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The submicroscopic structure of the balbiani-ring
Experimental
Cell Research,
THE
6, 195.-201
SUBMICROSCOPIC STRUCTURE THE BALBIANI-RING W.
Max Planck Institute
195
(1954)
lnstitut
for Cell
BEERMANN
and
fiir Meeresbiologie, Research, Karolinska Received
August
OF
G. F. BAHR Wilhelmshaven, Germany, and Institutet, Stockholm, Sweden 4, 1953
SINCE the work of Pease and Baker (9) several attempts have been made to obtain direct elec,tron optical proof of the polytene theory of giant chromosome organization. However, the evidence presented either in favour (9, 10, 11) or against (3, 8) this theory cannot be considered as convincing since the techniques used involved inadequate procedures, such as acetic acid treatment for squashing, dissolving the embedding medium after sectioning, and in most cases drying of the preparation. Only very recently techniques have been developed in electron microscopy which are satisfactory from the cytological point of view: fixation in buffered OsO,-solution according to Palade (7), embedding in methacrylate polymer according to Newman, Borysko and Swerdlow (6), and ultra-thin sectioning and investigation of the sections without dissolving the embedding medium as first proposed by Hillier and Gettner (4). Borysko (3) has used this method, but replaced the embedding medium by collodion. Freshly dissected salivary glands of larvae of Chironomus tentans Fabr., and one other undetermined Chironomus species, were placed over OsO,-vapour for 4 minutes and then fixed for 2 hours in 1 per cent 0~0, in distilled water, buffered at pH 7.2 with veronal. After embedding in methacrylate the glands were sectioned with a Spencer No. 820 rotary microtome adjusted to 0.1 p, using specially sharpened steel blades.1 After stretching on 50°C warm distilled water, the sections were transferred to collodion-coated specimen holders. The instrument used was an RCA EMC’ type electron microscope. The advantages of the new combined technique are obvious from Fig. 1: The general appearance and especially the banding pattern of the salivary gland chromosomes as seen in the electron microscope correspond completely to the pictures obtained by means of the UV or ordinary light microscope. Remarkably enough, the thicker bands arc not compound, in the sense that they would consist of subunits of uniform thickness corresponding to the 1 1;. Sjiistrand, 1 li ~ 533705
Personal
communication. Experimenlnl
Cell Research
6
W. Beermann
and G. F. Bahr
thinnest bands to be seen. At least, no such subdivision is detectable. Some are known already double or triple bands may occur, but they generally On the other hand, the thinnest bands, from light microscopical studies. ranging from 0.1 p down to about 0.05 p (Figs. 2 and 3), certainly are not revealed light-optically, even in optimally stretched chromosome parts in squash preparations. Their number, however, does not seem to be very large, so that the number of bands in cytological maps of giant chromosomes would not have to be multiplied by a factor much larger than 2. The total would still not exceed gene numbers as estimated from genetic evidence. The main interest in this study was directed to the structure of certain regions of the 4th chromosome of C. tenfans which in the salivary glands constantly appear modified as “Balbiani-rings”. From earlier observations (2) it was known that these structural modifications are an expression of high physiological activity of the regions involved. In the light microscope, Balbiani-rings have a certain resemblance to nucleoli but differ from them in several aspects, mainly their diffuse though regular contour. The adjacent chromosome sections split up progressively until the thinnest strands disappear within the diffuse, predominantly peripheral, parts of the ring (1). On the basis of the polytene theory it has been assumed (2) that the individual elements (“chromonemata”) of the giant chromosome completely give up their lateral union for a short part of their length at these points, each one forming a loop before uniting again with other elements to resume its helical course within the chromosome -“cable”. This interpretation has now been checked electron-optically as shown in Figs. 5-8. The photograph in Fig. 5 has been obtained from an oblique section through the big Balbiani-ring of the 4th salivary chromosome of Chironomus tentans. The majority of the branchings of the adjacent chromosome regions running into the ring have been cut transversely or obliquely-if hit longitudinally they clearly show their banding (lower right of Fig. 5). Between these, the light-optically diffuse parts of the ring appear as a mass of extremely spongy, uniform elements which at the periphery clearly appear curved, at some points even show complete loops. It is suggested that these are the Fig. 1. Regions 16 and 17 of the 2nd salivary chromosome of Chironomus tentans in the UV (upper photograph) and in the electron microscope, both pictures at the same magnification, 4,700 x . The WV photograph is from an unstained, acetic acid fixed squash preparation mounted in glycerol, the electron photograph from an ultra-thin section (see text for technique.) Fig. 2. Region 17 B of the 2nd chromosome, in the UV and in the electron microscope, 10,000 x . Fig. 3. Right end of the 2nd chromosome showing the “telomere”, a heterochromatic band of only about 0.05 p thickness, 30,000 X. Fig. 4. Partial section of one nucleolus together with its organizer, 12,000 x . Experimental
Cell Research
6
Submicroscopic
structure
of the Balbia
Experimental
Cell Research
6
198
Experimental
IV. Beermann
Cdl
Research
6
and G. F. Bahr
Submicroscopic
