生命科学 : 概念和技术

Yi Rao

饶毅

第一堂课的目的 :

1) 转变观念 :

你的中心任务 : 不再是“读书”而是“发现”

2) 给后来课稍铺垫

介绍一些常用技术

试试看 : 能否爱上生命科学研究

生命科学

思想

技术

生命科学思想

生命科学的思想和概念容易学习和理解 ,导致一种错觉 :以为生命科学的研究很容易 , 甚至以为只要有经费和出力气就行

生命科学的思想和概念难在于首先提出 , 或者证明 ( 最常见的困难 )或者接受

不同例子

从人类认识来说

1)已经清楚的 :从种瓜得瓜到遗传密码

2) 半懂不懂的 :从慢病毒到蛋白质催化的构相转

变

3) 还没入门的 : 对脸的识别

DNA as the Carrier of Genetic Information

Genetics

Mendel---Sutton---Morgan ---Beadle and Tatum

Biochemistry

Miescher---Kossel---Altmann---Levene---Chargaff---Mirsky

Convergence: Avery, MaCleod, and McCarty (1944)

Culmination: Watson and Crick (1953)

已经清楚的 :很容易学 DNA 和遗传

如果我们回到 1865 年 , 人类几乎是重复了几千年种瓜得瓜 种豆得豆

而到 1965 年 , 知道全部遗传密码

怎么发现和证明 DNA 是遗传信息的载体 ?

Mendel, G. (1865) Experiments in Plant Hybridization Read at the meetings of February 8th, and March 8th, 1865 http://www.mendel

web.org/Mendel.html

Mendel, Gregor (1866) "Versuche über Pflanzen-Hybriden." Verhandlungen des naturforschenden Vereines, Abhandlungen, Brünn 4, pp. 3-47

English translations appear at Blumberg (1997), in Matalová (1973), in Stern and Sherwood (1966), Bennet (1965), and Peters (1959). Mendel, G. (1866) Experiments in Plant Hybridization in Genetics: readings from Scientific American pp. 8-17. W.H. Freeman and Company, San Francisco-USA.

Correns, C.G. (1900). Mendels regel ber das Erhalten der Nachkommenschaft der Rassenbastarde. Ber. deutsch. Bot. Ges. 18: 158-168.

De Vries, H. (1900). Sur la loi de disjonction des hybrides. Compt. Rend. Ac. Sc. Paris 130: 845-847.

Tschermak, E. (1900). er k nstliche Kreuzung bei Pisum sativum. Ber. deutsch. bot. Ges. 18: 232-239.

Bateson, William (1902). "The Facts of Heredity in the Light of Mendel's Discovery." Report of the Evolution Committee of the Royal Society 1:125-160.

Gregor Mendel (1822-1884)

Sutton, W.S. (1902). On the morphology of the chromosome group in Brachystola magna. Biol. Bull. 4: 24-39.

Sutton, W.S. (1903) The chromosome in heredity. Biol. Bull. 4: 231-251.

Chromosomes as carriers of genetic information

Correlation of chromosome behavior with Mendel’s elementsin pairs equal segregating into gametes independent segregation

Walter S Sutton (1877-1916), a graduate student at ColumbiaTheodor Boveri (1862-1915) in Germany,

“I may finally call attention to the probability that the association of paternal and maternal chromosomes in pairs and their subsequent separation during the reducing division… may constitute the physical basis of the Mendelian law of heredity” [Sutton, W.S. (1902). ]

The first suggestion of a specific character to a chromosome:

accessory (X) is for male determination

McClung, C. E. (1901). Notes on the accessory chromosome. Anat. Anz 20:220-226.

The first correct assignment: Y for male beetles (Tenebrio) (males have XY and females have XX

Stevens, N. M. (1905). Studies in spermatogenesis with especial reference to the “accessory chrosome”. Carn. Inst. Wash., publ. 36,

Thomas H. Morgan and the Fly Room at Columbia (16 ft x 23 ft)Alfred H. Sturtevant, Calvin B. Bridges, Hermann J. Muller and othersDrosophila melanogaster (Aristotle?; Fallen, 1823; Meigen, 1830; Loew, 1862; C. W. Woodworth)

Morgan, T.H. (1910) Sex-linked inheritance in Drosophila. Science 32: 120-122.

Sturtvent, A.H. (1913) The linear arrangement of six sex-linked factors in Drosophila as shown by mode of association. J. Exp. Zool. 14: 39-45.

Muller, H. J. (1927). Artificial transmutation of the gene. Science 46:84-87.

“By 1915,…the group at Columbia was are ready to interpret the whole field of Mendelism in terms of the chromosome theory”

Morgan, A. H. Sturtevant, A. H., Muller, H. M., and Bridges, C. B. (1915). The mechanisms of Mendelian heredity. Henry Holt & Co., New York.

Quote from Sturtevant, A.H. (1965). A History of Genetics. Harper & Row, New York)

Johann Friedrich Miescher (1844-1895 ) discovered Nuclein 核素 in 1869 in what was previously used as a kitchen in the Castle of Tuebingen (Tübingen)

He was able to purify lots of it from salmon sperm in 1874.Miescher, J. F. 1871. Ueber die chemische Zusammensetzung der Eiterzellen.

Medisch-chemische Untersuchungen 4: 441-460. (submitted in 1869, delayed by the editor Hoppe-Seyler who wanted to confirm the results)

Miescher, Johann Friedrich. 1874. Die Spermatozoen einiger Wirbelthiere. Ein Beitrag zur Histochemie. Verhandlungen der Naturforschenden Gesellschaft in Basel 6: 138-208.

Nuclein, 1869

Albrecht Kossel (1853-1927, Nobel 1910)separated nuclein into proteins and nucleic acid (1881)found adenine, cytosine, guanine, thymine (1893) Kossel, A. (1881) Untersuchungen über die Nukleine und ihre S

paltungsprodubte (Investigations into the nucleins and their cleavage products).

The term “nucleic acid” introduced by Richard Altmann (1852-1900) Ueber Nucleinsäuren . Archiv für Anatomie und Physiologie. Physiologische Abteilung. Leipzig, 1889, 524-536.

