The effects of kinetin and contaminating bacteria on the incorporation of 32P-orthophosphate into various fractions of nucleic acid extracted from radish leaves

Summary By comparing ³²P-orthophosphate incorporation into nucleic acid extracts of sterile and non-sterile radish leaf discs, it was shown that contaminating bacteria cause a 2–4 fold increase in the rate of precursor incorporation and alter the pattern of label distribution after fractionation of the extracts by sedimentation through sucrose gradients or chromatography on MAK¹ columns. Using sterile senescing radish leaf discs, a stimulation of ³²P-orthophosphate incorporation into various fractions of nucleic acid was observed as a result of kinetin treatment.     

The effects of abscisic acid,kinetin and 5-fluorouracil on ribonucleic acid and protein synthesis in senescing radish leaf disks

Summary The effects of abscisic acid and kinetin on RNA synthesis in senescing radish leaf disks were investigated using the improved resolution afforded by polyacrylamide gel electrophoresis. Kinetin stimulated and abscisic acid inhibited incorporation of radioactivity into cytoplasmic ribosomal RNA and soluble RNA. Chloroplast ribosomal RNA synthesis appeared to be confined to the period of leaf expansion and was not detected in fully mature leaves. The effects of kinetin in retarding and of abscisic acid in accelerating leaf senescence were not altered by the inhibition of cytoplasmic ribosomal RNA synthesis with 5-fluorouracil. Following inhibition of cytoplasmic ribosomal RNA synthesis with 5-fluorouracil, kinetin stimulated and abscisic acid inhibited incorporation of radioactivity into polydisperse RNA. These results are discussed in relation to the possible mode of action of kinetin and abscisic acid in senescing leaf tissue.     

Incorporation of labelled precursors into the electrophoretic fractions of rat-liver ribonucleic acid

1. Incorporation of [(32)P]orthophosphate and of [2-(14)C]orotic acid into rat-liver RNA was studied by agar-gel electrophoresis by using u.v.-densitometry and radioautography of dried agar electrophoretograms. 2. During the electrophoresis some low-molecular-weight contaminants, including inorganic phosphate present in the RNA preparations, were separated from the RNA fractions. Since nucleoside mono-, di- and tri-phosphates still interfered, the RNA preparations had to be subjected to a purification procedure [Sephadex G-25 or Dowex 1 (X8)]. 3. In RNA extracted from cytoplasm, isolated microsomes or ribosomes, whatever variations were made in the phenol procedure no special rapidly labelled RNA fraction was detected other than ;soluble' RNA and the ribosomal RNA components. 4. When the whole homogenate or cytoplasmic fraction was treated only with phenol (pH6) a considerable part of the cytoplasmic RNA was not extracted. The treatment of the cytoplasmic fraction with sodium dodecyl sulphate before the addition of phenol increased the yield of the high-molecular-weight RNA and at the same time a higher specific activity was found for the faster ribosomal RNA component. 5. The presence of four distinct rapidly labelled RNA fractions was established in the RNA not extracted by phenol, and they moved slower than the ribosomal RNA. They were extracted only with the use of phenol-sodium dodecyl sulphate at an elevated temperature.     

The effects of abscisic acid on senescence in leaf discs of radish,Raphanus sativus L.

Summary After a 6 day incubation period, abscisic acid (ABA) at 10^-4 M retarded the decline in pigment levels and promoted the decline in protein levels of radish leaf discs. ABA treatment also retarded the rise in the specific activity of the RNA fraction (calculated by counts per minute incorporated of ¹⁴C-8-adenine as a fraction of optical density at 260 nm) observed in water-treated control discs. The results indicated that ABA was primarily effective in enhancing senescence in the early stages following leaf excision. Thus the increase in RNA specific activity during an initial 24 h incubation period was especially pronounced with ABA treatment although there was no effect of the hormone on RNA level. Moreover, in contrast to control discs, the pigment levels declined markedly in ABA-treated discs in this period. When the discs had been incubated in water (preaged) for 3 or 5 days prior to ABA treatment, however, the hormone then had little effect on RNA metabolism and protein and pigment levels relative to the water control.Data are collated from different experiments to show the changes in RNA, pigment and protein with ABA treatment during a 6 day senescence period.It is considered that ABA is speeding up the natural changes in RNA metabolism possibly by affecting both RNA synthesis and degradation.     

