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Pinocytosis in centrifuged and bisected amoebae
Centrifuged and bisected amoebae late telophase also suggest that constitutive heterochromatin may remain partially condensed in interphase. The localization of constitutive heterochromatin in the giant sex chromosomes of M. agrestis and its variable expression in cells of different types makes this species a unique and valuable model for the study of the biochemical nature and ultrastructure of this chromatin fraction as well as the factors involved in its heterochromatization. This investigation was supported by NIH grant HD 01962.
REFERENCES 1. Bianchi, N, Lima-de-Faria, A & Jaworska, H, Hereditas 51 (1964) 207. 2. Brown, S W, Science 151 (1966) 417. 3. Carr, D H & Walker, J E, Stain technol36 (1961) 233. 4. Hansen-Melander, E, Hereditas 52 (1964) 357. 5. Heitz, E, Jahrb wiss bot 69 (1928) 762. 6. Hsu, T C, J cell biol 23 (1964) 53. 7. Hsu, T C & Kellogg, D S, J natl cancer inst 25 (1960) 221. 8. Lee, J C & Yunis, J J, Exptl cell res 59 (1970) 339. 9. Lima-de-Faria, A & Jaworska, H, Nature 217 (1968) 138. 10. Sachs, L, J heredity 7 (1953) 227. 11. Schmid, W, Arch Klaus-Stift Vererb Forsch 42 (1967) 1. 12. Schmid, W, Smith, D W & Theiler, K, Arch Klaus-Stift Vererb Forsch 40 (1965) 35. 13. Wolf, U, Flinspach, G, Bohm, R & Ohno, S, Chromosoma 16 (1965) 609. 14. Yasmineh, W G & Yunis, J J, Exptl cell res 59 (1970) 69. 15. - Exptl cell res. In press. 16. Yunis ,J J & Yasmineh, W G, Science 168 (1970) 263. Received April 6, 1970 Revised version received May 22, 1970
Pinocytosis in centrifuged and bisected amoebae E. J. SANDERS,l Department of Zoology, of Southampton, Southampton, England.
University
461
pinocytosed intensely, possessed abundant plasmaLemma and contained many vesicles. Centrifugal halves, which failed to pinocytose, were bounded tightly by membrane which obliterated the hyaline layer. These halves contained vesicles with membrane similar to the plasmalemma. The results are discussed in relation to the amount of plasmalemma, or vesicular membrane precursor, available for invagination and the effect of the absence of the hyaline layer. It was shown that the centrifugal halves did not recover, and electron microscopical examination revealed an increase of free cytoplasmic membranes.
The synthesis and circulation with cells of vesicles or preformed membrane subunits which subsequently fuse with the surface, thereby adding newly fabricated membrane to the existing plasma membrane has been suggested for several cell types including amoebae [l-6]. An alternative mode of surface formation involves the interpolation of molecules or micelles of membrane material into the surface and has generally been the favoured mechanism for plasmalemma production in amoebae [7-91. The basis for this preference has been the absence of microscopical evidence to show fusion of vesicles with the surface. In the present investigation amoebae were centrifuged in order to stratify and identify electron microscopically any vesicular precursor material. The presence of presumptive plasmalemma was also tested for by applying pinocytosis inducers to the separated halves of the cells. Since it has been proposed that pinocytosis in amoebae is dependent on the amount of membrane available for invagination [lo, 111,it was considered that the intensity of pinocytotic response would be an indicator of the existence of quantities of membrane precursor localized by the centrifugation in one of the two fragments. Materials and Methods
Summary
was cultured and handled in modified Chalkley medium and fed on washed Tetrahymena.
