第 12 章 核酸通论

1. 核酸的发现和研究简史

核酸的发现 1868 年瑞士科学家 F.Miescher 由脓细胞分离得到细胞核，并从中提取出一种含磷量很高的酸性化合物，称为核素（ nuclein ）。因此，Miescher 被认为是细胞核化学的创始人和 DNA的发现者。

核酸（ DNA 和 RNA ） 脱氧核糖核酸 DNA 是细胞中一种十分重要的高分子化合物，其分子量大几十万到几百万，但个头很小，用电子显微镜也看不清楚。一个核酸分子的直径只有五百万分之一厘米， 25万根 DNA 并在一起，才和人的一根头发那么粗。 DNA 很细，但长度却有 1 米多长。 DNA 在细胞中的含量很少，但它却是生命的“主宰者”。

核糖核酸 RNA 也是由磷酸二酯键连接成的无分支的长链大分子，但它只有一条核酸链。主要包括核糖体 RNA （ rRNA ），转运 RNA （ tRNA ）和信使 RNA （ mRNA ）。

DNA 主要存在于细胞核中，同时在细胞质的线粒体和植物细胞的叶绿体也含有少量。 RNA 可以从细胞核进入到细胞质中发挥信息传递作用。

• 1868 年， F. Miescher 发现脱氧核糖核酸 • 核酸占细胞干重的 5~15% ，分核糖核酸和脱

氧核糖核酸两种• 核酸的遗传作用在 1944 年由 Avery 等通过肺

炎球菌 转化试验证明• 1950 年， Chargaff 分析不同生物 DNA 的碱

基组成，发现 A=T,C=G• 1953 年， Watson 和 Crick 阐明了 DNA 的

双螺旋结构• 20 世纪 70 年代建立了 DNA 重组技术

• Information encoded in a DNA molecule is transcribed via synthesis of an RNA molecule

• The sequence of the RNA molecule is "read" and is translated into the sequence of amino acids in a protein.

Nucleic Acids Are Essential For Information Transfer in Cells

Nucleic Acids• First discovered in 1869 by Miescher.• Found as a precipitate that formed when

extracts from nuclei were treated with acid.

• Compound contained C, N, O, and high amount of P.

• Was an acid compound found in nuclei therefore named nucleic acid

19231923- Use of Schiff reagent, which stains - Use of Schiff reagent, which stains DNA red, shows that it is located almostDNA red, shows that it is located almostexclusively within the exclusively within the chromosomeschromosomes

Nucleic Acids

• 1944 Oswald, Avery, MacLeod and McCarty demonstrated that DNA is the molecule that carrier genetic information.

• 1953 Watson and Crick proposed the double helix model for the structure of DNA

Nucleic Acids

• Nucleic acids are long polymers of nucleotides.

• Nucleotides contain a 5 carbon sugar, a weakly basic nitrogenous compound (base), one or more phosphate groups.

• Nucleosides are similar to nucleotides but have no phosphate groups.

Avery.MacLeod和McCarty的肺炎球菌的转导实验

DNA 是主要的遗传物质

1923 – Frederick Griffith performed studies1923 – Frederick Griffith performed studieswith with Streptococcus pneumoniaeStreptococcus pneumoniaeTwo forms : smooth (Two forms : smooth (SS) which is wild-type) which is wild-type

rough (rough (RR) mutant ) mutant S are virulent, R are notS are virulent, R are not

Transformation of bacteriaTransformation of bacteria

i). DNA transformation: in vivo experimentMice are injected either with Type R, non-virulentStreptococcus or with heat-killed, virulent Type S cells.

The mice are healthy.

X

• Mice are injected with both Type R, non-virulent and heat-killed, Type S Streptococcus

• DNA carrying genes fromthe virulent, heat-killed cellstransforms the non-virulentbacterial cells, making themlethal to the mice

TransformationTransformation

• The ability of foreign DNA to change the genetic The ability of foreign DNA to change the genetic characteristics of an organismcharacteristics of an organism

1931 – 1931 – Oswald AveryOswald Avery was able to achieve was able to achieve transformation without an animal host (transformation without an animal host (in vitroin vitro), ), simply by growing simply by growing livelive R-type bacteria in medium R-type bacteria in medium in the presence of components from in the presence of components from deaddead S-forms S-forms

DNA transformation: in vitro experiment

Type R cells Type R colonies

Type S cells Type S colonies

Mixture ofType R and Type S

colonies

Type R cells+ DNA fromType S cells

Next question : What component is Next question : What component is responsible for this phenomenon ?responsible for this phenomenon ?

Gene SS forcapsule formation

Mutant gene forcapsule formation

Bacterial transformation is caused by DNABacterial transformation is caused by DNA

In vitroIn vitro

Avery, McLeod and McCartyAvery, McLeod and McCarty

Evidence that genes are DNAEvidence that genes are DNA

1952 – Experiments of Martha Chase and1952 – Experiments of Martha Chase andAlfred Hershey, infecting bacterial cellsAlfred Hershey, infecting bacterial cellswith viruses termed bacteriophageswith viruses termed bacteriophages

Evidence that genes are DNAEvidence that genes are DNA

Bacteriophage are very Bacteriophage are very smallsmall, and are, and arecomposed of approximately equal weightscomposed of approximately equal weightsof of protein and DNAprotein and DNA. They depend on. They depend onthe host cell machinery for their replicationthe host cell machinery for their replication

The question:The question: What substance directs the What substance directs theproduction of new phage particles, DNAproduction of new phage particles, DNAor protein ? or protein ?