Disappearance of radioactivity from the various ribonucleic acid pools and acid-soluble fractions of mouse liver and kidney after a single injection of labelled orotic acid. The effect of castration

1. An attempt was made to study the rate of synthesis as well as the distribution of RNA in the various cellular fractions in the livers and kidneys of normal and castrated mice. 2. The tissue was fractionated by the procedure of Blobel & Potter (1967). By using this method it was not possible to find any pronounced difference in the relative proportions of RNA in isolated subcellular fractions when kidneys from normal and castrated mice were compared. On the other hand there was an indication of a shift toward the bound ribosomes in livers from normal mice in comparison with livers from castrated mice. 3. Disappearance of the radioactivity followed the pattern of the first-order reaction. Comparing the half-lives of RNA in liver and kidneys it was found that in the latter in both groups of animals half-lives were shorter no matter which cellular fraction was studied. 4. The half-lives for total homogenate RNA, total ribosomal RNA and low-molecular-weight RNA from kidneys of castrated mice were approximately 20-25% longer than the half-lives for the corresponding fractions from normal mouse kidneys. 5. An explanation is put forward for the anomalous finding that RNA from the castrated-mouse kidneys has a higher specific radioactivity than that isolated from normal mice.     

Non-uniformities in the metabolism of excised leaves and leaf discs

Summary In intact tobacco and Chinese-cabbage (Brassica pekinensis) leaves an ageing process begins as soon as the leaves are excised. The terminal few millimetres of the petiole increasingly preempt materials such as phosphate and uracil taken up by the leaf. Actinomycin D treatment blocks this process and leads to increased uptake of such materials into the lamina.Immediately after excision there is a radial geographical gradient, in the ability of excised leaf discs to accumulate phosphate and uracil from solution. Tissue near the cut edge accumulates much more of these materials than that near the centre, and total nucleic acids isolated from the outer zone have a higher specific activity.Over the first day after excision there is a marked increase in this ability of the outer tissue of discs to accumulate labelled precursors but the changes taking place with time are complex and variable. Specific activity of total nucleic acids increases somewhat less than the increased uptake of labelled precursor. Actinomycin D becomes very unevenly distributed in leaf discs floated on solutions of the drug. These various effects are of sufficient magnitude to affect the interpretation of studies using excised leaf tissues.     

Inhibitory effects of orotate on precursor incorporation into nucleic acids.

The influence of orotic acid on the incorporation of precursors into nucleic acids was studied in mice and rats and in isolated cells. In vivo, orotate levels were modified by two diets which are known to increase the rate of pyrimidine nucleotide synthesis in rat liver. Of these diets, a 1% orotate diet had greater inhibitory effects than an arginine-deficient diet on the incorporation of [3H]orotate into RNA of mouse kidney than mouse liver. This contrasted with the situation in the rat where there was a greater effect in the liver than the kidney. The situation in the rat was more readily interpreted than in the mouse in terms of previously established effects of these diets on ribonucleotide pool sizes. However, studies using [3H]adenosine as a precursor for incorporation into RNA suggested that even in the mouse the effects of orotate were on pool sizes rather than an inhibitory effect on RNA synthesis. The incorporation of [3H]thymidine into DNA was inhibited by orotate to a similar degree in cultured HTC hepatoma cells and a line of rat liver epithelial cells. An effect on DNA synthesis rather than solely on pool sizes was suggested by the observation that the pool size of dTTP was not increased by 5 mM orotate under conditions in which there was a four-fold increase in the level of UTP in HTC cells. An inhibitory effect of orotate on DNA synthesis was further supported by an observation of decreased incorporation of [3H]deoxyadenosine into DNA and a lower rate of cellular proliferation.