Изучение экспрессии и взаимодействий белка Stellate в семенниках Drosophila melanogaster тема диссертации и автореферата по ВАК РФ 03.01.03, кандидат биологических наук Егорова, Ксения Сергеевна

• Егорова, Ксения Сергеевна
• кандидат биологических наук
• 2010

Оглавление диссертации кандидат биологических наук Егорова, Ксения Сергеевна

Список сокращений.

1. Общая характеристика работы.

1.1 Актуальность проблемы.

1.2 Задачи исследования.

1.3 Научная новизна результатов исследования.

1.4 Практическая ценность.

1.5 Апробация работы.

2 Обзор литературы.

2.1 Экспрессия тандемных кластеров генов Stellate в семенниках Drosophila melanogaster.

2.1.1 Система crystal-Stellate.

2.1.2 Гены Stellate являются мишенью piPHK-сайленсинга.

2.1.3 Происхождение генетической системы crystal-Stellate.

2.1.4. Изучение особенностей белка Stellate.

2.2 Протеинкиназа СК2.

2.2.1 Роль СК2 в клетках различных организмов.

2.2.2 Структурно-функциональные характеристики СК2.

2.2.3 Мишени СК2.

2.2.4 CK2fi: внутривидовые особенности.

2.2.5 Мишени СК2 у D. melanogaster.

2.3 Метилирование негистоновых белков по остаткам лизина как способ регуляции их стабильности и функциональности.

2.3.1 Метилирование гистонов и концепция гистонового кода.

2.3.2 Метилирование транскрипционных факторов и других ядерных белков.

2.3.3 Метилирование белков трансляционного аппарата.

2.3.4 Белки, узнающие метилированные остатки лизинов («метиллизин-ридеры»).

2.3.5 Итоги и дальнейшие перспективы изучения метилированных белков.

3. Материалы и методы.

3.1 Линии Drosophila melanogaster, использованные в работе.

3.1.1 Схема скрещивания для получения мух с делецией локуса crystal, гетерозиготных по инсерции в ген гистонметилтрансферазы Su(Var)3-9.

3.2 Экспрессия и очистка рекомбинаптного белка Stellate.

3.3 Получение антител к белку Stellate.

3.4 Иммунофлуоресцентное окрашивание и конфокальная микроскопия.

3.5 Электрофорез в денатурирующих условиях в ДСН-ПААГ и Вестерн-блот-анализ.

3.5.1 Приготовление модифицированного полиакриламидного геля с измененной процентностью сшивки.

3.5.2 Окрашивание ПААГ «Кумасси».

3.5.3 Окрашивание ПААГ серебром.

3.6 Антитела.

3.7 Субклеточное фракционирование.

3.8 Фракционирование в градиенте сахарозы.

3.9 Субъядерное фракционирование.

3.10 Иммунопреципитация.

3.10.1 Ковалентная пришивка антител к протеин-С-сефарозе.

3.11 Аналитическая очистка белка Stellate и идентификация сайта метилирования.

3.12 Химическая сшивка белков in nucleo.

3.13 Фосфатазный анализ.

3.14 Иммуноферментный сорбционный анализ (ELISA).

4. Результаты.

4.1 Получение антител к белку Stellate.

4.2 Субклеточная локализация белка Stellate.

4.3 Поиск партнеров белка Stellate.

4.4 Stellate подвергается метилированию по остатку лизина.

4.5 Разделение белков Stellate на фракции, кодируемые эухроматиновыми и гетерохроматиновыми повторами Stellate.

5. Обсуяедение.

5.1 Субклеточная локализация белка Stellate.

5.2 Взаимодействие Stellate с СК2а.

5.3 Триметилирование Stellate.

5.4 Модель патогенеза при сверхэкспрессии Stellate.

6. Выводы.

7. Литература.

Благодарности

Список сокращений

АБТС — 2,2'-азино-бис(3-этилбензтиазолин-6-сульфоновая кислота)

ГЛМТ - гистонлизинметилтрансфераза

Да (кДа) - дальтон (кило дальтон)

ДМСО - диметилсульфоксид

ДСН - додецилсульфат натрия

ДСН-ПААГ — полиакриламидный гель, содержащий додецилсульфат натрия

ДТТ - дитиотреитол н. - нуклеотидов т.п.н. — тысяч пар нуклеотидов

ФМСФ - фенилметилсульфонилфторид

ФСБ - фосфатно-солевой буфер

ЭГТУ - этиленгликольтетрауксусная кислота (динатриевая соль)

ЭДТУ - этилендиаминтетрауксусная кислота (динатриевая соль)

DAPI - 4',6-диамидино-2-фенилиндол

DMP - диметилпимелимидат

DSP - дитиобиссукцинимидилпропионат

ELISA - enzyme-linked immunosorbent assay (иммуноферментный сорбционный анализ)

НЗК4те2 - гистон НЗ, диметилированный по остатку лизина К4 НЗК9те2 - гистон НЗ, диметилированный по остатку лизина К9 НЗК9теЗ - гистон НЗ, триметилированный по остатку лизина К9 НЗК27шеЗ - гистон НЗ, триметилированный по остатку лизина К27 IPTG - изопропил-р-О-тиогалактозид piPHK - Piwi-ассоциированные короткие РНК PVDF - поливинилиденфторид Su(Ste) — Supressor ofStellate

1. Общая характеристика работы

Заключение диссертации по теме «Молекулярная биология», Егорова, Ксения Сергеевна

6. Выводы

1. Охарактеризована субклеточная локализация белка Stellate в сперматоцитах D. melanogaster при генетической дерепрессии кластеров генов Stellate; Stellate формирует кристаллы преимущественно в цитоплазме сперматоцитов, в то время как в нуклеоплазме сперматоцитов присутствует также в некристаллической растворимой форме.

2. При дерепрессии кластеров генов Stellate выявляются белковые продукты как эухроматинового, так и гетерохроматинового кластеров Stellate.

3. Эндогенный белок Stellate взаимодействует с каталитической а-субъединицей протеинкиназы СК2.

4. Белок Stellate, как растворимый, так и кристаллический, несет посттрансляционную модификацию — триметилирование по остатку лизина К92; в метилировании Stellate принимает участие гистонлизинметилтрансфераза dSETDB 1.

5. Триметилированный по остатку лизина К92 белок Stellate специфически узнается антителами к модифицированному гистону НЗК9теЗ, демонстрируя структурную мимикрию соответствующего эпитопа гистона.

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