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`NATURE VOL. 302 24 MARCH 1983
`
`
`
`3“
`
`Table 3 Expression of Lyt
`
`.1 and Lyt 2.1 antigens by FACS analysis
`
`Control mice
`
`% Lymph node cells expressing
`Lyt 1.1
`Lyt 2.1
`83.6:1.l
`24.4-i 1.4
`
`24.2t0.7
`90.1 $0.6
`VAA-fed mice
`Non significant
`P < 0.001
`Student’s t-test
`difference
`
`lrnrnunofluorescently stained CBA lymph node cells wereszargalysed
`using FACS-II (Becton-Dickinson) as described previously '
`, but
`with the argon ion laser set at 300 mW, 488 nm and the photomultiplier
`tube (EMI 9524A) at 750 V with a fluorescence gain of 8 and a light
`scatter gain of 4. Data for each individual mouse were derived from
`the analysis of 5 x 10‘ lymph node cells. The fluorescein isothiocyanate-
`coupled rabbit anti-mouse immunoglobulin (obtained from Nordic) was
`selected for its binding to viable cells expressing immunoglobulin at
`the cell surface while producing negligible staining of other cells”. The
`values in the table were calculated after subtracting the count for
`immunoglobulin-positive cells and represent the mean ts.e. calculated
`from-seven determinations. These results have been repeated in two
`subsequent experiments.
`
`Our data are consistent with recent studies of Loveland and
`
`McKenzie2°'2‘ who have clearly shown that Lyt 1* T cells alone
`(depleted of Lyt 2* T cells) were sufiicient to restore immune
`responses generated against H-2 and non-H-2 alloantigens in
`ATXBM (adult thymectomized, irradiated and bone-marrow
`reconstituted) mice. Our data also corroborate their view“ that
`Lyt 1* cells have a central role in allograft responses. In either
`case, vitamin A acetate seems to be unique in that it enhances
`immune responses against an allograft when given by mouth
`and the mechanism of this action will require much further
`attention.
`
`In summary, these findings support the hypothesis" that the
`anti—cancer action of vitamin A acetate "'22 is mediated through
`an immunological process and we have shown that it acts by
`increasing the representation of the Lyt 1"2‘ phenotype in the
`T-cell population.
`We thank Dr G. L. Asherson for the gift of the F(ab’)2 rabbit
`anti-mouse immunoglobulin and Prof. I. F. C. McKenzie for
`the monoclonal antibodies. We thank Drs E. Simpson
`and B. E. Loveland for discussions and Mrs Pat McFarlane
`for preparation of the manuscript. This work was supported by
`the _CRC and MRC.
`'
`Received 1 December 1982; accepted 21 January 1983.
`
`_
`
`._...-.—.—“""!".°.“-79°>'9‘!-"
`
`1. Moore, T. in The Vitamin: (eds Sebrell, W. H. & Harris, R. S.) 245-266 (Academic, New
`York, 1967).
`2. Goodman, D. S. Fedn Prac. 38, 2501-2503 (1979).
`3. Bollag, W. in Rerinaids, Advances in Basic Research and Therapy (eds Orfanos, C; E. at
`al.) 5-11 (Springer. Berlin, 1981).
`.
`.5
`Spcrn. M. B. in Retinoids, Advances in Basic Research and Therapy (eds Orfanos, C. E.
`et al.) 73-76 (Springer. Berlin, 1981).
`Pawson, B. A. Ann. N. Y. Acad. Sci. 359, 1-8 (1981).
`Dresser, D. W. Nature 217, 527-529 (1968_).
`Floersheim, G. L. & Bollag, W. Transplantation 15, 564-567 (1972).
`Jurin. M. & Tannock, I. F. Immunology 23, 283-287 (1972).
`Cohen, B. E. & Cohen, 1. K. J. Immun. 111, 1376-1380 (1973).
`Blalock, J. E. & Gifiord, G. E. Prac. narn. Acad. Sci. U.S.A. 74, 5382-5386 (1977).
`Dennert, G. & Lotan, R. Eur. J. Immun. 8, 23-29 (1978).
