Взаимодействие ядерного актина и NAP57/дискерина с белками, специфически связывающими сателлитную ДНК тема диссертации и автореферата по ВАК РФ 03.00.25, кандидат биологических наук Кукалев, Александр Сергеевич

• Кукалев, Александр Сергеевич
• кандидат биологических наук
• 2004

Оглавление диссертации кандидат биологических наук Кукалев, Александр Сергеевич

1 СПИСОК ИСПОЛЬЗУЕМЫХ СОКРАЩЕНИЙ.

1. ВВЕДЕНИЕ.

1.1. Цели и задачи исследования.

1.2. Основные положения, выносимые на защиту.

2. ЛИТЕРАТУРНЫЙ ОБЗОР.

2.1 Организация генома эукариот.

2.2. Особенности организации сателлитных ДНК.

2.3. Белки, специфически связывающие сатДНК.

2.4. Гетерохроматин.

3. МАТЕРИАЛЫ И МЕТОДЫ.

3.1 Биохимические методы.

3.1.1. Получение препарата ядерного матрикса.

3.1.2. Получение ядерного экстракта из клеток культуры HeLa.

3.1.3. Аффинная очистка актин-связывающих белков наДНКазе I.

3.1.4. Аффинная очистка ядерного актина.

3.1.5. Получение и использование радиоактивно меченных фрагментов аминокислотной последовательности белка SAF-A/hnRNP U.

3.1.6. Изучение степени аффиности пептидов к ядерному актину.

3.1.1. SDS-электрофорез и окрашивание гелей.

3.2 Молекулярные методы.

3.2.1. Плазмиды.

3.2.2. Выделение плазмидной ДНК, электрофорез ДНК.

3.2.3. Полимеразная цепная реакция (ПЦР).

3.2.4. Праймеры.

3.2.5. Клонирование фрагментов ДНК.

3.3. Иммунологические методы.

3.3,1. Антитела.

3.3.2. Получение политональных антител.

3.3.3. Иммунопреципитация.:.

3.3.4. Иммуноблотинг.

3.4. Цитологические методы.

3.4.1. Культивирование клеток.

3.4.2. Использование диотиобис-сукцингшидилпропионата.

3.4.3. Трансфекция клеток культуры.

3.4.4. Синхронизация клеток.

3.4.5. Иммунофлуоресценция.

3.4.6. Микроинъекции.

3.4.7. Выявление сайтов транскрипции.

3.5. Методы компьютерного анализа.

РЕЗУЛЬТАТЫ.

4.1 Получение и тестирование поликлональных антител против белка NAP5 7/D yskerin.

4.2. Локализация белков ШРР140 и NAP57/Dyskerin.

4.3. Внутриядерная локализация белка NAP57/Dyskerin.

4.4. Локализация белка NAP57 и РНК-геликазы р68 семейства DEAD.

4.5. Доменный анализ первичной последовательности белка SAF-A/hnRNPU.

4.6. Изучение функциональной активности и характера локализации отдельных доменов белка SAF-A, меченных GFP.

4.7. Белок SAF-A взаимодействует с ядерным Р-актином in vitro и in vivo

4.8. Локализация белка SAF-A и ядерного актина в интерфазных и митотических клетках.

4.9. Белок SAF-A взаимодействует с Р-актином в ядре через С-концевой домен белка.

4.10. Белок SAF-A взаимодействует с Р-актином в ядре через пяти аминокислотную последовательность QRTQK на С-конце.

4.11. Комплекс SAF-A с Р-актином необходим для транскрипции РНК-полимеразойII.

5. ОБСУЖДЕНИЕ РЕЗУЛЬТАТОВ.

ВЫВОДЫ.

Введение диссертации (часть автореферата) на тему «Взаимодействие ядерного актина и NAP57/дискерина с белками, специфически связывающими сателлитную ДНК»

Со структурной точки зрения, классический конститутивный гетерохроматин представляет собой повторяющиеся последовательности тандемно расположенных мономеров сателлитных ДНК (сатДНК), ассоциированные со специфическими белками. У высших эукариот последовательности сатДНК концентрируются в центромерных и теломерных районах хромосом, однако их функциональная роль в процессе реализации генетической информации до сих пор не ясна.

