoyan, M.A., Rubtsov, N.B., Bochkarev, M.N., Serov, O.L., Rattazzi, O., Scandalios, J.G., Whitt, G., and Markert, C.L.: Regional assignment of the genes for TK1, GALK, ALDC and ESD on chromosome 8 in the American mink by chromosome-mediated gene transfer. Mol Gen Genet 200: 433--438, 1985. Holmes, R.S.: Genetics and ontogeny of alcohol dehydrogenase isozymes in the mouse: evidence for a cis-acting regulator gene (Adt-1) controlling C2 isozyme expression in reproductive tissues and close linkage of Adh-3 and Adt-1 on chromosome 3. Biochem Genet 17: 461--472, 1979. Ikeuchi, T.: Inhibitory effect of ethidium bromide on mitotic chromosome condensation and its application to high-resolution chromosome banding. Cytogenet Cell Genet 38: 56--61, 1984. Koch, G.A., Eddy, R.L., Haley, L.L., Byers, M.G., McAvoy, M., and Shows, T.B.: Assignment of the human phosphoserine phosphatase gene (PSP) to the pter-q22 region of chromosome 7. Cytogenet Cell Genet 35: 67-69, 1983. Littlelield, J.W.: Selection of hybrids from matings of fibroblasts in vitro and their presumed recombinants. Science 145: 709-710, 1964. Mullakandov, M.R., Gradov, A.A., Zakijan, S.M., Rubtsov, N.B., and Serov, O.L.: Peptidases A, B, C, D and S in the American
S.D. Pack et al.: New hybrid mink-mouse clone panel mink: polymorphism and chromosome localization. Theor Appl Genet 73: 272-277, 1986. Nesterova, T.B., Borodin, P.M., Zakijan, S.M., and Serov, O.L.: Assignment of the gene for adenine phosphoribosyltransferase on the genetic map of mouse chromosome 8. Biochem Genet 25: 563-568, 1987. Pack, S.D., Zhdanova, N.S., Sukoyan, M.A., and Serov, O.L.: Chromosomal and regional localization of the genes for UMPH2, APRT, PEPD, PEPS, PSP and PGP in mink: comparison with man and mouse. Cytogenet Cell Genet 50: 127-131. 1989. Parr, C.W., Bagster, I.A., and Welch, S.G.: Human red cell glyoxalase I polymorphism. Biochem Genet 15: 109-113, 1977. Povey, S., Inwood, M., Tanyar, A., and Bobrow, M.: The expression of creatine kinase isozymes in human cultured cells. Ann Hum Genet 43: 15-26, 1979. Rubtsov, N.B., Radjabli, S.I., Gradov, A.A., and Serov, O.L.: Chinese hamster • American mink somatic cell hybrids: characterization of a clone panel and assignment of the mink genes for malate dehydrogenase, NADP-1 and malate dehydrogenase, NAD-1. Theor Appl Genet 60: 99-106, 1981a. Rubtsov, N.B., Radjabli, S.I., Gradov, A.A., and Serov, O.L.: Chromosome localization of the three syntenic gene pairs in the American mink (Mustela vison). Cytogenet Cell Genet 31: 184187, 1981b. Rubtsov, N.B., Gradov, A.A., and Serov, O.L.: Chromosome localization of the loci GOTI, PP, NP, SODI, PEPA and PEPC in the American mink (Mustela vison). Theor Appl Genet 63: 331336, 1982. Serov, O.L. and Pack, S.D.: American mink (Mustela vison). In S.J. O'Brien (ed.), Genetic Maps (Locus Maps of Complex Genomes), pp. 4126--4128, 5th edition, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y., 1990. Serov, O.L., Gradov, A.A., Rubtsov, N.B., Zhdanova, N.S., Pack, S.D., Sukoyan, M.A., Mullakandov, M.R., and Zakijan, S.M.: Genetic map of the American mink: gene conservation and organization of chromosomes. In Isozymes: Current Topics in Biological and Medical Research, vol. 15, Genetics, Development, and Evolution, pp. 179-215, Alan R. Liss Inc., New York, 1987. Sukoyan, M.A., Matveeva, N.M., Belyaev, N.D., Pack, S.S., Gradov, A.A., Shilov, A.G., Zhdanova, N.S., and Serov, O.L.: Cotransfer and phenotypical stabilization of syntenic and asyntenic mink genes into mouse cells by chromosome-mediated gene transfer. Mol Gen Genet 196: 97-104, 1984. Wijnen, L.M,M., Grzeschik, K.H., Pearson, P.L., and Meera Khan, P.: The human PGM-2 and its chromosomal localization in man-mouse hybrids. Hum Genet 37: 271-278, 1977. Wilson, D.E., Swallow, D.M., and Povey, S.: Assignment of the human gene for uridine 5'-monophosphate phosphohydrolase (UMPH2) to the long arm of chromosome 17. Ann Hum Genet 50: 223-227, 1986. Yermolaev, V.I., Karasik, G.I., Khlebodarova, T.M., Matveeva, N.M., MuUakandov, M.R., Nayakshin, A.M., Shumny, T.V., Rubtsov, N.B., Serov, O.L., and Baranov, O.K.: Localization of the a2-macroglobulin gene and Lpm gene family on mink chromosome 9. Theor Appl Genet 78: 93-96, 1989.
