Изучение структурно-функциональной организации генома вируса натуральной оспы тема диссертации и автореферата по ВАК РФ 03.00.03, кандидат биологических наук Тотменин, Алексей Владимирович

• Тотменин, Алексей Владимирович
• кандидат биологических наук
• 2004

Оглавление диссертации кандидат биологических наук Тотменин, Алексей Владимирович

ВВЕДЕНИЕ.

1. ОБЗОР ЛИТЕРАТУРЫ.

1.1. Молекулярно-биологическая организация ортопоксвирусов.

1.2. Организация генома ортопоксвирусов.

1.3. Гены круга хозяев.

Ц 1.4. Молекулярные факторы вирулентности ортопоксвирусов.

1.5. Структурные перестройки в геномах ортопоксвирусов.

1.6. Модели возникновения дупликаций, транспозиций и делеций концевых последовательностей.

1.7. Гомологичная рекомбинация.

2. МАТЕРИАЛЫ И МЕТОДЫ.

2.1. Материалы.

2.2. Выделение и анализ плазмидной ДНК.

2.3. Получение компетентных клеток Е.соИ.

2.4. Трансформация компетентных клеток Е.соИ.

2.5. Получение геномных библиотек в космидном векторе.

2.6. Компьютерный анализ данных секвенирования.

3. РЕЗУЛЬТАТЫ И ОБСУЖДЕНИЕ.

3.1. Создание плазмидных и космидных клонотек фрагментов ДНК ВНО

Щ штамма Индия-1967.

3.1.1. Получение и характеризация коллекций гибридных плазмид, содержащих НтбIII- и А7ю1-фрагменты ДНК ВНО штамма Индия-1967.

3.1.2. Получение и характеризация библиотеки космид, содержащих ///жШ1-фрагменты ДНК ВНО штамма Индия-1967.

3.1.3. Использование полученных клонотек фрагментов ДНК ВНО-ИНД для секвенирования полной кодирующей последовательности генома данного вируса.

3.2. Получение и характеризация коллекции гибридных плазмид, содержащих Л7/о1-фрагменты генома ВНО штамма Гарсия-1966.

3.3. Создание международного репозитория гибридных плазмид, содержащих фрагменты ДНК ВНО.

3.4. Организация генома вируса variola major ВНО-ИНД.

3.5. Сравнение геномов вирусов ВНО major и ВОВ.

3.6. Организация генома штамма Гарсия-1966 ВНО minor alastrim.

3.7. Сравнение геномов вирусов ВНО и вируса оспы верблюдов.

3.8. Сравнение организации молекулярных факторов вирулентности ВНО, ВОВиВОВБ.

3.9. Интегральная схема преодоления вирусом натуральной оспы защитных барьеров организма.

3.10. Молекулярная эволюция ортопоксвирусов.

3.10.1. Различия в нуклеотидных последовательностях ДНК вирусов натуральной оспы и осповакцины.

3.10.2. Механизмы рекомбинационных перестроек ортопоксвирусных ДНК.

4. ВЫВОДЫ.

Введение диссертации (часть автореферата) на тему «Изучение структурно-функциональной организации генома вируса натуральной оспы»

Актуальность проблемы. В 1980г. Всемирная Ассамблея Здравоохранения известила мир о ликвидации оспы на земном шаре. Это достижение явилось результатом осуществлявшейся Всемирной Организацией Здравоохранения (ВОЗ) и продолжавшейся свыше 20 лет беспримерной международной программы. Завершающим шагом этой программы предполагалось уничтожение хранящихся в двух лабораториях мира (Сотрудничающие центры ВОЗ по ортопоксвирусам Российской Федерации и США) штаммов вируса натуральной оспы (ВНО). Однако, в целях сохранения информации об этом уникальном вирусе ВОЗ сочла необходимым предварительно осуществить исследования по расшифровке структуры его генома.

До начала этих исследований не существовало научно обоснованных заключений об эволюционном происхождении и взаимосвязях ортопоксвирусов, патогенных для человека. Отсутствовала информация о молекулярных факторах патогенности ВНО. Большинство исследований в этом направлении были выполнены на вирусе осповакцины (ВОВ).

Наиболее прямым подходом к решению стоящих вопросов является секвенирование и сравнительный компьютерный анализ организации геномов ВНО и ВОВ.

Цель и задачи исследования. Целью настоящего исследования было создание и характеризация клонсггек фрагментов ДНК высоко- и низковирулентного штаммов вируса натуральной оспы и выполнение работы по секвенированию и компьютерному анализу организации вирусных генов и их белковых продуктов.

В процессе работы решались следующие задачи:

1. Получение представительных клонотек фрагментов генома вируса натуральной оспы штаммов Индия-1967 (ВНО-ИНД) и Гарсия-1966 (ВНО-ГАР) в плазмидных и ко см ид пых векторах. Надежная консервация полученного банка клонов фрагментов ДНК двух изученных штаммов вируса натуральной оспы в составе Международного репозитория ДНК ВНО.

2. Проведение секвенирования полных последовательностей нуклеотидов геномов ВНО-ИНД и ВНО-ГАР.

