UNCLASSIFIED ORNL 483 ORNU-P-482 imit / MASTER ON THE CHEMISTRY OF CHROMOSOME CONTINUITY* Sheldon Wolff. Biology Division, Oak Ridge National Laboratory, Oak Ridge, Tennessee, U.S.A, -LEGAL NOTICE Two report m. yro parad v an account of Covenant aponsored work. Welther We Unid Haloo, nor and Condinolom, nor any puro kung on behalf of the Coamaalan: A. Makes my verrasty or repreneaualloa, expondus lapied, mu rospect to the scou. racy, compluwaon, or wechdoc. of the laboranuon contand u we report, or that the of my ladalinalon, apparsta, tod, or procesu di loud In No report wy hot latring primately owned righus; or D. A may liabilitar viu roopcl to the wool, or for mere roles frou une w of way to daratto, wel, method, or prorsus disclosed ja to report. As we are bore, "person una ball of the Coonho" clamy - ways or contractor of the Counum, or uploys of such contractor, hohe ostmet wat such uploys or contractor of the Cousinslon, or neployof much contractor wparna, dowuwu, or prorkes «cow to, may information purtat to vo opogut or contract with the Commission, or We employees with such contractor. *Research sponsored by the U. S. Atomic Energy Commission under contract with the Union Carbide Corporation. PNV Chromosomes of higher organisms consist of nucleic acids and proteins. This much is fact. If, however, we try to be more specific and describe the architectural relations of the chromosome's component molecules, we are soon involved in fancy, for we lave no precise knowledge of how the chromosome 18 constructed either linearly or laterally. That 18, we do not know if the linear continuity of the chromosome is maintained by nucleic acid, protein, both individually, or by a nucená protein complex, nor do we even know how many strands comprise a chromosome. Many models have been proposed for chromosome structure and each of these has been based on some, but not all, of the available experimental data. Consequently, these models all differ from one another. Some of these models maintain that the chromosome has a backbone, that may or may not be protein, from which deoxyribonucleic acid (DNA) extends (Taylor, 1957). Others maintain that the linear continuity of the chromosome is maintained by DNA (Taylor, 1959, 1960) or by alternating pieces of DNA and protein (Schwartz, 1958). The models differ more grossly in that some represent a chromosome consisting of several units that are separated laterally to form a multistranded unit (see Kaufmann, et. al, 1960; Steffensen, 1959, 1960; Ris, 1961), whereas others represent a chromosome that is single stranded. Over the years we have performed experiments that have given some information about chromosome structure. One type of experiment on the metabolic requirements for the rejoining of radiation-induced breaks can be interpreted as indicating that protein contributes to the linear continuity of the chromosome. A second type of experiment on the type of chromosome aberrations induced by radiation indicates that the chromosome 18 miltistranded. A third type of experiment on enzyme 8 Oule digestion of isolated fixed Vicia faba chromosomes has indicated that the mitotic chromosome depends upon protein for its linear continuity and is multistranded. - Evidence from experiments on chromosome brood heat rejoining or repair X-ray dose fractionation experiments were performed on two-break chromosome aberrations such as dicentrics and rings. These aberrations follow two-hit dose kinetics with x-rays, that 18, they increase approximately as the square of the dobe. If the dose is fractionated into two parts such that the breaks from the first dose cannot rejoin or restitute (rejoin in the original configuration) before the breaks from the second dose are introduced, then both groups of breaks will be present concurrently and capable of rejoining with one another to form two-break aberrations. If, however, the time between the doses is sufficient for restitution of breaks to occur then there will be no possible interaction between the two groups of breaks, and the yield of aberrations will be the sum of the aberrations induced by the first dose HUI On plus that induced by the second. We have performed an extensive series of experiments on G, cells in root tips of soaked seed of Vicia faba. The two-dose fractions were separated by enough time to allow rejoining of the