9 Springer-VerlagNew York Inc. 1992
Characterization of a new hybrid mink-mouse clone panel: chromosomal and regional assignments of the GLO, ACY, NP, CKBB, ADH2, and ME1 loci in mink (Mustela vison) Svetlana D. Pack, Vladimir M. Bedanov, Olga V. Sokolova, Natalia S. Zhdanova, Natalia M. Matveeva, and Oleg L. Serov Laboratory of Developmental Genetics, Institute of Cytology and Genetics, Academy of Sciences of the USSR, Siberian Branch, 630090, Novosibirsk-90, Russia Received August 26, 1991; accepted December 2, 1991
Abstract. To expand the mink map, we established a new panel consisting of 23 mink-mouse clones. On the basis of statistical criteria (Wijnen et al. 1977; Burgerhout 1978), we developed a computer program for choice of clones of the panel. Assignments of the following mink genes were achieved with the use of the hybrid panel: glyoxalase (GLO), Chromosome (Chr) 1; acetyl acylase (ACY), Chr 5; creatine phosphokinase B (CKBB), Chr 10; alcohol dehydrogenase-2 (subunit B) (ADH2), Chr 8. Using a series of clones carrying rearrangements involving mink Chr 1 and 8, we assigned the gene for ME1 to the short arm of Chr 1 and that for ADH2 to Chr 8, in the region 8p12-p24. Mapping results confirm the ones we previously obtained with a mink-Chinese hamster panel. However, by means of an improved electrophoretic technique, we revised the localization of the gene for purine nucleoside phosphorylase (NP), which has been thought to be on mink Chr 2. It is reassigned to mink Chr 10.
Introduction The genetic map of mink now includes more than 60 biochemical markers, of which 16 were assigned by regional localization (Serov and Pack 1990). Furthermore, two linkage groups located on Chrs 7 and 9, respectively, were identified (Serov et al. 1987; Yermolaev et al. 1989). In most of these localizations we used a panel of American mink-Chinese hamster clones (Rubtsov et al. 1981a; Serov et al. 1987). To expand the mink map, we set up a new minkmouse hybrid clone panel. We thereby hoped to confirm and revise our previous gene assignments made Offprint requests to: S.D. Pack
with the use of the mink-Chinese hamster clones. A characterization of the new mink-mouse clone panel, its efficiency, and prospects as a mapping tool are presented. By means of the new panel, we determined the chromosomal localization of the genes for ACY, CKBB, GLO, and ADH2 and also checked the accuracy of our previous assignments of ten loci based on the mink-Chinese hamster panel. The revised localization of the mink NP gene is now on Chr 10; it had been previously localized to Chr 2; the regional localization for the ME1 gene is on the short arm of Chr 1 and that for the ADH2 gene is on Chr 8, in the region 8p12-p24.
Materials and methods
Cell lines and cultivation conditions Somatic cell hybrids obtained by fusion of mouse cells L M T K (series LM) or A9 (series AB) with mink bone marrow cells were used (Pack et al. 1989). All the hybrid clones were cultivated in selective HAT medium (13.6 ~g/ml hypoxanthine; 0.4 ~g/ml aminopterin; 12.5 p~g/ml thymidine; and 7.5 I~g/ml glycine (Littlefield 1964) supplemented with 10% calf fetal serum.