structure of the Balbiani-ring
ultimate longitudinal units of the giant chromosbme, though obviously in a special structural state: certainly they are. not just smooth fibrils. In highresolution photographs (Figs. 7 and 8) each individual element resolves itself into a loose system of irregularly zig-zagging threads giving the impression of a “lamp-brush” (not to be confused with the “lamp-brush” chromosomes of amphibian oocytes). The existence of a continuous axial tibre can only be guessed, whereas numerous branchings of about 10 rnp thickness are clearly visible. They bear characteristic spherical bodies of a maximum diameter of about 30 rnp, apparently attached to their ends. These globules never appear at other points of the chromosomes. The whole “chromonema” in the condition described has an average diameter of about 0.2 p, twice as much as the finest still compact, hence light-optically discernible, branchings of the chromosome, cross sections of which appear as dark spots a few micra distant from the periphery of the ring. As judged from this distance the length of the “lamp-brush” segments of the chromonemata may reach 4 p. Continuous pieces of about 2 p are clearly seen at some points of the section in Fig. 5. These observations may be interpreted as follows: As has already been inferred from light-optical studies, the “active” segments of the chromonemata within the Balbiani-ring are in an extremely extended state, possibly more than 5 times their normal length, because the development of Balbiani-rings can be traced back to single bands or interband regions of no more than 0.5 to 1 TVthickness under normal circumstances. Since the giant chromosomes themselves have up to 10 times the length of the pachytene chromosomes, this means that the “lamp-brushes” are 50 times longer than the corresponding segments at pachytene, and yet, functionally, they may constitute nothing but one single gene locus. In addition, the “lamp-brushes” show a considerable degree of lateral “puffing” as judged from the fact that the immediately neighbouring branchings of the chromosome are only half as thick and still probably contain 2 or more of the ultimate elements. Unfortunately, points of transition between the finest branchings of normal structure and the “lamp-brushes” could not be demonstrated as yet. The blown-up condition of each individual “Balbiani-locus” has to be considered as the main cause
Fig. 5. Part of an oblique section through the big Balbiani-ring of the 4th chromosome of C. tentans, 12,000 x. Fig. 6. Peripheral part of the Balbiani-ring of another undetermined Chironomus species, 20,000 x . Fig. 7. High-resolution photograph of the periphery of a Balbiani-ring, 42,000 x . Fig. 8. Part of Fig. 5 at a higher magnification, showing the “lamp-brush” structures referred to in the text, 38,000 x. 12 - 633705
Experimental
Cell Research
6
200
W. Beermann
and G. F. Bahr
of “puffing” of the whole Balbiani-ring, as discussed by Mechelke (5), and certainly has some causal relationship to the formation of the characteristic globules mentioned above. Sufficiently thin sections of normal bands show what seem to be closely packed thread-like elements of the same dimensions as those composing the “lamp-brushes”. Failure to demonstrate the individual within the normally built regions of giant chromosomes “chromonemata” may then be explained as a consequence both of their complicated structure and close union. The question of the degree of polyteny of giant chromosomes has often been discussed. The number of “lamp-brushes” within the total area of the particular section, part of which is shown in Fig. 5, may roughly be estimated to lie between 1,000 and 2,000. Since the “lamp-brush” segments are rather short (2-4 p) as compared to the diameter of the Balbiani-ring (about 20 p), obviously only part of them is included in the section. The total may amount to 10 times as much, which means that the paired salivary chromosome of a full-grown Chironomus larva may contain more than 10,000 longitudinal elements. This value is in agreement with earlier estimates based on nuclear measurements (a), if the number of chromonemata within the original telophase chromosome, before it becomes polytene, is assumed to be very small. Some accidental observations on the salivary gland nucleoli of Chironomus tentans may be added here. Light-microscopically, the nucleolar substance often, but not always, is seen to consist of two zones, a central clear one and a peripheral, granular one. Islands of the granular substance may be included in the clear zone. The nucleolar organizers are diffusely staining (acetocarmine) interband regions of about 0.5 to 1 p thickness. In the electron microscope, after osmium “staining”, the central zone of the nucleoli appears highly absorbing and finely granular, while the peripheral substance is seen to consist of loosely packed, short, rod-like particles (Fig. 4). Points of transitional structure may be found. With its central, denser portion the nucleoli are attached to the organizers which have a slightly lesser absorption than the nucleolus itself. The preliminary observations reported here are in full accordance with the polytene theory in general, and with the concepts of Balbiani-ring structure proposed earlier (2, 5). OsO,-fixation may not be able to preserve or demonstrate all details of living structure, and in the interpretation this has to be kept in mind. However, no better fixative is known at present, and, by generally accepted cytological standards, the structures it does preserve are in excellent condition. Experimental
Cell Research
6
Submicroscopic
201
structure of the Balbiani-ring REFERENCES
1. 2. 3. 4. 5. 6. 7. 8. 9. 10. 11.
