DIRECT GENOME SEQUENCING--MYCOPLASMA CAPRICOLUM

直接基因组测序--山羊支原体

• 批准号: 3333153
• 负责人: WALTER GILBERT
• 金额: $ 173.72万
• 依托单位: HARVARD UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 1990
• 资助国家: 美国
• 起止时间: 1990-08-01 至 1993-07-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/3333153
• 关键词: Mycoplasma; bacterial genetics; chromosome walking; genetic transcription; genome; molecular cloning; nucleic acid sequence; open reading frames; polymerase chain reaction

We shall sequence Mycoplasma capricolum which has an 800 kilobase chromosome. This will provide the smallest model genome for a free-living organism capable or growing in a defined medium. Since mycoplasma has only about 500 genes, one can hope to develop a complete understanding of its biology as a result of the genome sequencing. M. capricolum is a pathogen for goats, and its related to a human pathogen, M. pneumoniae; the understanding of the organism will also shed light on its mechanism of infectivity. This organism will be sequenced by a direct technique that does not involve cloning and mapping: multiplex genomic walking, an oligonucleotide-based procedure that reveals the sequence of chromosomal DNA. PCR (polymerase chain reaction) methods will be used to resolve difficult regions and any sequence ambiguities. One round of shotgun cloning and sequencing will be needed to establish about 800 potential initiation points for the walking strategy. A database of information about this organism will be developed that will contain the genomic sequence, all transcription possibilities, all open reading frames, and the identification of most of those reading frames by sequence homologies. The database will contain information about related genes in other organisms and, eventually, will include information about all of the genes of mycoplasma. This database will be distributed on compact discs. This technician-based group specializing in the direct sequencing of microorganisms should achieve a rate of one megabase/year of finished double-stranded sequence by the second year. Sequencing methods will be developed and simplified so that a rate of two to three megabases/year will be achieved by the third year. After completing the mycoplasma sequence, these same direct methods will be applied to sequence a large chromosome from yeast or an other simple eukaryote.

我们将对山羊支原体进行测序， 染色体 这将提供最小的模型基因组为自由生活 能够在确定的培养基中生长的有机体。 由于支原体仅具有 大约500个基因，人们可以希望发展一个完整的理解， 基因组测序的结果。 M.摩羯座是一种病原体 对山羊的感染，以及与人类病原体M. 肺炎 了解有机体也将阐明其机制， 传染性 这种微生物将通过直接技术进行测序， 克隆和作图：多重基因组步移，一种基于多核苷酸的 揭示染色体DNA序列的程序。 PCR（聚合酶 链式反应）方法将用于解决困难的区域和任何 序列模糊性 一轮鸟枪克隆和测序将是 需要建立大约800个潜在的步行起始点 战略 将建立一个关于这种生物的信息数据库， 包含基因组序列，所有的转录可能性，所有开放的 阅读帧，以及通过以下方式识别大多数这些阅读帧 序列同源性 数据库将包含有关 其他生物的基因，最终，将包括有关 支原体的所有基因 该数据库将在 光盘。 这个以技术人员为基础的小组专门从事直接测序， 微生物应达到1兆/年的完成率， 第二年就形成了双链序列 测序方法将是 发展和简化，以便每年两到三百万的速度将 在第三年实现。 完成支原体测序后， 这些相同的直接方法将被应用于对大染色体进行测序 酵母或其他简单的真核生物。