- Email: [email protected]
Frequency of “Y chromatin body” in human skin fibroblasts in tissue culture, and its relation to growth phase
“Y chromatin body” frequency
473
number of biologists. Indeed, the monetary output for such a system may be minimal, depending upon the needs of the investigator (construction of a complete system may range from $1 200 to $15 000).
investigating if the frequency of the Y chromatin positive interphases varies with growth phase, as has been shown to be the case with the frequency of the X chromatin positive (Barr positive) interphases [6].
This work was supported in part by grant NSF GB 24 457, and American Cancer Society Institutional Grant IN-40K. Gratitude is expressed to James Mite, Steve O’Neill, and Wanny Cheng for aid in construction of the instrument.
Material
REFERENCES 1. Bessis, M, Gires, F, Mayer, G & Nomarski, G, Compt rend acad sci 225 (1962) 1010. 2. Saks, N M & Roth, R C, Science 141 (1963) 46. 3. Berns, M W, Olson, R S & Rounds, D E, Nature 221 (1969) 74. 4. Berns, M W, Rounds, D E & Olson, R S, Exptl
cell res 56 (1969) 292. 5. Berns, M W, Ohnuki, Y, Rounds, D E & Olson,
R S, Exptl cell res 60 (1970) 133. 6. Berns, M W & Rounds, D E, Sci Am 222 (1970) 98. 7. - Ann NY acad sci 168 (1969) 550.
Received November 11, 1970
Frequency of “Y chromatin body” in human skin fibroblasts in tissue culture, and its relation to growth phase A. J. THERKELSEN Institute of Human Aarhus, Denmark
and G. BRUUN PETERSEN, Genetics,
Unic%ersity oj Aarhus,
It has been shown recently by Pearson, Bobrow & Vosa [3] that the presence of a Y-chromosome in a cell can be demonstrated in interphase by fluorescence microscopy after staining with an aqueous solution of quinacrine dihydrochloride (“Atebrin”, G. T. Gurr). In 25 to 50 “/b of cells in buccal smears from normal males the Y-chromosome showed as an intensely fluorescent body with a diameter of approx. 0.25 pm. As the staining properties of the Y-chromosome in males and that of the heterochromatic X-chromosome in females seem different with Atebrin [2] as well as with quinacrine mustard [l], we found it worthwhile 31 -
711805
and Methods
Primary cultures of skin fibroblasts were established as described elsewhere [4]. The experiments were performed with cultures in the third passage in Leighton tubes [5]. Cultures from a normal male and a normal female were grown simultaneously. The growth curve was determined by counting the cells in three cultures from each person each day using a celloscope (Model 202, AB Lars Ljungberg, Stockholm). The frequency of cells with a Barr body in the female cultures was determined by counting 100 interphases each day in a Feulgen light green stained culture. As regards the staining of the Y chromatin body we used the following procedure. The cultures were fixed in methanol for 60 min, stained with an 0.5 % aqueous solution of Atebrin for 5 min, destained in running tap water for 3 min. embedded in MacIlvaine buffer- (pH 4.1) and thereafter counted as soon as possible. The frequency of cells with a Y chromatin body was determined by-both of us, except on day 1 of expt 197 when the counts were performed by one of us only. Both of us counted the number of cells with a Y chromatin-like body among 100 cells in each of four cultures per day: two from the normal male and two from the normal female. The countings were done blindfold. A Zeiss standard microscope with incident light from a ‘HBO 200 mercury vapour lamp was used. The excitor filter was a 4 mm ‘BG 12’ and a 500 nm filter was used as barrier. All countings were done with an oil immersion objective ( ‘: 100).
Results After a couple of preliminary experiments the two experiments illustrated in fig. 1 were performed. The figure shows the cell number per culture each day of the experiments for the normal male and female together with the frequency of Y chromatin positive cells in the male and the frequency of X chromatin positive cells in the female. It was possible by counting Y chromatin-like bodies to distinguish blindfold between male and female cultures in all cases but one: in one of the male cultures from expt 198, day 1, none of the 100 cells counted had a Y chromatin body. The average for the four male cultures on that day was 6.8 per 100 cells (fig. 1). The Exptl Cd Res 65
474
A. J. Therkelsen & G. Bruun Petersen
Table 1. Distribution of the Y chromatin body as regards localization positive cells counted Marginal Phase of growth Logarithmic (days 2, 3,4) Intermediate (days 5, 6) Postlogarithmic, (days 7, 8, 9 10) Total
Intermediary
Juxtanucleolar
No.