BAUER, H., and BEERMANN, W., Chromosoma, 4, 630 (1952). BEERMANN, W., ibid., 5, 139 (1952). BORYSKO, E., Bull. Johns Hopkins Hosp., 92, 151 (1953). HILLIER, J., and GETTNER, M. D., Science, 112, 520 (1950). MECHELKE, F., Chromosoma, 5, 511 (1953). NEWMAN, S. B., BORYSKO, E., and SWERDLOW, M., Sciende, 110, 66 (1949). PALADE, G. E., J. Expfl. Med., 95, 285 (1952). PALAY, S. L., and CLAUDE, A., ibid., 89, 431 (1949). PEASE, D. C., and BAKER, R. F., Science, 109, 8 (1949). SCHULTZ, J., MACDUFFEE, R. C., and ANDERSON, T. F., Science, 110, 5 (1949). YASUZUMI, G., ODATE, Z., and OTA, Y., Cytologia, 16, 233 (1951).
Experimental
Cell Research
6
Cell Research,
THE
6, 195.-201
SUBMICROSCOPIC STRUCTURE THE BALBIANI-RING W.
Max Planck Institute
195
(1954)
lnstitut
for Cell
BEERMANN
and
fiir Meeresbiologie, Research, Karolinska Received
August
OF
G. F. BAHR Wilhelmshaven, Germany, and Institutet, Stockholm, Sweden 4, 1953
SINCE the work of Pease and Baker (9) several attempts have been made to obtain direct elec,tron optical proof of the polytene theory of giant chromosome organization. However, the evidence presented either in favour (9, 10, 11) or against (3, 8) this theory cannot be considered as convincing since the techniques used involved inadequate procedures, such as acetic acid treatment for squashing, dissolving the embedding medium after sectioning, and in most cases drying of the preparation. Only very recently techniques have been developed in electron microscopy which are satisfactory from the cytological point of view: fixation in buffered OsO,-solution according to Palade (7), embedding in methacrylate polymer according to Newman, Borysko and Swerdlow (6), and ultra-thin sectioning and investigation of the sections without dissolving the embedding medium as first proposed by Hillier and Gettner (4). Borysko (3) has used this method, but replaced the embedding medium by collodion. Freshly dissected salivary glands of larvae of Chironomus tentans Fabr., and one other undetermined Chironomus species, were placed over OsO,-vapour for 4 minutes and then fixed for 2 hours in 1 per cent 0~0, in distilled water, buffered at pH 7.2 with veronal. After embedding in methacrylate the glands were sectioned with a Spencer No. 820 rotary microtome adjusted to 0.1 p, using specially sharpened steel blades.1 After stretching on 50°C warm distilled water, the sections were transferred to collodion-coated specimen holders. The instrument used was an RCA EMC’ type electron microscope. The advantages of the new combined technique are obvious from Fig. 1: The general appearance and especially the banding pattern of the salivary gland chromosomes as seen in the electron microscope correspond completely to the pictures obtained by means of the UV or ordinary light microscope. Remarkably enough, the thicker bands arc not compound, in the sense that they would consist of subunits of uniform thickness corresponding to the 1 1;. Sjiistrand, 1 li ~ 533705
Personal
communication. Experimenlnl
Cell Research
6
W. Beermann
and G. F. Bahr
thinnest bands to be seen. At least, no such subdivision is detectable. Some are known already double or triple bands may occur, but they generally On the other hand, the thinnest bands, from light microscopical studies. ranging from 0.1 p down to about 0.05 p (Figs. 2 and 3), certainly are not revealed light-optically, even in optimally stretched chromosome parts in squash preparations. Their number, however, does not seem to be very large, so that the number of bands in cytological maps of giant chromosomes would not have to be multiplied by a factor much larger than 2. The total would still not exceed gene numbers as estimated from genetic evidence. The main interest in this study was directed to the structure of certain regions of the 4th chromosome of C. tenfans which in the salivary glands constantly appear modified as “Balbiani-rings”. From earlier observations (2) it was known that these structural modifications are an expression of high physiological activity of the regions involved. In the light microscope, Balbiani-rings have a certain resemblance to nucleoli but differ from them in several aspects, mainly their diffuse though