3. Построение полных генетических карт геномов двух изученных штаммов ВНО.

4. Осуществление подробного сравнительного анализа структурно-функцио-нальной организации изучаемых геномов вируса натуральной оспы и вируса осповакцины штаммов Копенгаген (ВОВ-КОП) и Вестерн Резерв (ВОВ-\У11).

5. Выполнение детального сравнительного анализа внутривидовых и межвидовых структурных перестроек ДНК ортопоксвирусов и выявление эволюционных взаимосвязей ВНО и ВОВ.

Научная новизна и практическая значимость работы. Получена представительная библиотека гибридных плазмид и космид, несущих фрагменты генома ВНО-ИНД и ВНО-ГАР. Впервые определена полная нуклеотидная последовательность генома высоковирулентного (ВНО-ИНД) и низковирулентного (ВНО-ГАР) штаммов вируса натуральной оспы. Осуществлен подробный анализ организации геномов ВНО-ИНД и ВНО-ГАР и сравнение их с геномами вирусов осповакцины и оспы верблюдов. Выявлены потенциальные гены вирулентности ВНО. Выполнен детальный ф сравнительный анализ внутривидовых и межвидовых структурных перестроек ДНК ортопоксвирусов и сформулирована модель их возникновения. Впервые на основании анализа спектра коротких делеций в геноме ВНО относительно ВОВ сделан вывод об их видоспецифичности и независимом эволюционном происхождении ВНО и ВОВ от общего прародителя. Проведена надежная консервация полученных клонотек фрагментов ДНК двух изученных штаммов вируса натуральной оспы в составе Международного репозитория ДНК ВНО, что дает возможность использовать их в последующих научных исследованиях.

Публикации и апробация работы. По материалы диссертации опубликованы 17 статей в реферируемых изданиях. Результаты работы были представлены на международных конференциях: 9-th International Conference on Poxviruses and Iridoviruses (Les Diablerets, Switzerland, 1992), 9th International Congress of Virology (Glasgow, Scotland, 1993), 10th International Conference on Poxviruses and Iridoviruses (Banff, Alberta, Canada, 1994), 11th International Meeting on Poxviruses and Iridoviruses (Toledo, Spain, 1996), 12th International Poxvirus Simposium (St. Thomas, USA, 1998), International Conference on Bacterial and Viral Virulence Factors (Smolenice, Slovakia, 2000), 13th International Poxvirus and Iridovirus Symposium (Montpellier, France, 2000).

Вклад автора. Создание и характеризация клонотек фрагментов геномной ДНК штаммов Индия-1967 и Гарсия-1966 ВНО, компьютерный анализ организации генома штамма Гарсия-1966 ВНО, анализ структурных перестроек в нуклеотидных последовательностях ДНК вирусов натуральной ^ оспы и осповакцины выполнены лично автором; анализ организации генома штамма Индия-1967 ВНО выполнен автором совместно с В.М. Блиновым и сотрудниками лаборатории теоретической биологии и вирусологии ГНЦ ВБ «Вектор»; секвенирование ДНК вирусов натуральной оспы выполнено сотрудниками отдела молекулярной биологии геномов и отдела молекулярной вирусологии ГНЦ ВБ «Вектор» при участии автора.

1. ОБЗОР ЛИТЕРАТУРЫ

Заключение диссертации по теме «Молекулярная биология», Тотменин, Алексей Владимирович

4. ВЫВОДЫ

Щ 1. Получены и охарактеризованы клонотеки гибридных плазмид и космид, содержащих Hindlll- и Л7?о1-фрагменты ДНК вируса натуральной оспы штамма Индия-1967 (ВНО-ИНД) и покрывающих в совокупности весь вирусный геном. Создана и охарактеризована коллекция гибридных плазмид, содержащих Л7?о1-фрагменты ДНК вируса натуральной оспы штамма Гарсия-1966 (ВНО-ГАР). id Клонотеки гибридных ДНК ВНО паспортизованы и сданы на хранение в Международный репозиторий штаммов и ДНК ВНО (ГНЦ ВБ «Вектор»).

2. Впервые секвенированы геномы высоковирулентного ВНО-ИНД и низковирулентного ВНО-ГАР. Нуклеотидные последовательности ВНО-ИНД (185578 п.н.) и ВНО-ГАР (186 986 п.н.) аннотированы и зарегистрированы в Международном банке данных EMBL Data

Library под номерами Х69198 и Y16780.

3. Построены полные генетические (трансляционные) карты геномов ВНО-ИНД и ВНО-ГАР, содержащие соответственно 200 и 206 потенциальных открытых рамок трансляции. Осуществлен сравнительный анализ структурно-функциональной организации геномов вирусов натуральной оспы, осповакцины (ВОВ) и оспы верблюдов (ВОВБ). Впервые выявлены видоспецифичные различия генетических стратегий ВНО, ВОВ и ВОВБ.

4. Выполнен детальный сравнительный анализ внутривидовых и межвидовых структурных перестроек ДНК на примере двух штаммов ВНО и двух штаммов ВОВ, на основании которого сделано заключение, что ВНО и ВОВ эволюционировали от общего вируса-прародителя независимыми путями.

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