Cytogenetic analysis of mink-mouse clones Preparation and G-banding of the metaphase chromosomes were as described (Pack et al. 1989). To produce prometaphase chromosomes, ethidium bromide (5-8 ~g/ml) (Ikeuchi 1984) was added to the culture 2 h before fixation. During the last 10 min, colcemid (0.07 p~g/ml) was added, followed by hypotonic treatment with 0.56% KC1 and fixation in 3:1 methanol/acetic acid. The fixative was renewed four times; the cells were carefully resuspended each time the fixative was changed. The first cytogenetic analysis of the hybrid clones was based on examination of 15-25 metaphase plates. A clone was discarded when a chromosome rearrangement was detected in 5-10 metaphase
S.D. Pack et al.: N e w hybrid m i n k - m o u s e clone panel
113
Table 1. List of isozyme markers examined in mink-mouse hybrids.
Isozyme
Chromosomal localization
Conditions of electrophoretic analysis
ME1 PP PEPA IDH1 SOD1 LDHA LDHB NP MDH1 PEPC G6PD ESD GLO ACY PEPS PEPD APRT UMPH2 ADH2
malate dehydrogenase-1 (NADP dependent) inorganic pyrophosphatase peptidase A isocitrate dehydrogenase- 1 superoxide dismutase-1 lactate dehydrogenase A lactate dehydrogenase B purine nucleoside phosphorylase malate dehydrogenase-1 (NAD dependent) peptidase C glucose-6-phosphate dehydrogenase esterase D glyoxalase acetyl acylase peptidase S peptidase D adenine phosphoribosyltransferase uridine monophosphate phosphohydrolase-2 alcohol dehydrogenase B
1 2 4 4 5 7 9 10 11 13 X 8 1 5 6 7 7 8 8
Rubtsov et al. 1981a, b, 1982
CKBB
creatine phosphokinase B
10
PGP PSP
phosphoglycolate phosphatase phosphoserine phosphatase
14 14
plates; the second analysis of the clones for setting up the panel was based on examination of 30 metaphase or prometaphase spreads.
Analysis of isozyme markers The list of isozyme markers examined in hybrid cells, as well as conditions of analysis, are given in Table 1.
" " " " " Gradov et al. 1985 Parr et al. 1977 Mullakandov et al. 1986 " " Nesterova et al. 1987 Wilson et al. 1986 Holmes 1979; Parr et al. 1977 Chern et al. 1980; Povey et al. 1979 Baker and Hopkinson 1978 Koch et al. 1983
much more than five clones showing discordant segregation, which made up 22% of the total clone number, the highest value being 87% when 20 clones were discordant (Table 2). The efficiency of the panel was estimated by localization of the genes for ME1, PP, PEPA, PEPC, IDH1, SOD1, LDHA, LDHB, G6PD, MDH1, NP, and
Results and discussion
To establish the clone panel, we used 82 clones of LM and 19 clones of AB series. All the clones were of independent origin. The results of our previous cytogenetic and biochemical analyses demonstrated that the only segregating chromosomes were mink (Pack et al. 1989). Chromosome rearrangements of mink chromosomes were detected in 21 of 78 hybrid clones (27%) by cytogenetic analysis, and these were excluded from further analysis. After the first step, characterization of chromosome constitution, 52 clones that seemed promising for setting up the panel were chosen. A computer program based on the criteria of Wijnen and colleagues (1977) and Burgerhout (1978) guided our choice of the clones. As a result, a panel consisting of 23 hybrid clones was established (Fig. 1). The condition to be met in the choice was not less than five clones with any chromosome pair segregating discordantly. Estimation of the efficiency of an established clone panel can be based on the determination of the chromosomal localization of the relevant genes. This is feasible when a marker and a chromosome segregate concordantly in the majority of the clones in a panel, and, in the ideal situation, there would be no clones with a detectable chromosome and an absent relevant gene. For most chromosome pairs, the panel contained
Fig. 1. Distribution of mink c h r o m o s o m e s in the panel of hybrid m i n k - m o u s e clones. Designations for mink c h r o m o s o m e : filled square, presence; open square, absence. Left, designations of individual clones.
