Chaper 6 Inheritance analysis of bacteria

YANGTZE NORMAL UNIVERSITY

Chaper 6 Inheritance analysis of bacteria

• Cell and Chromosome of Bacteria

• Mutant and its screening of E. coli

• Sex of E. coli

• Disruption Hybrid and Recombinant Mapping• Sex-Induction of F/ Primer Factor

• Mapping of Transformation and Transduction


第六章细菌的遗传分析

• 6.1 细菌的细胞和染色体• 6.2 大肠杆菌的突变型及筛选• 6.3 大肠杆菌的性别• 6.4 中断杂交与重组作图• 6.5 F/ 因子的性导 • 6.6 转化与转导作图


Bacterial biological characters1. Short reproductive cycles2. Easy to mange and pure culture3. Haploid is useful to study mutation4. Large population5. Recombination easy to occur

6. Our extensive knowledge of bacteria and their resident plasmids has made for their widespread use in DNA cloning and other recombinant DNA studies

细菌作为遗传分析材料的优点：1. 细菌结构简单、繁殖周期短、易获得各种类突变型、多种病毒的宿主、易于培养、便于建立纯系2. 外源目标基因的载体3. 遗传学研究的常见材料，具有重要的遗传学研究意义


6.1 Cell and Chromosome of Bacteria


The internal structure of a bacteria cell does not regionalize and is surrounded by membranes and walls. Each cell, in average, has 1 to 2 chromosomes adhering to the membrane, but not packed, which is called nucleoidThe most of chromosomes in bacteria is a closed double-strand DNA circle, neither binding with histone or other proteins nor forming nucleosomes. Significantly, it is easy to insert a gene or DNA fragment from the same or different species into a bacterial chromosome. The bacterial chromosomes duplicate along with the cell division without mitosis and meiosis


6.1 细菌的细胞和染色体YANGTZE NORMAL UNIVERSITY

1. 细菌细胞：由一层或多层膜和壁所包围，其内部结构没有达到区域化程度，平均每个细胞中含 1---2 条染色体，每条染色体附着在细胞膜上的一定区域，但无核膜包裹，称为拟核。

不存在有丝分裂和减数分裂，染色体随细胞分裂而复制并分配到子细胞中

2. 细菌染色体：细菌染色体大都是双链 DNA 分子所组成的一个封闭的环，染色体裸露，没有组蛋白及其他蛋白质结合，也不形成核小体结构，细菌染色体有一个显著特点就是易于接受带有相同或不同物种的基因或 DNA 片段的插入


6.2 Mutant and its screening of E. coli 大肠杆菌的突变型及筛选

• 1. The anabolic functional mutant: The wild strain of E. coli has the function to synthesize complex organic compounds essential to its gowth and metabolism but becomes nutritional deficiency mutant nutrition, such as Met-

• 2. The catabolic functional mutant: The wild strain of E. coli can decompose the complex carbon while the mutant such as Lac- can not decompose lactose and can not grow on the medium with the lactose being only carbon source

• 3. The resistant mutant: Bacteria resistant to antibiotic and phages is due to the gene mutation, such as T1

r 、 Strs(sensitive to streptomycin)• 1. 合成代谢功能的突变型：其野生型品系在基本培养基上有合成代谢和生长

所必需的复杂有机物的功能，而因某一基因突变而成为营养缺陷型，如 Met -

( 甲硫氨酸营养缺陷型 )• 2. 分解代谢功能的突变型：野生型品系能分解复杂的不同碳源为已利用，而

如 Lac- 不能分解乳糖，不能生长在以乳糖为唯一碳源的培养基中 • 3. 抗性突变型：因基因突变而产生抗性，如 T1

r 、 Strs( 链霉素敏感 )


Bacterial mutation and growth

Mutation: Spontaneous mutation is the primary source of genetic variation in bacteriaPrototroph: is a bacterium that can synthesize all essential organic compound on minimal mediumAuxotroph: is a bacterium that loses, through mutation, the ability to synthesize one to more organic components, and that can not live on minimal medium