P. A. Levene (1869-1940) ribose, deoxyribose, phosphate composition of a nucleotide and linkage of nucleotides in the nu

cleic acidT in thymus nucleic acid (DNA)U in yeast nuclear acid (RNA)

Feulgen & Rossenbeck: staining for DNA---and DNA in the nucleus Feulgen R. and Rossenbeck, H. (1924). Z. Physiol. Chem. 135: 203-248. Torbjorn Caspersson (1940, 1941) Jean Brachet (1942):

RNA in the cytoplasm (Caspersson: Protein-RNA-Protein)

Levene, P. A., and W. A. Jacobs (1911). Ber. Chem. Ges., 1911, xliv, 1027.

Levene, P. A., and W. A. Jacobs (1912). On the structure of thymus nucleic acid. J. Biol. Chem. 12: 411 – 420.

Linkage of nucleotides

Tetranucleotide Hypothesis

and its implications

A:T:G:C=1:1:1:1

DNA: unlikely as a carrier of genetic information

before 1944

Conceptually DNA: a monotonous structure, a tetranucleotide repeat

more enthusiasm for proteins, crystal structure in the 30s, varied structures.J. D. Bernal & D. Crowfoot Hodgkin (pepsin structure 1934, 1st X r

ay photograph of a protein structure)

Technicallydifficulty in obtaining homogeneous DNA for detailed chemical analysis

Smooth "S" Cells Rough "R" Cells

Streptococcus pneumoniae (pneumococcal) cells  "R" form (unencapsulated), non-virulent

"S" form (encapsulated), virulent

Heat-inactivate R cells, mix with S cells –--- virulent

Transformation

The Griffith Transformation Experiment 1928

Griffth, F. (1928) The significance of pneumococcal types. J. Hyg. 27: 113-159.

Oswald T. Avery Colin MacLeod Maclyn McCarty (1877-1955) (1909-1972) (1911-)

194467

Avery, O. T., MacCleod, C. M., and McCarty, M (1944). Studies on the chemical nature of the substance inducing transformation of pneumococcal types. J. Exp. Med. 79: 137-158.

What is the nature of the “transformation principle” in S cells?

Oswald T. Avery, Colin McLeod, Maclyn McCarty at Rockefeller (1944)Used S cell extracts to transform R cellsMultiple assays for the R and S forms: enzymatic, chemical, and serological analysesMultiple assays for DNA and protein: electrophoresis, UV spectroscopy, and ultracentrifugation

S strain bacteria first had the large cellular structures removed.

TransformationProteases: + RNase: + DNase: _

Rollin Hotchkiss (1949) purified the sample to <0.02% prot. without loss of transforming ability

McCarty, M. & Avery, O. T. Studies of the chemical nature of the substance inducing transformation of pneumococcal types II. Effect of desoxyribonuclease on the biological activity of the transforming substance. J. Exp. Med. 83, 89–96 (1946).

McCarty, M. 1946 Chemical nature and biological specificity of the substance inducing transformation of pneumococcal types. Bacterial. Rev. 10: 63-71.

Hershey, A, and Chase, M. (1952). Independent functions of viral proteins and nucleic acid in growth of bacteriophage. J. Gen. Physiol. 36: 39-56.

Hotchkiss, R. D., and Marmur, J., (1954). Double marker transformations as evidence of linked factors in desoxyribonucleate transforming agents. Proc. Natl. Acad. Sci. USA 40: 55-60.

…the substance evoking the reaction and the capsular substance produced in response to it are chemically distinct, each belonging to a wholly different class of chemical compounds.

The inducing substance, on the basis of its chemical and physical properties, appears to be a highly polymerized and viscous form of sodium desoxyribonucleate...The experimental data presented in this paper strongly suggest that nucleic acids, at least those of the desoxyribose type, possess different specificities as evidenced by the selective action of the transforming principle.

It is, of course, possible that the biological activity of the substance described is not an inherent property of the nucleic acid but is due to minute amounts of some other substance adsorbed to it or so intimately associated with it as to escape detection.... If the results of the present study ... are confirmed, then nucleic acids must be regarded as possessing biological specificity the chemical basis of which is as yet undetermi

ned

Avery, MaCleod and McCarty:

Uncertainties about DNA as a carrier of genetic information

after 1944

Previous Conclusions in the Field and Colleagues at Rockefeller

Phoebus Aaron Levene: tetranucleotide hypothesis

Alfred Mirsky: protein not excluded

Previous mistake: exclusion of as proteins as enzymes

A Conceptual Jump from Transformation Experiments to Genetics“Nothing was known of chromosomes or genes in bacteria at that time: a certain leap of faith was required to relate the transformation (and therefore, in turn, DNA) to mendelizing genes”from Lederberg, J. (1994). The Transformation of Genetics by DNA: An Anniversary Celebration of AVERY, MACLEOD and MCCARTY (1944). Genetics 136: 423-426.

Multiple Interpretations of the Transformation Experiments

Phoebus Aaron Levene had been at the Rockefeller, active until his death in 1940, the world authority on the chemistry of DNA and originator of the tetranucleotide hypothesis, by which repetitive scheme DNA could not possibly specify diversity.

Levene, P. A (1909). Biochem. Z. 17:121.

Levene, P. A., and W. A. Jacobs (1911). Ber. Chem. Ges., 1911, xliv, 1027.

Levene, P. A., and W. A. Jacobs (1912). On the structure of thymus nucleic acid. J. Biol. Chem. 12: 411 – 420.

Levene P. A. (1917). The structure of yeast nucleic acid. J. Biol. Chem. 31: 591 – 598.

Horace F. Judson: The Eighth Day of Creation. Expanded Ed. CSHP, 1996

William Astbury (1898-1961)

X ray crystallographer, working on 3 D structure of DNA in the 40s

Astbury, W. T. (1947). X-ray studies of nucleic acids. Sym. Soc. Exp. Biol. 1:66-76. 

3.4 A base stacking vs 3.7 A peptide bond

Chargaff's Ratios

1949-1953 - Erwin Chargaff (stimulated by Avery, MacCleod, and McCarty, 1944)

Chargaff 1) new paper chromatography techniques 2) multiple organisms

Yeast - 18%G, 18%C, 32%A, 32%T [i.e. 36%(G+C)]

Tubercle bacilli - 62-70 %(G+C) depending on strain

disproved Levene's hypothesisChargaff, E. (1950). Chemical specificity of nucleic acids and mechanism of their enzymic degradation. Experientia 6:201-209.