No.
s:
164
(24.8)
217
(32.8)
281
(42.4)
662
208
(17.2)
533
(44.0)
469
(28.8)
1210
446 818
(15.2)
1 358 2 108 -
(46.1)
1 139 1 889
(38.7)
2 943 4 815
198
1
%
total
No.
localization of the Y chromatin body was noted in all positive cells and for the two males the added results of all the countings appear in table 1. Three localizations were distinguished: (1) marginal, i.e. adjacent to the nuclear membrane; (2) juxtanucleolar, i.e. adjacent to a nucleolus; (3) intermediary, i.e. neither 1 nor 2. 2000 1197
in the nucleus for all
16
The frequency of Y chromatin-like bodies in the two females appears from table 2. Our main conclusions from the experiments are the following. (1) The frequency of Y chromatin positive cells varies with the growth phase of the cultures, being low (minimum 6.8 %) in the logarithmic and close to 100% in the postlogarithmic phase (maximum in the two experiments 97.3 % and 96.5 %, respectively). (2) When the frequency is at the minimum, the distinction between male and female cultures based on the counting of Y chromatin positive cells may be difficult or impossible (cf day 1 of expt 198 and table 2). (3) The localization of the Y chromatin body changes during growth as the frequency of bodies located away from the nuclear membrane (i.e. intermediary fjuxtanucleolar bodies) rises significantly (cf table 1, xi = 35.5, P
female; O-O, male. Showing cell number for male and female cultures, frequency of Y chromatin positive interphases in male cultures and frequency of X chromatin positive interphases in female cultures.
Number of “Y chromatin positive” cells per 100 cells 4 5 6 Total 0 1 23
Fig. 1. Abscissa: days O--O,
Exptl Cell Res 65
Number of cultures
29 22 17 5
2
1
2
78
Identification found for the localization similar experiments [5].
of the Barr body in
This investigation was supported by grants from the Danish State Research Foundation and the Research Foundation of the University of Aarhus, Denmark.
REFERENCES 1. Caspersson, T, Zech, L & Johansson, C, Exptl cell res 60 (1970) 315. 2. George, K P, Nature 226 (1970) 80. 3. Pearson, P L, Bobrow, M & Vosa, C G, Nature 226 (1970) 78. 4. Therkelsen, A J, Acta path01 microbial Stand 61 (1964) 317: 5. - Ibid A 78 (1970) 295. 6. Therkelsen, A J & Petersen, G B, Exptl cell res 48 (1962) 681. Received January 15, 1971
Identification of human chromosomes in a mouse/human hybrid by fluorescence techniques T. CASPERSSON,t L. ZECH,’ H. HARRIS2 F. WIENER3 G. KLEIN,3 IInstitute for Medical Cell Research and Genetics,. Medical Nobel Institute, Karolinska Institutet, 104 01 Stockholm 60, =Sir William Dunn School of Pathology, University of Oxford, 0X1, England, and 3Department of Tumor Biology, Karolinska Institutet, 104 01 Stockholm, Sweden
The mouse fibroblast line A9 which lacks the enzyme inosinic acid pyrophosphorylase, is being used extensively for experiments involving hybridization with human cells. The identification of human chromosomes in the hybrid karyotypes is thus a problem of practical significance. This report concerns the identification of human chromosomes in hybrids of A9 and Daudi cells. The A9 are C3H derived L cells. Daudi cells came from a lymphoblastoid line showing surface IgM and were maintained in suspension culture for 4 years since derivation from a Burkitt lymphona biopsy [I]. The hybrid cell was made by fusing the A9 cell with the Daudi cell by means of the inactivated Sendai virus technique [2]. Daudi
of human chromosomes 475
cells grow in suspension and A9 cells do not grow in appropriate selective media. Selection of the hybrids thus presented no problems. The Hl-A and H-2 reactivity of the hybrid and its IgM production will be described elsewhere. Metaphase plates prepared from parent and hybrid cells after one year cultivation were examined by the quinacrine mustard fluorescence technique [3] to identify human chromosomes. The fluorescence patterns of the Daudi chromosomes (fig. 1) agree with those previously published for the normal human karyotype [4,5]. A narrow strongly flourescent band close to the centromere on the long arm of one chromosome number 13 (arrow) can be considered as a normally occurring individual variation. One or two extra chromosomes can be present. The A9 chromosomes had distinct patterns of fluorescent cross-striation (fig. 2). These patterns are sufficiently clear to allow an analysis of the whole karyotype if a large enough number of metaphases is investigated, A complete analysis of the A9 karyotype is not, however, necessary for the separation of the human chromosomes of mouse/human hybrids. The fluorescence patterns of human chromosomes belonging to the A group are so characteristic that they can be readily distinguished from the metacentric and submetacentric A9 chromosomes in fluorescence photographs. The patterns of the B, C and D groups are also distinctive. The small metacentric and submetacentric human chromosomes of groups E and F differ from the smaller metacentric mouse chromosomes and are unlikely to be confused with the acrocentrics. Human chromosomes 21 and 22 are morphologically similar to the shortest A9 chromosomes but the fluorescence patterns make discrimination possible. Exptl
Cell Res 65
473
number of biologists. Indeed, the monetary output for such a system may be minimal, depending upon the needs of the investigator (construction of a complete system may range from $1 200 to $15 000).
investigating if the frequency of the Y chromatin positive interphases varies with growth phase, as has been shown to be the case with the frequency of the X chromatin positive (Barr positive) interphases [6].