`. Dennert, G., Crowley, C., Kouba, J. & Lotan, R. J. nam. Cancer Inst. 62, 89-94 (1979).
`. Goldfarb, R. H. & Herberman, R. B. J. Immun. 126, 2129-2135 (1981).
`14. Abb, 1., Abb, H. & Deinhardt, F. Imrnunapharmacalagy 4. 303-310 (1982).
`I5. Sporn, M. B. & Newton, D. L. Fedn Pine. 38, 2528-2534 (1979).
`16. Bollag, W. & Matter, A. Ann. N. Y. Acad. Sci. 359, 9-23 (1981).
`17. Medawar, P. B. & Hunt, R. Immunology 42, 349-353 (1981).
`18. Medawar, P. B., Hum. R. & Merlin, 1. Proc. R. Soc. Lond. B206, 265-280 (1979).
`19. Malkovsky, M, er al. J. Irnmun. 130, 785-790 (1983).
`20. Loveland, B. E., Hogarth, P. M., Ceredig, Rh. & McKenzie, I.F.C. J. exp. Med. 153,
`1044-1057 (1981).
`21. Loveland, B. E. & McKenzie, I. F. C. Transplantation 33, 217-221 (1982).
`22. Moon, R. C., Grubbs, C. J. & Sporn, M. B. Cancer Res. 36. 2626-2630 (1976).
`23. Malkovsky, M., Asherson, C. L., Stockinger, B. & Watkins, M. C. Nature 300, 652-655
`(1982).
`24. Hughes, W. L. 81' al. Fedn Prac. 23, 640-648 (1964).
`25. Julius, M. 1-1., Simpson, E. & Herzenberg, L. A. Eur. J. Immun. 3, 645-649 (1973).
`26. Mage, M. G., MCI-lugh, L. L. & Rothstein, T. L. J. Immun. Math. 15, 47-56 (1977).
`27. Mags, M. 21 al. Eur. J. Immun. 11, 228-235 (1981).
`28. Zembala, M. A., Asherson, G. L., James, B. M. B.. Stein, V. E. & Watkins. M. C. J.
`Imn-um. 129, 1823-1829 (1982).
`29. Gorini, G., Medgyesi, G. A. 8: Doria, G. J. Imrnun. 103, 1132-1142 (1969).
`30. Abehsira, 0., Edwards, A. & Simpson, E. Eur. J. Immun. 11, 275-281 (1981).
`31. Simon, M. M. er al. Eur. J. Immun. 11, 246-250 (1981).
`
`Transier of a cloned immunoglobulin
`light-chain gene to
`mutant hyhridorna cells
`restores specific antibody production
`
`Atsuo Ochi, Robert G. llawley,
`Marc E. Shulman* & Nohumichi Hozumi
`
`Ontario Cancer Institute and Department of Medical Biophysics,
`University of Toronto, 500 Sherboume Street, Toronto,
`Canada M4X 1K9
`* Rheumatic Disease Unit, Wellesley Hospital, and
`Department of Medical Biophysics, University of Toronto,
`Toronto, Canada M4Y 113
`
`The expression of immunoglobulin (lg) genes is regulated at
`several
`levels. For example, although at-chain production
`requires a DNA rearrangement that juxtaposes variable and
`joining segments‘, this rearrangement is not suficient for K-
`chain gene expression; that is, some cell types do not permit
`immunoglobulin production“. The mechanisms responsible
`for the regulation of the expression.of rearranged immuno-
`globulin genes are poorly understood. The technique of modify-
`ing cloned genes in vitro and transferring the modified genes
`to cells in culture provides a tool for identifying the structural
`features required for gene expression. To analyse immuno-
`globulin genes in this manner, however, it is iirst necessary to
`use, as recipients, cells that normally permit immunoglobulin
`production. We report here that a cloned ac-chain gene is
`expressed in immunoglobulin-producing hybridoma cells. Fur-
`thermore, the product oi the transferred K-chain gene is capable
`of restoring specific antibody production to the transformed
`cells.