Sourcemol % of bases  Ratios

%GC A  G  C  T  A/T  G/C

X-174  24.0  23.3  21.5  31.2  0.77  1.08 44.8

Maize  26.8  22.8 17.0  27.2  0.99  0.98 46.1

Octopus  33.2  17.6  17.6  31.6  1.05  1.00 35.2

Chicken  28.0  22.0  21.6  28.4  0.99  1.02 43.7

Rat  28.6  21.4  20.5  28.4  1.01  1.00  42.9

Human  29.3  20.7  20.0  30.0  0.98  1.04  40.7

Mirsky, A. E., and Pollister, A. W. (1946). Chromosin, a desoxyribose nucleoprotein complex of the cell nucleus. J. Gen. Physiol. 30: 117-148.Mirsky, A. E. (1951). Some Chemical Aspects of the Cell Nucleus,” in Leslie C. Dunn, ed., Genetics in the 20th Century (New York), 127-153.

Alfred Mirsky (1900-1974) (also at Rockefeller)

Mirsky proposed with Linus Pauling (1936) proposed that role of hydrogen bonding in native proteins the nature of denaturation.

Mirsky A. E. and Pauling, L. (1936). On the Structure of Native, Denatured, and Coagulated Proteins,” in Proceedings of the National Academy of Sciences, 22: 439-447 (1936)

From 1940 to 1950, a major figure in purifying the chromosomes (with Arthus W. Pollister, of Columbia University, and later also with Hans Ris.

Over 90 percent were found to consist of nucleohistone containing DNA diploid somatic cells of an organism contain identical amounts of DNA, twice that of ha

ploid germ cells Even in 1951, Mirsky still thought that DNA was part of the gene substance, unconvinc

ed that DNA itself was the sole genetic material, pointing out the insensitivity of the assay and difficulty of assuring that minute quantities of protein are not attached to the DNA.

Lederberg called Mirsky “a dogged critic of the claim that DNA, alone, had been proven to be the exclusive chemical substance of transforming activity”.

Lederberg: “My stance was sympathetic to Mirsky’s: I felt that so crucial a claim should not be impulsively engrafted into the corpus of science as if by first intention.”

Previous mistakeIn the early twenties, Richard M. Willstätter (Nobel Chem 1915, pl

ant pigments, especially chlorophyll) in Munich, a specialist in enzymes, had claimed that he had gotten enzymatic, catalytic action with preparations that were free of protein. On his evidence, many scientists accepted that the biological specificity of solutions containing enzymes was not due to protein.

In 1926, James B. Sumner of Cornell crystalized urease (from the b

ean Canavalia ensiformis) (Nobel Chem 1946). In 1930, John H. Northrop (Nobel Chem 1946) at the Rockefeller Institute crystallized pepsin (from gastric juice) and showed that it was protein (also trypsin and chymotrypsin from the pancreas). Northrop and his associates developed precise techniques for correlating enzyme activity with the quantity of protein present, and showed conclusively that Willstätter's experiments had been contaminated by slight traces of protein.

A laboratory colleague of Avery's for many years, René Dubos, when asked about the effect of Willstätter’s bungle on Avery, replied, "It was on everybody's mind."

Horace F. Judson: The Eighth Day of Creation. Expanded Ed. CSHP, 1996

Interpretation of the Transformation Experiment

7 interpretations summarized in Lederberg J (1956). Genetic transduction. Am. Sci. 44: 264-280:

a specific mutagen to direct a particular gene to mutate in a definite direction (T. Dobzhansky);

a polysaccharide autocatalyst for polysaccharide synthesis.a bacterial virus provoking capsular synthesis as a host reaction;an autonomous cytoplasmic gene or a morphogenetic inducer.acted at a distance without penetrating the bacterium.a fragment of the genetic makeup of the bacterium, the only one to have be

en testedan element sui generis for which no general conception should be adduced.

More Experiments

e. g.,

Hershey, A, and Chase, M. (1952). Independent functions of viral proteins and nucleic acid in growth of bacteriophage. J. Gen. Physiol. 36: 39-56.

Hotchkiss, R. D., and Marmur, J., (1954). Double marker transformations as evidence of linked factors in desoxyribonucleate transforming agents. Proc. Natl. Acad. Sci. USA 40: 55-60.

Hershey, A, and Chase, M. (1952). Independent functions of viral proteins and nucleic acid in growth of bacteriophage. J. Gen. Physiol. 36: 39-56.A. D. Hershey---Nobel 1969

The Hershey-Chase Experiment (1952)

Avery, MacCleod and McCarty (1944)

stimulated Edwin Chargaff

and J. D. Watson

Watson and Crick (1953)

Watson, J.D. and Crick, F.H.C. (1953). A structure for deoxyribose nucleic acid. Nature 171:737-738.

http://content.nejm.org/content/vol348/issue17/images/large/15f1.jpeg

Watson and Crick vs Franklin

Significance of DNA recognized not fully (assigned, left the field)

Drive to solve the structure very strong moderate

X ray data none yes

Helix yes yes (and also Wilkins)

Double Helix yes yes (A. Klug found in her notebook)

Base inside first: pointed out to W & C

Model building yes no: data will pop out the structure

Base-pairing yes did not realize

Communication extensive little

Wilkins also has data, though initially not as good as those of FranklinA bit techincal detail: B (high humidity) easier than A (low himidity) to solve, Franklin detoured on A for a while)

Watson’ Double Helix should be balanced by others

Watson, J. D. The Double Helix: A Personal Account of the Discovery of the Structure of DNA (Athenaeum, New York, 1968).

Sayre, A. (1975). Rosalind Franklin and DNA. W. W. Norton, New York.

Klug, A. (1968) Nature 219:808-810; 843-844.Klug, A. (1974). Rosalind Franklin and the double helix. Nature 248:787-788.

 

Judson, H.F. (1996) - The Eighth Day of Creation: The Makers of the Revolution in Biology, Cold Spring Harbor, NY: Cold Spring Harbor Laboratory Press (Expanded Edition).

Franklin, R. and Gosling, R.G. (1953). Molecular configuration in sodium thymonucleate. Nature 171:740-741.