Evidence that genes are made of DNAEvidence that genes are made of DNA

Hershey and Chase Experiment:Hershey and Chase Experiment:

Hypothesis:Hypothesis:• ghosts left on outside are made of protein• genetic material injected into bacteria is DNA

How to Prove?How to Prove?• Radioactively label T2 phage protein with S35

• Radioactively label T2 phage DNA with P32

• DNA high phosphorous, no sulfur• Proteins high sulfur, no phosphorous

• phage does not enter bacterial cell• ghost (empty coat) remains on outside of cell• genetic material injected into bacteria

• this “material” controls phage cell cycle

Why use recombinant Proteins?

• Proteins are often only available in small amounts in a given tissue

• Tissue sources may not be readily available

• It is time consuming and expensive to purify protein from tissues

• It is difficult to obtain absolutely pure protein

Insulin

• Was first purified from human pancreas from cadavers and then from pig pancreas.

• Genentec expressed insulin gene in microbial host

• Can grow microbes in large fermenters to produce unlimited supply of insulin.

Product name  Protein type Application Company

Adagen (Adenosine deaminase )

An enzyme Severe combined immunodeficiency disease (SCID)

Enzon

Genotropin (Recombinant growth horm

one)

A hormone Growth hormone deficiency (GHD)

in children

Pharmacia & Upjohn

Humalog (Recombinant human insulin)

A hormone Diabetes Eli Lilly

Nabi-HB (Anti-Hepatitis B)

 An antibody Hepatitis-B Nabi

Novo Seven (Recombinant coagulation f

actor VIIa)

A modified factor Hemophillia patients with inhibitors

Novo Nordisk

Ontak (Diphtheria toxin-interleukin-2)

A fusion protein Cutaneous T-cell lymphoma (CTCL)

Ligand Pharmaceuticals

Roferon-A (Recombinant interferon alf

a-2a)

A modifier Hairy cell leukemia or AIDS-related Kap

osi's sarcoma

Hoffmann-La Roche

Recombinant proteins are also important to research

• For enzyme analysis need pure protein

• For structural analysis need lots (milligram amounts) of very pure protein

• Need pure proteins to make diagnostic tools such as antibodies

How to produce a recombinant protein

10 to 70% of cellular protein

0.1 to 1% of

cellular protein

Genetic Modification of Higher Organisms

• Can introduce gene into animals and plants

• These modified organism are powerful research tools to study the effect of a specific gene product on metabolism, development etc….

• Has also been used to develop improved agricultural products

Genetically Engineered SalmonIs Bigger Better?

http://www.agwest.sk.ca/sabic_index_tp.shtml

Plant Genetic EngineeringImproved Agricultural Production

A. Herbicide Resistance

B. Pest Resistance

Improved Nutrition

A. Vitamins - Golden Rice, Vitamin E

B. Increase essential Amino Acid Content

Chemical Synthesis

A. Bio-plastics

B. Bio-diesel

C. Lubricants/detergents

D. Rubber

Using isolated genes as diagnostic tools

• Track genetic disorders

• Look at patterns of gene expression to diagnose disease state.

• Gene Therapy

Gene Expression Tools

• Can determine how highly a gene is expressed using nucleic acid hybridization techniques

• Classic method called Northern Blotting – can analyze the expression of one to a few genes in a single experiment.

• Microarray – can analyze the expression of several thousand genes at one time

Northern BlotRNA

TransferRNA

Microarrays

• Can look at the expression of many different genes simultaneously

• Spot DNA of known genes on glass slides (up to 20,000 genes per slide)

• Can compare two sets of RNAs at one time (control Vs treatment)

http://www.bio.davidson.edu/courses/genomics/chip/chip.html

• The expression of 1733 different genes analyzed

• Compared expression in 84 different tumor types.

• Red = induced• Green = repressed

人类基因组计划的启动

1986 年诺贝尔奖获得者 R.Dulbecco 提出人

类基因组计划——测出人类全套基因组的 DNA 碱

基序列（ 1n: 3 X 109 b p ）

美国政府决定于 1990 年正式启动 HGP ，预计用 15 年时间，投入 30 亿美元，完成 HGP 。

由国立卫生研究院和能源部共同组成“人类基因组研究所（ NHGRI ）” 逐渐地， HGP 扩展为多国协作计划。参与者包括：欧共体、日本、加拿大、俄罗斯、巴西、印度和中国等国的科学家。

人类基因组计划的进展状况

（ 1 ）截至 1998 年 10 月，完成 1.8 X 108b ，占

计划的 6% 。

（ 2 ）完成一系列模式生物全基因组测定。

这些模式生物全基因组测定的完成有重大理论与

现实意义。

理论意义酵母—第一次揭示真核生物全基因组。 已大致确定： 5885 个编码蛋白基因 140 个 rRNA 基因 40 个 SnRNA 275 个 tRNA 基因 实践意义 病源微生物——病理机制 药物、疫苗

DNA 测序技术飞速提高

1998.5.9 J.C. Venter 等宣布，组建商

业公司，投入 3 亿美元， 3 年内完成。

接着又有若干家公司成立， 总共投入资金约几十亿美元，

形成 “公”“私”并进 格局

2000.6 完成并公布

人类基因组工作草图。

2001 年 2月 16日

人类基因组计划（ HGP ）完成

同时发表两套报告

Science, Vol. 291, No. 5507

Nature , Vol.409, p.860

Celera 等的论文 （ Science , 2001, 291:1304-1351 ） <作者 274 人，其中中国名字 57 人 >