Russian Original Vol. 21, No. 1, Part 1, January-February, 1987
`. July, 1987
`.
`·,
`
`ISSN 0026-8933
`
`1 HEALTH SCIENCES LIBRAf\Y
`University of Wisconsin
`1 ~n1 Linden Dr .. fvl::lrfison, w,·s. 5 37 ,r-;
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`
`MOLBBJ 21(1) 1-122 (1987)
`
`·~
`\
`
`MOLECULAR
`BIOLOGY
`
`MOflEKYflHPHAH 6~onorKH
`(MOLEKULYARNAYA BIOLOGIYA)
`
`TRANSLATED FROM RUSSIAN
`
`\ .
`
`®
`
`CONSULTANTS BUREAU, NEW YORK
`
`Illumina Ex. 1100
`IPR Petition - USP 10,435,742
`
`

`

`SUBSTRATE INHIBITORS OF DNA BIOSYNTHESIS
`
`A, A, Kraevskii, M. K. Kukhanova,
`A, M. Atrazhev, Z. G. Chidzhavadze,
`and R, Sh, Bibilashvili
`
`UDC 577.123
`
`Data are presented on an analysis of the inhibition of DNA biosynthesis by sub(cid:173)
`strate analogs in s.olutions containing DNA.polymerases of. different origin. An
`attempt has been made to suggest reasons for inhibitor specificity in regard to
`DNA synthesis catalyzed by different DNA polymerases.
`
`Three approaches are currently ell\ployed to study the mode of operation of enzymes and
`multienzyme complexes catalyzing replication, reverse transcription, genetic recombination,
`reparation, and other processes, One of these strategies is the study of the properties and
`composition of multi-protein complexes isolated from cells. A second approach is the recon(cid:173)
`struction of multienzyme complexes synthesizing DNA which have been obtained from purified
`protein components and the study of these enzymes at each stage in the assembly process. The
`third approach is an inhibitor analysis of the processes involved in DNA biosynthesis. By
`means of this approach it would be possible to compare complexes which synthesize DNA in cells
`and in cell-free solutions. A comparative analysis of these processes involving the use of
`monoclonal antibodies would be of great significance,
`The current knowledge of the problem of inhibitor analysis of various processes involved
`in DNA biosynthesis using substrate inhibitors will be examined in thts communication.
`It is known that most enzymes which catalyze the polycondensation of dNTP into the DNA
`chain are template-dependent enzymes, An exception is terminal deoxynucleotidyl transferase,
`with catalyzes the synthesis of DNA in a cell-free system in the absence of a specific program.
`, Special attention will be devoted to template DNA polymerases.
`Information currently available on DNA polymerases is quite fragmented. DNA polymerases
`from Esaheriahia aoU and certain phages parasitizing this bacterium have been studied in con(cid:173)
`siderable detail. Much attention has also been focused on n:tammalian DNA polymerases, which
`may be arbitrarily separated into replication (a), reparative (S), and mitochondrial (y) poly(cid:173)
`Investigations have also been made into DNA polymerases a and the mammalian terminal
`merases,
`deoxynucleotidyl transferase, although the biological functions of these enzymes are not clear.
`More detailed information on these enzymes may be found in review articles [1, 2].
`A comparative study of mammalian and mammalian virus DNA polymerases would be of particu(cid:173)
`lar importance. The differences in the properties of these enzymes provide an opportunity
`to block selectivity the reproduction of the viral genome in mammalian and human cells. Among
`the viral DNA-polymerases the DNA polymerases of herpes virus, variola vaccine, and adeno(cid:173)
`viruses, and among the retroviruses the reverse transcriptase of fowl myeloblastosis virus
`have been studied in the greatest detail. These polymerases markedly differ from each other, es-
`: pecially in regard to substrate analogs.
`Substrate inhibitors of DNA biosynthesis have different modes of action at the molecular
`level,
`
`1. The competitive inhibitors may compete with substrates for binding with the DNA-syn(cid:173)
`thesizing complex, which, in a cell-free system containing purified DNA polymerases, is usually
`the template-primer-DNA polymerase complex,
`In this case the competitive inhibitors are not
`incorporated into the DNA chain, but merely replace the dNTP in the complex by occupying a
`position either directly at the site at which dNTP is bound or close to this site.