Centrifuged amoebae were bisected to produce halves with different pinocytotic ability. Centripetal halves
1 Present address: Department of Zoology, University of Alberta, Edmonton, Alberta, Canada.
Amoebaproteus
30*-
701816
Exptl Cell Res 61
462
E. J. Sanders
The amoebae were starved for one or two days and washed in clean culture medium before use. Amoebae were centrifuged at 7 600 g for 10 min, excluding acceleration and deceleration, at 3°C. The cells were supported during centrifugation on 0.3 M sucrose solution and afterwards were pipetted into cold culture medium and quickly rinsed. Amoebae thus treated possessed two distinct halves separated by a conspicuous waist where the cells were bisected free-hand using glass micro-needles. After bisection the halves were left in culture medium for 20 or 30 min before testing for pinocytotic activity. Pinocytosis was quantitated by the channelcounting technique of Chapman-Andresen [12] with slight modification. The number of channels occurring in the microscope field was counted at various times after immersion in the pinocytosis inducer and the mean calculated for up to forty specimens. A measure of relative activity was obtained by taking the mean of the total number of channels observed in each amoeba. The inducer used was 0.125 M sodium chloride solution at pH 6.1-6.4. The amoebae were prepared for electron microscopy individually and fixed in 2 % osmium tetroxide in 0.01 N Sorensen phosphate buffer pH 6.98, at 3°C. The cells were washed and dehydrated in graded concentrations of ethanol, and embedded in Araldite. Sections were mounted on uncoated copper grids, stained with uranyl acetate and lead citrate and examined using a Philips EM300 electron microscope.
Results Light microscopical examination of the two halves produced by bisection showed that the centrifugal half was very granular, spherical and bounded by apparently unwrinkled membrane. The centripetal half, however, was very much less granular and extensive digitation was evident at the extreme centripetal end. The digitations were optically empty. The nucleus was positioned at the extreme centrifugal end of the cell.
The results of pinocytosis tests on these halves are expressed in table 1 where they are compared with similar tests on noncentrifuged halves. The table also shows the results obtained from two control experiments. Five-hour-old amoebae were used as a control for the size, since cells at this age are approximately half the size of a G2 phase cell. In the second control, amoebae were tested which had been subjected to a mimic cut, which consisted of removing a small piece of cytoplasm from a G2 phase cell and testing the remainder. From these control experiments it is clear that the results cannot be explained by reason of volume differences or of the effect of cutting. The time required for the recovery of pinocytotic activity of the centrifugal halves compared with uncentrifuged nucleated halves was determined (fig. 1). The nucleate halves returned to, and maintained, high activity after 5 h, but in the case of the centrifugal halves, pinocytosis tests were made up to 100 h after bisection and after a period of apparent recovery, attachment deteriorated, pseudopod extension became limited and cytolysis eventually ensued. Electron microscopy of centrifugal halves 24 h after bisection (fig. 2) revealed the presence of many free cytoplasmic membranes which were apparently discontinuous. The organelles occurring in the centrifugal
Table 1. Effect of centrifugation and bisection on the pinocytotic with 0.125 A4 sodium chloride pH 6.0-6.4
G2 Phase cell Centripetal half (Enucleate) Centrifugal half (Nucleate) Enucleate half Nucleate half Size control (5 h old cells) Mimic cut control Exptl Cell Res 61
Time after cutting (min)
No. of cells tested
Activity f S.E.
30 30 30 30 30 30
40 10 10 IO 10 7’
103.9 k9.1 184.6t34.2 1.5 kO.8 125.lk9.5 18.2k4.7 102.5 46.8 111.6k11.3
activity of A. proteus, tested
Enucleate/ nucleate ratio 123.1 : 1 6.9 : 1
Whole cell activity (X) 100 177.9 1.4 120.5 17.5 98.7 107.5
Centrifuged and bisected amoebae 130-
463
*\...A
120 110 -
100 90 --
i
80 70 60 50 40 30 -
Fig. 1. Abscissa: hours after bisection; ordinate: mean channel number. O-0, centrifugal halves. The extent of recovery of pinocytotic activity by bisected amoebae.
half were the nucleus, crystal vacuoles, food vacuoles, mitochondria, Golgi bodies and large vacuoles up to 5 ,um in diameter. Also localized in these fragments were flattened or angular vesicles bounded by membrane bearing filamentous material on the vacuolar face (fig. 3). The contents of the centripetal half comprised lipid droplets, contractile vacuole and a large number of small vesicles up to 450 nm in diameter which contributed about 95 % of the total volume (fig. 4).