first group of breaks. We have found that after the first dose was administed, that is after the first group of breaks were induced in the cells, we could inhibit metabolism in such a way that breaks would remain open instead of restituting. Thus, after the second dose of radiation, all breaks would be in the cell together, be able to rejoint with one another, and the yield of aberrations now would be proportional to the square of the total dose administered. If & found that cellular respiration and the production of ATP wau necessary for breaks to close. If we inhibited respiration by lowering the temperature, adding cyanide, adding 95% carbon mono::ide plus 5% oxygen in the dark (but not in the light), or by adding dinitrophenol to upcouple ATP synthesis from cellular respiration, then breaks remained open. We have similarly found that the addition of exogenous ATP seemed to cause breaks to close faster as did treatments with manganese chloride that possibly stimulated the production of endogenous ATP. All this showed that a source of energy was necessary when "honds were formed during chromosome break repair. We wondered about the nature of the bonds. Because chromosomes consist of protein and nucleic acids, it seemed worthwhile to see if the synthesis of any or all of these components was involved in the rejoinir.g or repair. We, therefore, performed experiments with the protein synthesis inhibitor chloramphenicol. It hed no effect on the yield induced after a dose of radiation to G, cells of soaked seeds of Vicia faba nor did it have an effect when given before a dose. When, however, it was administered between two doses, it prevented the breaks from the first dose from rejoining before the second was given. The breaks of the two doses were thus in the cell concurrently and could interact with one another to form aberrations. Since these experiments were performed in G, cells, DNA synthesis was thought not to occur and this was confirmed in experiznents in which we found that tritiated thymidine was not incorporated into those cells at the time the experiments were performed. We also found RNA was not affected by chloramphenicol treatment by finding that it did not change the incorporation of HPIZO into the nucleic acids. Chloraphenicol did, however, inh:init protein synthesis in Vicia. At the concentrations used Sit Here: (300 mg/ml) chloramphenicol was found to bring about a 30% reduction in incorporation of labelled amino acids into the cells' total protein. We have subsequently checked only nuclear proteins by incubating the cells with tritiated lysine, extracting the nuclei, and making autoradiographo of nuclei uncontaminated with cytoplasmic proteins. Counts of the grains in the autoradiographs showed that there is a 46% inhibition of incorporation of labelled amino acids into nuclear protein SO TO and, therefore, presumably a 46% inhibition of protein synthesis. Because of the effect of chloramphenicol, and because rejoining can occur throughout the cell cycle (even in stages where DNA synthesis does not normally occur), we postulated that the honds formed in the repair of chromosome breaks were proteinaceous and not in nucleic acid (Wolff, 1960, 1961). We recognized, however, that the studies on protein inhibition might only have indicated that synthesis of an enzyme, which of course would be a protein, was necessary. The formal possibility still existed that the repair occurred in DNA and required so little DNA synthesis that It could not be picked up in our experiments with tritiated thymidine. Sandra Bell and I (1964) then performed experiments with 5-fluorodeoxy- uridine (FUAR), a compound that inhibits DNA synthesis in bacterial systems. We found it did not prevent breaks in G, cells from rejoining. When it was administrered between two doses of x-rays, the yields were the same as in the controls, that 18, simply the sum of the yields produced by the two Individual doses. That the colipound can inhibit DNA synthesis in Vicia faba was shown by Bell in experiments in which she treated actively growing roots that had cells in S. Autoradiographs of FUIR treated roots showed no incorporation of tritiated deoxycytidine into DNA. In toto then the observations indicate repair occurs in those portions of the cell cycle in which DNA synthesis does not occur and that DNA synthesis inhibitors do