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第 12 章 核酸通论

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 ）

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第 12 章 核酸通论

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  1. 第12章 核酸通论

  2. 1. 核酸的发现和研究简史

  3. 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。

  4. 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。

  5. 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

  6. Nucleic Acids Are Essential For Information Transfer in Cells • 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.

  7. 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 1923- Use of Schiff reagent, which stains DNA red, shows that it is located almost exclusively within the chromosomes

  8. 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

  9. 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.

  10. DNA 是主要的遗传物质 Avery.MacLeod和McCarty的肺炎球菌的转导实验

  11. 1923 – Frederick Griffith performed studies with Streptococcus pneumoniae Two forms : smooth (S) which is wild-type rough (R) mutant S are virulent, R are not

  12. Transformation of bacteria

  13. i). DNA transformation: in vivo experiment Mice are injected either with Type R, non-virulent Streptococcus or with heat-killed, virulent Type S cells. The mice are healthy.

  14. Mice are injected with both Type R, non-virulent and heat-killed, Type S Streptococcus X • DNA carrying genes from • the virulent, heat-killed cells • transforms the non-virulent • bacterial cells, making them • lethal to the mice

  15. Transformation • The ability of foreign DNA to change the genetic characteristics of an organism 1931 – Oswald Avery was able to achieve transformation without an animal host (in vitro), simply by growing live R-type bacteria in medium in the presence of components from dead S-forms

  16. DNA transformation: in vitro experiment Type R cells Type R colonies Type S cells Type S colonies Type R cells + DNA from Type S cells Mixture of Type R and Type S colonies

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

  18. Gene S for capsule formation Mutant gene for capsule formation

  19. Bacterial transformation is caused by DNA In vitro Avery, McLeod and McCarty

  20. Evidence that genes are DNA 1952 – Experiments of Martha Chase and Alfred Hershey, infecting bacterial cells with viruses termed bacteriophages

  21. Evidence that genes are DNA Bacteriophage are very small, and are composed of approximately equal weights of protein and DNA. They depend on the host cell machinery for their replication The question: What substance directs the production of new phage particles, DNA or protein ?

  22. Evidence that genes are made of DNA

  23. Hershey and Chase Experiment: • 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 • Hypothesis: • ghosts left on outside are made of protein • genetic material injected into bacteria is DNA • 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

  24. 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

  25. 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.

  26. Product name  Protein type Application Company Adagen (Adenosine deaminase ) An enzyme Severe combined immunodeficiency disease (SCID) Enzon Genotropin (Recombinant growth hormone) 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 factor 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 alfa-2a) A modifier Hairy cell leukemia or AIDS-related Kaposi's sarcoma Hoffmann-La Roche

  27. 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

  28. How to produce a recombinant protein 0.1 to 1% of cellular protein 10 to 70% of cellular protein

  29. 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

  30. Genetically Engineered Salmon Is Bigger Better?

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

  32. Plant Genetic Engineering Improved Agricultural Production • Herbicide Resistance • Pest Resistance Improved Nutrition • Vitamins - Golden Rice, Vitamin E • Increase essential Amino Acid Content Chemical Synthesis • Bio-plastics • Bio-diesel • Lubricants/detergents • Rubber

  33. Using isolated genes as diagnostic tools • Track genetic disorders • Look at patterns of gene expression to diagnose disease state. • Gene Therapy

  34. 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

  35. Northern Blot RNA TransferRNA

  36. 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)

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

  38. The expression of 1733 different genes analyzed • Compared expression in 84 different tumor types. • Red = induced • Green = repressed

  39. 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）

  40. 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。

  41. 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。

  42. 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

  43. 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

  44. 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

  45. 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>

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