TIMING OF INCORPORATION OF TRITIATED NUCLEOSIDES INTO DNA AND RNA OF EMBRYONIC CELLS OF RANA PIPIENS

Abstract

The incorporation of tritiated nucleosides into DNA and RNA has been examined in partially synchronized cells of Rana pipiens embryos at the neurula and tailbud stages. Tritiated thymidine and deoxyguanosine are incorporated into the DNA in two maxima, or waves, during the S phase at both stages. More DNA replicates in the early maximum at the neurula stage than at the tailbud stage. A comparison of the degree of incorporation of labelled deoxyguanosine to labelled thymidine into DNA suggests that earlier replicating DNA at both stages may be GC-rich compared to later replicating DNA. The incorporation of tritiated uridine into RNA during the S phase also differs between the neurula and tailbud stages. Pulse and continuous label experiments indicate that at the neurula stage the highest rate of RNA synthesis occurs late in the S phase whereas at the tailbud stage the higher rate of RNA synthesis has shifted to an interval earlier in the S phase.The mechanism by which the emergence of new synthetic pathways occurs in differentiating cells seems to be related to DNA replication (9). It has been proposed that changes in DNA replication, or in a replication-related process, could provide a basis for the programming of differential gene expression in eukaryotic cells (1 1, 19). The phenomenon of obligatory DNA synthesis for gene expression and cytodifferentiation has been described in a number of eukaryotic cell systems (9,11,15,17). The dependance of gene expression on DNA replication is a common feature of the development of the larger bacterial and animal viruses (5,23). With the problem of the significance of DNA replication for differential transcription in mind, we have examined changes in the timing of DNA and RNA synthesis during the DNA synthetic (S) phase of embryonic neurula and tailbud cells of Rana pipiens. The data show a coincidence of changes in the synthesis of DNA and RNA during the S phase in embryonic cells, and they are consistent with the hypothesis that changes in DNA replicationa re related to changes in the timing of transcription in the embryonic cell. MATERIALS AND METHODSThe procedures have been described in detail previously (18). Neurula and tailbud embryos of Ranapipiens were used in this study. Stage 13 neurulae and stage 18 tailbuds were cut into dorsal axial ectoderm-mesoderm and belly endoderm regions in order to facilitate the entry of isotopes and to separate cell types with different prospective pathways of development (Fig. I). Groups of 50 cut explants were incubated in NIU-TWITTY solution (16) containing penicillin G(100 units) and streptomycin sulfate (50 pg/ml).M 5-fluorodeoxyuridine (FUdR) to inhibit DNA synthesis (8) after which lo-' M unlabelled thymidine was added to release the block of DNA synthesis. By this method cells accumulated at the beginning of the S phase traversed the rest of the cell cycle in synchrony.To induce cell synchrony the explants were incubated at 20°C for 14 hr in 11

Replication timing and cell differentiation

“…However, both autoradiographic (Stambrook and Flickinger, 1970) and biochemical experiments (Remington and Flickinger, 1971) show an increase of late‐replicating DNA from gastrulation to the tailbud stage. Furthermore, much of the DNA that becomes late replicating during this period is AT rich (Flickinger and Richman, 1983; Remington and Flickinger, 1978). Further work is required to learn if the increase of cellular histone H1 (Dimitrov et al, 1993) during this time has a role in the shift of AT‐rich DNA from early to late phase replication.…”

“…If HMG‐I/Y has a role in establishing replicon origins within AT‐rich isochores, then reduction of the rate of HMG‐I/Y synthesis may determine in part the shift of AT‐rich DNA to late replication during early development (Flickinger and Richman, 1983; Remington and Flickinger, 1978). The level of total protein synthesis (Stanners and Becker, 1971) and that of HMG‐I/Y (Lanahan, 1992; Reeves, personal communication) is reduced in confluent cultured cells.…”

“…There is an increase of late‐replicating DNA during this period (Remington and Flickinger, 1971; Stambrook and Flickinger, 1970). Much of the DNA that becomes late replicating is AT‐rich (Flickinger and Richman, 1983; Remington and Flickinger, 1978). Autoradiographs show that all DNAs except those of telomeres are early replicating in neural plate cells of early neurulae (Remington and Flickinger, 1971).…”

Possible role of H1 histone in replication timing

“…Early work: AT-rich DNA becomes late replicating in developing frog embryos Autoradiography and quantitative determinations of labeled DNA synthesis in partially synchronized explants from early frog embryos showed an increase of late replicating DNA during early development (Remington & Flickinger 1971). Subsequent work indicated a shift of labeled thymidine, but not deoxyguanosine, incorporation into late replicating DNA during this period (Remington & Flickinger 1978). Isolated nuclei in an in vitro system displayed a significant increase of labeled thymidine triphosphate (TTP), but not dCTP, incorporation into late replicating DNA during early development, (Table 1), (Flickinger & Richman 1983).…”