not prevent repair, whereas protein synthesis inhibitors do. We, thus, still favor the interpretation that the bonds formed in chromosome rejoining are proteinaceous. Evidence from radiation experiments Radiation studies on the types of induced chromosomal aberrations have long indicated that in late prophase and metaphase an aberration can be induced that ostensibly represents the interaction of but half chromatids (Swanson, 194%, Crouse, 1954, Wilson, et al., -959). This Indicates that the chronos ome 18 at least two stranded. We have carried out studies on the types of aberrations induced in ( Sinem chonestatis illud gedaalid different parts of the cell cycle (Wolff and Luippold, 1964). Actively Everno y (19 growing lateral roots of Vicia faba vere treated with tritiated thymidine for 15 minutes to label those cells in the S portion of interphase. The • w 1 . roots were then irradiated with 150 r of 250 KVP x rays. The cells were sampled every three hours and metaphase cells were scored to see the types of aberrations induced and whether or not the cells were labelled. We found that the first cells to reach metaphase came from Go, that is they were unlabelled. These cells all had chromatid aberrations. Later samplings showed labelled cells from S at metaphase. These, too, all had chromatid aberrations. When cells from G, reached metaphase, they were found to be unlabelled and to have chromosome aberrations - the type in which both strands are similarly affected. The presence of this type of 11 . aberration indicates that the chromosome was effectively single at the time of x irradiation when the break was produced and at the time of 27 chromosome replication the break, too, 18 replicated. In an attempt to find just when the chromosome did replicate (act 2 . 10 double to radiation) in reference te the time of DNA synthesis, we performed an experiment in which we irradiated first and then administered tritiated thymidine either immediately or after a delay. It was thought that the delay would allow chromos anes that were broken in G, (pre S interphase) to proceed to S where they would be labelled upon duplication. This should give rise to labelled chromosome aberrations. Roots were fixed at successive three hour intervals in an attempt to sample cells that were in different portions of the cell cycle at the time of irradiation and especially to insure that cells were sampled from very early S. Results from such an experiment are presented in Table 1, in the column isted short-exposure. The table shows that if the label was administered immediately after the radiation that no labelled chromosome sberrations appeared even at fixations up to 24 hours. If, however, the application of label was delayed for one hour after radiation, then an occasional cell Vreded nitrifero. oeeurred that had labelled chromosome aberrations. It was not until the application of label was delayed for two hours, however, that a significant number of cells broken in G, seemed to proceed to S where they could pick up label to give rise to labelled chromosome as contrasted to chromatid aberrations. The simplest interpretation for such results is that in irradiated material the chromosome is already acting double to radiation for a period of two hours before s. Thus, irradiation of chromosomes before s could give rise to chromatid aberrations that become labelled when the cells move into's. It is as though the chromosome were single two hours before s and double from then on. Such an interpretation, however, presupposes an orderly progression of cells through the cell cycle and presupposes there would be no radiation 11 induced delay of mitosis thet would affect the order in which ells arrive at metaphase. Such, however, is not the case: After irradiation, 100% of the metaphases are never labelled as would be expected if there were an orderly progression of cells through the mitotic cycle (see Wimber, 1960). Furthermore, some experiments were performed in which autoradiographs were exposed for very long periods of time so that the number of grains over the nuclei increased to the point where heavily labelled cells could no longer be scored for aberrations because grains obscured the chromosomes. We found that among the cells that could be scored, 1. e., were only very lightly labelled, labelled chromosome aberrations did appear