Cold thymidine

Trowbridge removed the pulse by washing the cells, Kettman et al. by chasing with excess of cold thymidine. 

Lark et al. (1966) have examined the segregation of labeled and unlabeled chromosomes in mouse primary fibroblasts and Chinese hamster fibroblasts after variable periods of growth in tritiated thymidine, followed in some cases by growth in cold thymidine. The mouse cells provided clear evidence of nonrandom segregation of old and new DNA this result implies attachment of the mitotic chromosomes to a structme upon which they can be oriented with respect to one another, and the author suggests that this structure is the nuelear membrane. Similar data were obtained for the diploid hamster line, but the results were not as convincing. 

Thymidine Uptake Studies. Tritiated thymidine (52 mCi/ anole 0.1 /rCi/ml) was added to cells for 60 min at 37°C. Cells were then washed with PBS, incubated at 4°C for 15 min in the presence of ice cold 5% TCA, rinsed with TCA and scraped from the flasks with a rubber policeman. Cells were again washed with PBS, and solubilized in 0.1N NaOH overnight. Aliquots were assayed for protein (62) and radioactivity (scintillation fluid 100 ml Biosolve (Beckman, Fullerton, CA.), 7g of PPO and 0.6 g of POPOP per liter of toluene). 

Fig. 12.1. Equilibration of tritiated thymidine with the acid-soluble pool and incorporation into DNA. Mouse L929 cells, growing in 5 cm dishes, were incubated with [3H]thymidine (0.7/iCi/ml 5/tM) for the indicated times after which the cells were quickly washed three times with ice-cold BSS. The acid-soluble material (AS) was extracted into cold i% TCA and after further acid and ethanol washes the cells were solubilised in 0.3 N NaOH and incorporation into DNA measured. (Reproduced from Adams, 1969a, with kind permission of the publisher.)...

Bacterial production was estimated by incorporation of 3H-thymidine (Fuhrman and Azam 1982). Duplicate 10 ml subsamples were incubated for 1 h in the dark at 2°C in the presence of 20 nM of 3H-thymidine (Amersham 40-50 Ci mol ), filtered on 0.2-pm cellulose acetate membrane filters (Sarto-rius) and extracted with ice-cold 5% Trichloroacetic acid (TCA) (Becquevort and Smith 2001). Thymidine incorporation was converted into bacterial production using conversion factors of Ducklow et al. (1999) established for the Ross Sea bacterial communities (i.e., 8.6 X 1017 bacteria produced per mole of thymidine incorporated in the cold TCA insoluble material). For bacterial production, the relative standard deviation was 9.3% (n = 20). The specific growth rate was estimated from bacterial production and bacterial biomass. 

Cultured cells are rinsed with ice-cold PBS and were then mechanically removed in the presence of 1 mM MgCl2, 50 mM HEPES pH 7.5, 0.3 mM phenylmethylsulfonly fluoride, 30 ig/ml leupeptine, 1 mM dithiothreitol, 10 mM thymidine, 5 M [ P]NAD,... 

Preparation of Crude Extracts The parasite larvae or adult forms were homogenized by grinding with sand in a mortar (placed on ice) with three to five volumes of ice-cold 0.05 M phosphate buffer, pH 7.5, containing 0.1 M KCl and 0.01 M 2-mercaptoethanol, except for assays of thymidine kinase activity, when phosphate buffer was substituted by Tris-HCl buffer at the same concentration and pH. The suspension was sonicated and centrifuged to obtain crude extract. 

DNA in spores is densely packed and covered by small acid-soluble spore proteins. The DNA is strongly dehydrated and exits in a more A-Hke conformation. This dense packing enables spores to survive even extreme conditions like extreme cold and heat or severe dryness. Irradiation of spores with UV Kght causes formation of yet another photolesion, which is exclusively formed in spores, possibly due to the unusual A-like conformation of the DNA. > The formation of this lesion (Scheme 5) can be mimicked by irradiation of a thin film of dry thymine or of a frozen thymidine solution with a UV-C hght source (254 nm), as depicted in Scheme 6(a). ... 

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