`
`The hybridoma Sp603 produces IgM(r<) specific for the hap-
`ten 2,4,6-trinitrophenyl (TNP)5. The rearranged gene encoding
`the TNP—specific K chain (K-nvp) has been cloned . As recipient
`cells, we used the mutant cell line igk-14 which was derived
`from Sp603 and does not produce the Km chain. As shown
`in Fig. 3, the Km gene is apparently deleted from the igk-14
`cell line. Because the igk—14 cells still produce the TNP—specific
`p. heavy chain, it would be expected that the expression of the
`Km; gene in these cells would restore the production of TNP-
`specific IgM. To select for cells that have taken up the K1-Np
`gene, the cloned K-mp gene, designated TK1, was inserted into
`the plasmid pSV2-neo, which contains a bacterial phos-
`photransferase gene (neo) and confers resistance to the amino-
`glycoside antibiotic G418 (ref. 7). In the present study, we have
`used two recombinant plasmids: in the plasmid pT-TK1, TK1
`was inserted so that the direction of transcription would be in
`the same direction as transcription initiated from the SV40
`early promoter;
`in pR-TKI,
`the orientation of the gene is
`reversed (Fig. 1).
`To transfer the DNA, bacteria harbouring the pT—T;<1 and
`pR—T:<1 plasmids were converted to protoplasts and fused with
`igk-14, after the method of Schafiner“. G418-resistant transfor-
`mants of igk—14 were then selected as described in the legend
`to Table 1. The frequency of G418-resistant cells ranged from
`5 X 107" to 10'5 per input igk-14 cell, and was comparable for
`the PT-TK1, pR-TK1 and pSV2—neo plasmids. The resistant
`cells were then tested for the ability to make TNP—specific
`plaques. By this criterion about 10% of the G418-resistant
`transformarrts from PT-TK1 and about 30% of the transfor-
`mants from pR-TK1 made TNP—specific IgM. By contrast, of
`30 G418-resistant transformants from the pSV2-neo vector
`alone, none made TNP—specific plaques.
`_
`Representative transformants were selected, cloned by limit-
`ing dilution and studied further. The production of the K-rNP
`chain has been assayed by TNP—specific haemagglutination and
`
`0028-0836/83/120340-0350‘ 00
`
`Genzyme Ex. 1021, pg 598
`
`© 1983 Macmillan Journals Lid
`
`Genzyme Ex. 1021, pg 598
`
`

`
`' NATURE VOL. 302 24 MARCH 1983
`
`————-— i"(')'|\:,'.‘;'IUR”‘E
`
`
`
`DER 322 ori
`
`5V4o°"
`
`Fig. 1 Structure of T.-<1 transducing plasmids. The TKJ fragment
`(9.6 kb)5 was inserted into the BamHI site of pSV2-neo (donated
`by P. Berg)7 and transfected into Escherichia coli cells (C600) to
`obtain the two types of recombinant, pT-TK1 (TK1 inserted in
`tandem with the SV40 early promoter) and pR-Tk 1 (T7c1 orienta-
`tion reversed with respect to the SV40 early promoter). The
`directions of transcription of the K'rNp gene and SV4O early region
`are indicated by arrows.
`
`'
`
`‘ plaque formation (Table 1). Synthesis of the Km; chain has
`also been measured by SDS-polyacrylamide gel electrophoresis
`: (PAGE) (Fig. 2). The recipient cells igk-14 still produce the K
`, chain from the myeloma X63-Ag8 used to generate the parental
`_, Sp603 hybridoma, and this myeloma K chain migrates more
`slowly than the Kmp protein. Low level production of the Km,
`chain by R11L3 can be detected both as low titre haemagg1uti-
`nation and as a weak band on SDS-PAGE. In general, the
`intensities of the bands corresponding to the KTN}: chain are
`roughly proportional to the TNP-specific haemagglutination
`titres.
`To analyse the Kmp genes in the transformed cell lines,
`transformant DNA was digested with the restriction endonu-
`‘_ clease BamHI, fractionated by agarose gel electrophoresis and
`transferred to nitrocellulose. The blot was then probed with a
`cDNA clone of the K constant-region gene segment (Fig. 3).
