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The Use of Tritium-Labelled Thymidine in Studies on the Synthesis of Deoxyribonucleic Acids; Emploi de la Thymidine Tritiee Dans L'etude de la Synthese de l'Acide Desoxyribonucleique; 0418 0441 043f 043e 0414 ; Empleo de Timidina Tritiada para Estudiar la Sintesis de los Acidos Desoxirtibonucleicos

Abstract

In the course of studies on the biosynthesis of DNA some experiments were performed using Ehrlich ascites cells, examining the uptake and incorporation of H{sup 3}-thymidine. After in-vitro incubation of the cells with this compound, autoradiographs of the cells were made and DNA was also isolated and assayed for H{sup 3} activity using a flow-counter. A comparison of the two methods of assay showed a marked discrepancy; the H{sup 3} activity per cell, calculated from the autoradiographs always appeared to be greater than the activity of the isolated DNA. Subsequent flow-counter assay of the activity of washed, homogenized whole cells gave figures which agreed with the autoradiographic assay. It thus appeared that, in this system, the use of autoradiography as a measure for DNA synthesis was open to criticism. Analysis of the bound, non-DNA activity has been made and similar studies of total cellular H{sup 3} activity and the activity of isolated DNA have been undertaken on other cell types; these have also shown similar effects. From this information it has been possible to divide the synthetic process into various stages: (1) The initial incorporation of thymidine into the cell; (2) Subsequent phosphorylation in at least two steps; (3) Polymerization  More>>
Authors:
Bianchi, P. A.; Crathorn, A. R.; Shooter, K. V. [1] 
  1. Chester Beatty Research Institute, Institute of Cancer Research, London (United Kingdom)
Publication Date:
Feb 15, 1962
Product Type:
Conference
Resource Relation:
Conference: Symposium on the Detection and Use of Tritium in the Physical and Biological Sciences, Vienna (Austria), 3-10 May 1961; Other Information: 10 refs., 2 tabs., 2 figs.; Related Information: In: Tritium in the Physical and Biological Sciences. Vol. II. Proceedings of the Symposium on the Detection and Use of Tritium in the Physical and Biological Sciences| 456 p.
Subject:
60 APPLIED LIFE SCIENCES; ASCITES; AUTORADIOGRAPHY; BIOSYNTHESIS; DNA; FLOW COUNTERS; IMAGES; IN VITRO; INCUBATION; PHOSPHORYLATION; POLYMERIZATION; THYMIDINE; TRACER TECHNIQUES; TRITIUM; TRITIUM COMPOUNDS; UPTAKE
OSTI ID:
22190104
Research Organizations:
International Atomic Energy Agency, Vienna (Austria); Joint Commission on Applied Radioactivity of the International Council of Scientific Unions, Paris (France)
Country of Origin:
IAEA
Language:
English
Other Identifying Numbers:
Other: ISSN 0074-1884; TRN: XA13M3707014589
Submitting Site:
INIS
Size:
page(s) 269-274
Announcement Date:
Feb 13, 2014

Citation Formats

Bianchi, P. A., Crathorn, A. R., and Shooter, K. V. The Use of Tritium-Labelled Thymidine in Studies on the Synthesis of Deoxyribonucleic Acids; Emploi de la Thymidine Tritiee Dans L'etude de la Synthese de l'Acide Desoxyribonucleique; 0418 0441 043f 043e 0414 ; Empleo de Timidina Tritiada para Estudiar la Sintesis de los Acidos Desoxirtibonucleicos. IAEA: N. p., 1962. Web.
Bianchi, P. A., Crathorn, A. R., & Shooter, K. V. The Use of Tritium-Labelled Thymidine in Studies on the Synthesis of Deoxyribonucleic Acids; Emploi de la Thymidine Tritiee Dans L'etude de la Synthese de l'Acide Desoxyribonucleique; 0418 0441 043f 043e 0414 ; Empleo de Timidina Tritiada para Estudiar la Sintesis de los Acidos Desoxirtibonucleicos. IAEA.
