Список сокращений
Введение
1. Обзор литературы
1.1. Структура и функции теломер
1.2. Репликация теломерных областей
1.3. Структура хроматина теломерных областей
1.4. Транскрипция теломерных областей и РНК TERRA.
1.5. Теломерные последовательности вне теломер и их функции
1.6. Подходы к изучению теломерных последовательностей
2. Цель работы
3. Задачи работы
4. Материалы и методы.
4.1. Материалы.
4.1.1 Штаммы, использованные в работе
4.1.2. Среды, использованные в работе
4.1.3. Плазмиды, использованные в работ
4.1.4. Олигонуклеотиды, использованные в работе
4.2. Выделение белков, взаимодействующих с РНК, содержащей человеческие
теломерные повторы в дрожжевой системе
4.3. Масс-спектрометрия.
4.4. Метод ПЦР
4.5. Рестрикция плазмид UIRL-lacO-25-12-8 и UIRL-lacO-25-12-5 рестриктазой SmiI
4.6. Дрожжевая трансформация
4.7. Бактериальная трансформация
4.8. Гель-фильтрация SUMO-LacI
4.9. Анализ взаимодействия SUMO-LacI с ДНК, содержащей теломерные повторы и
последовательность lacO.
4.10. Описание модельной системы для выделения белков, взаимодействующих с
человеческими теломерными повторами в ДНК в дрожжевой системе.......................29
4.11. Фрагментация хроматина
4.12. Анализ структуры белка in silico
5. Результаты
5.1. Выделение белков, взаимодействующих с РНК, содержащей человеческие
теломерные повторы в дрожжевой системе
5.2. Анализ структуры белка Ten1 in silico
5.3. Выделение белков, взаимодействующих с теломерными последовательностями в
ДНК.
5.3.1. Очистка SUMO-LacI и анализ условий взаимодействия SUMO-LacI c lacO..38
5.3.2. Конструирование штаммов S.cerevisiae AAY51 и AAY52. ..............................42
5.3.3. Фрагментация хроматина
6. Обсуждение результатов
7. Выводы.
8. Благодарности
8. Список литературы
Теломеры представлены нуклеопротеиновыми комплексами, сформированными концевыми участками линейных хромосом и взаимодействующими с ними белками, основной функцией которых является защита концов хромосом от систем рекомбинации и репарации двунитевых разрывов ДНК. Последовательность ДНК теломер у большинства видов эукариот представлена короткими гуанин-богатыми тандемными повторами, которые также обнаруживаются во внутренних частях хромосом и известны как интерстициальные теломерные последовательности (от англ.
interstitial telomeric sequences, ITSs). Теломерные последовательности ассоциированы с
областями с повышенной геномной нестабильностью, а также в силу высокого содержания гуанина и особенностей хроматиновой укладки являются сложными матрицами для работы систем репликации и репарации ДНК. Теломерные последовательности на концевых и внутренних участках хромосом вовлечены в взаимодействия с различными белками, участвующими в регуляции транскрипции проксимальных и дистальных генов и других внутриядерных процессах. Изучение теломер и теломерных последовательностей является непростой задачей для исследователей. В последнее время накапливается все больше данных о разнообразных белках, вовлеченных в процессы функционирования теломерных последовательностей в геноме, но многие участники и детали этих процессов до сих пор остаются мало изученными. Разработка методов для эффективного выделения и идентификации белков, вовлеченных в функционирование теломерных последовательностей, является одной из важных задач современной науки. Основой для таких методов могут служить относительно простые и удобные для генетических манипуляций дрожжевые системы. Подобные методы были разработаны и использованы в данной работе для выделения и идентификации белков, взаимодействующих с теломерными последовательностями в ДНК и РНК.
1. В ходе работы разработан и оптимизирован метод выделения и идентификации белков, взаимодействующих с теломерными последовательностями в РНК.
2. Отработана идентификация выявленных белков, взаимодействующих с
человеческими теломерными последовательностями в РНК, методом массспектрометрии. Среди идентифицированных белков, взаимодействующих с человеческими теломерными последовательностями в РНК, обнаружен белок Ten1, участвующий в регуляции длины теломер.
3. Разработан метод выделения и идентификации белков, взаимодействующих с теломерными повторами в ДНК. Показано специфическое взаимодействие SUMO-LacI с последовательностями, содержащими теломерные повторы и lacO в модельной дрожжевой системе.
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