Выявление генов дифференциально экспрессирующихся при регенерации планарий тема диссертации и автореферата по ВАК РФ 03.00.03, кандидат биологических наук Богданова, Екатерина Андреевна

• Богданова, Екатерина Андреевна
• кандидат биологических наук
• 1999

Оглавление диссертации кандидат биологических наук Богданова, Екатерина Андреевна

СОДЕРЖАНИЕ

ВВЕДЕНИЕ

1. ЛИТЕРАТУРНЫЙ ОБЗОР

1.1. Типы репаративной регенерации

1.2. Планарии как модельный объект при иссследованиях регенерации

1.2.1. Морфологические и функциональные особенности пресноводных 5 планарий

1.2.2. Общие аспекты регенерации планарии

1.3. Молекулярно- биологические иследования регенерации (Методы 9 идентификации регуляторных генов и их применение к исследованию регнерации)

1.3.1. Методы, основанные на мутационном анализе

1.3.2. Поиск генов с использованием их эволюционного-.консерватизма

1.3.3. Ограничения методов поиска генов, основанных на их эволюционном 20 консерватизме

1.3.4. Методы прямого поиска дифференциально экспрессируюгцихся генов

1.4. Заключение

2. РЕЗУЛЬТАТЫ И ИХ ОБСУЖДЕНИЕ

2.1. Модельный эксперимент

2.2. Получение библиотек кДНК, обогащенных последовательностями 30 специфически экспрессирующимися в ходе регенерации различной полярности

2.2.1. Стратегия вычитающей гибридизации

2.2.2. Получение образцов амплифицированной кДНК из регенерационных 33 бластем планарии

2.2.3. Приготовление образцов кДНК трейсера и драйвера

2.2.4. Вычитающая гибридизация

2.3. Дифференциальный скрининг

2.3.1. Описание метода in vitro клонирования

2.3.2. Применение клонирования in vitro для анализа образцов кДНК, 35 обогащенных последоватльностями специфическими для регенерации различной полярности

2.4. Структура выделенных фрагментов

2.4.1. Нозерн-блот гибридизация

2.4.2. Выявление полной кодирующей последовательности кДНК гена scarf

2.4.3. Анализ структуры гена scarf

2.4.4. Scarf - представитель новой группы лектинов С-типа

2.5. Анализ экспрессии генов scarf и collar в теле нерегенерирующих 43 планарии

2.5.1. Whole mount in situ гибридизация

2.5.2. Анализ распределения мРНК выделенных генов вдоль дорсо- 45 вентральной оси

2.5.3. RT-ПЦР анализ

2.6. Анализ экспрессии генов scarf и collar в ходе регенерации 47 3. ЭКСПЕРИМЕНТАЛЬНАЯ ЧАСТЬ

3.1. Оборудование и материалы

3.2. Методы исследования 56 ВЫВОДЫ 66 СПИСОК СОКРАЩЕНИЙ 67 СПИСОК ЛИТЕРАТУРЫ

Введение диссертации (часть автореферата) на тему «Выявление генов дифференциально экспрессирующихся при регенерации планарий»

ВВЕДЕНИЕ

Одно из важнейших свойств живых существ - способность восстанавливать целостность организма после ранения или утраты части тела. Это свойство может быть обнаружено у самых разных животных - от простейших до высших млекопитающих. В отличиии от большинства систематических групп животных, являющихся модельными объектами биологии развития, пресноводные планарии обладают способностью к восстановлению утраченного паттерна даже из небольших фрагментов тела и являются удобным объектом для изучения генетических основ репаративной регенерации.

Одним из наиболее эффективных подходов к пониманию механизмов регенерационных процессов является исследование генов, контролирующих эти процессы.

Целью представляемой работы была идентификация генов, дифференциально экспрессирующихся в ходе регенерации различной полярности (восстановление головной или хвостовой недостающей части тела) и могущих служить генетическими маркерами при изучении процесса восстановления нового паттерна. Для решения этой задачи мы применили стратегию вычитающей гибридизации. В результате нами были сконструированы библиотеки кДНК, обогащенные последовательностями, специфичными для регенерационных почек различной полярности. В ходе дифференциального скрининга этих библиотек было выявлено два гена (названные нами scarf и collar ), специфических для постериорной регенерации. Анализ структуры идентифицированных генов показал, что последовательность гена scarf соответствует белковому продукту, относящемуся к новому семейству лектинов С-типа. Были проведены исследования экспрессии выявленных генов в интактных и регенерирующих планариях.

Заключение диссертации по теме «Молекулярная биология», Богданова, Екатерина Андреевна

выводы

1. С помощью метода вычитающей гибридизации созданы кДНК библиотеки, обогащенные последовательностями, специфическими для головной и хвостовой регенерационных почек планарии Dugesia tigrina.

2. Идентифицированы два новых гена - scarf и collar, дифференциально экспрессирующихся в ходе регенерации различной полярности планарий.

3. Показано, что ген scarf является представителем нового семейства генов, продуктами которых являются растворимые полидоменные лектины С-типа.

4. Проведен анализ экспрессии выявленных генов в интактных и регенерирующих планариях. Показан регион-специфический характер их экспрессии в теле нерегенерирующих планарий и падение уровня экспресии при антериорной регенерации. Продемонстрирована принципиальная возможность использования выявленных генов как маркеров для наблюдения за восстановительными процессами.

Список литературы диссертационного исследования кандидат биологических наук Богданова, Екатерина Андреевна, 1999 год

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