1、基本概念
- Mutation: A heritable change in the genome.基因组中的可遗传性的变化。
- Mutant: a strain carrying mutation.携带突变的菌株
- Genotype: the sequence of the genome.基因组的序列
- Phenotype: the observable properties of a strain, relative to the parental strain.
品系相对于亲本品系的可观察到的特性。
- Wild-type: the natural isolate.自然分离得到的
Genotype变化不一定导致Phenotype的变化，但Phenotype的变化一般是由Genotype变化导致的。
2、有些性状是有明显选择性的，有些是没有明显选择性的
Selectable mutation在某些条件下有明显的生长优势，如抗生素等
None-selectable mutation 没有明显的生长优势，如色素等
3、auxotrophs：营养缺陷型，a mutant with a nutritional requirement for growth
Prototroph：原养型微生物
4、点突变
Missense：错义突变
Silent：沉默突变
Nonsense：无义突变
Transition：嘌呤变成嘌呤
Transversion：嘌呤变成嘧啶
5、移码突变Frameshift
Insertion插入突变、Deletion缺失突变
6、定点突变Site-directed mutagenesis——mutation at specific sites.
Reversions (back mutations)回复突变: point and insertion are reversible.
- Deletion (>3 bp) is generally regarded to be irreversible.
- True Reversions: the same site revertant, restores the original sequence.
- Suppressor：抑制:第二个突变补偿原来的突变，恢复原来的表型
7、突变速率Mutation Rate
自发突变Spontaneous
细胞的微生物：10-6 to 10-7 per kb per round of replicaiton，有proofreading、碱基互补、修复功能
更高等的生物：10-7 to 10-8
DNA virus：10至1000倍
RNA virus：105倍，因为没有校对功能，也没有修复功能
8、Induced mutations诱变: Mutations occur at a greatly increased rate in the presence of mutagens诱变剂 (chemical, physical, and biological agents)
Nucleotide base analogs核苷酸碱基类似物；
Chemical modifications化学修饰；
Alkylating agents烷基化剂，Nitrosoguanidine亚硝基胍
extremely efficient modifies DNA.
Intercalating agents插入剂: Acridine吖锭, Ethidium bromide (EB)溴化乙锭
Nitrosoguanidine亚硝基胍, acts by adding alkyl groups to the O6 of guanine and O4 of thymine, which can lead to transition mutations between GC and AT.
Introduce changes in nonreplicating DNA.
Intercalating agents插入剂: Acridine, planar molecules able to insert DNA-frameshift移码突变. Ethidium bromide (EB)
Radiation辐射:
1) Nonionizing, UV, 260nM (maximum absorption) 导致pyrimidine dimers嘧啶二聚体. A dose of UV (50- 90% death rate) for mutagenesis.
2) Ionizing radiation电离辐射 (X-rays, γ)导致 OH• on macromolecules.
9、Repair system修复系统
1) Error-free直接修复成原来的状态
a) Direct reversal, photolyase光修复酶, alkylated bases烷基化;
b) ssDNA damage, cut the damaged and synthesize new;
2) Error-prone紧急修复
dsDNA recombination mechanism; -- SOS system
由LexA and RecA调控
10、Gene Transfer
Transformation转化 by free DNA (fragment or plasmid);
Transduction转导, mediated by a virus;
Conjugation接合, DNA exchange via cell-cell contact.
被转移的DNA的命运：
1) Degraded by restriction enzymes;被降解
2) Replicate by itself (but only if it possesses its own origin of replication such as a plasmid or phage genome);复制
3) Combine with the host chromosome.整合到宿主染色体
Homologous Recombination:
1) Single Crossover单次交叉, whole plasmid (circular) integration to the chromosome;微生物常用
2) Double Crossover, the fragment of linear DNA between two crossover sites exchanged.
重组的detection：用selection medium，外源导入selectable marker
11、转化
Transformation: Free DNA (self-replicable or Not; Transfection if DNA from a virus);
Host cells must be competent感受态 (natural or induced);
Methods: chemical induced, electroporation.
12、转导
Transduction: DNA transfer mediated by a virus.
1) Generalized transduction广义的转导,any portion of the host genome;宿主基因组的任何部分。
2) Specialized transduction, DNA from a specific region of the host genome.特定区域
专门化(Specialized):噬菌体DNA在特定位点整合到宿主染色体中，促进邻近基因的转移。
Gene transfer agents (GTAs) : phage-like entities that contain a random piece of the genome of the producing cell. 基因转移剂(GTAs):噬菌体样的实体，包含产生细胞基因组的随机片段。
13、接合
Conjugation: Genetic transfer via direct cell-to-cell contact mediated by pili (F pili). 由菌毛(F菌毛)介导的细胞间直接接触的遗传转移。
DNA is synthesized by rolling circle replication. 通过滚环复制合成的。
TraI, encoded by the tra operon of the F plasmid, has helicase activity; DNA polymerase; Membrane proteins.
F+ + F-=F++F+含F因子的细胞染色体DNA一般不被转移
Hfr (High frequency of recombination) strain.
F+ strain (F plasmid) -Hfr when F integrated into chromosome (Single Crossover)
Transferable chromosome.在接合过程中可以把部分或全部的染色体DNA都转移。
如果Hfr+F-接合，那还是Hfr+F-
Hfr的应用Chromosome Mapping染色体作图: organization of a chromosome
14、Complementation回补试验
rescue defective phenotype caused by mutation用质粒或者之类的转移DNA的方法
in trans: in the different molecules
in cis: in the same molecules
15、Gene Transfer in Archaea
转化、转导、接合在古菌中都发现
困难1:unusal growth conditions，培养古菌难
困难2:many antibiotics are ineffective，筛选古菌难
可用的抗生素：Novobiocin (a DNA gyrase inhibitor), mevinolin (an inhibitor of isoprenoid biosynthesis), puromycin and neomycin (both protein synthesis inhibitors)
新诺贝菌素(一种DNA旋转酶抑制剂)、甲氧诺林(一种类异戊二烯生物合成抑制剂)、嘌呤霉素和新霉素(两种蛋白质合成抑制剂)
16、Conjugation in Archaea
Fertility plasmid可育质粒、Cell aggregation细胞聚合、Nanotubes纳米管、Cytoplasmic
Bridges胞质桥
17、Mobile DNA
discrete segments离散片段 of DNA that moves as units以一个单位移动 from one location to another within other DNA molecules. This process called transposition.转座
Transposable elements转座元件
two essential components两个关键组分: short inverted terminal repeats 短的倒置的末端重复序列and transposase.转座酶
有两类1) IS (insertion sequences)插入序列, 2) transposons转座子, contain other genes.
Transposition Mechanisms转座机制: Conservative 保留型的and replicative.复制型的
Mutagenesis with transposons:转座会引发诱变
1) gene interrupted by the insertion转座子插入导致基因失活
2) gene untranscribe by the insertion转座子插入导致基因不转录