Graduate Thesis Or Dissertation
 

Isolation and partial characterization of temperature sensitive division mutants of Chlamydomonas reinhardtii

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  • The primary objective of this study was to develop methods for the selection of temperature sensitive division (TSD) mutants of Chlamydomonas reinhardtii. Such methods were developed and four temperature sensitive (TS) mutants were isolated and partially characterized. Wild type (WT) cells of the plus mating type were synchronized by a 12 hr light-12 hr dark regime at 25 C in culture tubes containing a high salt minimal medium buffered with PIPES (Piperazine-N, N'- bis (2-ethane sulfonic acid) monosodium monohydrate). Cells were mutagenized by irradiation with 1725 ergs/mm² of 254 nm ultraviolet light (UV) during the first half of the light period (G₁). The irradiated cells were kept in the dark for 24 hr to prevent photoreactivation, and then treated with a 10 mM concentration of hydroxyurea (HU) for 24 hr at an elevated temperature of 33 C. It was determined that this concentration of HU and this duration of treatment effectively kills WT cells at some stage i.n the middle of the generation cycle (presumably S phase). Temperature sensitive mutants that cannot progress into the HU-sensitive phase at 33 C were expected to survive. The combined treatment with UV and FU reduced the population to approximately 1% and increased the yield of possible TSD mutants. (UV alone reduced the population to 7% and HU to about 6.9%.) The cells were then plated on agar plates made with minimal medium and returned to 33 C for 5 days. The colonies that appeared during this time were considered to have arisen from unmutated cells or non-TSD mutants that had somehow survived the selection treatment; these colonies were marked and the agar plates placed at 25 C for an additional 5 days. The colonies that now formers were considered possible TSD mutants. Of 116 such colonies, only four were found to be temperature sensitive after replating and testing them at 33 and 25 C, the permissive temperature in this study. The TS mutants isolated were partially characterized on the basis of their responses to the restrictive temperature. The mutants, along with the WT cells, were observed in microdrops of minimal or complete medium covered with paraffin oil and on minimal or complete agar plates. The percentages of cells having divided once, twice, and three or more times were recorded at various times during continuous incubation at 25 C and during and after a 24 hr exposure to 33 C. In addition, the mutants were observed for viability and morphological changes. The effects of light intensities of 0, 200, and 600 foot candles were also studied. It was concluded that no ideal TSD mutant had been isolated. An ideal TSD mutant would be one in which division could be reversibly blocked by a restrictive temperature at a specific stage of the generation cycle. Although such mutants were not isolated, three of the four TS mutants (TSLt-1, TSLt-2, and TSGL) appear to show responses at the 33 C which suggest that some specific phase of the generation cycle is impaired, but the effect is not reversible and the cells die. The fourth mutant (TSL) is killed by 33 C at all phases of its generation cycle. The mutants all formed normal colonies, or nearly so, at 25 C, although TSL had a somewhat reduced growth rate. Light intensity and medium composition play a significant role i.n the lethal response of the mutants to the 33 C temperature: TSLt-1 and TSLt-2 show enhanced survival on minimal medium or at low light intensities (0 and 200 foot candles) when compared to cells exposed to 33 C on complete medium or at a higher light intensity (600 foot candles). TSGL shows a moderate light intensity effect. Giant cell formation was observed in TSGL and to a lesser extent i.n TSLt-2. The responses of the mutants suggest that continued growth while some division process is blocked by 33 C (imbalanced growth) may be lethal. Studies of the division characteristics of WT cells indicate that individual cell growth (as determined by the number of daughter cells bursting from the mother cell at the end of the generation cycle) is only very loosely coupled to the division process (as determined by generation time, i. e. , the time between successive bursts). The generation time appears to be fairly constant under a variety of conditions, but the burst size varies from 2 to 32. Further studies with these mutants and with others that may be isolated by the techniques developed in this study may aid the understanding of the interrelationships of various processes involved in the division of cells.
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file details WolffDaroldMelvin1972.pdf 2017-08-07 Pubblico Scaricare