This work was supported in part by grant NSF GB 24 457, and American Cancer Society Institutional Grant IN-40K. Gratitude is expressed to James Mite, Steve O’Neill, and Wanny Cheng for aid in construction of the instrument.
Material
REFERENCES 1. Bessis, M, Gires, F, Mayer, G & Nomarski, G, Compt rend acad sci 225 (1962) 1010. 2. Saks, N M & Roth, R C, Science 141 (1963) 46. 3. Berns, M W, Olson, R S & Rounds, D E, Nature 221 (1969) 74. 4. Berns, M W, Rounds, D E & Olson, R S, Exptl
cell res 56 (1969) 292. 5. Berns, M W, Ohnuki, Y, Rounds, D E & Olson,
R S, Exptl cell res 60 (1970) 133. 6. Berns, M W & Rounds, D E, Sci Am 222 (1970) 98. 7. - Ann NY acad sci 168 (1969) 550.
Received November 11, 1970
Frequency of “Y chromatin body” in human skin fibroblasts in tissue culture, and its relation to growth phase A. J. THERKELSEN Institute of Human Aarhus, Denmark
and G. BRUUN PETERSEN, Genetics,
Unic%ersity oj Aarhus,
It has been shown recently by Pearson, Bobrow & Vosa [3] that the presence of a Y-chromosome in a cell can be demonstrated in interphase by fluorescence microscopy after staining with an aqueous solution of quinacrine dihydrochloride (“Atebrin”, G. T. Gurr). In 25 to 50 “/b of cells in buccal smears from normal males the Y-chromosome showed as an intensely fluorescent body with a diameter of approx. 0.25 pm. As the staining properties of the Y-chromosome in males and that of the heterochromatic X-chromosome in females seem different with Atebrin [2] as well as with quinacrine mustard [l], we found it worthwhile 31 -
711805
and Methods
Primary cultures of skin fibroblasts were established as described elsewhere [4]. The experiments were performed with cultures in the third passage in Leighton tubes [5]. Cultures from a normal male and a normal female were grown simultaneously. The growth curve was determined by counting the cells in three cultures from each person each day using a celloscope (Model 202, AB Lars Ljungberg, Stockholm). The frequency of cells with a Barr body in the female cultures was determined by counting 100 interphases each day in a Feulgen light green stained culture. As regards the staining of the Y chromatin body we used the following procedure. The cultures were fixed in methanol for 60 min, stained with an 0.5 % aqueous solution of Atebrin for 5 min, destained in running tap water for 3 min. embedded in MacIlvaine buffer- (pH 4.1) and thereafter counted as soon as possible. The frequency of cells with a Y chromatin body was determined by-both of us, except on day 1 of expt 197 when the counts were performed by one of us only. Both of us counted the number of cells with a Y chromatin-like body among 100 cells in each of four cultures per day: two from the normal male and two from the normal female. The countings were done blindfold. A Zeiss standard microscope with incident light from a ‘HBO 200 mercury vapour lamp was used. The excitor filter was a 4 mm ‘BG 12’ and a 500 nm filter was used as barrier. All countings were done with an oil immersion objective ( ‘: 100).
Results After a couple of preliminary experiments the two experiments illustrated in fig. 1 were performed. The figure shows the cell number per culture each day of the experiments for the normal male and female together with the frequency of Y chromatin positive cells in the male and the frequency of X chromatin positive cells in the female. It was possible by counting Y chromatin-like bodies to distinguish blindfold between male and female cultures in all cases but one: in one of the male cultures from expt 198, day 1, none of the 100 cells counted had a Y chromatin body. The average for the four male cultures on that day was 6.8 per 100 cells (fig. 1). The Exptl Cd Res 65
474
A. J. Therkelsen & G. Bruun Petersen
Table 1. Distribution of the Y chromatin body as regards localization positive cells counted Marginal Phase of growth Logarithmic (days 2, 3,4) Intermediate (days 5, 6) Postlogarithmic, (days 7, 8, 9 10) Total
Intermediary
Juxtanucleolar
No.