想象力和创造性 : 碱基配对

Watson, J.D. and Crick, F.H.C. (1953). A structure for deoxyribose nucleic acid. Nature 171:737-738.

Wilkins, M.H.F., Stokes, A.R., and Wilson, H.R. (1953). Molecular structure of deoxypentose nucleic acids. Nature 171:738-740.

Franklin, R. and Gosling, R.G. (1953). Molecular configuration in sodium thymonucleate. Nature 171:740-741.

Watson, J.D. and Crick, F.H.C. (1953). Genetical implications of the structure of deoxyribonucleic acid. Nature 171:964-967.

Franklin, R. and Gosling, R.G. (1953). Evidence for 2-Chain Helix in Crystalline Structure of Sodium Deoxyribonucleate. Nature 172:156-157.

There was only theory (based on indirect, circumstantial evidence), no data

碱基配对

想象力和创造性 : 碱基配对

Theory Data

several indirect hints no direct evidence

Chargaff’s rule no data in X ray

Hydrogen bonding to suggest or support

Constraint place by double helix base-pairing

the most significant feature of the model beautiful, but no data

Faith: it is so beautiful, it has to be true

Pauling, L. and Corey, R. (1953). A proposed structure for the nucleic acids. PNAS 39:84-97.

思考理论模型 对 结果资料自然明了

Watson and Crick vs Franklin

Ernest Rutherford vs. the Curies

On the other hand, 结果对提出理论起很强作用的时候 : 比如

有些遗传筛选 : Nusslein and Wieschaus遗传密码的最终破译

The Genetic CodeInformation

George Gamow

Francis Crick

Sydney Brenner

Paul C. ZamecnikRobert Holley

Marshall NirenbergSevero Ochoa

Gobind Khorona

Protein SynthesisBiochemistry

思考 and 资料

Coding and DecodingInformation Flow

Energy and material assembly

William T. Astbury (1898-1961)

Astbury, W. T. (1947). X-ray studies of nucleic acids. Sym. Soc. Exp. Biol. 1:66-76. 

Ideas about nucleotide sequence and amino acids

Caldwell, P. C and Hinshelwood C (1950). Some considerations on autoshythesis in bacteria. J Chem Soc. 3156-9.

Dounce, A. L. (1952). Duplicating mechanism for peptide chain and nucleic acid synthesis. Enzymologia 15:251-253.

Dalgliesh, C. E. (1953). The template theory and the role of transpeptidation in protein biosynthesis. Nature 171:1027-8.

A dichotomy presented by Campbell and Work (1953)

Stepwise coupling of small peptides vs. synthesis on templates (perhaps genes)

“It is impossible with our present knowledge to choose between these two theories”

Campbell, P. N., and Work, T. S. (1953). Biosynthesis of proteins. Nature 171:997-1001.

The Genetic Code

The Genetic Code: translating DNA sequence into protein sequenceGeorge Gamow (1954)

DNA StructureWatson and Crick (1953)

Insulin SequenceFred Sanger (1951, 1953)

Gamow, G (1954). Possible relation between deoxyribonucleic acid and protein structures. Nature 173:318.

George Gamow (1904-1968)

Gamow, G (1946). Expanding Universe and the Origin of Elements. Phys. Rev. 70 (1946) 572.

Alpher, R. A. Bethe, H. A. and Gamow, G. (1948) Phys. Rev 73:803.

Gamow, G (1954). Possible relation between deoxyribonucleic acid and protein structures. Nature 173:318.

Gamow, G., and Ycas, M. (1955). Statistical correlation of protein and ribonucleic acid composition. PNAS 41:1011-1019.

Text books: Matter, Earth & Sky (1958, rev 1965) and with J.M. Cleveland: Physics: Foundations & Frontiers (1960, rev 1969).

Mr. Tomkins in Wonderland (1936)One, Two, Three ... Infinity (1947)Mr. Tompkins Learns the Facts of Life (1953)The Creation of the Universe (1952; revised edition 1961) A Planet Called Earth (1963)A Star Called the Sun (1964).

Gamow, G (1954). Possible relation between deoxyribonucleic acid and protein structures. Nature 173:318.

20 different cavities

20 aa

Exact match?Pure coincidence?

Astbury: 碱基间距离 (3.4) and 氨基酸间距 (3.7)

Watson and Crick: 碱基对间氢键距离符合双螺旋结构空间限制

Gamow: 20 cavities, 20 aa

(Gamow 当时不知道确切有多少种 , 当时大家知道是 20 到 30,Crick and Watson 立即对照资料 , 猜哪些是必需的基本氨基酸 ,

列出 20 个 )

数字正好对上

不能不重要 , 还是纯粹碰巧 ?

Crick disproved Gamow’s model with the known sequences of insulin(violating restrictions predicted by Gamow’s model on neighboring am

ino acids)

But the Gamow paper stimulated work on the coding problemnucleic acid bases---amino acid residues minimum of 3 (4x4x4) to encode 20 essential residues

Endoplasmic reticulum (ER) by A. Claude, K. R. Porter and G. Palade (1953)

Microsomes as sites of protein synthesis (Keller EB, Zamecnik PC, and Loftfield RB (1954) J Histochem Cytochem 2:378-86 )

RNA: perhaps the more likely template20 cavities on RNA?Or

Gamow: coding by 3 consequitive bases, the central one playing predominant role

Edward Teller: 2 bases plus the proceeding aa

RNA: structure pursued by Jim Watson and Alex Rich

Crick: “one should not abandon a good theory because of a few contradictory facts”

This is hard to practiced even by Watson (Feb 13, 1954) wrote to Crick: “in RNA from all other species the ratios are complementary. This is not an obvious fact as much of the data is sloppy but good papers show the ratios and are the ones to be considered” (Judson HF, p267)

(AT, GC ratios)

Structure of RNA was hard to be solved and once solved, did not provide as much information as the structure of DNA

Crick and Adaptors (tRNA) (1954)

Summer 1954, Crick conceived a kind of small molecule, not yet known to biochemists, had 2 ends,

one for sticking to the nucleic acid using only base-pairing (hydrogen bonding)

one for sticking to an amino acidusing enzymes specifying the match between aa and the sm

all molecule(s)

No requirement for direct and stereochemical matching between the nucleic acid template and the amino acid residues

it “discourages a purely structural approach….allows one to construct, in theory, codes of bewildering variety,…I have no stomach for decoding”

Crick, FHC (1954). On degenerate templates and the adaptor hypothesis. A note for the RNA Tie Club.