Bianchi, P. A., Crathorn, A. R., and Shooter, K. V. 1962. "The Use of Tritium-Labelled Thymidine in Studies on the Synthesis of Deoxyribonucleic Acids; Emploi de la Thymidine Tritiee Dans L'etude de la Synthese de l'Acide Desoxyribonucleique; 0418 0441 043f 043e 0414 ; Empleo de Timidina Tritiada para Estudiar la Sintesis de los Acidos Desoxirtibonucleicos." IAEA.
@misc{etde_22190104,
title = {The Use of Tritium-Labelled Thymidine in Studies on the Synthesis of Deoxyribonucleic Acids; Emploi de la Thymidine Tritiee Dans L'etude de la Synthese de l'Acide Desoxyribonucleique; 0418 0441 043f 043e 0414 ; Empleo de Timidina Tritiada para Estudiar la Sintesis de los Acidos Desoxirtibonucleicos}
author = {Bianchi, P. A., Crathorn, A. R., and Shooter, K. V.}
abstractNote = {In the course of studies on the biosynthesis of DNA some experiments were performed using Ehrlich ascites cells, examining the uptake and incorporation of H{sup 3}-thymidine. After in-vitro incubation of the cells with this compound, autoradiographs of the cells were made and DNA was also isolated and assayed for H{sup 3} activity using a flow-counter. A comparison of the two methods of assay showed a marked discrepancy; the H{sup 3} activity per cell, calculated from the autoradiographs always appeared to be greater than the activity of the isolated DNA. Subsequent flow-counter assay of the activity of washed, homogenized whole cells gave figures which agreed with the autoradiographic assay. It thus appeared that, in this system, the use of autoradiography as a measure for DNA synthesis was open to criticism. Analysis of the bound, non-DNA activity has been made and similar studies of total cellular H{sup 3} activity and the activity of isolated DNA have been undertaken on other cell types; these have also shown similar effects. From this information it has been possible to divide the synthetic process into various stages: (1) The initial incorporation of thymidine into the cell; (2) Subsequent phosphorylation in at least two steps; (3) Polymerization of the phosphorylated thymidine into DNA. Thus, although the assumption that the incorporation of thymidine into the cell gives a measure of DNA synthesis is an oversimplification, it would seem that considerable information about the preliminary stages in the process can be obtained by use of this tracer. (author) [French] Au cours d'etudes sur la biosynthese de l'ADN, l'auteur a fait certaines experiences, a l'aide de cellules eosinophiles ascitiques, en vue de mesurer l'absorption et l'incorporation de la thymidine tritiee. Apres incubation in vitro des cellules avec ce compose, on a fait des autoradiographies; on a, d'autre part, isole l'ADN et determine son activite due au tritium a l'aide d'un compteur a balayage continu. Les deux methodes de mesure ont donne des resultats sensiblement differents. Pour chaque cellule, l'activite due au tritium, calculee d'apres les autoradiographies, a toujours paru superieure a celle de l'ADN isole. En mesurant ensuite a l'aide du compteur a balayage continu l'activite de cellules entieres, apres lavage et homogeneisation, on a obtenu des chiffres qui concordaient avec les resultats de l'autoradiographie. Il semble donc, en l'occurrence, que l'autoradiographie ne permette pas de mesurer de maniere satisfaisante la synthese de l'ADN. On a etudie l'activite associee, autre que celle de l'ADN, et entrepris sur d'autres types de cellules des etudes analogues de l'activite totale due au tritium et de l'activite de l'ADN isole; des effets similaires ont ete observes. A partir de ces donnees, on a pu distinguer plusieurs phases dans le processus de synthese : 1 Degree-Sign incorporation initiale de la thymidine a la cellule; 2 Degree-Sign phosphorylation en deux etapes au moins; 3 Degree-Sign formation d'ADN par polymerisation de la thymidine phosphorylee. Ainsi, on simplifierait a l'exces en supposant que l'incorporation de thymidine