114
S.D. P a c k et al.: N e w hybrid m i n k - m o u s e clone panel
Table 2. Discordancy percentage in a mink-mouse clone panel for paired comparisons of mink chromosomes. Chr B ChrA
1
1
0
2 3 4 5 6 7 8 9 10 11 12 13 14 X
57 43 39 43 30 35 47 30 56 39 26 30 35 56
2
3
4
5
6
7
8
9
10
11
12
13
14
0 30 52 83 39 47 43 70 52 52 56 52 56 43
0 30 61 26 43 22 56 39 47 52 47 35 65
0 47 47 47 35 43 52 43 47 43 39 52
0 70 43 47 30 30 56 43 30 61 47
0 43 47 52 56 39 35 47 43 56
0 30 30 30 47 43 39 43 56
0 35 35 61 70 52 39 87
0 52 52 39 43 56 61
0 61 56 43 39 52
0 22 61 56 52
0 39 52 30
0 47 35
65
ESD, which we have previously mapped by means of mink-Chinese hamster hybrids (Serov et al. 1987) As Fig. 2 shows, complete concordant segregation was observed for 9 of the 12 isozyme markers; MEI, Chr 1; PP, Chr 2; PEPA and IDHI, Chr 4; SOD1, Chr 5; ESD, Chr 8; LDHB, Chr 9; MDH1, Chr 11; and G6PD, Chr X. A low discordancy level of 4% (a single discordant clone of the studied 23) was observed in two instances: LDHA, Chr 7; PEPC, Chr 13. The complete concordant segregation of the NP gene with Chr 10 (Fig. 2) deserves comment. This gene is located on Chr 2 according to previous data (Rubtsov et al. 1982). However, in further analysis of a series of subclones of a mink-mouse hepatoma hy-
X
0
0
brid clone carrying a rearrangement of Chr 2, we revealed discordant segregation of NP with Chr 2, as well as that of NP with the other markers of this chromosome (Serov et al. 1987). In analysis of the subclones of another mink-mouse hepatoma clone, we observed apparent cosegregation of NP with PP, hexokinase-1 (HK1), and glutamate-oxaloacetate transaminase-1 (GOT1). The cytogenetic analysis in both series of the subclones was, regretfully, incomplete (Serov et al. 1987). Thus, in the previous study we could not explain adequately the discordancy between NP and Chr 2 and its enzyme markers (Serov et al. 1987). Figure 3A compares the electrophoretic NP pat-
Fig. 2. A synopsis of c h r o m o s o m a l localizations. T w e n t y - t w o ge ne s l o c a l i z e d w i t h t he use of 23 m i n k - m o u s e hybri d clones. Left, clone designations; top, ge ne s y m b o l s ; bottom, c h r o m o s o m e n u m b e r ; filled s qua re , p r e s e n c e of c h r o m o s o m e and ma rke r; open square, a b s e n c e of c h r o m o s o m e and m a r k e r ; hatched square, d i s c o r d a n t clones: marker+/chromosome - (+) and marker-/ c h r o m o s o m e + ( - ).
S.D.
Pack
et al.:
New
Fig. 3.
Electrophoretic
ADH2
(D) in parent
ster
(1); channel hybrid
clone
hybrid
clone
2, mouse;
mink
ACY;
ACY.
(C) Channel
mouse mouse
hybrid
channel for mink for mink
channel
2, mink;
ADH2; ADH2.
for
mink
negative
channels
for
hybrid
2, mink;
for mink
channels
CKBB; CKBB. ( D )
negative
for
for mink
3,5,6, mink-
channel
4,
channel
1, m o u s e ;
hybrid
4,5, mink-mouse
a mink1, m i n k ;
positive
mink
ham-
4, a mink-
clone
clone
3,6, mink-mouse
channels
channel
clones
hybrid
bearing Chr 10) and LM48 (30% of cells bearing it) were lower than in the remaining cells positive for mink NP (the percentage of cells with Chr 10 varied from 80% to 123% in these clones). Two discordant mink-Chinese hamster clones, L26 (NP +/Chr 2 -) and F9B (NP -/Chr 2 + ), are noteworthy (Rubtsov et al. 1982; Serov et al. 1987) in that mink Chr 10 is present in the former and absent in the latter. The efficiency of the mink-mouse panel was tested with the use of 11 markers (ME1, PP, PEPA, PEPC, IDH1, SOD1, LDHA, LDHB, G6PD, MDH1, and ESD) with known chromosomal localization. Of the 253 tested marker-chromosome combinations, just 2 segregated discordantly. This less than 1% discordancy frequency testifies to the efficiency of the panel. The revised localization of the NP gene on Chr 10, with reliance on the new panel, appears