Discussion In order to attempt to explain the high pinocytotic activity of the centripetal halves in terms of the presence of large amounts of membrane freely available for invagination, two observations were considered. Firstly these fragments possessed excess membrane already at the surface in the form of extensive digitation at the centripetal end. This almost certainly contributed to the high activity. Secondly the abundant vesicles present could have helped produce the large quantity of
nucleate halves; V-V,
surface required by fusion with the plasmalemma. It is clear that the membrane of these
vesicles as shown in fig. 4 is quite unlike the coated plasma membrane of amoebae [13]. However, it is possible that they comprised a membrane pool, some or all of which were awaiting glycosidation prior to incorporation into the plasmalemma in the manner suggested by Cook [6]. This possibility is rendered unlikely perhaps since the Golgi bodies, absent from the centripetal halves, are thought to participate in the process. The membrane of the fringed vesicles of the centrifugal half was exceedingly similar to that of the plasmalemma and must be considered as possible surface precursor. In noncentrifuged cells these vesicles were readily identified closely below the plasmalemma but, in common with previous investigations [lo, 121, were not observed in fusion with it. Judged by their angular or flattened shape these vesicles did not appear to have been recently derived from the plasmalemma by pinocytosis, and it has been shown that vesicles invaginated in such a way actually lose Exptl Cell Res 61
464
E. .7. Sanders
Fig. 2. Electron micrograph showing the cytoplasm of the centrifugal half of A. proteus 24 h after centrifugation and bisection. Note the presence of free cytoplasmic membranes (arrows). x 14 300. Fig. 3. Electron micrograph showing fringed vesicles from the centrifugal half of A. proteus. x 44 290. Fig. 4. Electron micrograph showing the type of vesicle which packed the centripetal half of A.proteus. x 37 530.
Exptf Cell Res 61
Isoenzymes
the filamentous surface material [2]. Despite these microscopical findings the centrifugal halves failed to pinocytose. The reason for this was sought in the observation that the plasmalemma of these halves was stretched tightly around the cytoplasm forming a spherical fragment with no membrane at the surface available for invagination. The membrane was applied so close to the cytoplasm that the usual hyaline layer separating the membrane from the ectoplasm was obliterated, the hyaline fluid having been displaced into the centripetal half. Goldacre [14] has demonstrated
that contact
of the plasmagel
and the cell membrane causes contraction of the former. This may account for the inability to pinocytose since this phenomenon itself is thought to involve cytoplasmic contraction dependent
on metabolic
energy [15].