The Biochemistrical ApproachPaul Zamecnik, MGH and Robert Holley, Cornell

Cell free protein synthesis (Zamecnik, 1951-1954)Activation of aa (high energy P from ATP) (1957) (Holley; Zamecnik)Purification of tRNA (Zamecnik,1960; Holley, 1961)The enzyme (Paul Berg, 1958; Holley, 1959, 1962)

Zamecnik PC, and Keller EB (1954). Relation between phosphate energy donors and incorporation of labeled amino acids into proteins. J Biol Chem 209:337-54.

Littlefield JW, Keller EB, Gross J, Zamecnik PC. (1955). Studies on cytoplasmic ribonucleoprotein particles from the liver of the rat. J Biol Chem 217:111-23.

Holley, R. W. (1957). An alanine-dependent, ribonuclease-inhibited conversion of AMP to ATP, and its possible relationship to protein syntehsis. J Amer Chem Soc 79:658-662.

Hoagland, M. B., Zamecnik, PC and Stephenson, ML (1957). Intermediate reactions in protein biosynthesis. Biochim Biophys Acta. 24:215-6.

Hoagland, M. B., Stephenson, ML, Scott, JF, Hecht, LI, Zamecnik, PC (1958). A soluble ribonucleic acid intermediate in protein synthesis. J Biol Chem. 231:241-57.

Energy, amino acids and enzymes

Information flow

Hoagland and Zamecnik: put radiolabelling leucine into the protein synthesis system, also found RNA attached to the radiolabelled leucine

Watson visited them around Christmas, 1956, told them that they had found what Crick predicted

“This is the interpretation of your results”

Hoagland’s feeling of resentment “that Jim would be telling me how to interpret my results”

When a theory helps experimentalists

Brenner, S. (1957). On the impossibility of all overlapping triplet codes in information transfer from nucleic acid to proteins. PNAS 43:687-694.

But no theory can crack the code, only experiments could

Decoding by Nirenberg and Ochoa

using the cell free protein synthesis system and defined RNA

Nirenberg, M. W., and Matthaei, J. H. (1961). The dependence of cell-free protein synthesis in E. coli upon naturally occurring or synthetic polyribonucleotides. Proc. Natl. Acad. Sci. U. S. A. 47: 1588–1602.

Lengyel, P, Spever, JF and Ochoa, S (1961). Synthetic polynucleotides and the amino acid code. Proc Natl Acad Sci USA. 47:1936-42.

1968, with Khorona and Holley 1959, with Arthur Kornberg

Nirenberg, M. W., and Matthaei, J. H. (1961). The dependence of cell-free protein synthesis in E. coli upon naturally occurring or synthetic polyribonucleotides. Proc. Natl. Acad. Sci. U. S. A. 47: 1588–1602.

Poly(U) encodes phenylanine

FIG. 6.-Stimulation of U-C14-L-phenylalanine incorporation by polyuridylic acid. *without polyuridylic acid; A 10 ,g polyuridylic acid added.

Lengyel, P., Speyer JF, and Ochoa S (1961). Synthetic polynucleotides and the amino acid code. Proc Natl Acad Sci USA. 47:1936-42.

If poly U codes for polyphenylalanine, a short sequence of three or more U residueswould be the code letter for phenylalanine. If for the sake of simplicity we assume a triplet code, the ratio of UUU to UUC (or UCU, or CUU) triplets in a random copolymer of the composition of poly UC would be the same as the U:C ratio of the polymer, i.e., 5:1. The ratio of UUU to UCC (or CUC, or CCU) triplets would be 25:1. From Table 4 the ratio of phenylalanine to serine incorporation with poly UC was 4.4: 1. On the basis of this result the triplet code letter for serine would be either UUC, UCU, or CUU. The ratio of phenylalanine to tyrosine incorporated with poly UA (U:A = 5:1) was 4.0. This would make either UUA, UAU, or AUU as the likely triplet code letter for tyrosine.

The theoretical frequencies of RNA codons in randomly ordered poly(AC) preparations that contain different proportions of A and C, compared with the observed frequencies of incorporation of radioactively labeled amino acids into protein. The codon for histidine contains one A and two Cs, and the codons for asparagine and glutamine contain two As and one C. These results showed that the code was a triplet code.

Nirenberg M. W. et al. (1963).On the coding of genetic information. Cold Spring Harb. Symp. Quant. Biol. 28 (1963), pp. 549–557.

Jones, O. W. and M.W. Nirenberg, M. W. (1966). Degeneracy in the amino acid code. Biochim. Biophys. Acta 119 (1966), pp. 400–406.

Decoding by using RNA of defined sequences

Nirenberg, M. and Leder, P. (1964). RNA codewords and protein synthesis. I. The effect of trinucleotides upon the binding of sRNA to ribosomes. Science 145:l399–l407.

Nishimura S, Jones DS, Khorana HG. Studies on polynucleotides. 48. The in vitro synthesis of a co-polypeptide containing two amino acids in alternating sequence dependent upon a DNA-like polymer containing two nucleotides in alternating sequence. J Mol Biol. 13:302-24.

Söll, D., Ohtsuka, E., Jones, D S., Lohrmann, R., Hayatsu, H., Nishimura, S., Khorana, H. G. (1965). Studies on polynucleotides, XLIX. Stimulation of the binding of aminoacyl-sRNA's to ribosomes by ribotrinucleotides and a survey of codon assignments for 20 amino acids. Proc. Natl. Acad. Sci. U. S. A. 54:1378–1385.

遗传密码 : 理论和实验的相互依存 , 相互促进

遗传密码概念的提出 : Gamow 简单 , 且可以实验资料检验的理论

tRNA 的提出 : 敏锐理论思维的作用without the adaptor hypothesis, experimentalists did not know why t

hey were getting RNA attached to amino acid residues

遗传密码的破译 : 实验资料最后解决问题

Without direct experimentation, it was not possible to solve the coding problem or predict the exact codes

2) 半懂不懂的 :从慢病毒到蛋白质催化的构相转变

生命科学概念容易发现和接受吗 ?