to be more correct than the previous localization on Chr 2 with the use of the mink-Chinese hamster panel. The more recent analyses of NP in a series of mink-Chinese hamster clones confirmed that NP is, indeed, located on Chr 10 (Table 3). The new panel was utilized for determining the chromosomal localization of the genes for ACY, GLO, CKBB, and ADH2. As seen in Fig. 2, the expression of mink GLO is concordant with the presence of mink Chr 1 and its marker ME1 in all the 23 clones of the mouse-mink panel. It may be inferred that the GLO gene is located on mink Chr 1. The distribution for ACY in the cells of mouse, mink, and several mouse-mink clones is shown in Fig. 3B. From the data of Fig. 2 it follows that there is a 100% concordancy segregation of the mink ACY gene with Chr 5 and its marker, SOD1. Analysis of mink ACY in the mink-Chinese hamster clone panel also provided evidence for the assignment of the AC Y gene to mink Chr 5. The electrophoretic patterns of CKBB from mink, mouse, and hybrid clone cells are shown in Fig. 3C. CKBB activity was not detected in the L M T K - and A9 cells under the conditions used, as in the case reported by Povey and co-workers (1979). For this reason, the clones negative for mink CKBB show no
(C), and
1, C h i n e s e
NP; a n d c h a n n e l 5 , NP. ( B ) C h a n n e l
hybrid
channel
positive
115
ACY(B), CKBB
3, mouse;
3, mink-mouse
1, m o u s e ;
clone
(A),
(A) Channel
4, mink-mouse
clones
panel
cells.
for mink
negative
clone
ofNP
channel
positive
channel
hybrid
hybrid
2, mink;
mouse
mink-mouse
patterns and
mouse channel
hybrid
mink-
positive
clones
negative
terns yielded by mink, mouse, Chinese hamster, and several mink-mouse hybrids. Clearly, electrophoresis separates mink and mouse NP much better than mink and Chinese hamster NP. In this way, we obtained assurance that NP segregates concordantly with mink Chr 10, and not with mink Chr 2 (Fig. 2). We have further analyzed eight additional mink-mouse clones not included in the established mink-mouse clone panel. Here again, the concordancy results are consistent with the assignment of NP to mink Chr I0. There was another line of evidence for the association of the NP gene and mink NP activity. The evidence came from the relation between the activity level of mink NP and number of cells carrying Chr 10 in the clones. Thus, judging by visual estimation, the activity levels of mink NP in clones LM7 (23% of cells
T a b l e 3. S e g r e g a t i o n o f the m i n k
NP, ADH2, ACY1, a n d
Mink marker
mink chromosomes
Chromosome
in t h e m i n k - C h i n e s e
hamster cell hybrid clones. NT means not tested.
of mink
Clone
NP
ADH2
ACY
1
2
3
4
5
6
7
8
9
10
II
12
13
14
X
F3M F12B-1 FD9M KO2-1 L22-1 KOI-I D7BI D12M D13M K12-1 L15-1 L25-1 R14-1 D3M DllB
+ + + + + + + + + + NT
+ + + + + + + . NT
+ + + + + . +
+ + + + + + + + . .
+ + + -
+ + . + + + + +
+ +
+ + . + + +
+ -
+ -
+ + + + + + _ + + +
+ _ + +
+ + _ + + + _ + _ -
+ + + + + + + + + ~ _ _ _ +
+ _ + _ + + + . + + + + + .
_ _ + _ _ + + _
_ + + + + _ + +
+ _ + + + + _ _
+ _ + + +
_ + _ + _
+ + + + _ + _ _ + + + _ _ _ +
+ + + + + + + + + + + + + + +
.
. .
.
. .
+ .
.
. .
.
. + + + + + +
.
. .
.
. + .
.
-
.
.
.
.
+ + _ _ _ .
.
.
116
S . D . P a c k et al.: N e w h y b r i d m i n k - m o u s e
F i g . 4. E l e c t r o p h o r e t i c A D H p a t t e r n s in d i f f e r e n t m i n k t i s s u e s : s t o m a c h (1,4), k i d n e y (2,5), a n d M V c e l l s (3,6). 1 , 2 , 3 - - s t a i n i n g w i t h the use of butanol as a substrate; 4,5,6---staining with the use of e t h a n o l as a s u b s t r a t e . ADH1, ADH2, a n d A D H 3 a r e A D H i s o z y m e s having presumably subunit compositions AA, BB, and CC, respect i v e l y . It is s e e n t h a t M V cells c o n t a i n ADH2 i s o z y m e .
