Table Of ContentProgress in Molecular and Subcellular Biology 3
Progress in Molecular
and Subcellular Biology
3
By
A. S. Braverman· D. J. Brenner· B. P. Doctor· A. B. Edmundson
K. R. Ely • M. J. Fournier . F. E. Hahn . A. Kaji . C. A. Paoletti
G. Riou . M. Schiffer . M. K. Wood
Editorial Board
F. F. Hahn· T. T. Puck· G. F. Springer
W. Szybalski . K. Wallenfels
Managing Editor
F. E. Hahn
With 58 Figures
Springer-Verlag New York· Heidelberg. Berlin 1973
ISBN-13: 978-3-642-65580-7 e-ISBN-13: 978-3-642-65578-4
DOl: 10.1007/978-3-642-65578-4
This work is subject to copyright. All rights are reserved, whether the whole or part of the material is concerned,
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publisher, the amount of the fee to be determined by agreement with publisher.
@ by Springer-Verlag Berlin· Heidelberg 1973. Library of Congress Catalog Card Number 75-79748.
Softcover reprint of the hardcover 1st edition 1973
The use of registered names, trademarks, ctc. in this publication does not imply, even in the absence of a specific
statement, that such names are exempt from the relevant protective laws and regulations and therefore free for
general usc.
Contents
FRED E. HAHN: Reverse Transcription and the Central Dogma ............ 1
I. Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1
II. Cryptology and the Central Dogma ........... . . . . . . . . . . . . . . . . . . . . 2
III. Reverse Transcription: Experimental Evidence. . . . . . . . . . . . . . . . . . . . . 3
IV. Reverse Transcription and Cancer ................................ 5
V. Is There Reverse Transcription in Bacteria? . . . . . . . . . . . . . . . . . . . . . . . . 6
VI. Biological Significance of Reverse Transcription. . . . . . . . . . . . . . . . . . . . 7
VII. Cryptography and the Central Dogma . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 8
VIII. Conclusion .................................................... 9
IX. Glossary of Terms. ...... ... ... ............ . . . ... .. ........... .. 10
References .................................................... 11
X. Addendum.................................................... 13
MAURILLE J. FOURNIER, Jr., DON J. BRENNER, and B. P. DOCTOR: The Isolation
of Genes: A Review of Advances in the Enrichment, Isolation and in vitro
Synthesis of Specific Cistrons ...................................... 15
I. Introduction. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 15
II. Procedures for the Preparation of Specific Cistrons ................. 16
ill. Conclusion.................................................... 74
References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 78
AKIRA KAJI: Mechanism of Protein Synthesis and the Use ofInhibitors in the
Study of Protein Synthesis. ........................................ 85
I. Introduction ... . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 85
II. Initiation of Protein Synthesis ................................... 85
III. Chain Elongation. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 95
IV. Chain Termination ............................................. 136
V. Epilogue ................................. . . . . . . . . . . . . . . . . . . . . . 141
References .................................................... 143
ALLEN B. EDMUNDSON, MARIANNE SCHIFFER, KATHRYN R. ELY and MICAL
K. WOOD: Structural Features of Immunoglobulin Light Chains ........ 159
I. Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 159
II. Association of Light Chains ..................................... 161
ill. Thermal Behavior of Bence-Jones Proteins ........................ 161
IV. Distribution of Polar and Apolar Residues in Amino Acid Sequences of
Light Chains .................................................. 161
VI Contents
V. Criteria of Purity in the Bence-Jones Protein Used in the Crystallographic
Study......................................................... 168
VI. Crystallography of the Mcg Bence-Jones Protein ................... 171
VIT. Discussion .................................................... 177
VITI. Summary ..................................................... 178
References .................................................... 178
ALBERT S. BRAVERMAN: The Thalassemia Syndromes: Genetically Determined