Transmissible spongiform encephalopathies (TSEs)The classical triad of spongiform vacuolation (affecting any part of the cerebral grey matter), neuronal loss, and astrocytic proliferation. Or Prion diseases

Scrapie: sheep and goats transmissible mink encephalopathy hronic wasting disease of mule deer and elk bovine spongiform encephalopathy (BSE) feline spongiform encephalopathy

Human:Creutzfeldt-Jakob disease (CJD), Gerstmann-Sträussler syndrome (GSS) kuru

Soto, C. and, Castilla, J. (2004) The controversial protein-only hypothesis of prion propagation. Nat Med 10 Suppl:S63-7.

1936: scrapie transmissible by inoculation between sheep (and goats) Cuillé J, and Chelle PL. 1936. La maladie dite tremblante du mouton es

t- elle inocuable? C. R. Acad. Sci. 203:1552-54.

1959: similarities between kuru and scrapie at the neuropathological, clinical, and epidemiological levels

Hadlow WJ. (1959). Scrapie and kuru. Lancet 2:289-90.

1966: kuru transmission by intracerebral inoculation with brain homogenates into chimpanzees

Gajdusek DC, Gibbs CJJr , and Alpers MP (1966). Experimental transmission of a kuru-like syndrome to chimpanzees. Nature 209:794-96.

1968: transmission of CDJGibbs CJJr , Gajdusek DC, Asher DM, Alpers MP, Beck E, et al. (196

8). Creutzfeldt-Jakob Disease (spongiform encephalopathy): transmission to the chimpanzee. Science 161:388-89.

Nucleic Acid or Protein?1966 small size (~200 kD)

Alper T, Haig DA, Clarke MC. (1966). The exceptionally small size of the scrapie agent. Biochem. Biophys. Res. Commun. 22:278-84

1967 no nucleic acid? Resistance to UV and ionizing radiationAlper T, Cramp WA, Haig DA, Clarke MC (1967). Does the agent of scrap

ie replicate without nucleic acid? Nature 214:764-66.

1967 protein-only hypothesisGriffith JS. (1967). Self replication and scrapie. Nature 215:1043-44.

1982 Prions“small proteinaceous infectious particles that resist inactivation by procedures which modify nucleic acids”

Prusiner SB. 1982. Novel proteinaceous infectious particles cause scrapie. Science 216:136-44

After infection and a prolonged incubation period, the scrapie agent causes a degenerative disease of the central nervous system in sheep and goats. Six lines of evidence including sensitivity to proteases demonstrate that this agent contains a protein that is required for infectivity. Although the scrapie agent is irreversibly inactivated by alkali, five procedures with more specificity for modifying nucleic acids failed to cause inactivation. The agent shows heterogeneity with respect to size, apparently a result of its hydrophobicity; the smallest form may have a molecular weight of 50,000 or less. Because the novel properties of the scrapie agent distinguish it from viruses, plasmids, and viroids, a new term "prion" is proposed to denote a small proteinaceous infectious particle which is resistant to inactivation by most procedures that modify nucleic acids. Knowledge of the scrapie agent structure may have significance for understanding the causes of several degenerative diseases.

Bolton DC, McKinley MP, and Prusiner SB. (1982). Identification of a protein that purifies with the scrapie prion. Science 218:1309-11.

Purification of prions from scrapie-infected hamster brain yielded a protein that was not found in a similar fraction from uninfected brain. The protein migrated with an apparent molecular size of 27,000 to 30,000 daltons in sodium dodecyl sulfate polyacrylamide gels. The resistance of this protein to digestion by proteinase K distinguished it from proteins of similar molecular weight found in normal hamster brain. Initial results suggest that the amount of this protein correlates with the titer of the agent.

PrPSc and PrPC

1985 encoded by a cellular gene (not a viral gene)Oesch B, Westaway D, Walchli M, McKinley MP, Kent SB, et al. 1985. A cel

lular gene encodes scrapie PrP 27-30 protein. Cell 40:735-46.1990 PrPSc derived from PrPC

Borchelt DR, Scott M, Taraboulos A, Stahl N, Prusiner SB. 1990. Scrapie and cellular prion proteins differ in their kinetics of synthesis and topology in cultured cells. J. Cell Biol. 110:743-52.

Caughey B, Raymond GJ. 1991. The scrapie-associated form of PrP is made from a cell surface precursor that is both protease- and phospholipase-sensitive. J. Biol. Chem. 266:18217-23.1993 no differences detected by mass spec or sequencing

Stahl N, Baldwin MA, Teplow DB, Hood L, Gibson BW, Burlingame AL, Prusiner SB. (1993). Structural studies of the scrapie prion protein using mass spectrometry and amino acid sequencing. Biochemistry 32, 1991–2002 (1993). 1993 helix to sheet conversion

Pan KM, Baldwin M, Nguyen J, Gasset M, Serban A, Groth D, Mehlhorn I, Huang Z, Fletterick RJ, Cohen FE, and Prusiner SB (1993). Conversion of -helices into -sheets features in the formation of scrapie prion poteins. Proc. Natl. Acad. Sci. USA 90:10962–10966.

PrPSc vs PrPC Infectivity yes noProtease relatively resistant non-resistantDetergent insoluble solubleDegradation slow rapidDistribution in the brain: different-sheet content high (43%) low (3% )-helix relatively low (30%) high (42%)

Evidence for Prion in InfectionAnti-Prion Antibody neutralization of infectivityGabizon, R., McKinley, M.P., Groth, D. and Prusiner, S.B. (1988). Immunoaffinity pur

ification and neutralization of scrapie prion infectivity. PNAS 85:6617–6621. PrP Mutations in Human Neurodegenerative DieseasesOwen, F., Poulter, M., Lofthouse, R., Collinge, J., Crow, T.J., Risby, D., Baker, H.F., R

idley, R.M., Hsiao, K. and Prusiner, S.B. (1989) Insertion in prion protein gene in familial Creutzfeldt-Jakob disease. Lancet 1:51-52.

Hsiao, K., Baker, H.F., Crow, T.J., Poulter, M., Owen, F., Terwilliger, J.D., Westaway, D., Ott, J. and Prusiner, S.B. (1989) Linkage of a prion protein missense variant to Gerstmann-Straussler syndrome. Nature 338:342-345.