CKBB activity, while those positive for it displayed CKBB activity of the mink type (Fig. 3C). The results of segregation analysis of mink chromosomes and CKBB in the hybrid clone cells are summarized in Fig. 2. CKBB segregated concordantly with mink Chr I0, as well as its MDH1 marker in all 23 hybrid clone cells. Based on the obtained results, the CKBB gene may be assigned to mink Chr 10. The ADH patterns were obtained from mink tissues and MV cells. When butanol was used as substrate, three forms of ADH are revealed: one anodal B (stomach), two cathodal A (liver, kidneys), and C, with low activity (stomach) (Fig. 4). When the substrate was ethanol, no ADHB activity was detectable (Fig. 4). This is the feature distinguishing the B from the A and C forms of ADH after incubation with ethanol. The electrophoretic patterns of ADH2 from mink, mouse, and a number of hybrid clone cells are presented in Fig. 3D; mink ADH2 activity is detectable, whereas the activity of mouse ADH2 is undetectable in L M T K - and A9 cells. Mink ADH2 is clearly seen in the hybrid clones positive for it, and mink ADH2 is missing in those negative for it. Figure 2 shows how the activity of mink ADH2 is distributed in the hybrid clone cells. As also evident from Fig. 2, in 96% of cases the presence (or absence) of mink ADH2 in the hybrid mink-mouse clones is concordant with the presence (or absence) of mink Chr
clone panel
8. It may be important that, in all the clones positive for mink ADH2, the activities of UMPH2 and ESD, the other markers of mink Chr 8, were also detected. Furthermore, hybrid clone LM6, which contained in the background of mouse chromosomes a single mink Chr 8, was also positive for ADH2 (Table 4). Analysis of ten more clones not included in the panel and of the mink-Chinese hamster clone panel (Table 3) persuasively confirmed concordancy between ADH2 and Chr 8. Thus, taken together, the results demonstrate the localization of the ADH2 on mink Chr 8. In our efforts to localize the ADH2 gene to mink Chr 8, we analyzed transformant clones obtained by transfer of mink genes to mouse cells (Sukoyan et al. 1984) and also mouse-mink clones in which different rearrangements involving mink Chr 8 were detected (Pack et al. 1989). The translocation t(1;8) (lqterlcen::8cen-8pter) identified in clone LM24 involved the apparently intact long arm of Chr 1 and the short one of Chr 8 (Fig. 5). The breakpoints are located in the centromeric regions of the corresponding chromosomes. LM55 contained translocation t(1;8)(lqterlq21::8q24-p26) (Fig. 5), which involved translocation o f the long arm of Chr 1 to Chr 8, whose breakpoint was on segment 8q24. Both clones, LM24 and LM55, showed activity of ADH2 (Table 4). The results of segregation analysis of the enzyme markers of Chr 1 and 8 and their fragments are summarized in Table 4. Chromosomal localization of the TKI, GALK (galactokinase), ALDC (aldolase C), and ESD genes was determined with the use of minkChinese hamster hybrid clones (Gradov et al. 1985), and that of the UMPH-2 and ADH2 genes with the panel of mink-mouse clones (Pack et al. 1989; this communication). From the data for the segregation of the markers and fragments of mink Chr 8 (Table 4) it follows that ADH2 activity correlates with the retention of the integrity of a region 8p24-p12 of the short arm of Chr 8. Fragment 8pter-pl2 was present in clone STT13-1 cells expressing mink A D H 2 . Clones STT16-8 and STT16-3, which contained respectively regions p25-cen and p24-q24 of mink Chr 8, as well as STT13-1, showed expression of ADH2. This suggested that the ADH2 gene is localized in the 8p24-p12 region of mink Chr 8. The genes in the short arm of Chr 8 may b e o r d e r e d as c e n - ( A D H 2 , T K 1 ) - G A L K (ALDC,HOX-2)-UMPH2 (Fig. 6). We further regionally localized the gene for ME1, which resides on mink Chr 1. The clones used for this
Table 4. Cytogenetic and biochemical characterization of hybrid and transformed mouse-mink clones containing different fragments of mink Chrs 1 and 8. Enzyme markers of mink Clone
Chr 8 and its fragments
TK1
GALK
ALDC
UMPH-2
ESD
ADH2
ME1
LM6 STT13-1 a STT16-8 a STT16-3 a LM24 LM55
intact pter--pl2 p25--cen p24---q24 t(1;8)(lqter-lcen::8cen-8pter) t(1 ;8)(lqter-I q21 ::8q24-8p26)
+ + + + + +
0 + + . 0 0
+ + -
+ + -
+ -
+ -
-
+ + + + 4 +
0 0 0 0 -
Presence ( + ) or absence ( - ) of marker; (0) not examined. a For detailed cytogenetic characterization of the clones, see Gradov et al. 1985.