Disorders of the Regulation of Protein Synthesis in Eukaryotic Cells . . . . . 183
I. Introduction. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .. 183
II. The Beta Thalassemias: Clinical Picture and Pathogenesis of Anemia. .. 184
III. The Molecular Basis of the Suppression of Beta Chain Synthesis in Severe
Beta Thalassemia ............................................... 192
Appendix I: The Alpha Thalassemias . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 196
Appendix II: The Lepore-Pylos Hemoglobins............ .......... 197
References .................................................... 198
CLAUDE A. PAOLETTI and GUY RIOu: The Mitochondrial DNA of Malignant
Cells.. ....... ...... .......................... .. ................. 203
I. Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .. 203
II. Morphology of Mitochondria in Malignant Cells ................... 204
III. Size and Structure of Mitochondrial DNA in Malignant Cells. . . . . . . . .. 205
IV. Synthesis of Mitochondrial DNA in Malignant Cells: Content, Rate
and Mechanism ................................................ 217
V. Is the Informational Content of Mitochondrial DNA Modified in Malig-
nant Cells? .................................................... 220
VI. Are the Changes in Mitochondrial DNA in Malignant Cells Under
Genetic Control? ...... . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .. 222
VIT. Are the Changes in Mitochondrial DNA Related to Some Energy Im-
balance in Mitochondria of Malignant Cells? ............ . . . . . . . . . .. 223
VIII. Are the Changes Observed in Mitochondrial DNA of Malignant Cells
Specific of Malignancy? ................. . . . . . . . . . . . . . . . . . . . . . . .. 225
IX. Concluding Remarks ........................................... 228
Appendix I. Isolation and Examination of Mitochondrial DNA ....... 229
Appendix II. DNA Circular Oligomers other than Mitochondrial ..... 234
References .................................................... 239
Subject Index ....................................................... 249
List of Contributors
ALBERT S. BRAVERMAN, New York Medical College, Metropolitan Hospital Center,
New York, New York 10029, USA
DON J. BRENNER, Walter Reed Army Institute of Research, Washington, D. C. 20012,
USA
BHUPENDRA P. DOCTOR, Walter Reed Army Institute of Research, Washington, D. C.
20012, USA
.Au.EN B. EDMUNDSON, Argonne National Laboratory, Argonne, Illinois 60439,
USA
KATHRYN R. ELy, Argonne National Laboratory, Argonne, Illinois 60439, USA
MAURILLE J. FOURNIER, Department of Biochemistry, University of Massachusetts,
Amherst, Massachusetts 01002, USA
FRED E. HAHN, Walter Reed Army Institute of Research, Washington, D. C. 20012,
USA
AKlRA KAJI, Department of Microbiology, School of Medicine, University of
Pennsylvania, Philaddphia, Pennsylvania 19104, USA
CLAUDE A. PAOLETTI, Institut Gustave Roussy, 94 Villejuif, France
GUY RIOu, Institut Gustave Roussy, 94 Villejuif, France
MARIANNE SCHIFFER, Argonne National Laboratory, Argonne, Illinois 60439, USA
MICAL K. WOOD, Argonne National Laboratory, Argonne, Illinois 60439, USA
Reverse Transcription and the Central Dogma
FRED E. HAHN
"Molecular biologists have a religion all of their own in which Nobel prize winner
Francis Crick is the prophet and the DNA molecule is the icon. Molecular biologists have
a 'trinity' of three kinds of molecules - DNA, RNA and the protein molecules - which
correspond to each other on a unit-for-unit informational basis. They have a 'dogma' (and
they call it a dogma) which says that 'information' - that is the molecular pattern - passes
from DNA to RNA to protein but does not pass in the reverse direction."
ParrER (1964)
I. Introduction
The Central Dogma of molecular biology which postulates the unidirectional
transmission of genetic specifications for protein biosynthesis was enunciated by
CRICK (1958) who proposed explicidy that "once 'information' has passed into
protein it cannot get o«t again. In more detail, the transfer of information from nucleic
acid to nucleic acid, or from nucleic acid to protein may be possible, but transfer from
protein to protein or from protein to nucleic acid is impossible. Information means
here the precise determination of sequence either of bases in the nucleic acids or of
amino acids in the protein."