`
`Institute of Molecular Biology, Academy of Sciences of the USSR, Moscow; Institute of
`Experimental Cardiology, All-Union Cardiological Research Center, Academy of Medical Sciences
`of the USSR, Moscow, Translated from ~olekulyarnaya Biologiya, Vol. 21, No. 1, pp. 33-38,
`January 1987, Original article submitted July 28, 1986.
`
`0026-8933/87/2101-0025$12.50
`
`© 1987 Plenum Publishing Corporation
`
`25
`
`(cid:1) (cid:1)(cid:2)(cid:3)(cid:4)(cid:5)(cid:6)(cid:7)(cid:8)(cid:9)(cid:10)(cid:3)(cid:7)(cid:11)(cid:5)(cid:6)(cid:7)(cid:12)(cid:5)(cid:13)(cid:9)(cid:5)(cid:14)(cid:10)(cid:15)(cid:8)(cid:9)(cid:16)(cid:8)(cid:9)(cid:17)(cid:5)(cid:13)(cid:12)(cid:5)(cid:18)(cid:15)(cid:14)(cid:12)(cid:10)(cid:3)(cid:19)(cid:2)(cid:8)(cid:5)(cid:11)(cid:7)(cid:20)(cid:5)(cid:21)(cid:1)(cid:3)(cid:8)(cid:11)(cid:9)(cid:5)(cid:22)(cid:23)(cid:5)(cid:24)(cid:25)(cid:26)(cid:25)(cid:5)(cid:18)(cid:15)(cid:17)(cid:9)(cid:27)(cid:1)
`
`

`

`;f,
`2, Terminator substrates also compete with substrates but they are incorporated into the
`DNA chain and then for various reasons arrest' subsequent elongation of the primer chain.
`3. Finally, substrate analogs, which also compete with substrates for binding with the
`DNA complex being synthesized, are incorporated in the DNA chain, but the rate of subsequent
`elongation of the DNA chain in• this case is significantly reduced.
`~,;';
`A number of systems are currently being used to study the properties of DNA substrate
`inhibitors. Firstly t~ere is t~e so-called acti~ated ?NA. Th~s is usually a do~ble-stranded.f
`DNA of random composition, and in one of the chains, with the aid of DNase, small nicks or gaps
`•~
`of tens or hundreds of nucleotide residues are formed (the selection of the chain for each J
`·.11".
`nick is random), These damages are "filled in" in the presence of dNTP with the aid of DNA-
`polymerases, but the results of such experiments provide oniy general information, and in thi~.'.;:
`case it i~ difficult to study the mode of action of the substrate inhibitors at the molecular··
`level.
`,"''
`Systems containing individual DNAs (phages, plasmids) are more convenient, Either singl~
`spiral circular DNAs (more frequently phage Ml3 DNA) with a synthetic primer or restrictase
`fragments in which one chain is partically hydrolyzed from the 3 1-end in the presence of E.
`aoU:exonuclease 111 are used in such systems, Additional systems, which are modifications of
`the systems described, are also used. With these systems it is possible to study the mode of{
`action of substrate inhibitors at the molecular level and also the effect of the primary strul·
`ture of the template on the processes involved in the synthesis,
`Finally in certain specific cases more complex experimental variants are used, Firstly,
`the above-mentioned systems with additional auxiliary replication proteins ( a protein which
`stabilizes single-spiral DNA and others) are used, Secondly, cell-free systems containing
`chromatin or cell nuclei are used,
`In such systems the .DNA polymerases are complexed with
`many other replication proteins but usually complete replicative complexes are not formed,
`Thirdly, there are cells which are permeable with' respect to dNTP as a result of various re(cid:173)
`actions and the resultant replicative complexes are apparently similar to complexes of normal, -
`cells. Although in this case one should take into account that at least in eukaryotic cells
`the substrate "flows" differ from.those which exist in normal replicating cells and that the
`relationship between the pathways of biosynthesis of different DNA substrates (de novo and ad~
`ditional) are markedly changed. Finally, there are cell cultures available with the aid of
`"!