The observation that the centrifugal halves of amoebae fail to recover is of some interest since normal
nucleated
fragments
apparently
suffer no ill effects apart from delayed onset of the next division [16]. The possibility that the centrifugal halves were deficient in lipid food reserve is unlikely in view of the finding that fat globules are used only shortly before death due to starvation [17, 181. Since the fragments did not appear to be suffering from an inability to osmoregulate, the evidence suggests that they were vitally missing the small vesicles or, more likely, inclusions not detected in this investigation. The increase in frequency of discontinuous membrane profiles in these fragments was very similar
to that
observed
in amoeabe
in human cell lines
465
REFERENCES 1. Palade, G E, J biophys biochem cytol, suppl. 2 (1956) 85. 2. Nachmias, V T & Marshall, J M, Biological structure and function (ed T W Goodwin & 0 Lindberg) vol. 2, p. 605: Academic Press, New York and London (1961). Revel. J P & Hav. E D. Z Zellforsch mikrosk Anat &l (1963) lib. ’ 4. Shaffer, B M, J cell sci 3 (1968) 151. Gross, L, J theoret biol 15 (1967) 298. 2: Cook, G M W, Biol rev 43 (1968) 363. I. Roth, L E, J protozool 7 (1960) 176. 8. Daniels. E W. Z Zellforsch mikrosk Anat 64 (1964) 3,s. ’ 9. Nachmias, V T, Exptl cell res 43 (1966) 583. 10. Chapman-Andresen, C, Progress in protozoology; uroc 1st int tong on nrotozoolonv, Prague (ed J Ludwig, J Lam- & J‘ Vavra) p-67. Academic Press. New York and London (1963). 11. Sand&s, E J & Bell, L G E, Exptl cell res 63 (1970). In press. 12. Chapman-Andresen, C, Compt rend trav lab Carlsberg 33 (1962) 73. 13. Nachmias, V T, Exptl cell res 38 (1965) 128. 14. Goldacre, R J, Exptl cell res, suppl. 8 (1961) 1. 15. Chapman-Andresen, C, Protoplasma 63 (1967) 103. 16. Prescott, D M, Exptl cell res 11 (1956) 94. 17. Holter, H & Zeuthen, E, Compt rend trav lab Carlsberg ser chim 26 (1948) 277. 18. Cohen, A I, J biophys biochem cytol3 (1957) 923. 19. Flickinger, C J, J cell biol 37 (1968) 300. Received April 27, 1970
Isoenzyme stability in human heteroploid cell lines N. AUERSPERG*
and S. M. GARTLER2, Tamer Research Centre, University of British Columbia, Vancouver, B.C., Canada, and 2Departments of Medicine and Genetics, University of Washington, Seattle, Wash. 98105. USA.
after
enucleation [19], although in the present case the centrifugal halves were nucleated. It appears that the occurrence of such membranes is a feature of degenerating amoebae. The author acknowledges receipt of an MRC studentship for the duration of this work.
The value of isoenzyme patterns as a means of tracing the origin of tissue-culture cell lines depends on the stability of these genetic markers. Of particular interest in this regard is the polymorphic isoenzyme variant, glucose-6-phosphate dehydrogenase (G6PD), which permits distinctions among human cell populations at the intraspecies level. In norExptl Cell Res 61
REFERENCES 1. Bianchi, N, Lima-de-Faria, A & Jaworska, H, Hereditas 51 (1964) 207. 2. Brown, S W, Science 151 (1966) 417. 3. Carr, D H & Walker, J E, Stain technol36 (1961) 233. 4. Hansen-Melander, E, Hereditas 52 (1964) 357. 5. Heitz, E, Jahrb wiss bot 69 (1928) 762. 6. Hsu, T C, J cell biol 23 (1964) 53. 7. Hsu, T C & Kellogg, D S, J natl cancer inst 25 (1960) 221. 8. Lee, J C & Yunis, J J, Exptl cell res 59 (1970) 339. 9. Lima-de-Faria, A & Jaworska, H, Nature 217 (1968) 138. 10. Sachs, L, J heredity 7 (1953) 227. 11. Schmid, W, Arch Klaus-Stift Vererb Forsch 42 (1967) 1. 12. Schmid, W, Smith, D W & Theiler, K, Arch Klaus-Stift Vererb Forsch 40 (1965) 35. 13. Wolf, U, Flinspach, G, Bohm, R & Ohno, S, Chromosoma 16 (1965) 609. 14. Yasmineh, W G & Yunis, J J, Exptl cell res 59 (1970) 69. 15. - Exptl cell res. In press. 16. Yunis ,J J & Yasmineh, W G, Science 168 (1970) 263. Received April 6, 1970 Revised version received May 22, 1970
Pinocytosis in centrifuged and bisected amoebae E. J. SANDERS,l Department of Zoology, of Southampton, Southampton, England.