`Previous work has shown that this probe detects three K-chain
`A genes in DNA from the Sp603 hybridoma—the bands at 5.9
`and 5.4 kilobases (kb) correspond to K-chain genes donated by
`the myeloma parent; the band at 9.6 kb represents the K-mp
`gene“, and this band is not observed in the case of igk-14. For
`» all the G418—resistant transformants tested, including the T1L2
`cells which do not make TNP-specific IgM, a band at 9.6 kb
`was observed with this probe. Results from DNA blot analysis
`of high molecular weight transformant DNA before and after
`digestion with the restriction endonuclease EcoRI
`(which
`cleaves the recombinant plasmids once) suggest that the trans-
`ferred sequences are integrated into cellular DNA as tandem
`oligomers (data not shown). These results are consistent with
`those obtained by other investigators using the pSV2 transfer
`vectors7'9"°.
`We can estimate the copy number of the K-nqp gene in the
`transformants by comparing the intensity of the band corres-
`ponding to the Km; gene in DNA from the transform-ants with
`the intensity of the band corresponding to the Kfivp gene in the
`DNA of Sp603 which apparently contains one copy of the Km;
`gene per cells. By this criterion, the transformants T3L2 and
`R31L4 have multiple copies. of the Km,» gene per cell, while
`A T1L2 and R11L3 contain about 1 copy per cell. It is interesting
`that the transformant R31L4 makes more K;-Np chain than does
`T3L2, although T3L2 has more copies of the Kn.-P gene. Fur-
`thermore, R31L4 makes about 10-fold less K-mp chain per gene
`copy than does the wild-type hybridoma, although it should be
`pointed out that we do not know if all copies of the Kmp gene
`in R31L4 function equally efficiently. This variability in gene
`
`.
`
`
`
`Identification of romp-chain production in hybridoma cell
`Fig. 2
`lines and pT-TK1 and pR-TK1 transformants. Lane a, igk-14;
`lane b, T1L2; lane c, T3L2; lane :1’, R11L3; lane e, R31L4; lane
`f, Sp603. Secreted immunoglobulin was radiolabelled by incubat-
`ing cells for 18h in leucine-free Dulbecco’s modified Eagle’s
`medium containing “C-leucine (5 u.Ciml_l) and dialysed fetal
`calf serum (5%). Immunoglobulin was reduced and culture super-
`natants analysed by SDS-PAGE as described previouslys.
`
`Table 1 Secretion of TNP-specific lgM by G418-resistant ,
`transformants
`
`Vector
`pT-TK1
`
`pR-TK1
`
`Cell lines
`
`Sp603
`igk-14
`Transformants
`T1I.2
`T3L2
`T4L1
`T12e
`Tl7l.1_
`T19L1
`'I‘21L2
`RZL6
`R9L3
`R10L18
`R1 113
`R20L1
`R22L1
`R31L4
`
`Haemagglutination
`titre
`
`TNP plaque
`formation
`
`1 / 1,280
`< 1 /2
`
`<1/2
`1/160
`< 1/2
`<1 /2
`1/20
`<1 /2
`<1 /2
`1/1,280
`<1 /2
`<1 /2
`1/20
`< 1/2
`1 / 640
`1/320
`
`+
`-
`
`—
`+
`—
`—
`—
`—
`—
`+
`-
`—
`-
`—
`+
`+
`
`The igk-14 cells were fused with bacterial protoplasts containing
`either the pT-TK1 or pR-TK1 plasmids. The methods for increasing
`the plasmid copy -number and making protoplasts have been described
`elsewhere”. About 101° protoplasts were collected by centrifugation.
