Молекулярно-генетические механизмы повышенной устойчивости бактерий к потенциально-летальным повреждениям ДНК
Диссертация
Наши данные показывают, что репарация радиационно-индуцированных повреждений ДНК (вероятно, включая двунитевые разрывы как основные летальные повреждения) ассоциирована с активностями RecBCDи RecF-путей рекомбинационной репарации не только в клетках дикого типа, но и в радиоустойчивых мутантах. Очевидно, адаптация Е. coli к ДНК-повреждающим! агентам, включая у-излучение, не связана с i появлением… Читать ещё >
Содержание
- ВВЕДЕНИЕ.б
- Глава 1. ОБЗОР ЛИТЕРАТУРЫ
- 1. 1. РЕАКЦИЯ БАКТЕРИЙ НА ПОВРЕЖДЕНИЕ ДНК, SOS-OTBET,
- РЕКОМБИНАЦИОННАЯ РЕПАРАЦИЯ И СВЯЗЬ С РЕПЛИКАЦИЕЙ ДНК
- 1. 1. 1. Основные категории генов, участвующих в защите генома
- 1. 1. 2. Однонитевые повреждения ДНК и их репарация
- 1. 1. 3. Алкилирование ДНК и адаптивный ответ
- 1. 1. 4. Реакция на окислительный стресс
- 1. 1. 5. Механизмы эксцизионной репарации
- 1. 1. 6. Коррекция ошибочно спаренных оснований
- 1. 1. 7. SOS-ответ coli на повреждение ДНК
- 1. 1. 8. Происхождение двунитевых повреждений ДНК и их репарация
- 1. 1. 9. Ранние модели репарации двунитевых повреждений ДНК
- 1. 1. 10. Пути рекомбинационной репарации у Е. col
- 1. 1. 11. Репарация двунитевых концов ДНК в отсутствие RecBCD
- 1. 1. 12. RecE-путь рекомбинационной репарации
- 1. 1. 13. Репарация ДНК по RecF-пути рекомбинации
- 1. 1. 14. Репарация двунитевых разрывов ДНК при участии фермента RecBCD
- 1. 1. 15. Рекомбинационная репарация, зависящая от репликации, и белок РпА
- 1. 1. 16. Модель репарации, сопряженной с репликацией ДНК
- 1. 1. 17. Подготовка двунитевых концов ДНК нуклеазой RecBCD
- 1. 1. 18. Гены, кодирующие экзонуклеазу V
- 1. 1. 19. Деградация ДНК после облучения ионизирующей радиацией и УФ-светом
- 1. 1. 20. Деградация ДНК как способ контроля хромосомной репликации
- 1. 1. 21. Роль белка RecA в рекомбинационной репарации
- 1. 1. 22. Клеточные процессы, зависящие от RecA
- 1. 1. 23. Контроль активности RecA
- 1. 1. 24. Значение рекомбинационной репарации
- 1. 1. 25. Клеточные процессы, сопровождающиеся рекомбинационную репарацию
- 1. 1. 26. Унифицированный механизм репарации двунитевых разрывов
- 1. 2. ДОПОЛНИТЕЛЬНЫЕ СТРЕССОВЫЕ СИСТЕМЫ КЛЕТКИ И РАДИОУСТОЙЧИВОСТ
- 1. 2. 1. Реакция бактериальной клетки на тепловой шок во время облучения
- 1. 2. 2. Система постэкспоненциальных белков
- 1. 2. 3. Стрессовый ответ холодового шока
- 1. 3. ГИПЕРРАДИОРЕЗИСТЕНТНАЯ БАКТЕРИЯ Deinococcus radiodurans И РОЛЬ RecA-ПОДОБНОГО БЕЛКА
- 1. 3. 1. Характеристика радиочувствительности D. radiodurans
- 1. 3. 2. Роль рекомбинанационной репарации и белка RecA в радиоустойчивости D. radiodurans
- 1. 3. 3. Ингибироваиие радиационно-индуцированной деградации у D. radiodurans
- 1. 3. 4. Дополнительные факторы радиоустойчивости D. radiodurans
- 1. 4. СВОЙСТВА МУТАНТОВ Escherichia coli К-12 GAMr
- 1. 4. 1. Радиобиологическая характеристика мутантов Gamr
- 1. 4. 2. Предварительная характеристика мутантных аллелей Gamr
- 1. 4. 3. Сравнение мутантов Е. coli Gamr и D. radiodurans
- 1. 5. ПРИРОДНЫЕ ПЛАЗМИДЫ, ПОВЫШАЮЩИЕ РАДИОУСТОЙЧИВОСТЬ БАКТЕРИЙ
- 1. 6. ВЫВОДЫ ПО ГЛАВЕ 1
- Глава 2. МАТЕРИАЛЫ И МЕТОДЫ
- 2. 1. Штаммы Escherichia coli, Salmonella derby, Deinococcus radiodurans, фаги и плазмиды
- 2. 2. Получение рекомбинантных плазмид
- 2. 3. Среды
- 2. 4. Трансдукция
- 2. 5. Спот-тест для качественной оценки уровня радиочувствительности
- 2. 6. Изучение зависимости выживаемости клеток от дозы у-излучения
- 2. 7. Клонирование локусов Gamr
- 2. 8. Определение экзонуклеазной активности
- 2. 9. Введение радиоактивной метки в белки