even without a delay between irradiation and the ON addition of tritiated thymidine. The appearance of lightly labelled chromosome aberrations under these conditions indicates that the chromosome i£ effectively single in very early s before much label becomes incorporated into the chromosomes. Shortly after this, however, the chromosomes, indeed, are effectively double as evidenced by the fact that those cells that are heavily labelled and so observable with short exposures, always have only chromatid aberrations in them. We were not able to distinguish whether the lightly labelled cells that had chromosome aberrations in the long exposure experiments had been in S at the time they were broken or had been in G, and then picked up & later when the cells proceed into s) a small amount of label that could not be washed from the cells. The resolution of the method, therefore, is only good enough to allow the statement that the chromosome acts double to radiation either before s or in very early s before much DNA has been duplicated. In either case we are left with an indication that the chromosome is at least two stranded. 12 Chu (1965) has carried out studies on chromatid aberrations irduced by ultraviolet Irradiation of mammalian cells in tissue culture. lle has found that the action spectrum for the efficiency of various wavelengths of ultraviolet in inducing aberrations has a broad peak between 2400 A and 2800 Ă. If chromosome breaks were induced only in DNA then we might expect the peak of the action spectrum to more closely resemble the UV absorption spectrum for DNA and to drop sharply to 2800 A. Since his action spectrum did not drop sharply at this wavelength, it indicates that the energy that breaks chromosomes can be absorbed in both DNA and protein, perhaps even in a nucap protein complex. Chu has also found that irradiation with 2690 A induces chromosome breaks, whereas irradiation at 2800 A causes what he calls chromosome demolition. If he irradiates at do A, a wavelength that is efféciently absorbed by DNA, and subsequently treats with puromycin, a protein synthesis inhibitor, then he obtains demolished chromosomes much like that found when he irradiates at 2800 A. This possibly implicates breaks in protein even when irradiations are carried out at 2650. Studies on enzyme digestion of chromosomes Trosko, who has been working in my laboratory, has recently treated - fixed isolated formalin, chromosomes of Vicia faba with various enzymes (Trosko and Wolff, 1965). The enzymes were other trypsin (0.1 mg trypsin/ml of 0.01 M phosphate buffer pH 7.21 DNase (1.0 mg DNase/m2 of 0.003 M Magnesium sulfate in 0.1 M phosphate buffer pH 6.8), RNase (1.0 mg RNase/ml of 0.01 M phosphate buffer at pH 7.0), or pepsin (1 mg pepsin/ml of 0.1 N HCl at pH 1.3). One drop of isolated nuclei and chromosomes in phosphate buffer was mixed with three drops of enzyme solution on a micro- slide which was then incubated in a moist chamber at 35°C for one hour. At tim unit of this time the preparations were air dried and stand for ) Wer DNA by the feulgen procedure for RNA with azure B, or for histones by the alkaline fast green method of Alfert and Geschwind (1953). The latter was modified in that the material was hydrolized in 5% trichloroacetic acid for one hour instead of only 15 minutes. The isolation procedure gave preparations of nuclei that were relatively free of cytoplasmic contamination (Figure 1). In Figure 2 we see a portion of the preparation containing both free nuclei and clumped metaphase chromosomes that are caught in cytoplasm. A summary of the effects of various enzyme treatments is presented in Table 2 It may be seen that pepsin, DNase and RNase do not appreciably affect the morphology of the nuclei and chromosomes. This is is. true even though pepsin removes histones as evidenced in that the preparations became alkaline fast green negative. DNase and RNase botte respectivant removed bombtt DNA and RNA as shown either by feulgen negativity or azure B negativity of the slides. Trypsin hydrolysis for one hour, however, had a considerable effect on chromosome and nuclear morphology, in addition to rendering the preparations alkaline fast green negative. If interphase nuclei and chromosomes were watched under the microscope while they were being treated with trypsin, the nuclei could be seen to swell and to become progressively less refractile so that eventually they could not be seen with the light microscope. The enzymes seem to digest away some of the material in these nuclei. If after one hour of treatment and air drying with the nuclei were observed under phase contrast microscopy, they were found to have stretched to about three times their original diameter (Figure 3). 