`which replication, reparation, reverse transcrip'tion, and other processes in the presence of
`substrate inhibitors may be quite conveniently studied·; However, in thes~ systems it is pos-·
`sible to use only 2 '-deoxynucleosides, since dNTPs do not penetrate the cell wall. One should
`also take into account the possibility that analogs of the nucleosides in whole ce11 systems
`may be modified by certain metabolic enzymes and may exert their action in the form of other
`chemical structures,
`The principle of selecting substrates with matrix-primer-DNA-polymerase complexes as a
`physicochemical process has not been studied in detail, One aspect of this selection pro-
`cedure has _been investigated, i.e., the interaction between the substrate base and the tern-
`plate base leading to the formation of hydrogen bonds according to the Watson-Crick law, The
`triphosphate residue of the substrate which interacts with the enzyme is known to play an im-;
`portant role, but it is not known how significant the remaining elements in the dNTP molecule
`are, or whether certain other atoms in the bases and sugars are essential for the direct bind;
`ing with the complex, This information would be much more important in regard to the constru~(cid:173)
`tion of highly-specific inhibitors of viral DNA-polymerases, which are currently prepared in ·
`·
`an empirical manner.
`The 6-aryl-aminouracils, 6-arylhydrazinouracils, 6-arylhydrazinocytosines, N2 -n-butyl(cid:173)
`phenylguanine, 2-butylphenylaminoadenine, their 2 1-deoxyribosides, and also the 5 1-tri(cid:173)
`phosphates of the latter [3] are among·the most characteristic and potent representatives of.
`the first type of inhibitor (competitive inhibitors), These agents inhibit the synthesis of i
`DNA catalyzed by mammalian DNA-polymerases a, and by fowl by fowl myeloblastosis reverse tran,
`criptase, and also by a number -of bacterial and phage DNA-polymerases. These ;compounds in ··
`the fonl). of bases, 2 1-deoxynucleosides and 2 '-deoxynucleoside-5 '-triphosphates bind with the ::
`template-primer-DNA-polymerase ·complex and compete with substrates containing the analogous
`•··
`base, However, these inhibitors are not incorporated into the DNA chain,
`Such compounds have a selective action on DNA synthesis catalyzed by certain DNA poly-
`merases, since they form a strong complex with the template (three hydrogen bonds) which is
`
`.
`'
`
`.,,
`:
`
`', 2p
`
`

`

`TABLE 1, Terminator Activity of Substrate Analogs in the Bio(cid:173)
`. synthesis of DNA
`
`dNTP analog·
`
`-
`
`f
`
`'(3
`frorii
`·a, from
`I from
`E. coli :calf
`rat
`;thymusc liver
`--
`
`-
`
`. -·
`
`DNA-po l.ymerase
`Terminal Reverse
`trans-
`nucleo-
`tidyl
`cri¥tase PhageT4
`of owl
`trans-
`m1elo-
`ferase
`b astosis
`from
`virus
`calf
`thymus
`+
`+
`+
`+
`+
`+
`+
`
`ddNTP
`dNTP (3'NH2)
`dNTP (3'NHacet)
`dNTP (3'NHbio)
`dNTP(3'Ns)
`·dNTP(3'F)
`dNTP (3'0Me)
`araNTP (3'NH2)
`araNTP (3'N 3)
`dNTP(3'NH2) (a-S)
`dNTP (3'N 3) (a-S)
`dNTP (3'F) (a-S)
`
`-
`-
`-
`
`+
`+
`
`+
`+
`+
`-
`+
`+
`+
`+
`+
`+
`+
`+
`+
`-
`+-
`-
`+
`-
`+
`+
`-
`+
`+
`-
`-
`-
`+
`+
`+
`+
`-
`-
`-
`+
`+
`+
`-
`+-
`-
`+
`-
`-
`+
`+
`+
`(+), termination; (-), absence of termination; (+ -),
`Note.
`termination when there is a 10 2 -10 3-fold excess of analog over
`substrate; ddNTP, 2' ,3'~dideoxynucleoside-5'-triphosphates;
`dNTP (3'NH 2 ) , 31-amino-2',3'-dideoxynucleoside-5'-triphosphates;
`dNTP (3'NHacet) and dNTP (3'NHbio), 3'-acetyl- and 3'-biotinyl(cid:173)
`derivatives of dNTP (3'NH 2 ) respectively; dNTP (3'F), 3'-fluoro-
`2' ,3'-dideoxynucleoside-5 1-triphosphates; araNTP (3'NH 2 ) , 3'(cid:173)
`amino-3'-amino-31-deoxynucleoside-5'-triphosphates; araNTP(3'N3),
`Ji-azido-3'-deoxynucleoside-S'~triphos'phates; dNTP (3'0Me), 3'-
`0-methyl-dNTP; (a-S) denotes that a-phosphate has been replaced
`by a-thiophosphate.