- 2. 10. Введение радиоактивной метки в клеточную ДНК
- 2. 11. Определение относительной эффективности посева мутантного фага
- 2. 12. Определение частоты замещения хромосомного аллеля
- 2. 13. Выделение плазмидной ДНК
- 2. 14. Электрофоретическое разделение плазмидной ДНК и белков
- 2. 15. Полимеразная цепная реакция, гибридизация и секвенирование ДНК
- 2. 16. Математическая обработка результатов опытов
- Глава 3. ЭКСПЕРИМЕНТАЛЬНЫЕ РЕЗУЛЬТАТЫ
- 3. 1. КОНСТИТУТИВНЫЕ И ИНДУЦИБЕЛЬНЫЕ ПУТИ РЕПАРАЦИИ И РАДИОУСТОЙЧИВОСТ
- 3. 1. 1. Роль RecBCD-пути рекомбинационной репарации, сопряженной с репликацией, в радиоустойчивости мутантов Gamr Escherichia coli. Ill
- 3. 1. 2. Роль индивидуальных генов RecF- и RecBC-путей рекомбинационной репарации в радиационной устойчивости Е. col
- 3. 2. МУТАНТНЫЕ АЛЛЕЛИ РАДИОРЕЗИСТЕНТНОСТИ ИЗ ШТАММА ESCHERICHIA COLI GAMR444: КЛОНИРОВАНИЕ И ХАРАКТЕРИСТИКА
- 3. 2. 1. Клонирование и селекция рекомбинантных плазмид
- 3. 2. 2. Свойства клонированной рецессивной мутации gam
- 3. 2. 3. Свойства клонированных доминантных аллелей gam
- 3. 2. 4. Аддитивность радиозащитного действия двух классов аллелей Gamr
- 3. 3. КОНСТИТУТИВНОЕ ИНГИБИРОВАНИЕ ДЕГРАДАЦИИ ДНК, ВЫЗВАННОЙ ФЕРМЕНТОМ RECBCD, В МУТАНТЕ GAMR 444 Е. COLI
- 3. 3. 1. АТФ-зависимая деградация ДНК в клеточной фракции, содержащей экзонуклеазу V
- 3. 3. 2. Оценка экзонуклеазной активности RecBCD с помощью мутанта фага Т4 2″
- 3. 3. 3. Предварительное картирование мутантного аллеля gam26 из радиоустойчивого штамма Gamr
- 3. 4. СВОЙСТВА МУТАНТНОГО АЛЛЕЛЯ GAM
- 3. 4. 1. Тестирование радиозащитного эффекта плазмиды pGaml8 на RecE- и
- 3. 1. КОНСТИТУТИВНЫЕ И ИНДУЦИБЕЛЬНЫЕ ПУТИ РЕПАРАЦИИ И РАДИОУСТОЙЧИВОСТ
- 3. 4. 2. Компенсация дефекта гена uvrD, кодирующего ДНК-геликазу П, мутантным аллелем gaml
- 3. 5. ПРИРОДНАЯ ПЛАЗМИДА pSD89 (Cm1) ИЗ Salmonella derby К89, ПОВЫШАЮЩАЯ РАДИОУСТОЙЧИВОСТЬ ШТАММОВ Escherichia coli К
- 3. 5. 1. Радиочувствительность штаммов Salmonella derby
- 3. 5. 2. Электрофоретическое разделение плазмидных ДНК из S. derby К
- 3. 5. 3. Изменение радиочувствительности штаммов Е. coli, трансформированных плазмидой pSD89 (Cm1)
- 3. 5. 4. Способность плазмиды pSD89 (Cm1) поддерживать размножение ро/Л-зависимых репликонов
- 3. 6. РОЛЬ СТРЕССОВОЙ СИСТЕМЫ БТШ В РАДИОУСТОЙЧИВОСТИ БАКТЕРИЙ И БАКТЕРИОФАГОВ
- 3. 6. 1. Влияние мутаций в структурных генах белков теплового шока на радиорезистентность штамма дикого типа Escherichia col
- 3. 6. 2. Зависимость повышенной радиорезистентности мутантов Gamr от состояния структурных генов белков теплового шока
- 3. 6. 3. Влияние белков теплового шока на пострадиационную деградацию ДНК
- 3. 6. 4. Рост радиоустойчивости бактериофагов как результат усиления экспрессии стрессовых систем клетки-хозяина
- 3. 6. 4. 1. Определение экспрессии стрессовых систем у радиоустойчивых мутантов
- 3. 6. 4. 2. Реактивация и W-реактивация у-облученных фагов
- 3. 6. 4. 3. Радиочувствительность фагов, облученных в комплексе с клетками
- 3. 6. 4. 4. Пострадиационная деградация фаговой ДНК
- 3. 7. ИССЛЕДОВАНИЕ СВОЙСТВ ГЕНА RECA D. RADIODURANS В Е. COLI
- 3. 7. 1. Роль гена recA+ D. radiodurans в радиоустойчивости Е. col
- 3. 7. 2. Радиоустойчивость штаммов Е. coli гесА' с клонированными мутантными аллелями гена гесА Е. coli и D. radiodurans
- 3. 8. ВКЛАД БЕЛКОВ ХОЛОДОВОГО ШОКА В РАДИОУСТОЙЧИВОСТ
- 3. 8. 1. Влияние пнсерционной мутации в гене cspA главного белка холодового шока на радиорезистентность клеток Е. col
- 3. 8. 2. Роль мутации в кластере генов cspH-cspG в радиоустойчивости
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