Results of the serial dilution technique and subsequent culture of bacteria


Imprint culture


大肠杆菌突变型的筛选

不同的突变型用相应的筛选方法1. 糖发酵性突变株 Lac- 选择：在 EMB 培养基

上 Lac+ 形成紫色菌落， Lac- 形成白色菌落2. 抗性突变型的选择：经诱变处理的菌株培养

在含噬菌体或抗菌素的培养基上，检测菌落克隆的存在与否

3. 营养缺陷型突变的选择：先在完全培养基上培养，然后用印迹法


6.3 Sex of E.coli

Discovery of the E. coli sex Strain hybridization and genetic recombination in E. coli carried ou

t in 1946 led to the sex in E. coli 1. Strain A （ strs ） X strain B （ strr ） can obtain recombinant

wild colonies on the medium with or without streptomycin 2. Strain B （ strr ） X strain A can not obtain recombinant wild c

olonies on the medium with streptomycin 3. Reason: The strain B as a receptor is resistant to streptomycin an

d its cytokinesis and receiving alien genetic material is not affected by the streptomycin in the medium.

The E. coli sex is conferred by a F factor, so there is the differentiation between receptor and donor


6.3 大肠杆菌的性别

1. 大肠杆菌性别的发现： 1946 年，大肠杆菌品系间可杂交并发生基因重组

2. 品系 A （ strs ） X 品系 B （ strr ），在含（与不含）链霉素的培养基上，均可得到重组的原养型菌落

3. 品系 B （ strr ） X 品系 A （ strs ）在含有链霉素的培养基上，不能产生原养型菌落

4. 原因：品系 B （ strr ）作为受体对链霉素有抗性，培养基中的链霉素不影响其细胞分裂和接受外源遗传物质

5. F 因子赋予了大肠杆菌的性别，从而有受体与供体的区别


F Factor and its Relavant Concepts

F factor: Sex factor, a plasmid episome, a DNA circle

F+ cell: the cell with F factorF- cell: the cell without F factorF/ factor ： A circular F factor

with the bacterial geneHfr: high frequency recombinat

ion, the strain has a integrationsal F factor in the chromosome

Lfr: low frequency recombination, the strain with F+


F 因子及其相关概念F 因子：性因子，一种质

粒附加体，为环状 DNA

F+ 细胞：含有 F 因子的细胞

F- 细胞：不含有 F 因子的细胞

F/ 因子：带有插入细菌基因的环状 F 因子

Hfr ：高频重组，其染色体中带有一个整合的 F因子的品系

Lfr ：低频重组， F+ 品系


Conjugation


Evidence supporting genetic recombination


F factorCell contact is a essential step


Cojugation and the behavior of F factor


Hfr X F-


F factor and its behavior


Character of bacterial recombination

• 1. F factor confers the F- cell a small chromosomal fragment of the donor cell not itself

• 2. The F- cell receiving only a small chromosomal fragment of the donor cell is called partial diploid or merozygote. If a single cross-over occurres between exogenote conferred by the donor and endogenote by receptor the cell can not reproduce, as the circular chromosome is destroyed and can not copy. Bacterial chromosomes are kept only by even cross-over, and the genetic recombination is produced


细菌重组的特点

• 1. F- 细胞得到的只是供体细胞染色体的一部分，而 F 因子并没有发生转移

• 2. F- 受体细胞只接受部分的供体染色体，这样的细胞就称为部分二倍体或称为半合子，供体提供的部分基因组称为外基因子，而受体提供完整的基因组，称内基因子，如果内、外基因子之间只发生单交换，则环状染色体被破坏，形成的线状染色体不能复制，因而细胞就不能繁殖，只有偶数次的交换才能保持细菌染色体的完整性，产生有活性的重组子，而不出现相反的重组子


6.4 Interrupted mating and recombinant Mapping

The gene is orderly transferred from Hfr cell to F- cell. The gene mapping is based on the time and order of the gene into the cell. The steps are 1) to sample the bacteria in conjugation at different time when stirring the sample to interrupt the conjugation of the bacterias, 2) analysize the genotypes of the receptor bacteria

The interrupted mating is a method to position genes in bacteria such as Escherichia coli

Interrupted mating principle


6.4 中断杂交与重组作图

基因从 Hfr 细胞按次序转入 F- 细胞，可根据基因进入F- 细胞的时间和次序制作基因图谱；其基本步骤为：把结合中的细菌在不同时间取样，并把样品猛烈搅拌以中断接合中的细菌，然后分析受体细菌的基因型。

中断杂交是大肠杆菌等细菌中用来测定基因位置的一种方法

中断杂交实验原理


Interrupted mating technique


Interrupted mating mapping ( 中断杂交作图 ) With the time being unit, genes are located based on the order of gene transferring

