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

5)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?
 Gregor Mendel (1822-1884)

Mendel, G. (1865) Experiments in Plant Hybridization

Read at the meetings of February 8th, and March 8th, 1865
http://www.mendelweb.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.
Chromosomes as carriers of genetic information

Correlation of chromosome behavior with Mendel’s elements

in pairs
equal segregating into gametes
independent segregation

Walter S Sutton (1877-1916), a graduate student at Columbia

Theodor 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). ]

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.
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 others
Drosophila 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)
 Nuclein, 1869

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.
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
Spaltungsprodubte (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
nucleic acid
T 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)
 Linkage of nucleotides

Tetranucleotide Hypothesis
and its implications

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

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.
 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
ray photograph of a protein structure)

Technically
difficulty in obtaining homogeneous DNA for detailed chemical analysis
 The Griffith Transformation Experiment 1928
Streptococcus pneumoniae (pneumococcal) cells

"R" form (unencapsulated), non-virulent

"S" form (encapsulated), virulent

Smooth "S" Cells Rough "R" Cells

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

Transformation

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-)

67
1944

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 cells
Multiple assays for the R and S forms: enzymatic, chemical, and serological
analyses
Multiple assays for DNA and protein: electrophoresis, UV spectroscopy, and
ultracentrifugation

S strain bacteria first had the large cellular structures removed.

Transformation
Proteases: +
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.
Avery, MaCleod and McCarty:
…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 undetermined
 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
Astbury, W. T. (1947). X-ray studies of nucleic acids. Sym. Soc. Exp. Biol. 1:66-76.

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

3.4 A base stacking vs 3.7 A peptide bond

William Astbury (1898-1961)
 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 hypothesis

Chargaff, E. (1950). Chemical specificity of nucleic acids and mechanism of
their enzymic degradation. Experientia 6:201-209.
 mol % of bases  Ratios
Source %GC
 A  G  C  T  A/T  G/C
φX-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
 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
haploid germ cells
Even in 1951, Mirsky still thought that DNA was part of the gene substance,
unconvinced 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.”

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.
 Previous mistake
In the early twenties, Richard M. Willstätter (Nobel Chem 1915,
plant 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
bean 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
been tested
an 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.
 The Hershey-Chase Experiment (1952)

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
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.
 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 structur

Base-pairing yes did not realize

Communication extensive little

Wilkins also has data, though initially not as good as those of Franklin
A 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
 碱基配对

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

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.
 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 Code Protein Synthesis
Information Biochemistry

George Gamow Paul C. Zamecnik

Robert Holley
Francis Crick
Marshall Nirenberg
Sydney Brenner Severo Ochoa

Gobind Khorona

思考 and 资料

Coding and Decoding Energy and material assembly

Information Flow
 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

DNA Structure Insulin Sequence

Watson and Crick (1953) Fred Sanger (1951, 1953)

The Genetic Code: translating DNA sequence into protein sequence

George Gamow (1954)

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).
 20 different cavities

20 aa

Exact match?
Pure coincidence?

Gamow, G (1954). Possible relation between deoxyribonucleic acid and protein structures. Nature 173:318.
 数字正好对上

不能不重要 , 还是纯粹碰巧 ?

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

But the Gamow paper stimulated work on the coding problem

nucleic 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 template

20 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 acid

using enzymes specifying the match between aa and the
small 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 Approach
Paul 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
 When a theory helps experimentalists

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”
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

 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.
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.
If poly U codes for polyphenylalanine, a short sequence of three or more U residues
would 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.

Lengyel, P., Speyer JF, and Ochoa S (1961). Synthetic polynucleotides and the
amino acid code. Proc Natl Acad Sci USA. 47:1936-42.
 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
they 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
 生命科学概念容易 发现 和接受 吗 ?
2) 半懂不懂的 :
从慢病毒到蛋白质催化的构相转
变
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
est- 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 CDJ

Gibbs CJJr , Gajdusek DC, Asher DM, Alpers MP, Beck E, et al.
(1968). 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 radiation

Alper T, Cramp WA, Haig DA, Clarke MC (1967). Does the agent of
scrapie replicate without nucleic acid? Nature 214:764-66.

1967 protein-only hypothesis

Griffith 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
cellular 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 α helix to β 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 no
Protease relatively resistant non-resistant
Detergent insoluble soluble
Degradation slow rapid
Distribution in the brain: different
β-sheet content high (43%) low (3% )
α-helix relatively low (30%) high (42%)
 Evidence for Prion in Infection
Anti-Prion Antibody neutralization of infectivity
Gabizon, R., McKinley, M.P., Groth, D. and Prusiner, S.B. (1988). Immunoaffinity
purification and neutralization of scrapie prion infectivity. PNAS 85:6617–6621.
PrP Mutations in Human Neurodegenerative Dieseases
Owen, F., Poulter, M., Lofthouse, R., Collinge, J., Crow, T.J., Risby, D., Baker, H.F.,
Ridley, 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 prion
Hsiao KK, Scott M, Foster D, Groth DF, DeArmond SJ, Prusiner SB. (1990)
Spontaneous 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 Phenomenon
Wickner, R.B. (1994). [URE3] as an altered URE2 protein: evidence for a prion analog
in Saccharomyces cerevisiae. Science 264, 566–569.
 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

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.
 PrPSc Propogation in Cultured Cells

PC12 cells
Rubenstein, 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 brans
Race, 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 Prion
King, 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 Prion

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.

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

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

?
 Conversion of α-helices into β-sheets

Original hypothesis:
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 α-
helices into β-sheets features in the formation of scrapie prion poteins. Proc.
Natl. Acad. Sci. USA 90:10962–10966.

Figure from
Collinge J. (2001). Prion diseases of humans and animals: their causes and
molecular basis. Annu Rev Neurosci 24:519-50.
 不同例子

从人类认识来说

5)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.
 transplantation

Nicole Le Dourin
从法国中学老师到
世界著名发育生物学家
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
 生命科学技术应用
用常规的 cDNA cloning 技术 解决问题

David Julius
UCSF
 Old Technology in New
Gurdon JBBreakthroughs
(1975) Attempts to analyse the biochemical basis of regional
differences 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 Cloning
Julius 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, 1994

Brake 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
  
D (1997)  
  
  
JD, Julius

The capsaicin
  
  
a heat-activated
  
receptor:
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.