24 They ciearly showed a network of fibers within the nucleus filho Similarly, clured metaphase chromosomes were observed to separate from one another and stretch by a factor of three. After air drying, the stretched and partially digested metaphase chromosomes are found to be multistranded. Each chromatid consists of two strands or hall chromatids that are relationally coiled about one another. The enzym treatment evidently reduce the numbers of coils and in many instances we have found that the two half chromatids essentially lie side by side (Fig. 5, lig. 6). in certain favorable cases (Fig. 6) we can see that each half chromatid is in itseli composed of two interwound strands, indicating that at the light microscopic level the chromos one is at least quadripartite (See also Gimenez-Martin, 1963). We have occasiorially obtained preparations in which the uncoiling has gone on to the extent to which we noe. can find the chromatids difurcating. We have been able to follow some of the bifurcations to the point at which we can see the four parts of the individual chromatid (Fig. 7). Although the stretched nuclei (nuclear skeletons) and elongated chromosomes ieft aiior trypsin digestion are fast green negai:ve indicating that most of their histones liave been removed, they are still feulgen positive, indicating that DNA is still present. Som DNA has been released from the nuclei au chromosomes, however, as evidenced by the fact that in the feuigen preparations the general background is now light pink. It should be pointed out that although some WA 18 left behind in the skeletons and chromosomes, DNA does not appear to be the architectural comercrome retvorotorous component on which the linear continuity of these shtetures dependí For instance, in those trypsin treated preparations that were stained with (90°c). The preparations were feuigen negative, azur B negative, and fast green negative, yet the skeletons and chromosomes were unchanged and could still be seen with phase-contrast microscopy. Since pepsin, which also leads to alkaline fast green preparations, does not cause stretching and splitting of nuclei and chromosomes, we do not believe that the trypsin effect is caused by a simple removal of histones. Trosko and I interpret these experiments to indicate that nonhistone protein contributes to the linear continuity of these mitotic cirromosomes and that the chromosome is a multistranded structure that is embedded in a matrix of nonhistone protein. The regular appearance of multistranded chromatids at metaphase, confirms observations that have been made with the light microscope in the past. Kaufmann (1931) and Nebel (1932, 1937) both observed at least two and on occasion four strands in a chromatid. Similarly, Manton (1945) found that chromatids of the fern Todea to be bipartite and Gimenez-Martin, et al. (1963) have found the chromatids of Sicillato consist of two interwoven strands. The extensive uncoiling the.t occurs after trypsin treatment in Trosko's experiments, however, can lead to a total unwinding of chromatid strands so that the multistranded nature of the chromosomes 18 particularly clear. Taylor (1962) has proposed a model of the chroms ome (based on reese's 1958 model) that could explain the appearance of half chromatids from what is basically a single-stranded chromsome. His model consists of two chains of links that are parallel. These are interconnected by a rung-like arrangement of DNA molecules. If, under some conditions, only the two parallel chains of links were observable, the chromatid would have the appearance of being bipartite. The appearance of four strands with. 1 a chromosome, however, and the appearance of doubly M 16 bifurcating chromatids do not seem to be consistent with such a model. The appearance of multistranded chromatids not only 18 consistent with previous observations of chromosone 8, it also fits the results of radiation experiments in which half chromatid aberrations were induced