University
461
pinocytosed intensely, possessed abundant plasmaLemma and contained many vesicles. Centrifugal halves, which failed to pinocytose, were bounded tightly by membrane which obliterated the hyaline layer. These halves contained vesicles with membrane similar to the plasmalemma. The results are discussed in relation to the amount of plasmalemma, or vesicular membrane precursor, available for invagination and the effect of the absence of the hyaline layer. It was shown that the centrifugal halves did not recover, and electron microscopical examination revealed an increase of free cytoplasmic membranes.
The synthesis and circulation with cells of vesicles or preformed membrane subunits which subsequently fuse with the surface, thereby adding newly fabricated membrane to the existing plasma membrane has been suggested for several cell types including amoebae [l-6]. An alternative mode of surface formation involves the interpolation of molecules or micelles of membrane material into the surface and has generally been the favoured mechanism for plasmalemma production in amoebae [7-91. The basis for this preference has been the absence of microscopical evidence to show fusion of vesicles with the surface. In the present investigation amoebae were centrifuged in order to stratify and identify electron microscopically any vesicular precursor material. The presence of presumptive plasmalemma was also tested for by applying pinocytosis inducers to the separated halves of the cells. Since it has been proposed that pinocytosis in amoebae is dependent on the amount of membrane available for invagination [lo, 111,it was considered that the intensity of pinocytotic response would be an indicator of the existence of quantities of membrane precursor localized by the centrifugation in one of the two fragments. Materials and Methods
Summary
was cultured and handled in modified Chalkley medium and fed on washed Tetrahymena.
Centrifuged amoebae were bisected to produce halves with different pinocytotic ability. Centripetal halves
1 Present address: Department of Zoology, University of Alberta, Edmonton, Alberta, Canada.
Amoebaproteus
30*-
701816
Exptl Cell Res 61
462
E. J. Sanders
The amoebae were starved for one or two days and washed in clean culture medium before use. Amoebae were centrifuged at 7 600 g for 10 min, excluding acceleration and deceleration, at 3°C. The cells were supported during centrifugation on 0.3 M sucrose solution and afterwards were pipetted into cold culture medium and quickly rinsed. Amoebae thus treated possessed two distinct halves separated by a conspicuous waist where the cells were bisected free-hand using glass micro-needles. After bisection the halves were left in culture medium for 20 or 30 min before testing for pinocytotic activity. Pinocytosis was quantitated by the channelcounting technique of Chapman-Andresen [12] with slight modification. The number of channels occurring in the microscope field was counted at various times after immersion in the pinocytosis inducer and the mean calculated for up to forty specimens. A measure of relative activity was obtained by taking the mean of the total number of channels observed in each amoeba. The inducer used was 0.125 M sodium chloride solution at pH 6.1-6.4. The amoebae were prepared for electron microscopy individually and fixed in 2 % osmium tetroxide in 0.01 N Sorensen phosphate buffer pH 6.98, at 3°C. The cells were washed and dehydrated in graded concentrations of ethanol, and embedded in Araldite. Sections were mounted on uncoated copper grids, stained with uranyl acetate and lead citrate and examined using a Philips EM300 electron microscope.
Results Light microscopical examination of the two halves produced by bisection showed that the centrifugal half was very granular, spherical and bounded by apparently unwrinkled membrane. The centripetal half, however, was very much less granular and extensive digitation was evident at the extreme centripetal end. The digitations were optically empty. The nucleus was positioned at the extreme centrifugal end of the cell.
