DIRECT GENOME SEQUENCING--MYCOPLASMA CAPRICOLUM

直接基因组测序--山羊支原体

基本信息

批准号：
3333155
负责人：
WALTER GILBERT
金额：
$ 200.44万
依托单位：
HARVARD UNIVERSITY
依托单位国家：
美国
项目类别：
财政年份：
1990
资助国家：
美国
起止时间：
1990-08-01 至 1993-10-31
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/3333155
关键词：
Mycoplasma bacterial genetics chromosome walking genetic transcription genome molecular cloning nucleic acid sequence open reading frames polymerase chain reaction

项目摘要

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 800 KB 的山羊支原体进行测序染色体。这将为自由生活提供最小的模型基因组能够在确定的培养基中生长的生物体。由于支原体仅大约 500 个基因，人们有望对其有一个完整的了解生物学是基因组测序的结果。山羊支原体是一种病原体对于山羊，其与人类病原体肺炎支原体有关；这对有机体的了解也将揭示其机制传染性。该生物体将通过不涉及的直接技术进行测序克隆和作图：多重基因组行走，一种基于寡核苷酸的方法揭示染色体 DNA 序列的程序。 PCR（聚合酶链式反应）方法将用于解决困难地区和任何序列歧义。一轮鸟枪法克隆和测序将需要建立大约 800 个潜在的步行起始点战略。将开发有关该生物体的信息数据库，该数据库将包含基因组序列，所有转录可能性，全部开放阅读框，以及大多数阅读框的识别序列同源性。该数据库将包含相关信息其他生物体中的基因，最终将包括有关支原体的所有基因。该数据库将分布在光盘。这个以技术人员为基础的团队专门从事直接测序微生物应达到每年一兆碱基的速率到第二年就形成双链序列。测序方法将是开发和简化，以便每年两到三个兆基地的速度到第三年才能实现。完成支原体序列后，这些相同的直接方法将用于对大染色体进行测序来自酵母或其他简单的真核生物。