at metaphase and chromatid aberrations at the end of G, or very beginning of s. These results do not, however, fit the observations of Gall (1963) on the kinetics of DNase-induced chromosome breaks of lampbrush chromosome. Gara has found that within loops of the chromosome DNase breaks chromosomes with approximately two-hit kinetics. This 18 interpreted as indicating that, in the loop the chromosome consists of but a single molecule of double stranded DNA, 1. e., the loop 18 single stranded. In the interloop regions of the lampbrush chromosome where the strands from two loops come together, breaks are produced with about four-hit kinetics. In addition, the results of our enzyme digestions do not parallel the results obtained by MacGregor and Callan (1962) on lampbrush chromosomes in which they free found that DNase and not proteases can break the chromosomes. We are left to wonder if meiotic lampbrush chromosomes differ from the usul mitotic chromosomes observed in plants and animals. If we make a balance sheet, it seems that most of the pertinent observations with the light microscope (Kaufmann, 1931; Nebel, 1932, 1937; Swanson, 1943; Manton, 1945; Wilson place on her cody bar (1960 et al., 1959; R18, 1961; Gimenez-Martin, 1963) indicate that the mitotic chromosome 18 multistranded, whereas experiments on lampbrush chromosome's do not necessarily fit this pattern (MacGregor and Callan, 1962; Gall, 1963). It should be pointed ut in spite of the experiments of Gall, and of MacGregor and callan, work by R18 on the same chromosomes has led to the interpretation that lampbrush chromosomes, too, are multistranded. - - .. . lhe semi-conservative distribution of DNA into daughter chromatids . . . as first discovered by Taylor, et al. (1957) 18 simple to visualize in terms of a single stranded chromatid, i. e., a chromatid consisting of but one double stranded DNA molecule. Peacock (1963) has recently repeated Taylor's autoradiographic studies and found that, although the majority of the chromosomal label is distributed in the fashion discovered by Taylor, very frequently two chromatids of the second division are labelled at the same level. 1. e., are 18olabelled. This led iim to believe that the chromosomes consist of at least two strands rather than one. (See, however, Wolff, 1964, for another possible interpretation of Peacock's results). Although it is easy to visualize Taylor's results in terms of a single stranded chromosome, it 18 possible to make schemes whereby chromatids that consist of more than one DNA double helix distributes its label so that both chromatide will be labelled at the first division and that only one chromatid would be labelled at any successive division. Such schemes have been proposed Wy (Schwartz, 1978; Steffensen, 1961, Cavallow and Rosenberg, 1961; Peacock, 1963). In Fig. 8 we present a model that is similar to one proposed by Uhl to explain crossing over. This model can account for the segregation of label among daughter chromatids when the chromatid 18 multistranded. In the model several strands of DNA run side by side longitudinally between links. Hon replication cach strand of the doub..e stranded DNA helix 16 attached to opposite sides of the plane of splitting of the link. Following one round of replication in label, cach strand of the multistranded chromatid will now be labelled and both chromatids will appear to be labelled. If these chromatide now replicate in the absence of label, all of the labelled ,-. IP: LAST DNA would be in ce chromatid, whereas the sister chromatid would be completely unlabelled. We do not intend that this model be taken as an indication that we believe the chromosome 18 constructed in this fasuion. It only demonstrates S that in the absence of any critical knowledge of how a chromosome is trikini, mwinSTORU constructed, it is easy to conceive of an arrangement of the DNA in multistranded chromosomes that could give the results first observed by Taylor and his co-workers, results that must be considered in any formulation voru.4.- of chromosome structure. ,. Unfortunately, a multistranded chromosome makes it difficult for us . 