At the time of that writing (1958), messenger RNA as a separate macromolecular
category had been neither proposed nor discovered (indications of the formation
of phage T2 messenger RNA obtained by VOLKIN and ASTRACHAN (1956) had gone
largely unrecognized). The transcription of RNA from DNA, in general, was awaiting
discovery and OCHOA (1958) still considered polycondensation of nucleoside di-
phosphates through reversal of the polynucleotide phosphorylase reaction to re-
present biosynthesis of RNA. Transfer RNAs, than called "soluble RNA", had not
yet been shown to be the set of amino acid adaptors excogitated by CRICK (1957),
and the cryptanalysis of the amino acid code was bogged down in abstract speculations
on the nature of symbols comprising a putative nucleic acid alphabet and on formal
reasons why an assumed alphabet of 43 nucleotide triplets might be intrinsically
restricted to the unambiguous designation of precisely 20 different amino acids,
i.e. of the standard set of constituents of proteins (CRICK, GRIFFITH and ORGEL, 1957).
Ifo ne accepts one of Webster's Seventh Collegiate Dictionary's definitions of "dogma"
as "a point of view or tenet put forth as authoritative without adequate grounds",
the Central Dogma of 1958 certainly was a dogma.
However, when POTTER (1964) wrote his spirited remarks above on the Central
Dogma of molecular biology, the cryptanalysis of the RNA code which determines
amino acid sequence in protein biosynthesis, was nearly completed, and the two-step
biochemical decipherment of structural genes of DNA through consecutive opera-
2 FRED E. HAHN
tions called "transcription" and "translation", was envisaged, at least in general
outline. At that time, molecular biologists were, therefore, justified to expand the
proposition of the Central Dogma to denote the unidirectional passage of "infor-
mation" concerning sequential molecular pattern from DNA through RNA into
protein.
II. Cryptology and the Central Dogma!
To molecular biologists interested in cryptology, it should have also been
apparent at that time that a plaintext, the linear covalent amino acid sequence
in protein, is superenciphered in the ciphertext of its determinant structural gene in
chromosomal DNA. The first encipherment consists of a substitution transformation
in which one set of symbols (the amino acids) is replaced by another set of symbols
(the codons in messenger RNA). The superencipherment involves a second sub-
stitution transformation in which the RNA codons are replaced by their comple-
mentary triplets in the transcribable DNA strand. The biological decipherment requires,
therefore, two separate procedures in reverse: (1) the decipherment of the second
substitution: this is known as "transcription" and yields messenger RNA in placode,
followed by (2) the decipherment of the first substitution, known as "translation"
which yields the amino acid plaintext. CRICK (1970) calls this "information transfer
from one polymer with a defined alphabet to another".
Since in substitution transformation the letters in the plaintext lose their identities
but retain their positions, the postulation of the "sequence hypothesis" (CRICK, 1958),
which assumed colinearity of amino acids in protein and of corresponding symbols
in nucleic acids, was tantamount to postulating that the genetic ciphertext must be
the result of a substitution transformation instead of a transposition in which the letters
retain their identities but change their positions. One might consider the three-dimen-
sional rearrangement of linear polypeptide chains into functional proteins which
brings topographically distant amino acids into proximity to represent an encipher-
ment by a transposition transformation; this process appears to be an inherent
deterministic function of key amino acid sequences and does not require the operation
of a separate ad hoc cryptographic machinery except in those instances in which
existing covalent bonds are broken or new covalent bonds are formed in order to
stabilize the eventual biologically active three-dimensional protein configuration.
Considering in cryptological terms the DNA ciphertext an encicode, there exists
no a priori formal reason why free passage of information in both directions, that is
decipherment and encipherment could not biologically occur. It is mechanistically
apparent, however, that a transmission of biological information in both directions
might require separate cryptographic machineries. This is obvious for the "trans-
lation" step for which no mutually deterministic relationship appears to exist between
the symbols of the codon alphabet and those of the amino acid alphabet; it is not so
obvious for the "transcription" step for which a deterministic relationship between
DNA and RNA symbols does exist with base complementarity as the key and the only
mechanistic requirement remains for polymerizing enzymes to link template-deter-
mined nucleoside triphosphates by repetitive condensations.