a la cellule permette de mesurer la synthese de l'ADN; il semble cependant qu'en utilisant cet indicateur on puisse obtenir des indications utiles sur les etapes preliminaires du processus. (author) [Spanish] En el curso de estudios sobre la biosintesis del acido desoxirribonucleico se han realizado algunos experimentos, utilizando celulas asciticas de Ehrlich, para investigar la fijacion e incorporacion de timidina-{sup 3}H. Despues de incubar in vitro las celulas marcadas con este compuesto, se han tomado autorradiografias de las mismas; tambien se ha aislado el acido desoxirribonucleico y se ha medido la actividad del {sup 3}H utilizando un contador de flujo. Al comparar los dos metodos de medicion, se advierte una marcada discrepancia; la actividad del {sup 3}H por celula, calculada a base de las autorradiografias, siempre parece mas alta que la del acido desoxirribonucleico aislado. Midiendo a continuacion, con ayuda de un contador de flujo, la actividad de celulas enteras, lavadas y homogeneizadas, se han obtenido valores numericos que concuerdan con los resultados de la determinacion autorradiografica. Parece pues que, en este caso particular, la utilizacion de autorradiografias para medir la sintesis del acido desoxirribonucleico es susceptible de critica. En otros tipos de celulas se ha analizado la actividad fijada, no procedente del acido desoxirribonucleico, y se han efectuado estudios analogos sobre la actividad del {sup 3}H en toda la celula y la actividad del acido desoxirribonucleico aislado, obteniendose resultados semejantes. Basandose en estos datos, es posible dividir el proceso sintetico en varias fases: 1) Incorporacion inicial de timidina en la celula; 2) Fosforilacion consecutiva, en dos etapas por lo menos; 3) Polimerizacion de la timidina fosforilada, con formacion de acido desoxirribonucleico. Por consiguiente, si bien la hipotesis de que la incorporacion de timidina en la celula constituye un indice de la sintesis del oxido desoxirribonucleico es demasiado simplista, el empleo de dicho indicador permite obtener aparentemente datos de considerable interes acerca de las fases iniciales del proceso. (author) [Russian] V hode izuchenija biosinteza DNK byli postavleny nekoto- rye opyty s ispol'zovaniem ascitovyh kletok Jerliha po izucheniju usvoenija i pogloshhenija mechennogo tritiem timidina. Posle vyderzhivanija v termostate v laboratornyh uslovijah kletok s takim soedineniem byli sdelany radioavtogrammy kletok, i DNK byla takzhe otdelena i proverena na aktivnost' tritija pri pomoshhi protochnogo schetchika. Sravnenie dvuh metodov analiza pokazalo zametnoe rashozhdenie. Aktivnost' tritija na kletku, rasschitannaja po radioavtogrammam, vsegda okazyvalas' bol'she, chem aktivnost' otdelennoj DNK. Posledujushhij analiz pri pomoshhi protochnogo schetchika aktivnosti vseh promytyh gomogenizirovannyh kletok dal cifry, kotorye soglasujutsja s radioavtograficheskim analizom. Takim obrazom, pred- stavljaetsja, chto v jetoj sisteme ispol'zovanie radioavtografii dlja izmerenija sinteza DNK mozhno postavit' pod somnenie. Byl proveden analiz svjazi, aktiv- nosti v otsutstvii DNK, i bylo takzhe predprinjato podobnoe izuchenie obshhej kletochnoj aktivnosti tritija i aktivnosti otdelennoj DNK na drugih tipah kletok; jeti opyty takzhe dali podobnye razul'taty. Blagodarja jetim dannym stalo vozmozhnym razbit' process sinteza na razlichnye stadii: 1. pervonachal'noe pogloshhenie timidina kletkoj, 2. posledujushhee fosforiliro- vanie po krajnej mere v dva priema, 3. polimerizacija fosforilirovannogo timidina s prevrashheniem v DNK. Takim obrazom, hotja predpolozhenie o tom, chto pogloshhenie timidina kletkoj javljaetsja merilom sinteza DNK, javljaetsja slishkom uproshhennym, vse zhe predstavljaetsja, chto blagodarja ispol'zovaniju jetogo indikatora mozhno polu- chit' mnogo svedenij o predvaritel'nyh stadijah processa. (author)}
place = {IAEA}
year = {1962}
month = {Feb}
}