1.. . ... .. . - to visualize many genetic phenomena, such as mutagenesis and crossingover. The l'act that it is more diff:nult to think of these processes in terms of multistranded chromosomes than : 1s in single stranded ones, however, does not constitute evidence that the chromosome 18 single stranded. Summary In conclusion then let me say that three different types of experiments performed in our laboratory, experiments of the metabolic requirements for the repair of radiation-induced chromosome breaks, experiments on the types of aberrations induced at various stages of the cell cycle, and experiments on enzyme digestion of isolated mitotic nuclei and chromosomes have indicated to us that the mitotic chromosomes of Vicia faba depend in the main upon nonhistone protein for their linear continuity and are multistranded structures. The results obtained with Vicia faba chromosomes, in general, seem to fit those obtained with many other organisms. The one major exception being the results obtained by some workers, but not all, on lampbrush chromosomes of the newt. REFERENCES Alfert, M. and I. Geschwind, A selective staining method for the basic protein of cell nuclei, Proc. Natl. Acad. Sci., 1953, 39, '991. Bell, S. and S. Wolff, Studies on the mechanism of the effect of fluorodeoxyuridine on chromosoines, Proc. Natl. Acad. Sci. US, 1964, 51, 195-202. . hank you Cavalieri, 1. F. and B. H. Rosenberg, The replication of DNA. III. Changes in the number of strands in, E. coli DNA during its replication cycle, Biophysical J., 1961, 1, 337. turtu Crouse, H. V., X-ray breakage of 11ly chromosomes at first meiotic metaphase, Science, 1954, 119, 485. . - - Evans, H. J. and J. R. K Savage, The relation between DNA synthesis and chromosome structure as resolved by X-ray damage, J. Cell Biol., 1963, 18, 525-540. Freese, E., The arrangement of DNA in the chromosome, Cold Spring Harbor Symposia Quant. Biol., 1958, 23, 13-18. Gimenez-Martin, G., J. F Lopez-Saez and A. Gonzalez-Fernandez, Somatic chromosome structure, Cytologia, 1963, 28, 381. Gall, J. G., Kinetics of deoxyribonucleasc action on chromosomes, Nature, 1963, 198, 36. 20 Kaufmann, B. P., Chromonemata in somatic and meiotic mitoses, Am. Naturalist, 1931, 65, 280. Kaufmann, B. P., H.Gay and M. McDonald, Organizational patterns within chromosoines, in Intern. Review of Cytology, 1960, 2, 77. · MacGregor, H. C. and H. G. Callan, The actions of enzymes on lampbrush chromosomes, Quarterly J. of Microscopical Science, 1962, 102, 173. Manton, I., New evidence on the telophase split in Todea barbara, Am. J. Botany, 1945, 32, 342. Nebel, B. R., Chromosome structure in Tradescantias I. Methods and morphology, Z. Zellforsch. u. mikroskop. Anat., 1932, 16, 251. Nebel, B. R., Chromosome structure XII. Further radiation experiments with Tradescantia, Amer. J. Bot., 1937, 24, 365. Peacock, W. J., Chromosome duplication and structure as determined by autoradiography, Genetics, 1963, 49, 93. R18, H., The annual mutation lecture: Utrastructure and molecular organization of genetic systems, Canad. J. of Genet. and Cytol., 1961, 3, 95. Schwartz, D., Deoxyribonucleic acid side-chain model of the chromosomes. Nature, 1958, 181, 1149. Steffensen, D., A comparative view of the chromosoma, In Brookhaven Symposia in Biology, 1959, No. 12, 103. · · .... " " . .. .. .. .. .. .-- - ... 21 Steffenson, D., Chromosome structure with special reference to the role of metal ions, in Intern. Review of Cytology, 1961, 12, 163. Swanson, C. P., X-ray and ultraviolet studies on pollen tube chromosomes. II. The quadripartite structure of the prophase chromosome of Tradescantia, Proc. Natl. Acad. Sci., 1947, 33, 229. Taylor, J. H., The time and mode of duplication of chromosomes, Am. Nat., 1957, 21, 209. Taylor, J. H., Chromosome Reproduction, Intern. Rev. Cytol., 1962, 13, 39. Taylor, J. H., P. S. Woods and W. L. Hughes, The organization and duplication of chromosomes as revealed by autoradiographic studies using tritium-labeled thymidine, Proc. Nat... Acad. Sci., 1957, 43, 122. . . . Trosko, J. E. and S. Wolff, Strandedness of Vicia faba chromosomes as revealed by enzyme digestion studies, In Press, 1965. ...... 1 T Wilson, G. B., A. H. Sparrow and V. Pond, Sub-chromatid rearrangements .. . . . in Trillium erectum. I. Origin and nature of configuration induced * by ionizing radiation, Am. J. Bot., 1959, 39, 309. 1 vi Wolff, S., Radiation studies on the nature of chromosome breakage, 2 . . Am. Nat., XCIV, 85-93. a rko 9 Wolff, S., Some postirradiation phenomena that affect the induction of chromosome aberrations. J. Cell and Comp. Phys., 1961, 58 suppl. 