`
`correctly orientated on account of the strong additional interaction between the aromatic
`(or other hydrophobic) substituent and the "pocket" of DNA polymerases present at this site,
`'This explanation of the facts is hypothetical. However, other DNA polymerases (f3, y, o) which
`do not have a corresponding "pocket" are not sensitive to arylnucleosides and their 5'-triphos(cid:173)
`'Phates, However, these inhibitors of DNA biosynthesis are not incorporated into the DNA chain,
`irrespective of the form in which they are added to the reaction mixture.
`Terminator substrates of DNA polymerases have already been studied in considerable de(cid:173)
`.. tail. There studies commenced in the laboratories of A. Kornberg and F. Sanger [ 4, 5], who
`. w~re the first research workers to determine the mode of action of dideoxynucleoside triphos(cid:173)
`phates (ddNTP) at the molecular level. When ddNTP molecules bound with the template-primer(cid:173)
`DNA-polyermase I complex from E. coli, the -nucleotide residues from ddNTP were incorporated
`a.t the 3'-end of the DNA chain in .accordance with the complementarity rule, but synthesis sub(cid:173)
`sequently ceased, since a hydroxyl group was ~ot present in the 3 1-position of these compounds
`and therefore a new phosphodiester bond could not be formed, The ddNTP molecules have been
`extensively used for determination of the primary structure of DNA by the polymerization method
`[5].
`
`More J;"ecent studies have shown that ddNTP molecules are not utilized by all DNA poly(cid:173)
`merases and are in fact not incorporated into the DNA chain by replicative-DNA-polymers [2],
`'!11is implied that there could be a possible route for creating new terminator substrates,
`,which are specific for DNA polymerases with different cellular functions,
`Extensive studies on such compounds have been carried out in recent years. Thes·e studies
`Were initiated when the synthesis of a number of dNTP (3'NH 2 ) compounds had been achieved, and
`,the latter were studied in experiments with purified DNA polymerases [6, 7], reparation chroma(cid:173)
`tin from liver [8], and sea urchin embryo replicating nuclei [9], and the corresponding nucleo(cid:173)
`sides were studied in experiments with mouse myeloma p 3 Ag653 cells in a sea urchin embryo cul-
`·ture [10], The arsenal of terminator substrates was later further extended, and dNTP(3'F)
`[11], araNTP(3 1NH 2 ) , araNTP (3'N 3 ) [12], dNTP(3'0Me) and dNTP(3 1N3 ) were studied. Data on
`these and certain other compounds have been combined and presented in Table 1 [2, 6, 7, 11,
`12].
`
`27
`
`

`

`Inhibitors of DNA Synthesis Catalyzed by Various
`TABLE 2.
`Mammalian and Mammalian Virus DNA-polymer~ses.
`DNA~poiymerases
`Terminal Various
`• nucleo-
`reverse
`Herpes
`tidyl
`trans-
`trans-
`criptases- viruses
`fe:tase
`-
`+
`-
`-
`
`\.-\i:ylnucleosides
`,faNTP
`' rdUTP
`. op-GTP
`. araNTP (2'N3)
`araNTP (2'F)
`
`I
`
`·+
`+
`-
`·-
`-
`+
`
`..
`
`+
`+
`+
`+
`
`Compounds
`
`a
`
`f3
`
`+
`+
`-
`-
`+
`+-
`
`-
`-
`+-
`-
`-
`
`"
`
`0
`
`-
`
`y
`
`-
`-
`+
`-
`
`araNTP, arabinonucleoside-·5 '-triphosphates; araNTP
`Note.
`(2'Ns), 2'-deoxy-2'-azido-araNTP;· araNTP (3'F), 2'-fluoro-
`21-deoxy-araNTP; BrdUTP, 5-(S-bromovinyl)-2'-deoxyuridine-
`(+), inhibition; (-), no inhibition; (+ -),
`5'-triphosphate.
`weakly pronounced inhibition. More detailed information may
`be found in the review [2].
`
`Certain conclusions can be drawn from the data in Table 1.
`1. None of the DNA polymerases has the ability to catalyze the formation of phosphoamide
`bonds •. This is apparent from the finding that dNTP(3'NH 2 ) and araNTP(3'NH 2 ) act as terminator,
`t1·
`of DNA synthesis and are not incorporated into the middle of the chain.
`2. DNA polymerase a possesses the highest specificity in comparison with other mammalian
`DNA polymerases examined (S and terminal deoxynucleotidyl transferase). Unfortunately data 011.~.