Neurodegeneration caused by GSS mutant of prionHsiao KK, Scott M, Foster D, Groth DF, DeArmond SJ, Prusiner SB. (1990) Spontane

ous neurodegeneration in transgenic mice with mutant prion protein. Science 250, 1587–1590 (1990).   

Resistance of PrP knockout mice to PrPSc

Bueler H, Aguzzi A, Sailer A, Greiner RA, Autenried P, Aguet M, Weissmann C. (1993). Mice devoid of PrP are resistant to Scrapie. Cell 73:1339–1347.

Yeast Prion-like PhenomenonWickner, R.B. (1994). [URE3] as an altered URE2 protein: evidence for a prion analog

in Saccharomyces cerevisiae. Science 264, 566–569.

Peretz D, Williamson RA, Kaneko K, Vergara J, Leclerc E, Schmitt-Ulms G, Mehlhorn IR, Legname G, Wormald MR, Rudd PM, Dwek RA, Burton DR, and Prusiner SB (2001). Antibodies inhibit prion propagation and clear cell cultures of prion infectivity. Nature 412, 739–743. Enari, M., Flechsig, E. and Weissmann, C. (2001). Scrapie prion protein accumulation by scrapie-infected neuroblastoma cells abrogated by exposure to a prion protein antibody. PNAS 98:9295–9299.

antibodies binding cell-surface PrPC inhibit PrPSc formation and inhibit prion propagation in cultured mouse neuroblastoma cells (ScN2a) infected with PrPSc

Bioassay: CD-1 Swiss mice were inoculated with antibody-treated (10 µg ml-1) and untreated ScN2a cells. Mice inoculated intracerebrally with Fab D18-, D13- or even R2-treated cells were free of disease after 265 days, whereas mice inoculated with untreated or R72-treated cells had a mean incubation time to disease of 169 and 165 days, respectively

PrPSc Propogation in Cultured Cells

PC12 cellsRubenstein, R., Carp, R.I. & Callahan, S.M. In vitro replication of scrapie agent in a neuronal model: infection of PC12 cells. J. Gen. Virol. 65, 2191–2198 (1984).

Mouse neuroblastoma cells receptive to homogenates from infected mouse, but not hamster bransRace, R.E., Fadness, L.H. & Chesebro, B. Characterization of scrapie infection in mouse neuroblastoma cells. J. Gen. Virol. 68, 1391–1399 (1987).   

Peretz D, Williamson RA, Kaneko K, Vergara J, Leclerc E, Schmitt-Ulms G, Mehlhorn IR, Legname G, Wormald MR, Rudd PM, Dwek RA, Burton DR, and Prusiner SB (2001). Antibodies inhibit prion propagation and clear cell cultures of prion infectivity. Nature 412, 739–743.

Enari, M., Flechsig, E. and Weissmann, C. (2001). Scrapie prion protein accumulation by scrapie-infected neuroblastoma cells abrogated by exposure to a prion protein antibody. PNAS 98:9295–9299.

In Vitro Biochemical Conversion of PrPC into PrPSc and Amplification

Kocisko, D.A. Come JH, Priola SA, Chesebro B, Raymond GJ, Lansbury PT, Caughey B. (1994). Cell-free formation of protease-resistant prion protein. Nature 370, 471–474. 

Kocisko DA, Priola SA, Raymond GJ, Chesebro B, Lansbury PTJr , Caughey B. (1995). Species specificity in the cell-free conversion of prion protein to protease-resistant forms: a model for the scrapie species barrier. PNAS 92:3923-27.

Bessen RA, Kocisko DA, Raymond GJ, Nandan S, Lansbury PT, and Caughey B. (1995). Non-genetic propagation of strain-specific properties of scrapie prion protein. Nature 375:698-700.

Hill A, Antoniou M, Collinge J. (1999). Protease-resistant prion protein produced in vitro lacks detectable infectivity. J. Gen. Virol. 80:11-14 .

Saborio, G.P., Permanne, B. and Soto, C. (2001). Sensitive detection of pathological prion protein by cyclic amplification of protein misfolding. Nature 411, 810–813. 

Bieschke J, Weber P, Sarafoff N, Beekes M, Giese A, and Kretzschmar H (2004). Autocatalytic self-propagation of misfolded prion protein. PNAS 101:12207-12211.

Difficulties with (or Evidences against) the Prion Hypothesis

Multiple strains: multiple protein conformation? how? cause or effect?

Correlation of PrPres with infectivity: not always

Presence of nucleic acid in infected samples, though not in infectious prion prep

Binding of PrPres to RNA

Other neurodegenerative diseases: no infectious misfolding

What Is the Most Important Evidence Missing So Far?

Direct evidence for the infectious activity of prion proteins

In Vitro Generation of Infectious Yeast PrionKing, C.Y. & Diaz-Avalos, R. Protein-only transmission of three yeast prion strains. Nature 428, 319–323 (2004). 

Tanaka, M., Chien, P., Naber, N., Cooke, R. & Weissman, J.S. Conformational variations in an infectious protein determine prion strain differences. Nature 428, 323–328 (2004). 

In Vitro Generation of Infectious Mammalian PrionLegname G, Baskakov IV, Nguyen HO, Riesner D, Cohen FE, DeArmond SJ, Prusiner SB.(2004). Synthetic mammalian prions. Science 305:673-6.

Legname G, Baskakov IV, Nguyen HO, Riesner D, Cohen FE, DeArmond SJ, Prusiner SB.(2004). Synthetic mammalian prions. Science 305:673-6.

Recombinant mouse prion protein (recMoPrP) produced in Escherichia coli was polymerized into amyloid fibrils that represent a subset of beta sheet-rich structures. Fibrils consisting of recMoPrP(89-230) were inoculated intracerebrally into transgenic (Tg) mice expressing MoPrP(89-231). The mice developed neurologic dysfunction between 380 and 660 days after inoculation. Brain extracts showed protease-resistant PrP by Western blotting; these extracts transmitted disease to wild-type FVB mice and Tg mice overexpressing PrP, with incubation times of 150 and 90 days, respectively. Neuropathological findings suggest that a novel prion strain was created. Our results provide compelling evidence that prions are infectious proteins.