regular contour. The adjacent chromosome sections split up progressively until the thinnest strands disappear within the diffuse, predominantly peripheral, parts of the ring (1). On the basis of the polytene theory it has been assumed (2) that the individual elements (“chromonemata”) of the giant chromosome completely give up their lateral union for a short part of their length at these points, each one forming a loop before uniting again with other elements to resume its helical course within the chromosome -“cable”. This interpretation has now been checked electron-optically as shown in Figs. 5-8. The photograph in Fig. 5 has been obtained from an oblique section through the big Balbiani-ring of the 4th salivary chromosome of Chironomus tentans. The majority of the branchings of the adjacent chromosome regions running into the ring have been cut transversely or obliquely-if hit longitudinally they clearly show their banding (lower right of Fig. 5). Between these, the light-optically diffuse parts of the ring appear as a mass of extremely spongy, uniform elements which at the periphery clearly appear curved, at some points even show complete loops. It is suggested that these are the Fig. 1. Regions 16 and 17 of the 2nd salivary chromosome of Chironomus tentans in the UV (upper photograph) and in the electron microscope, both pictures at the same magnification, 4,700 x . The WV photograph is from an unstained, acetic acid fixed squash preparation mounted in glycerol, the electron photograph from an ultra-thin section (see text for technique.) Fig. 2. Region 17 B of the 2nd chromosome, in the UV and in the electron microscope, 10,000 x . Fig. 3. Right end of the 2nd chromosome showing the “telomere”, a heterochromatic band of only about 0.05 p thickness, 30,000 X. Fig. 4. Partial section of one nucleolus together with its organizer, 12,000 x . Experimental
Cell Research
6
Submicroscopic
structure
of the Balbia
Experimental
Cell Research
6
198
Experimental
IV. Beermann
Cdl
Research
6
and G. F. Bahr
Submicroscopic
structure of the Balbiani-ring
ultimate longitudinal units of the giant chromosbme, though obviously in a special structural state: certainly they are. not just smooth fibrils. In highresolution photographs (Figs. 7 and 8) each individual element resolves itself into a loose system of irregularly zig-zagging threads giving the impression of a “lamp-brush” (not to be confused with the “lamp-brush” chromosomes of amphibian oocytes). The existence of a continuous axial tibre can only be guessed, whereas numerous branchings of about 10 rnp thickness are clearly visible. They bear characteristic spherical bodies of a maximum diameter of about 30 rnp, apparently attached to their ends. These globules never appear at other points of the chromosomes. The whole “chromonema” in the condition described has an average diameter of about 0.2 p, twice as much as the finest still compact, hence light-optically discernible, branchings of the chromosome, cross sections of which appear as dark spots a few micra distant from the periphery of the ring. As judged from this distance the length of the “lamp-brush” segments of the chromonemata may reach 4 p. Continuous pieces of about 2 p are clearly seen at some points of the section in Fig. 5. These observations may be interpreted as follows: As has already been inferred from light-optical studies, the “active” segments of the chromonemata within the Balbiani-ring are in an extremely extended state, possibly more than 5 times their normal length, because the development of Balbiani-rings can be traced back to single bands or interband regions of no more than 0.5 to 1 TVthickness under normal circumstances. Since the giant chromosomes themselves have up to 10 times the length of the pachytene chromosomes, this means that the “lamp-brushes” are 50 times longer than the corresponding segments at pachytene, and yet, functionally, they may constitute nothing but one single gene locus. In addition, the “lamp-brushes” show a considerable degree of lateral “puffing” as judged from the fact that the immediately neighbouring branchings of the chromosome are only half as thick and still probably contain 2 or more of the ultimate elements. Unfortunately, points of transition between the finest branchings of normal structure and the “lamp-brushes” could not be demonstrated as yet. The blown-up condition of each individual “Balbiani-locus” has to be considered as the main cause