`the inhibitor analysis of DNA-polymerase y are not available. Similar data have also been ob~·
`tain:d for the DNA-rep.licase from the Chinese silkworm, Bombyxmori/.[13]. Non.e of the compound_.
`studied from the ddNTP, dNTP(3'NH 2 ) , dNTP(3'N 3 ) , dNTP(3'F) group inhibited the DNA synthesis,;
`reaction. However, ddNTP, dNTP(3 1 NH 2 ) , and dNTP(3'F) markedly inhibited the synthesis of DNA;,
`catalyzed by the Chinese silkworm nuclear polyhedrosis virus DNA polymerase [13],
`';
`3. On the other hand the reverse transcriptase has the lowest specificity for the termi71
`nator subst:ates. For example all compounds tes7ed in the deoxy- and arabino series terminatr_l~
`DNA synthesis in the presence of reverse transcriptase, but three groups, dNTP(3'Ns), araNTP , .
`(3'Ns), and dNTP(3'0Me), were found to be highly specific inhibitors for this enzyme in com- I
`parison with the mammalian enzymes,'DNA polymerases a and~. Thus, by means of certain alter8j
`tion~ in t~e terminator substrates, it· is pos~ibl~ to con~truct specific inhibitors of DNA syn]
`thesis, which is catalyzed by reverse transcriptase. It is not known what laws govern the con·
`struction of specific inhibitors of reverse transcriptases in regard to physicochemical calcu~
`lations. It is also not known whether low substrate specificity with respect to substrate
`·
`analogs is a feature of different reverse transcriptases and not only the fowl myeloblastosis{
`virus enzyme. There are data in the literature which indicate that the reverse transcriptase:
`of the HTLVIII/LAV virus also has relatively low specificity. For example, 3'-azido-2',3'(cid:173)
`dideoxythymidine blocked viral reproduction in a culture of human thymocytes infected with
`RTL VIII [4].
`It is known that reverse transcriptases are less specific towards the template structure!
`(they utilize single-spiral DNA, RNA, and also RNA methylated at the 2 '-hydroxyl positions and\
`RNA in which all 2 '-hydroxyls have been replaced by fluorine) and the base structure, since tlj_ 1
`number of mismatches occurring during DNA synthesis catalyzed by reverse transcriptases is 2-3,
`orders of magnitude higher than in the case of DNA synthesized by DNA polymerases a and ~ [21\
`,
`-
`This property of the reverse transcriptases is partically exp'ressed, when phosphate is
`replaced by thiophosphate in terminator substrates, for example dNTP(3 1 NH 2 ) (a-S) (Table 1),
`The decrease in specificity with respect to all components in the DNA biosynthesis system is
`probably a common property of reverse transcriptases.
`Another group of terminators of DNA biosynthesis appears, when herpes virus DNA-polymer-;
`ases are used. The two most active preparations in this group, the triphosphates of 9-(2-
`·
`hydroxyethoxymethyl)-guanine and 9-(1,3-dioxy-2-propoxymethy~).-guanine (nor-GTP), are se-
`
`1_
`
` .. _.s,.
`
`28
`
`

`

`>~ '!tectively incorporated into. the 3' end of the DNA chain, when the process is catalyze~ by
`}"therpus virus and cytomega~ovirus DNA polymerases, but these agents bind very weakly with the
`·c:t DNA polymerases of mammalian cells ( [15], see [2]). Both preparations cause irreversible in(cid:173)
`hibition of viral DNA-polymerases but the mechanism of this inhibition is now known.
`Finally, the third group of substrate inhibitors includes substrate analogs which are in(cid:173)
`corporated into the DNA chain and either retard the polymerization p~0cess (kinetic inhibition)
`;r having been incorporated into the DNA render the latter functionally inactive, A consider(cid:173)
`~ble number of analogs of this type is known. The compounds in _this group which are selective
`for particular DNA-polymerases are of interest.