.
. + +
.
S.D. Pack et al.: New hybrid mink-mouse clone panel
Fig. 5. Intact mink Chr 8 (1) and 1 (4) and translocations t(1;8) detected (3) in LM24 t(l ;8)(lqter-lcen::8cen-8pter) and (2) in LM55 t(1 ;8)(lqter-lq21::8q24-8p26). Numbers 2 and 3 on the chromosomes denote break-points in clones LM55 and LM24.
117
tical criteria (Wijnen et al. 1977; Burgerhout 1978). With the use of the mink-mouse hybrid clones, we localized ten new mink genes to individual chromosomes; of these ten, localization of four genes--GLO, ADH2, ACY, and CKBB is described here (Fig. 2), and that of six others was done in our previous report (Pack et al. 1989; Fig. 2). Chromosomal localizations for ACY, ADH2, and PGP were amply supported by analysis of the mink-Chinese hamster hybrid panel (Pack et al. 1989; Table 3). Circumstantial evidence was obtained allowing us to reassign the NP gene to mink Chr 10. Independent confirmation of assignments, which we have achieved with a mink-Chinese hamster panel for 11 genes, was obtained. This comparative approach is advantageous because, to our
purpose were LM24 and LM55 containing translocations made up of parts of Chr 1 and 8 (Table 4). LM24 contains the entire long arm of Chr 1, and LM55 the distal part of the long arm, qter-q21 (Fig. 5). No ME1 activity was detectable in the two clones (Table 4). This suggested the assignment of the ME1 gene to the short arm of mink Chr 1 (Fig. 7). Thus, to map mink chromosomes, we set up a panel of 23 hybrid mink-mouse clones fulfilling statis-
m
4.3
2
i
!
U]V[PH-2 ALDC,HOX-2
]
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Fig. 6. Subchromosomal localization of the gene for A D H 2 on the short arm of mink Chr 8.
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Fig. 7. Subchromosomal localization of the gene for ME1 on mink Chr 1.
118
knowledge, there is no other strategy for directly checking the correctness of gene mapping. The segregation of each of the 22 markers to specific chromosomes provided us with a basis for estimation of the efficiency of the mink-mouse panel (Fig. 2). The number of hybrid clones tested for panel efficiency was 23. Of the 506 marker-chromosome combinations, only in 5 (less than 1%) was the segregation discordant, thereby yielding a 99% efficiency for the panel. This produces confidence in the mink-mouse panel as a tool in mink gene mapping. Acknowledgments. The authors are indebted to A. Fadeeva for translation of the paper from Russian into English and to I.B. Nosatova for technical assistance.
References Baker, R.F. and Hopkinson, D.A.: Genetic polymorphism of human phosphoglycolate phosphatase (PGP). Ann Hum Genet 42: 143151, 1978. Burgerhout, W.G.: A standard procedure for gene assignment by use of somatic cell hybrids. Cytogenet Cell Genet 22: 689--693, 1978. Chern, C.J., Tan, P., and Park, H.: Chromosomal mapping of human creatine kinase (brain type) using human-rodent somatic cell hybrids. Cytogenet Cell Genet 27: 232-237, 1980. Gradov, A.A., Pack, S.D., Sukoyan, M.A., Rubtsov, N.B., Bochkarev, M.N., Serov, O.L., Rattazzi, O., Scandalios, J.G., Whitt, G., and Markert, C.L.: Regional assignment of the genes for TK1, GALK, ALDC and ESD on chromosome 8 in the American mink by chromosome-mediated gene transfer. Mol Gen Genet 200: 433--438, 1985. Holmes, R.S.: Genetics and ontogeny of alcohol dehydrogenase isozymes in the mouse: evidence for a cis-acting regulator gene (Adt-1) controlling C2 isozyme expression in reproductive tissues and close linkage of Adh-3 and Adt-1 on chromosome 3. Biochem Genet 17: 461--472, 1979. Ikeuchi, T.: Inhibitory effect of ethidium bromide on