Are there important questions for you?

Yes

Do You Accept the Protein-Only Hypothesis?

How Do Prions Cause the Disease?

Mechanism

New Biochemistry of General Or Limited Significance (peculiar to prions)

漂亮 or 简单的理论 对 不漂亮 or 复杂的资料 :

Crick 的说法 : “one should not abandon a good theory because of a few contradictory facts”

Watson 也有用错的时候 : Watson (Feb 13, 1954) wrote to Crick: “in RNA from a

ll other species the ratios are complementary. This is not an obvious fact as much of the data is sloppy but good papers show the ratios and are the ones to be considered” (Judson HF, p267) (AT, GC ratios)

现在面对 prions, 你又怎么看 ? 接受 Protein-only 的理论吗 ?

Original hypothesis:Pan KM, Baldwin M, Nguyen J, Gasset M, Serban A, Groth D, Mehlhorn I, Huan

g Z, Fletterick RJ, Cohen FE, and Prusiner SB (1993). Conversion of -helices into -sheets features in the formation of scrapie prion poteins. Proc. Natl. Acad. Sci. USA 90:10962–10966.

Figure fromCollinge J. (2001). Prion diseases of humans and animals: their causes and molecu

lar basis. Annu Rev Neurosci 24:519-50.

Conversion of -helices into -sheets

不同例子

从人类认识来说

1)已经清楚的 :从种瓜得瓜到遗传密码

2) 半懂不懂的 :从慢病毒到蛋白质催化的构相转

变

3) 还没入门的 : 对脸的识别

生命科学技术发明和应用

为了解决重要问题 , 而发明或应用关键技术

细胞和分子水平Cell based: fate mapping, transplantation, nuclear transfer

DNA: genetics, manipulations of genes

RNA: hybridization, RNAi

Protein: antibodies, GFP

Chemistry: purification, synthesis

Physics: structural biology, modern imaging

fate mapping

Harfe BD, Scherz PJ, Nissim S, Tian H, McMahon AP, Tabin CJ (2004). Evidence for an expansion-based temporal shh gradient in specifying vertebrate digit identities. Cell 118:517-28.

Nicole Le Dourin从法国中学老师到世界著名发育生物学家

transplantation

transplantation

Nuclear transfer

Gurdon JB, Byrne JA, Simonsson S (2003). Nuclear reprogramming and stem cell creation. Proc Natl Acad Sci U S A. 100 (Suppl 1):11819-22

Genetics

Bacteria

Yeast: sequence, knockout, protein localization

George Streisinger

Choosing an Organism or System

Simplicity

Technological accessibility

General significance

DNA: cloning genes

生命科学技术应用

用 最新 或者 优美的 技术发现 或者 解决问题

用 旧 或者 不怎么优美的 技术发现 或者 解决问题

用常规的 cDNA cloning 技术 解决问题

生命科学技术应用

David JuliusUCSF

Old Technology in New Breakthroughs Gurdon JB (1975) Attempts to analyse the biochemical basis of regional differe

nces in animal eggs. Ciba Found Symp (29):223-39. Marbaix G et al. (1975). PNAS 1975 Aug;72(8):3065-7. Chan L, Kohler PO, O'Malley BW. (1976) J Clin Invest. 57:576-85.

Expression CloningJulius D, MacDermott AB, Axel R, Jessell TM. (1988). Molecular characterization of a functional cDNA encoding the serotonin 1c receptor.

Science 241:558-64. Science 244:1057-62, 1989 PNAS 86:6793-7, 1989.

Eating disorder and epilepsy in mice lacking 5-HT2c serotonin receptors.

Nature 374:542-6, 1995. Julius D, Molecular biology of serotonin receptors.

Annu Rev Neurosci. 1991;14:335-60.

An ionotropic ATP receptor Nature 371:519-23, 1994Brake AJ, Julius D (1996) Signaling by extracellular nucleotides.

Annu Rev Cell Dev Biol. 12:519-41.

Caterina MJ, Schumacher MA, Tominaga M, Rosen TA, Levine JD, Julius D (1997)

The capsaicin receptor: a heat-activated ion channel in the pain pathway

Nature 389:816-24.

                                                                                             

VR1 responds to purified vanilloids and pepper extracts. a, Activation of VR1 by capsaicin and resinferatoxin. Left, agonists were applied sequentially to the same Xenopus oocyte expressing VR1. Membrane currents were recorded in the whole-cell voltage-clamp configuration. Bars denote duration of agonist application. Right, concentration–response curve for capsaicin (filled squares) and resiniferatoxin (open circles). Membrane currents were normalized in each oocyte to a response obtained with 1 M capsaicin and expressed as a percent of maximal response to capsaicin. Each point represents mean values (s.e.m.) from five independent oocytes. The Hill equation was used to fit the response data. b, Antagonism by capsazepine (cpz) and ruthenium red (RR). Current tracing at top left shows reversible block of capsaicin (cap; 0.6 M) response by capsazepine (cpz; 10 M) after 2 min pretreatment. Slash marks represent washout periods of 2 and 3 min, respectively (n = 3). A capsazepine inhibition curve is shown to the right (n = 4 independent oocytes for each point). Current responses were normalized to that elicited by capsaicin alone in each oocyte. (0.6 M, open diamond). Current tracing at bottom left shows reversible block of a capsaicin (0.6 M)-evoked response by ruthenium red (RR; 10 M). Slash marks denote washout periods of 2 and 12 min, respectively (n = 3). c, Responses to capsaicin (10 M) and extracts derived from four varieties of peppers in oocytes expressing VR1 (30 s application). Bottom right, relative potencies of each pepper extract are plotted (mean s.e.m., n = 3). Values were normalized in each cell to responses obtained with capsaicin (10 M). Extracts evoked no responses in water-injected cells. Reported pungencies for pepper varieties (in Scoville units) are: Habanero (H), 100,000–300,000; Thai green (T), 50,000–100,000; wax (W), 5,000–10,000; and Poblano verde (P), 1,000–1,500 (ref. 23). Capsaicin (C) is rated as 16   106 units.

VR1 responds to purified vanilloids and pepper extracts

Activation of VR1 by capsaicin and resinferatoxin. Left, agonists were applied sequentially to the same Xenopus oocyte expressing VR1.