The results of pinocytosis tests on these halves are expressed in table 1 where they are compared with similar tests on noncentrifuged halves. The table also shows the results obtained from two control experiments. Five-hour-old amoebae were used as a control for the size, since cells at this age are approximately half the size of a G2 phase cell. In the second control, amoebae were tested which had been subjected to a mimic cut, which consisted of removing a small piece of cytoplasm from a G2 phase cell and testing the remainder. From these control experiments it is clear that the results cannot be explained by reason of volume differences or of the effect of cutting. The time required for the recovery of pinocytotic activity of the centrifugal halves compared with uncentrifuged nucleated halves was determined (fig. 1). The nucleate halves returned to, and maintained, high activity after 5 h, but in the case of the centrifugal halves, pinocytosis tests were made up to 100 h after bisection and after a period of apparent recovery, attachment deteriorated, pseudopod extension became limited and cytolysis eventually ensued. Electron microscopy of centrifugal halves 24 h after bisection (fig. 2) revealed the presence of many free cytoplasmic membranes which were apparently discontinuous. The organelles occurring in the centrifugal
Table 1. Effect of centrifugation and bisection on the pinocytotic with 0.125 A4 sodium chloride pH 6.0-6.4
G2 Phase cell Centripetal half (Enucleate) Centrifugal half (Nucleate) Enucleate half Nucleate half Size control (5 h old cells) Mimic cut control Exptl Cell Res 61
Time after cutting (min)
No. of cells tested
Activity f S.E.
30 30 30 30 30 30
40 10 10 IO 10 7’
103.9 k9.1 184.6t34.2 1.5 kO.8 125.lk9.5 18.2k4.7 102.5 46.8 111.6k11.3
activity of A. proteus, tested
Enucleate/ nucleate ratio 123.1 : 1 6.9 : 1
Whole cell activity (X) 100 177.9 1.4 120.5 17.5 98.7 107.5
Centrifuged and bisected amoebae 130-
463
*\...A
120 110 -
100 90 --
i
80 70 60 50 40 30 -
Fig. 1. Abscissa: hours after bisection; ordinate: mean channel number. O-0, centrifugal halves. The extent of recovery of pinocytotic activity by bisected amoebae.
half were the nucleus, crystal vacuoles, food vacuoles, mitochondria, Golgi bodies and large vacuoles up to 5 ,um in diameter. Also localized in these fragments were flattened or angular vesicles bounded by membrane bearing filamentous material on the vacuolar face (fig. 3). The contents of the centripetal half comprised lipid droplets, contractile vacuole and a large number of small vesicles up to 450 nm in diameter which contributed about 95 % of the total volume (fig. 4).
Discussion In order to attempt to explain the high pinocytotic activity of the centripetal halves in terms of the presence of large amounts of membrane freely available for invagination, two observations were considered. Firstly these fragments possessed excess membrane already at the surface in the form of extensive digitation at the centripetal end. This almost certainly contributed to the high activity. Secondly the abundant vesicles present could have helped produce the large quantity of
nucleate halves; V-V,
surface required by fusion with the plasmalemma. It is clear that the membrane of these
vesicles as shown in fig. 4 is quite unlike the coated plasma membrane of amoebae [13]. However, it is possible that they comprised a membrane pool, some or all of which were awaiting glycosidation prior to incorporation into the plasmalemma in the manner suggested by Cook [6]. This possibility is rendered unlikely perhaps since the Golgi bodies, absent from the centripetal halves, are thought to participate in the process. The membrane of the fringed vesicles of the centrifugal half was exceedingly similar to that of the plasmalemma and must be considered as possible surface precursor. In noncentrifuged cells these vesicles were readily identified closely below the plasmalemma but, in common with previous investigations [lo, 121, were not observed in fusion with it. Judged by their angular or flattened shape these vesicles did not appear to have been recently derived from the plasmalemma by pinocytosis, and it has been shown that vesicles invaginated in such a way actually lose Exptl Cell Res 61
464
E. .7. Sanders
Fig. 2. Electron micrograph showing the cytoplasm of the centrifugal half of A. proteus 24 h after centrifugation and bisection. Note the presence of free cytoplasmic membranes (arrows). x 14 300. Fig. 3. Electron micrograph showing fringed vesicles from the centrifugal half of A. proteus. x 44 290. Fig. 4. Electron micrograph showing the type of vesicle which packed the centripetal half of A.proteus. x 37 530.