mitotic chromosome condensation and its application to high-resolution chromosome banding. Cytogenet Cell Genet 38: 56--61, 1984. Koch, G.A., Eddy, R.L., Haley, L.L., Byers, M.G., McAvoy, M., and Shows, T.B.: Assignment of the human phosphoserine phosphatase gene (PSP) to the pter-q22 region of chromosome 7. Cytogenet Cell Genet 35: 67-69, 1983. Littlelield, J.W.: Selection of hybrids from matings of fibroblasts in vitro and their presumed recombinants. Science 145: 709-710, 1964. Mullakandov, M.R., Gradov, A.A., Zakijan, S.M., Rubtsov, N.B., and Serov, O.L.: Peptidases A, B, C, D and S in the American
S.D. Pack et al.: New hybrid mink-mouse clone panel mink: polymorphism and chromosome localization. Theor Appl Genet 73: 272-277, 1986. Nesterova, T.B., Borodin, P.M., Zakijan, S.M., and Serov, O.L.: Assignment of the gene for adenine phosphoribosyltransferase on the genetic map of mouse chromosome 8. Biochem Genet 25: 563-568, 1987. Pack, S.D., Zhdanova, N.S., Sukoyan, M.A., and Serov, O.L.: Chromosomal and regional localization of the genes for UMPH2, APRT, PEPD, PEPS, PSP and PGP in mink: comparison with man and mouse. Cytogenet Cell Genet 50: 127-131. 1989. Parr, C.W., Bagster, I.A., and Welch, S.G.: Human red cell glyoxalase I polymorphism. Biochem Genet 15: 109-113, 1977. Povey, S., Inwood, M., Tanyar, A., and Bobrow, M.: The expression of creatine kinase isozymes in human cultured cells. Ann Hum Genet 43: 15-26, 1979. Rubtsov, N.B., Radjabli, S.I., Gradov, A.A., and Serov, O.L.: Chinese hamster • American mink somatic cell hybrids: characterization of a clone panel and assignment of the mink genes for malate dehydrogenase, NADP-1 and malate dehydrogenase, NAD-1. Theor Appl Genet 60: 99-106, 1981a. Rubtsov, N.B., Radjabli, S.I., Gradov, A.A., and Serov, O.L.: Chromosome localization of the three syntenic gene pairs in the American mink (Mustela vison). Cytogenet Cell Genet 31: 184187, 1981b. Rubtsov, N.B., Gradov, A.A., and Serov, O.L.: Chromosome localization of the loci GOTI, PP, NP, SODI, PEPA and PEPC in the American mink (Mustela vison). Theor Appl Genet 63: 331336, 1982. Serov, O.L. and Pack, S.D.: American mink (Mustela vison). In S.J. O'Brien (ed.), Genetic Maps (Locus Maps of Complex Genomes), pp. 4126--4128, 5th edition, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y., 1990. Serov, O.L., Gradov, A.A., Rubtsov, N.B., Zhdanova, N.S., Pack, S.D., Sukoyan, M.A., Mullakandov, M.R., and Zakijan, S.M.: Genetic map of the American mink: gene conservation and organization of chromosomes. In Isozymes: Current Topics in Biological and Medical Research, vol. 15, Genetics, Development, and Evolution, pp. 179-215, Alan R. Liss Inc., New York, 1987. Sukoyan, M.A., Matveeva, N.M., Belyaev, N.D., Pack, S.S., Gradov, A.A., Shilov, A.G., Zhdanova, N.S., and Serov, O.L.: Cotransfer and phenotypical stabilization of syntenic and asyntenic mink genes into mouse cells by chromosome-mediated gene transfer. Mol Gen Genet 196: 97-104, 1984. Wijnen, L.M,M., Grzeschik, K.H., Pearson, P.L., and Meera Khan, P.: The human PGM-2 and its chromosomal localization in man-mouse hybrids. Hum Genet 37: 271-278, 1977. Wilson, D.E., Swallow, D.M., and Povey, S.: Assignment of the human gene for uridine 5'-monophosphate phosphohydrolase (UMPH2) to the long arm of chromosome 17. Ann Hum Genet 50: 223-227, 1986. Yermolaev, V.I., Karasik, G.I., Khlebodarova, T.M., Matveeva, N.M., MuUakandov, M.R., Nayakshin, A.M., Shumny, T.V., Rubtsov, N.B., Serov, O.L., and Baranov, O.K.: Localization of the a2-macroglobulin gene and Lpm gene family on mink chromosome 9. Theor Appl Genet 78: 93-96, 1989.