1; 151-162. C .....: M ...... Wolff, S. and H. E. Luippold, Metabolism and chromosome break rejoining, Science, 1955, 122, 231-232. Wolff, S. and H. E. Luippold, Chromsome splitting as revealed by combined x-ray and labell:ng experiments, Expti. Cell Res., 1964, 34, 548, 556. LEGENDS Figure 1. Feulgen-stained isolated Vicia faba nuclei. Figure 2. Feulgen-stained isolated Vicia faba nuclei and chromosomes (x). Figure 3. Feulgen-stained, midly trypsin treated isolated nucleus showing skeleton (x). Figure 4. Normal Vicia faba chromosome complement at metaphase. Figure 5. Feuigen-stained, trypsin treated isolated metaphase chromosomes. The M-chromosome (c) 16 seen with each of its chromatids (Ctd) split into two subchromatids (Scta). The satellite (s), secondary constriction (s.c.) and centromere (CM.) are also visible (x). Figure 6. Feulgen-stained 1solated metaphase chromosomes (x). Figure 7. Feulgen-stained, mildly trypsin treated metaphase chroniosome showing one chromatid separated into half-chromatids, with each of these bifurcating into quarter chromatids. - . . - - . Figure 8. A model 1llustrating that it is topologically possible for a multistranded chromosome to segregate labelled and unlabelled DNA at the second division following labelling. In the model 4 strands of DNA run from link to link along the length of the .... - - - ... - . . chromosome. Heavy lines indicate the original DNA. Dotted . . lines Indicate radioactive DNA. . . 7: 17 ..- . ' Table I Labolod aborrations after combined X-Ray and I _Thymidino Treatment Exporimontal Protocol: 150r, a dolay of 0,1.or 2 ar, and then r-Thymddine. 200 cells each point, porcentagos computed from the number of labeled aberrations/total no. scored motapha sos Timo Labeled Chromatid Labolod Chromo so mo Delay (hr) to fix (hr) lo . 33.5 30.5 23.5 : 29.5 0.5 30.5 24.5 0,5 - 1.5 22.5 18.5 27.0 2.5 Table to The effect of various enzymes on isolated nuclei and chromosomes of Vicia faba Alkaline Appearance of treated Treatment Fast Green Feulgen Azure B Bromide nuclei and chromosomes Trypsin (short hydrolysis) .* Chromosomes elongated and split into sub- chromatid strands; Nuclear matrix removed, leaving reticular skeleton Trypsin (long hydrolysis) - Structural Integrity completely disrupted DNase Chromosomes swollen, but seemingly intact RNase No apparent change in structure Pepsin Chromosomes and nuclei slightly shrunken * Alkaline fast green stained, trypsin-treated chromosomes exhibit no demonstrable stain, yet under phase optics, nuclear skeletons and chromosomes are intact (DNA is removed by the hot TCA hydrolysis used in this staining procedure). = negative staining reaction * = positive staining reaction 3, 022 2 اسمع - * - - . - حل ,3 و7 - - - - - . . . . . . . . ...... anco. -*-*- .. . - - - - - - - Ost'er options M-Chromosomos $.c.. S.C. ' C ::-... ' , . .- ...: , quipme n ... --. Scid 1/2 Seid. . .. : , 'create'i T... i 2. ...me . 13,*81 : . . 13,482 END VIEW S .CHROMOSOME ROMOSOME -1- FIRST REPLICATION IN Hz-THYMIDINE CENTROMERE; L FIRST DIVISION METAPHASE CHROMOSOME CROMATID kes kocht oc SHOX SECOND REPLICATION IN NON-RADIOACTIVE MIDINE 1 METAPHASE CHROMOSOME DATE FILMED 12/ 2 /164 WYNI w D SS. VI C 1.10 LEGAL NOTICE 21 This roport was propared as an account of Government sponsored work. Neither the United States, nor the Commission, nor any porson acting on behalf of the Commission: A. Makes any warranty or ropresentation, expressed or implied, with respect to the accu- racy, completeness, or usefulness of the information containod in this report, or that the use of any information, apparatus, mothod, or procesu disclosed in this report may not infringe privately owned rights; or B. Assumes any liabilities with respect to the use of, or for damages resulting from the use of any information, apparatus, method, or process disclosed in this report. As usod in the above, person acting on behalf of the Commission includes any em- ployee or contractor of the Commission, or employee of such contractor, to the extent that such employoo or contractor of the Commission, or employee of such contractor prepares, disseminates, or provides access to, any information pursuant to his employment or contract with the Commission, or his employment with such contractor, . I '. KA rat - i . I TE END X IL , O NA