Exptf Cell Res 61
Isoenzymes
the filamentous surface material [2]. Despite these microscopical findings the centrifugal halves failed to pinocytose. The reason for this was sought in the observation that the plasmalemma of these halves was stretched tightly around the cytoplasm forming a spherical fragment with no membrane at the surface available for invagination. The membrane was applied so close to the cytoplasm that the usual hyaline layer separating the membrane from the ectoplasm was obliterated, the hyaline fluid having been displaced into the centripetal half. Goldacre [14] has demonstrated
that contact
of the plasmagel
and the cell membrane causes contraction of the former. This may account for the inability to pinocytose since this phenomenon itself is thought to involve cytoplasmic contraction dependent
on metabolic
energy [15].
The observation that the centrifugal halves of amoebae fail to recover is of some interest since normal
nucleated
fragments
apparently
suffer no ill effects apart from delayed onset of the next division [16]. The possibility that the centrifugal halves were deficient in lipid food reserve is unlikely in view of the finding that fat globules are used only shortly before death due to starvation [17, 181. Since the fragments did not appear to be suffering from an inability to osmoregulate, the evidence suggests that they were vitally missing the small vesicles or, more likely, inclusions not detected in this investigation. The increase in frequency of discontinuous membrane profiles in these fragments was very similar
to that
observed
in amoeabe
in human cell lines
465
REFERENCES 1. Palade, G E, J biophys biochem cytol, suppl. 2 (1956) 85. 2. Nachmias, V T & Marshall, J M, Biological structure and function (ed T W Goodwin & 0 Lindberg) vol. 2, p. 605: Academic Press, New York and London (1961). Revel. J P & Hav. E D. Z Zellforsch mikrosk Anat &l (1963) lib. ’ 4. Shaffer, B M, J cell sci 3 (1968) 151. Gross, L, J theoret biol 15 (1967) 298. 2: Cook, G M W, Biol rev 43 (1968) 363. I. Roth, L E, J protozool 7 (1960) 176. 8. Daniels. E W. Z Zellforsch mikrosk Anat 64 (1964) 3,s. ’ 9. Nachmias, V T, Exptl cell res 43 (1966) 583. 10. Chapman-Andresen, C, Progress in protozoology; uroc 1st int tong on nrotozoolonv, Prague (ed J Ludwig, J Lam- & J‘ Vavra) p-67. Academic Press. New York and London (1963). 11. Sand&s, E J & Bell, L G E, Exptl cell res 63 (1970). In press. 12. Chapman-Andresen, C, Compt rend trav lab Carlsberg 33 (1962) 73. 13. Nachmias, V T, Exptl cell res 38 (1965) 128. 14. Goldacre, R J, Exptl cell res, suppl. 8 (1961) 1. 15. Chapman-Andresen, C, Protoplasma 63 (1967) 103. 16. Prescott, D M, Exptl cell res 11 (1956) 94. 17. Holter, H & Zeuthen, E, Compt rend trav lab Carlsberg ser chim 26 (1948) 277. 18. Cohen, A I, J biophys biochem cytol3 (1957) 923. 19. Flickinger, C J, J cell biol 37 (1968) 300. Received April 27, 1970
Isoenzyme stability in human heteroploid cell lines N. AUERSPERG*
and S. M. GARTLER2, Tamer Research Centre, University of British Columbia, Vancouver, B.C., Canada, and 2Departments of Medicine and Genetics, University of Washington, Seattle, Wash. 98105. USA.
after
enucleation [19], although in the present case the centrifugal halves were nucleated. It appears that the occurrence of such membranes is a feature of degenerating amoebae. The author acknowledges receipt of an MRC studentship for the duration of this work.
The value of isoenzyme patterns as a means of tracing the origin of tissue-culture cell lines depends on the stability of these genetic markers. Of particular interest in this regard is the polymorphic isoenzyme variant, glucose-6-phosphate dehydrogenase (G6PD), which permits distinctions among human cell populations at the intraspecies level. In norExptl Cell Res 61