Fig. 5. Part of an oblique section through the big Balbiani-ring of the 4th chromosome of C. tentans, 12,000 x. Fig. 6. Peripheral part of the Balbiani-ring of another undetermined Chironomus species, 20,000 x . Fig. 7. High-resolution photograph of the periphery of a Balbiani-ring, 42,000 x . Fig. 8. Part of Fig. 5 at a higher magnification, showing the “lamp-brush” structures referred to in the text, 38,000 x. 12 - 633705
Experimental
Cell Research
6
200
W. Beermann
and G. F. Bahr
of “puffing” of the whole Balbiani-ring, as discussed by Mechelke (5), and certainly has some causal relationship to the formation of the characteristic globules mentioned above. Sufficiently thin sections of normal bands show what seem to be closely packed thread-like elements of the same dimensions as those composing the “lamp-brushes”. Failure to demonstrate the individual within the normally built regions of giant chromosomes “chromonemata” may then be explained as a consequence both of their complicated structure and close union. The question of the degree of polyteny of giant chromosomes has often been discussed. The number of “lamp-brushes” within the total area of the particular section, part of which is shown in Fig. 5, may roughly be estimated to lie between 1,000 and 2,000. Since the “lamp-brush” segments are rather short (2-4 p) as compared to the diameter of the Balbiani-ring (about 20 p), obviously only part of them is included in the section. The total may amount to 10 times as much, which means that the paired salivary chromosome of a full-grown Chironomus larva may contain more than 10,000 longitudinal elements. This value is in agreement with earlier estimates based on nuclear measurements (a), if the number of chromonemata within the original telophase chromosome, before it becomes polytene, is assumed to be very small. Some accidental observations on the salivary gland nucleoli of Chironomus tentans may be added here. Light-microscopically, the nucleolar substance often, but not always, is seen to consist of two zones, a central clear one and a peripheral, granular one. Islands of the granular substance may be included in the clear zone. The nucleolar organizers are diffusely staining (acetocarmine) interband regions of about 0.5 to 1 p thickness. In the electron microscope, after osmium “staining”, the central zone of the nucleoli appears highly absorbing and finely granular, while the peripheral substance is seen to consist of loosely packed, short, rod-like particles (Fig. 4). Points of transitional structure may be found. With its central, denser portion the nucleoli are attached to the organizers which have a slightly lesser absorption than the nucleolus itself. The preliminary observations reported here are in full accordance with the polytene theory in general, and with the concepts of Balbiani-ring structure proposed earlier (2, 5). OsO,-fixation may not be able to preserve or demonstrate all details of living structure, and in the interpretation this has to be kept in mind. However, no better fixative is known at present, and, by generally accepted cytological standards, the structures it does preserve are in excellent condition. Experimental
Cell Research
6
Submicroscopic
201
structure of the Balbiani-ring REFERENCES
1. 2. 3. 4. 5. 6. 7. 8. 9. 10. 11.
BAUER, H., and BEERMANN, W., Chromosoma, 4, 630 (1952). BEERMANN, W., ibid., 5, 139 (1952). BORYSKO, E., Bull. Johns Hopkins Hosp., 92, 151 (1953). HILLIER, J., and GETTNER, M. D., Science, 112, 520 (1950). MECHELKE, F., Chromosoma, 5, 511 (1953). NEWMAN, S. B., BORYSKO, E., and SWERDLOW, M., Sciende, 110, 66 (1949). PALADE, G. E., J. Expfl. Med., 95, 285 (1952). PALAY, S. L., and CLAUDE, A., ibid., 89, 431 (1949). PEASE, D. C., and BAKER, R. F., Science, 109, 8 (1949). SCHULTZ, J., MACDUFFEE, R. C., and ANDERSON, T. F., Science, 110, 5 (1949). YASUZUMI, G., ODATE, Z., and OTA, Y., Cytologia, 16, 233 (1951).
Experimental
Cell Research
6