No.
s:
164
(24.8)
217
(32.8)
281
(42.4)
662
208
(17.2)
533
(44.0)
469
(28.8)
1210
446 818
(15.2)
1 358 2 108 -
(46.1)
1 139 1 889
(38.7)
2 943 4 815
198
1
%
total
No.
localization of the Y chromatin body was noted in all positive cells and for the two males the added results of all the countings appear in table 1. Three localizations were distinguished: (1) marginal, i.e. adjacent to the nuclear membrane; (2) juxtanucleolar, i.e. adjacent to a nucleolus; (3) intermediary, i.e. neither 1 nor 2. 2000 1197
in the nucleus for all
16
The frequency of Y chromatin-like bodies in the two females appears from table 2. Our main conclusions from the experiments are the following. (1) The frequency of Y chromatin positive cells varies with the growth phase of the cultures, being low (minimum 6.8 %) in the logarithmic and close to 100% in the postlogarithmic phase (maximum in the two experiments 97.3 % and 96.5 %, respectively). (2) When the frequency is at the minimum, the distinction between male and female cultures based on the counting of Y chromatin positive cells may be difficult or impossible (cf day 1 of expt 198 and table 2). (3) The localization of the Y chromatin body changes during growth as the frequency of bodies located away from the nuclear membrane (i.e. intermediary fjuxtanucleolar bodies) rises significantly (cf table 1, xi = 35.5, P
female; O-O, male. Showing cell number for male and female cultures, frequency of Y chromatin positive interphases in male cultures and frequency of X chromatin positive interphases in female cultures.
Number of “Y chromatin positive” cells per 100 cells 4 5 6 Total 0 1 23
Fig. 1. Abscissa: days O--O,
Exptl Cell Res 65
Number of cultures
29 22 17 5
2
1
2
78
Identification found for the localization similar experiments [5].
of the Barr body in
This investigation was supported by grants from the Danish State Research Foundation and the Research Foundation of the University of Aarhus, Denmark.
REFERENCES 1. Caspersson, T, Zech, L & Johansson, C, Exptl cell res 60 (1970) 315. 2. George, K P, Nature 226 (1970) 80. 3. Pearson, P L, Bobrow, M & Vosa, C G, Nature 226 (1970) 78. 4. Therkelsen, A J, Acta path01 microbial Stand 61 (1964) 317: 5. - Ibid A 78 (1970) 295. 6. Therkelsen, A J & Petersen, G B, Exptl cell res 48 (1962) 681. Received January 15, 1971
Identification of human chromosomes in a mouse/human hybrid by fluorescence techniques T. CASPERSSON,t L. ZECH,’ H. HARRIS2 F. WIENER3 G. KLEIN,3 IInstitute for Medical Cell Research and Genetics,. Medical Nobel Institute, Karolinska Institutet, 104 01 Stockholm 60, =Sir William Dunn School of Pathology, University of Oxford, 0X1, England, and 3Department of Tumor Biology, Karolinska Institutet, 104 01 Stockholm, Sweden
The mouse fibroblast line A9 which lacks the enzyme inosinic acid pyrophosphorylase, is being used extensively for experiments involving hybridization with human cells. The identification of human chromosomes in the hybrid karyotypes is thus a problem of practical significance. This report concerns the identification of human chromosomes in hybrids of A9 and Daudi cells. The A9 are C3H derived L cells. Daudi cells came from a lymphoblastoid line showing surface IgM and were maintained in suspension culture for 4 years since derivation from a Burkitt lymphona biopsy [I]. The hybrid cell was made by fusing the A9 cell with the Daudi cell by means of the inactivated Sendai virus technique [2]. Daudi
of human chromosomes 475
cells grow in suspension and A9 cells do not grow in appropriate selective media. Selection of the hybrids thus presented no problems. The Hl-A and H-2 reactivity of the hybrid and its IgM production will be described elsewhere. Metaphase plates prepared from parent and hybrid cells after one year cultivation were examined by the quinacrine mustard fluorescence technique [3] to identify human chromosomes. The fluorescence patterns of the Daudi chromosomes (fig. 1) agree with those previously published for the normal human karyotype [4,5]. A narrow strongly flourescent band close to the centromere on the long arm of one chromosome number 13 (arrow) can be considered as a normally occurring individual variation. One or two extra chromosomes can be present. The A9 chromosomes had distinct patterns of fluorescent cross-striation (fig. 2). These patterns are sufficiently clear to allow an analysis of the whole karyotype if a large enough number of metaphases is investigated, A complete analysis of the A9 karyotype is not, however, necessary for the separation of the human chromosomes of mouse/human hybrids. The fluorescence patterns of human chromosomes belonging to the A group are so characteristic that they can be readily distinguished from the metacentric and submetacentric A9 chromosomes in fluorescence photographs. The patterns of the B, C and D groups are also distinctive. The small metacentric and submetacentric human chromosomes of groups E and F differ from the smaller metacentric mouse chromosomes and are unlikely to be confused with the acrocentrics. Human chromosomes 21 and 22 are morphologically similar to the shortest A9 chromosomes but the fluorescence patterns make discrimination possible. Exptl
Cell Res 65