DNA fibres from human lymphocyte nuclei

642

Musao S. Sasaki and A. Norman

The most frequent regenerations, i.e. 50-70 per cent, were found in 2 per cent agar. With the decreasing concentration of agar the percentage of regenerations decreases (in 1.3 per cent agar only up to 5 per cent). Prolonged boiling or repeated thawing of agar gels lowers the amount of regenerations. Lowered regeneration also results from the inoculation of a thin suspension of protoplasts into agar. The different sorts of agar influence neither the amount of regenerations nor their morphology. The perfect embedding of protoplasts into agar is very important. Insufficiency of that is believed to be a cause of NeEas failure. In agar gels the protoplasts of both Saccharomyces carlsbergensis 48-63 and Candida utilis 29-38-2 (Chusas Bratislava) may regenerate under the same conditions as Saccharomyces cerevisiae protoplasts. The morphology of their regeneration corresponds to that in high percentage gelatin. [7] The question of specific effect of agar gel on the regeneration of the protoplasts is very important. Agar as a polysaccharide might be suspected to be furnishing a priming substance for cell wall polysaccharides. Contrary to this, however, are the facts that the regeneration may occur in higher percentage of agar only, the vast chemical difference between agar and cell wall mannans and glucans, and finally, the previous findings that the protoplasts are able to regenerate in high percentage gelatin too. Thus it seems that the action of agar on the regeneration process will be the same as that of gelatin, i.e. it acts physically. Similar results were published by Landman and Halle [a], who studied the action of gelatin and agar on the reversion of L-bodies of Bacillus subtilis. It is very probable that both gels act mainly during the synthesis of the new cell wall [6], but the mechanism is not yet known in detail. REFERENCES 1. EDDY, A. A. and WILLIAMSOX, D. H., Nafure 183, 1101 (1959). 2. LANDXAN, 0. E. and HALLE, S., .J. Mol. Hiol. 7, 721 (1963). 3. NE&AS, O., Nature 192, 580 (1961). 4. -Folin biof. (Praha) 8, 256 (1962). 5. -Personal communication. Jena 1965. Akademie6. NE~AS, 0. and SVOBODA, A., Symposium tiber Hefe-Protoplasten Verlag Berlin, 1966. 7. ROST, K. and VENNER, H., Arch. Mikrobiol. 51, 130 (1965). 8. SVOBODA, A., Symposium iiber Hefe-Protoplasten Jena 1965. Akademie-Verlag Berlin, 1966.

DNA

FIBRES

FROM

HUMAN

LYMPHOCYTE

NUCLEI

MASAO S. SASAKI AND A. NORMAN Department

of Radiology,

FmMhuman

of Medicine, Los Angeles, Cal& Received May 31, 1966

UCLA

School

U.S.A.

lymphocyte nuclei we have extracted DNA fibres with lengths ranging up to 2.2 cm. Such long fibres are to be expected if the interphase chromosome consists basically of a single DNA fibre. Experimenfal

Cell Research 44

DNA fibres from human lymphocyte

nuclei

643

Fig. l.-DNA autoradiogram of a portion of a tight bundle of fibres (at least 1.9 cm total length). was taken using Time in lysis medium was 1 hr. Exposure time was 1 month. Photograph bright field phase contrast.

Lymphocytes obtained from the peripheral blood of women were incubated for 72 hr in a culture medium (NCTC-109, fetal calf serum, PHA, penicillin and streptomycin) augmented for the last 24 hr with 0.2 mC/ml of SH-thymidine. The cells were washed and disrupted by syringing in detergent (0.25 vol. per cent Triton X-100, 0.25 M sucrose, 1 mIM MgCl,). The nuclear suspension was diluted 20 x into a 0.25 M sucrose, 1 mM MgCl, solution, filtered through cheese cloth and absorbent cotton, layered over a 0.5 M sucrose, 1 mll/I MgCl, solution, and centrifuged for 5 min at 500 rpm. The top, 0.25 M sucrose, layer was discarded and the nuclei were collected by centrifuging for 10 min at 2000 rpm. The chromatin was then extracted by incubating the nuclei at 37’C for 1 to 10 hr in a lysis medium (10-2&Z EDTA, 10-z M sodium citrate, pH 8). Using the surface spreading technique of Kleinschmidt and Zahn [4] the chromatin was prepared for autoradiography by suspending it in 2 M ammonium acetate solution containing 0.01 per cent cytochrome c and spreading the solution on the surface of distilled water held at 34-40°C in a Langmuir trough. The material spread on the water surface was picked up on microscope slides. The slides were dried, dipped into absolute ethanol, dried, soaked in 5 per cent TCA at 4°C for 30 min, and washed in running tap water for 1 hr. The slides were then coated with Kodak NTB-2 emulsion, exposed for l-3 months at 4’C, developed in Kodak Dektol, fixed, washed and mounted. The lengths of the DNA fibres were determined from photomicrographs of the autoradiographs and from camera lucida drawings. Autoradiographs of chromatin extracted from nuclei incubated for l-2 hr inlysis medium frequently show tight bundles of fibres. Some of th.ese bundles are several cm in length and show puff-like structures where the individual fibres spread out. An example of such a bundle is shown in Fig. 1. When the nuclei were incubated for 5-10 hr, the bundles were loosened and many individual fibres were found. The majority of individual fibres were short, measuring less than 100 ,W However, of the measured fibres 56 have lengths in excess of 0.5 mm and 12 of these are over 1 cm in length. The 12 fall into two groups, 5 circular fibres Rith lengths of 1.1, 1.4, 1.8, 2.0, and 2.2 cm and 7 linear fibres of length 1.0, 1.1, 1.3, 1.3, 1.6, 1.8, and 2.1 cm. None of the fibres less than 1 cm long were circular. The 2.0 cm circular fibre is shown in Fig. 2. The longest DNA fibres extracted from the human lymphocyte are about 10 times the length of the fibres obtained by Cairns [l] from bacteria and of the longest fibres obtained from Chinese hamster cells [3]. They are two and three orders of magnitude longer respectively than fibres extracted from sea urchin sperm [7] and boar sperm [2]. Experimental