`In this regard two groups of these analogs
`should be mentioned. One of these groups, arabinonucleoside-5 1-triphosphates, is incorporated
`into the DNA chain during catalysis in the presence of DNA polymerases a. Apparently 2'-sub(cid:173)
`stituted araNTPs (azide, halogen) are also incorporated. However, incorporation of mononucle(cid:173)
`otide residues of arabinonucleosides into the DNA chain inhibits the subsequent stages in the
`condensation process [2]. Another group of analogs includes the 5-substituted derivatives of
`2'-deoxyuridine-5'-triphosphate. The 2,;,..bromovinyl analog is 'the most selective of these com(cid:173)
`pounds [16] •
`Certain data on the different types of inhibitors are presented in Table 2. However, in
`contrast to the data _presented in Table 1, these data have been obtained in different labora(cid:173)
`tories and in different systems and the mode of action of these inhibitors at_the molecular
`level has not always been clearly determ-ined.
`- - -
`-
`In conclusion one might add that the creation and study of new substrate inhibitors of
`DNA synthesis has advanced considerably in dozens of laboratories throughout the world,
`Judg(cid:173)
`ing by the advances made in recent years one might hope that highly effective preparations
`with antiviral and anticancer activity may be developed.
`
`1.
`.. 2.
`
`3.
`
`'5.
`
`/6.
`. 7.
`
`12,
`
`13,
`
`.. 14,
`
`15,
`
`16,
`
`LITERATURE CITED
`A, Kornberg, DNA Replication, Freeman, San Francisco (1980), p. l-726.
`A,. A. Kraevskii and M.· K, Kukhanova, in: Advances in Science and Technology; Molecular
`Biology Series· [in Russian], All-Union Institute of Scientific and Technical, '.information,
`Moscow (1986), Vol, 22, p. 1-164.
`N, N. Khan, C, E, Wright, L. W. Dudycz, and N, C. Brown, Nucleic Acids Res., 13, 6331-
`6342 (1985),
`-
`M. R, Atkinson, M. P. Duetcher, A, Kornberg, A, F, Russell, and J, G, Moffatt, Biochemis-
`try, 8, 4897-4904 (1969),
`F, Sanger, S.· Neiclen, and A. R, Coulson, Proc. Natl, Acad, Sci, USA, J...i, 5463-5467
`(1977).
`z. Chidgeavadze, R, Beabealashvilli, A. Atrazhev, M. Kukhanova, A. Azhayev, and A.
`Krayevsky, Nucleic Acids Rev., 12, 1671-1686 (1984),
`R, Sh, Bibilashvili, Z, G, Chidzhavadze, A. A. Kraevskii, M. K. Kukhanova, A. M. Atrazhev,
`A. V, Azhaev, and T, V. Kutateladze, Biopolim. Kletka, .!., 293-306 (1985).
`A. Krayevsky, M. Kukhanova, A. Alexandrova, N. Belyakova, and V. Krutyakov, Biochim,
`Biophys. Acta, 783, 216-220 (1984).
`M, Kukhanova, A. Krayevsky, N. Terentyeva, and V. Rasskazov, Biochim. Biophys. Acta,
`783, 221-226 (1984).
`A. A, Kraevskii, S, V. Kochetkova, and A, V, Azhaev, Mol. Biol, 19, 173-176 (1985),
`z. Chidgeavadze, A. Scamrov, R. Beabealashvilli, E, Kvasyuk, G, Zaytseva, I. Mikhailipulo"
`G, Kowollik, and P, Langen, REBS Lett., 183, 275-278 (1975).
`A. V, Papchikhin, P. P, Purygin, A. V. Azhaev, A. A. Kraevskii, T. V. Kutateladze, Z. G.
`Chidzhavadze, and R, Sh, Bibilashvili, Bioorg. Khim., 11, 1367-1379 (1985).
`V. S, Mikhailov, K. A. Marlyev, D, 0, Ataeva, P. K. Kullyev, A. M. Atrazhev, and A, A.
`Kraevskii, Bioorg. Khim., 12, 620-632 (1986),
`R, L. Tolman, A, K. Field,J. D, Karkas, A. F. Wagner, J. J, Germershattsen, C. Crumpacker,
`and E, M. Scolnick, Biochim, Biophys. Res, Conunun., 128, 1329-1335 (1985),
`H. Mitsuya, K. J. Weinhold, P.A. Furman, M. N. Clair;-s. Lehrman, R. c. Gallo, D. Bolog'\"t'.
`nesi, D, W, Barry, and S. Broder, Proc. Natl, Acad, Sci, USA, 82, 7096-7100 (1985),
`H. S, Allaudeen, M, S, Chen, J, J, Lee, E, Declercq, and W. H.Prussoff, J, Biol, Chem,,
`ill, 603-606 (1982);
`
`1• •'
`
`29
`
`