Out of the cryptological framework of reference, the 1964 version of the Central
Dogma might have been restated to say that biological systems are only equipped (1)
1 A glossary of cryptological terms is printed at the end of this article.
Reverse Transcription and the Central Dogma 3
to decipher DNA (by transcription) but not to superencipher information (from RNA)
as to the sequence of symbols in DNA, and (2) to decipher the messenger RNA
placode (by translation) but not to encipher (from a plaintext amino acid sequence)
the sequence of codons in messenger RNA. The recent discovery of reverse tran-
scription shows that the first of these two tenets is not invariably valid.
III. Reverse Transcription: Experimental Evidence
In 1964 LEE-HuANG and CAVALIERI demonstrated the first instance of reverse
transcription in an in vitro model system by showing that a DNA polymerase pre-
paration from E. coli synthesized poly (dA + T) on a template of poly (U + rA); the
authors discussed their results only in terms of enzymology but did not interpret
them as to their possible biological significance. In the same year TEMIN [1964 (1)]
hypothesized that the replication of the RNA of RNA-containing tumor viruses
proceeds through a DNA intermediate. This would require the action of an enzyme
capable of catalyzing a reversed transcription, i.e. the biosynthesis of DNA on an RNA
template. Such enzymatic activity was discovered simultaneously by BALTIMORE (1970)
and by TEMIN and MIZUTANI (1970) in Rauscher mouse leukemia and Rous sarcoma
viruses. The enzymatic reaction was demonstrated by incubating suspensions of the
purified virions with the four deoxyribonucleoside triphosphates, including tritiated
thymidine triphosphate, and magnesium ions. In these experiments, using virus
particles as a source of both the RNA template and the reverse transcriptase enzyme,
tritium was incorporated into acid-insoluble, Le. polymeric products which were
susceptible to hydrolysis by deoxyribonuclease. The enzymatic reaction was precluded
by pretreating the virus suspensions with ribonuclease, suggesting that the RNA of
the virus particles was essential for the polymerization reaction.
TEMIN'S hypothesis [1964 (1)] further predicted that a DNA, complementary to
virus RNA, should appear in infected cells during the course of viral replication and
should be demonstrable by molecular hybridization techniques. In fact, he presented
some evidence in favor of this prediction [1964 (2)]. SPIEGELMAN, BURNY, DAs,
KEYDAR, SCHLOM, TRAVNICEK and WATSON [1970 (1)] proceeded to show not only
the occurrence of the RNA-dependent polymerase reaction catalyzed in vitro by six
different RNA-containing tumor viruses but they also demonstrated that these viruses
synthesized DNA-RNA hybrids using the single-stranded virus RNAs as templates;
finally, hybridization experiments proved that the DNA strands were, indeed, com-
plementary to the virus RNAs. The formation of hybrid DNA-RNA was soon confir-
med and is species-specific for the homologous virus RNA (ROKUTANDA, ROKUTANDA,
GREEN, FU]INAGA, RAY and GURGO, 1970; DUESBERG and CANAAN!, 1970; HATANAKA,
HUEBNER and GILDEN, 1971).
These discoveries were rapidly extended by numerous additional examples of
reverse transcriptase activities in tumor viruses (HATANAKA, HUEBNER and GILDEN,
1970; GREEN, ROKUTANDA, FU]INAGA, RAy, ROKUTANDA and GURGO, 1970; SCOLNICK,
RANDS, AARONSON and TODARO, 1970). A total of 27 isolated preparations of RNA
tumor viruses was shown to contain RNA-dependent DNA polymerase activity
(SCHLOM, HARTER, BURNY and SPIEGELMAN, 1971). The enzymatic activity is imbedded
in the core of the virus particles (GERWIN, TODARO, ZEVE, SCOLNICK and AARONSON,
1970), is unmasked by treatment of virus suspensions with non-ionic detergents such
4 FRED E. HAHN
as Nonidet P-40 or with ether and is enhanced more strongly by Mn2+ than by Mg2+
(GREEN et al., 1970; SCOLNICK et al., 1970). The product DNAs are of relatively small
molecular size, having sedimentation coefficients of 2-4 S (HATANAKA et aI., 1970)
or 7 S (GREEN et al., 1970).