据基因转移的先后次序，以时间为单位进行基因定位的方法


Interrupted mating and recombinant mapping

Recombinant mapping localizes genes based on the recombination frequency between genes. Interrupted mating and recombinant mapping are complementary each other. Interrupted mating is suitable to the distant genes. A minute in interrupted mating is equivalent to 20 map units in recombinant mapping and a map unit equivalent to 2000 bp in E. coli

重组作图是根据基因间的重组率进行基因定位的，这两种方法相互补充，如基因距离较远，则采用中断杂交作图。中断杂交 1min 相当于重组率测定的 20 个图距单位，在大肠杆菌中，一个图距 (m.u.)约等于 2000 bp


6.5 F’ factor and sexduction

F factor excision precision Imprecise excision

F’ factor production


Sexduction

Sexduction is process whereby a bacterium gains access to and incorporates foreign DNA brought in by a modified F factor during conjugation


Comparison between F- and F’

Hfr ： thr+leu+strs× F- ： thr-leu-strr→ F- ： thr+leu+strr(not infectious) F’ ： thr+leu+strs × F- ： thr-leu-strr→ F’ ： thr+leu+strr (infectious )


6.6 Transformation and mapping of bacteria

Griffith’s experiment


Transformation also provides a mechanism for recombining genetic information in some bacteria. In transformation, small pieces of extrcellular DNA are taken up by a living bacterium, ultimately leading to a stable genetic change in the recipient cell. Exposure of the cells or organisms to DNA isolated form a different genotypes is the critical step of transformationCompetence is a particular physiological state of bacterial cells that can take up DNA isolated


Transformation and mapping


Transduction and mapping

Transduction: A process whereby a cell can gain access to and incorporate foreign DNA brought in by a viral particle

The Lederberg

Zinder Experiment

using Salmonella


转导与作图

转导：转导就是以病毒作为载体把遗传信息从一个细菌细胞传到另一个细菌细胞，它又分为普遍性转导和局限性转导


Generalized transductionGeneralized transduction: Form of transduction in which any region of the host genome can be transduced

普遍性转导：供体基因组中所有基因有同等机会被转导，形成不同转导子的频率大致相等，噬菌体携带供体染色体片段是随机的


Transduction analysis of three factors(genes) 3 因子（基因）转导分析


Transduction analysis of three factors(genes)

Transductant Phage P1Genome with a length of 8.0 kb Donor: E. coli supC+ trpA+ pyrF+

Aceptor: E.coli supC- trpA- pyrF-

supC : markerResults: 1. supC+ trpA+ pyrF+ 　　３６　　　　 2. supC+ trpA+ pyrF- 　　　１１４　　　　 3. supC+ trpA- pyrF+ 　　　０　　　　 4. supC+ trpA- pyrF- 453 The gene changing the location locates between the other two genes by c

omparing the transductant genotype with the lowest frequency with the parental genotypes

The order of the 3 genes is supC　 trpA　 pyrF, based on (3)


三因子（基因）转导分析

转导噬菌体 P1基因组大小为８０ kb供体： E. coli supC+ trpA+ pyrF+

受体： E.coli supC- trpA- pyrF-

以 supC 为选择性标记结果 : 1.supC+ trpA+ pyrF+ 　　３６　　　　 2.supC+ trpA+ pyrF- 　　　１１４　　　　 3.supC+ trpA- pyrF+ 　　　０　　　　 4.supC+ trpA- pyrF- 453 转导子频率最低的基因型与亲本基因型比较，改变位置的基因即在

中间位置顺序：根据 (3)知３基因的顺序为 supC　 trpA　 pyrF


Specialized transduction

Specialized transduction: form of transduction based on faulty looping out by temperate phage. Only neighboring loci to the attachment site can be transduced

The gene Gal and bio in E. coli is transducted byλ phage

局限性转导：噬菌体只能转导供体基因组中的特定基因 -- λ 噬菌体专门转导大肠杆菌的 gal 或 bio 基因


Transduction frequency and physical map distance转导频率与物理图距

• d = L(1 – x1/3)• D ： Map distance between two genes at the same chro

mosome• L ： Average length of transductant DNA• X ： Co-transduction frequency of two genes

D ：同一染色体上两基因间的图距L ：转导 DNA 的平均长度X ：两基因共转导的频率

Chaper 6 Inheritance analysis of bacteria

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