Cell Research 44

Fig. 2.-DNA autoradiogram of a circular fibre 2.01 cm in length. Time in lysis medium was 3 hr. Exposure time was 1.5 months. Photograph was taken on reversal film using dark field phase contrast.

The DNA content of the human interphase nucleus is about 5.6 pg. Using the figure of 192 daltons/A for the DNA helix and assuming that each chromosome consists of a single DNA fibre the length of such fibres should range from about 1.7 cm for the shortest chromosome to about 7.5 cm for the longest human interphase chromosome. If an intact chromatin thread consists basically of a single DNA molecule, as seems likely from preliminary studies, then a 2 cm thread corresponds in DNA content either to a single short chromosome or to one arm of a median sized human chromosome. The finding of such long chromatin fibres gives support, therefore, to models that picture the interphase chromosome as a single DNA fiber. The fact that chromosomes of higher organisms have multiple replication centres [S, 61 is not inconsistent with the organization of the DNA into a single fibre. Certainly the observation that chromosome fragments produced by radiation or other treatment can replicate suggests that neither the centromere nor the normal chromosome ends have any special role in initiating replication. Experimental

Cell Research 44

Thymidine

645

metabolism of HeLa cells

At any rate, we are of the opinion that the human interphase chromosome is a single continuous DNA fiber. This work was supported in part by United States Energy Commission contract number AT(ll-l)-34. REFERENCES 1. CAIRNS, J., J. Mol. Biol. 6 208 (1963)., A., Proc. Natl Acad. Sci. 53, 356 (1965). 3. HUBER~~AN, J. A. and RIGGS, A. I>., Proc. Natf Acad. Sci. 55, 599 (1966). 4. KLEISSCHMIDT, A. and Z.~HN, R. K., %. Naturforsch. 14, 770 (1959). 5. PL.IUT, IV. and NASH, D., in 11. LOCKE (ed), The Role of Chromosomes p. 113. Academic Press, Sew York, 1964. 6. SCHMII~, W., Cvtogenefics 2, 175 (1963). 7. SOLARI, A. .J., Proc. Satf Acad. Sci. 53, 503 (1965). 2. HOTTA, Y. and BASSEL,

DETERMINATION

OF THYMIDINE

CELL CULTURES AND

BY A COMBINED

PAPER

W. LANGI, Institut

fiir

Medisinische

METABOLISM

D. MULLER

METHOD

and W. MAURERI

der Universitiit Wiirzburg Marburg, Deutschland

Received

IN HELA

ELECTROPHORETIC

CHROMATOGRAPHIC

Strahlenkunde Universitiit

in Dovelopnrr~!I

und Hygiene- Institut

der

June 6, 1966

W

HEN using 3H-TdR as a DNA precursor in long-term experiments with mammalian cells in culture, two main factors must be taken into account. Firstly, cultured mammalian cells are able to degrade TdR to T, DHT, and/or BUIB, which cannot-or only to a very low degree-serve as DNA precursors [6,16]. Secondly, as could be demonstrated for mouse fibroblasts in culture, several types of enzyme activity, necessary for the phosphorylation of TdR, decline before the cells pass from the exponential growth phase into the stationary phase [la, 13,211. Degradation of TdR could be demonstrated for human leucocytes [3,14,15], mouse thymocytes [ 31, and Ehrlich ascites cells [ll, 221. This paper deals with the TdR degradation in HeLa cell cultures over a period of 48 hr. HeLa cells were grown in Roux bottles (inoculated with 5 x 104 cells/ml) in 100 ml Eagle’s medium supplemented with 10 per cent calf serum and antibiotics (104 units penicillin/ml and 100 pg/ml streptomycin). 42 hr after inoculation the medium was replaced by 100 ml Eagle’s medium containing 0.25 ,uC/ml SH-TdR (spec. act. 120 mC/m&f). 3H-TdR with a spec. act. of 6700 mC/mlM (New England

Present address: Institut ftir Medizinische Isotopenforschung der Universitlt K61n. Abbreuiations: BAIB, P-amino-iso-butyric acid; BUIB, p-ureido-iso-butyric acid; DHT, dihydro-thymine; DNA, deoxyribonucleic acid; T, thymine; TMP, TDP, TTP, thymidine mono-, di-, and tri-phosphate; TdR, thymidine. Experimental

Cell Research 44