The Mn2+ preference of the polymerases and their susceptibility to rifamycin
derivatives (GALLO, YANG and TING, 1970; SCOLNICK, AARONSON, TODARO and
PARKS, 1971; GURGO, RAY, THIRY and GREEN, 1971) as well as to streptovaricins
(BROCKMAN and CARTER, 1971) are reminiscent of properties of bacterial RNA-poly-
merase enzymes. It should be noted that LEE-HuANG and CAVALIERI (1964) considered
their E. coli DNA polymerase which transcribed poly (dA+ T) from poly (V + rA)
to be a subunital hybrid of DNA and RNA polymerases. In contrast, mammalian
DNA-dependent RNA polymerases such as that of liver nuclei (WEHRLI, NUESCH,
KNtiSEL and STAEHELIN, 1968) or of Ehrlich ascites cells (MIZUNO, YAMAZAKI, NITTA
and VMEZAWA, 1968) are not inhibited by rifamycins or streptovaricins.
Viral DNA polymerases exhibit a bewildering lack of template specificity. Origi-
nally, the emyme was found to depend upon endogenous viral RNAs (BALTIMORE,
1970; TEMIN and MIZUTANI, 1970). The correct operational definition of this type
of enzyme remains, therefore, that of a polymerase which synthesizes DNA on a
single-stranded RNA template. In fact, SCHLOM, SPIEGELMAN and MOORE (1971)
insist that this definition and experimental proof of the formation of a DNA-RNA
hybrid be applied as stringent criteria in the evaluation of all instances of assumed
reverse transcription in different life forms.
Following the original discoveries of BALTIMORE (1970) and of TEMIN and
MIZUTANI (1970) it was found that native or denatured DNAs of different biological
origins were also utilized as templates [MIZUTANI, BOETTGER and TEMIN, 1970;
SPIEGELMAN et aI., 1970 (2); RiMAN and BEAUDREAU, 1970; McDONNELL, GARAPIN,
LEVINSON, QUINTRELL, FAN SHIER and BISHOP, 1970; FU]INAGA, PARSONS, BEARD,
BEARD and GREEN, 1970] as well as yeast RNA (BOSMANN, 1971), or certain synthetic
polynucleotides, foremost poly dA. dT, poly rA. dT, poly dA (MIZUTANI et aI., 1970),
poly dC.rG, poly rl.rC, poly dI.rC [SPIEGELMAN et aI., 1970 (3)], poly rA.dT
(SCOLNICK et al., 1971) or poly rA.rV (STONE, SCOLNICK, TAKEMOTO and AARONSON,
1971). RNA-dependent DNA polymerases show much greater activity with certain
synthetic polynucleotides than they exhibit with homologous RNAs. For this reason,
experiments with synthetic primers/templates are useful in the detection of such
enzymes.
DUESBERG, HELM and CANAANI (1971) succeeded in solubilizing and purifying a
DNA polymerase preparation from Rous sarcoma virus which utilized as templates
native homologous viral RNA and denatured salmon DNA but had low activity
with heat-dissociated homologous virus RNA or with the RNAs of influenza or
tobacco mosaic viruses. Similar studies have been reported by McDONNELL, TAYLOR,
LEVINSON and BISHOP (1971) who found that the purified enzyme did not function
with poly rA. rV as a template. While the broad range of template utilization could
suggest that more than one species of DNA polymerases might occur in the various
biological sources studied, DUESBERG et al. (1971) and McDONNELL et al. (1971)
observed homogeneity of enzyme activity in centrifugation analysis, which leads to
the inference that activities stimulated by different nucleic acid templates may reside
within biophysically homogeneous enzyme preparations. On the other hand, MIZU
