S.R. Pelc

Nuclear incorporation of P32 as demonstrated by autoradiographs

Abstract Roots of Vicia faba seedlings were grown for periods of 2 to 48 hours in solutions of P 32 in the form of NaH 2 PO 4 . Stripping-film autoradiographs were made of squashes and sectioned material from the meristem and from proximal segments where cell divison is rare. It is concluded (a) that P 32 enters into organically bound form (probably desoxyribonucleic acid) in the nucleus during the resting stage, but not during the actual division of the cell; (b) that P 32 is incorporated in this form only in nuclei which are preparing for division; (c) that P 32 in this form is inherited by the daughter cells.

Synthesis of Desoxyribonucleic Acid in Normal and Irradiated Cells and Its Relation to Chromosome Breakage

Summary(1) The normal mitotic cycle in the meristem of main roots of Vicia faba seedlings is estimated to take 30 h, of which 4 are spent in division, 12 between the end of division and the beginning of 32P uptake into new DNA, 6 in DNA synthesis as judged by 32P uptake, and 8 between the end of synthesis and the prophase of the next division.(2) A dose of 13 × 103 erg/g (140r) of X-rays does not immediately stop DNA synthesis in cells which are in or near the period of synthesis at the time of irradiation, but causes these cells to be delayed in entering division for a period of about 4 hours.(3) The same dose of X-rays causes cells which are at other stages of the mitotic cycle at the time of irradiation to be delayed in entering synthesis for a period of 10 h or more.(4) Neutron doses of 1·1, 2·4 and 4·9 × 103 erg/g and 13 × 103 erg/g of X-rays have, within the standard deviations, the same effect on the number of cells synthesising DNA during 12 h following irradiation.(5) The estimated delay in enter...

Effect of various doses of x-rays on the number of cells synthesizing deoxyribonucleic acid.

A number of recent papers, referred to below, have described the effect of irradiation on the rate of incorporation of P32 or of C14-labeled precursors into deoxyribonucleic acid (DNA). Since this rate is very low in nondividing tissues (Hevesy and collaborators, 1-4; Brues et al., 5), the incorporation seen in tumors and other actively dividing tissues has generally been ascribed largely to synthesis of new DNA. The methods used for measuring the effect of irradiation have been the biochemical separation of DNA from bulk tissue and the measurement of its specific activity relative to unirradiated controls. The measurements have thus been of the average behavior of all cells in the tissue, or more accurately the product of number of synthesizing cells times rate of synthesis per cell. Any inequality in the effect of X-rays on different cells in a tissue would escape detection by such methods but could be brought to light if the behavior of individual cells after irradiation could be studied. The autoradiograph method allows an estimate to be made of the proportion of cells in a tissue which incorporate tracer into DNA during a given period of time. This report describes an experiment in which this proportion was measured in Vicia faba root meristems after doses from 17.5 to 200 r of X-rays. The results, taken together with those of two other experiments on the same material (Howard and Pelc, 6, and unpublished) indicate that whether a cell is or is not inhibited from synthesizing DNA depends on its position in the mitotic cycle at the time of irradiation, and that the sensitive mitotic class is maximally affected by low doses of radiation, i.e., about 50 r.

Nuclear uptake of labelled adenine in the seminal vesicle of the mouse.