Достоверно известно, что сатДНК, образующие центромеру, обеспечивают формирование кинетохора, корректное спаривание и разделение хромосом в ходе митотического и мейотического делений клетки. Прицентромерные блоки сатДНК необходимы для правильной ассоциации сестринских хроматид.

В интерфазный период сатДНК участвует в трехмерной организации интерфазного ядра (Razin et al., 1995; Manuelidis, 1997), определяющую транскрипционный паттерн клетки. Известно, что характер распределения центромер в ядре меняется в ходе клеточного цикла (Ferguson, Ward, 1992) и эмбрионального развития (Longo et al., 2003). Изменение положения центромер в ядрах нейронов происходит при изменении транскрипционной активности при росте аксонов в условиях in vitro (Chon, De Boni, 1996). Хорошо известна способность центромерного гетерохроматина ингибировать транскрипционную активность генов, при ассоциации последних с районами гетерохроматина. С другой стороны, ингибирование РНК-полимеразы II приводит к деконденсации прицентромерного гетерохроматина (Haaf, Ward, 1996). Недавние исследования показали прямую связь между формированием гетерохроматина и постгранскрипционной регуляцией генной активности посредством механизма RNA interference (RNAi) (Agrawal et al., 2003), подтверждая участие гетерохроматиновых районов хромосом в метаболизме молекул РНК.

Активность сатДНК должна определяться белковыми факторами, способными специфически распознавать последовательности сатДНК в ядре интерфазной клетки. Между тем, белки связывающие сатДНК изучены недостаточно полно. Известные сатДНК-связывающие белки определены с помощью сыворотки аутоиммунных больных (Moroi et al., 1980; Earnshaw, Rothfield, 1985), либо методом ретардации в геле (Lobov et al., 1998), основанным на изменении подвижности ДНК-белкового комплекса в ПААГ.

Последний метод наиболее эффективен, поскольку позволяет точно определить специфичность распознавания использованных в качестве зонда последовательностей ДНК. Так с помощью метода ретардации в геле был установлен специфический характер взаимодействия РНК-геликазы р68 семейства DEAD с последовательностями альфоидной ДНК (а-сатДНК) человека и минорного сателлита (миСат) мыши (Podgornaya et al., 2003; Enukashvily et al., 2005), а также взаимодействия ядерного белка SAF-A/hnRNP U с мажорным сателлитом мыши (маСат) (Lobov et al., 2000; 2001).

Предметом настоящего исследования стало изучение белковых комплексов, в состав которых входят РНК-геликаза р68 и белок SAF-A/hnRNP U.

1.1. Цели и задачи исследования

Целью настоящей работы являлось изучение взаимодействия ассоциированных с сателлитными ДНК белков SAF-A/hnRNP U и РНК-геликазы р68 с нехроматиновыми белками - ядерным fJ-актином и белком N АР5 7/Dyskerin.

Были поставлены следующие задачи:

1. Получить антитела, специфически узнающие белок NAP57.

2. Изучить внутриклеточную локализацию сатДНК-связывающих и сопутствующих им белков.

3. Подтвердить взаимодействие пар молекулярных партнеров (геликаза p68/NAP-57 и SAF-A/p-актин) in vivo и in vitro

4. Идентифицировать необходимые для формирования белковых комплексов аминокислотные последовательности.

5. Определить функции изучаемых белковых комплексов.

Заключение диссертации по теме «Гистология, цитология, клеточная биология», Кукалев, Александр Сергеевич

выводы

1. Внутриядерная локализация белка NAP57/Dyskerin не зависит от фаз клеточного цикла и может быть разделена на два типа: ядрышковая и нуклеоплазменная.

2. Белок NAP57/Dyskerin специфически взаимодействует in vitro с РНК-геликазой р68 семейства DEAD, колокализуясь с ней во внутриядерных хранилищах факторов сплайсинга (спеклах) in vivo.

3. Белок SAF-A специфически взаимодействует с (3-актином в ядре, образуя единый комплекс с РНК-полимеразой II.

4. Белок SAF-A взаимодействует с ядерным (3-актином через пятиаминокислотную последовательность: глутамин-аргинин-треонин-глутамин-лизин на С-конце аминокислотной последовательности SAF-А.

5. Комплекс SAF-A с р-актином необходим для транскрипции РНК-полимеразой II.

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