Abstract Mice were injected with [8- 14 C]-adenine, killed 1 to 6 days later, organs were fixed in acetic acid-alcohol and formol saline, embedded in paraffin wax and sectioned. After deparaffinising some slides were treated with ribonuclease, deoxyribonuclease, cold perchloric acid or hot perchloric acid and autoradiographs were prepared by the stripping film technique. Both nuclei and cytoplasm of the epithelial cells of the seminal vesicle of mice killed one day after injection were found to be well labelled in unextracted slides. After extraction with cold perchloric acid or ribonuclease approximately 40 per cent of the nuclei are labelled, their cytoplasm is weakly labelled. No nuclear autoradiograph is shown after extraction with hot perchloric acid, whilst the cytoplasmic autoradiograph remains and probably represents incorporation into protein. Comparison of staining reactions and grain counts indicates that cold perchloric acid is effective in extracting ribonucleic acid. The nuclear autoradiographs observed after cold perchloric acid are therefore due to labelled adenine incorporated in deoxyribonucleic acid. The seminal vesicle of the mouse has to be regarded as non-dividing tissue and these results therefore indicate DNA synthesis independent of mitosis. The literature on incorporation of tracers in non-dividing tissues is reviewed. It is suggested that ( a ) synthesis or exchange of DNA takes place in some tissues as a metabolic function unconnected with cell division, and ( b ) that this metabolism is connected with the formation of certain proteins.

P32 Autoradiographs of Mouse Testis: Preliminary Observations of the Timing of Spermatogenic Stages

(1) Testis material from mice killed 2, 5, 9 and 12 days after injection of 200 μC of P32 was autoradiographed by the stripping film technique and examined cytologically. (2) Cells at different stages of spermatogenesis were found to take up P32 differently. Some preliminary estimates as to the time taken by various stages of spermatogenesis are (a) from spermatogonial metaphase to early meiotic prophase, between 3 and 9 days; (b) from diakinesis to second metaphase, 4 days or less; (c) from diakinesis to immature sperm, 7 days or more; (d) from spermatogonial metaphase to immature sperm, more than 10 days. (3) Leydig cells were observed to show a strong uptake of P32 within the first 2 days. Some possible explanations for this observation are put forward.

A difference between spermatogonia and somatic tissues of mice in the incorporation of [8-14C]-adenine into deoxyribonucleic acid.

Abstract C 57 black × C 3 H F 1 hybrid male mice about 5 months old were injected intraperitoneally with 20 μC [8- 14 C]-adenine-HCl and killed from 1 to 32 days later. Autoradiographs of squashes and sections of various organs were prepared after treatment with ribonuclease. The available evidence indicates that the autoradiographs obtained after this treatment are due to 14 C incorporated in deoxyribonucleic acid during synthesis. Visual inspection and cell counts show that a maximal number of nuclei is labelled after 1 day in those somatic tissues in which cell division is frequent. In the testis only 12.3 per cent of the spermatogonia are labelled 1 day after injection, rising to 63.3 per cent after 10 days. Grain counts and the distribution of labelled spermatogonia in the tubules indicate that the increase in percentage is due to new labelling and not to redistribution of DNA through cell division. Primary spermatocytes are unlabelled at 1, 2 and 4 days; some are labelled at 6 days. Labelled secondary spermatocytes and spermatids first appear at 8 days. Sperm in the testis are unlabelled at 10 days, but labelled at 25 days. After 32 days labelled sperm is found in the epididymis. The timing of spermiogenesis derived from these observations agrees with previous estimates. At present no satisfactory explanation can be given for the difference in DNA labelling of spermatogonia and of somatic tissues. It is suggested that a DNA precursor, either within the testis or elsewhere, becomes maximally labelled soon after injection, and is drawn upon by spermatogonia but not by cells of somatic tissues.

On the connection between the synthesis of RNA and DNA in the testis of the mouse.

Abstract The nucleic acids of various tissues were labelled by i.p. injection of [8- 14 C]-adenine and autoradiographs of 5 μ sections were prepared. It had previously been reported that spermotogonia synthesize labelled DNA for a period of up to eight days after a single injection whilst no additional labelling of DNA takes place later than one day in the corneal epithelium and small intestine. Observations on autoradiograhs of sections of mouse testes containing labelled RNA and DNA show that labelled adenine is incorporated into the RNA of most non-dividing spermotogonia within one day after injection. It is concluded that the observations can be explained if it is assumed that RNA acts as a precursor for DNA, as originally suggested by Brachet. The difference between testis and somatic tissues in the timing of the uptake of adenine into DNA indicates a different time-lag in the conversion of RNA to DNA.