`107 igk-14 cells, grown as described previouslys, were washed’ and
`resuspended in Gibco H21 medium, and centrifuged onto the protoplast
`pellet. The protoplasts and igk-14 cells were then fused with polyethyl-
`ene glycol 1000 (PEG), as described by Taniguchi and Miller for the
`production of T-cell hybridomas”. Briefly, after centrifugation, the
`cell-protoplast pellet was gently resuspended in 2 ml of H21 medium
`containing 40% PEG and 10% dimethyl sulphoxide. After 15 s, the
`mixture was added to an equal volume of H21 medium containing 50%
`PEG and mixing was continued for a further 15 s. The cells were then
`diluted to 50 ml with H21 medium containing 20% fetal calf serum,
`washed once in the same medium and plated in 24-well culture dishes
`at a cell density of 105 cells per well. After 24 h, the fused cells were
`selected in H21 medium containing 1n1gml'1 of 6418 (a gift of
`Schering Corporation). The transformants resistant
`to C-418 were
`screened for the production of TNP-specific lgM by testing for TN?-
`specific plaque formation. Representative transformants were then
`cloned by limiting dilution. Culture supernatants were tested for
`haemagglutination of TNP-coupled sheep red cells (TNP-SRC) as
`described elsewhere5. The sensitivity of the assay was enhanced by
`including monoclonalrat anti-p. antibody C2-23, as described else-
`where (M. Potash 8.’ al.,
`in preparation). Plaquing was done on
`~10“ cells, also as described previously’.
`
`
`
`Genzyme Ex. 1021, pg 599
`
`Genzyme Ex. 1021, pg 599
`
`

`
`342
`
`NATURE VOL. 302 24 MARCH 1983
`
`LETTERS TO NATURE
`11. Kelley. D. B., Coleclough, C. & Perry, R. 1’. Cell 29, 681-689 (1982).
`12. I-Iamlyn, P. H., Brownlee, G. G., Chang, C. C., Gait. M. J. & Milstein.-C. Cell 15.
`1067-1075 (1978).
`13. Falkner. F. G. & Zachau, H. G. Nature 298, 286-288 (1982).
`14. Sandri-Goldin, R. M., Goldin, A. I... Levine, M. & Glorioso. J. C. Molec. cell. Biol. 1,
`743-752 (1981).
`15. Taniguchi. M. & Miller. J. F. A. P. J. exp. Med. 148, 373-382 (1978).
`16. Southern. E. M. J. malec. Biol. 97, 503-517 (1975).
`17. Rice, D. & Baltimore. D. Prac. nam. Acad. Sci. U.$.A. 79, 7862-7865 (1982).
`18. Oi. V. T., Morrison, S. L.. Herzenberg, L. A. & Berg, P. Prac. narn. Acad. Sci. U.S.A.
`80, 825-829 (1983).
`
`
`Deduced amino acid sequence
`irons the bovine
`oxytocin-ueurophysin E
`precursor cDNA.
`
`H. Land*:i, M. Grez*:l:, S. Rupperf“, H. Schrnalei,
`M. Rehbeini‘, D. Richter‘? & G. Schiitz*
`* Institute of Cell and Tumor Biology, German Cancer Research
`Center, Im Neuenheimer Feld 280, D-6900 Heidelberg, FRG
`‘r Institut fiir Physiologische Chemie, Abteilung Zellbiochemie,
`Universitiit Hamburg, UKE, 2000 Hamburg 20, FRG
`
`The nonapeptide hormone oxytocin-like arginine-vasopressin
`(AVP)“‘ is synthesized as part of a larger precursor polypep-
`tide. The precursor also includes the neurophysin molecule with
`which the hormone is associated in the nenrosecretory granules
`of the hypothalamo-pituitary tract. A protein of molecular
`weight (M.) ~ 20,000 has been isolated from supraoptic nuclei
`of rat hypothalami which, after tryptic cleavage, released a
`neurophysin-like molecule of M,~ 10,000 and an oligopeptide
`related to oxytocins. This result was complemented by in uitro
`translation of bovine hypothalamic mRNA”. Among the
`primary
`translation
`products
`a
`single
`polypeptide
`of
`M,~16,500 was shown to contain antigenic determinants rec-
`ognized by specific antisera against bovine neurophysin I and
`oxytocin. Here we report the amino acid sequence of the bovine
`oxytocin—neurophysin I (OT-Npl) precursor which was derived
`from sequence analysis of the cloned cDNA. As is the case for
`the bovine arginine-vasopressin-neurophyin II (AVP-Npll)
`precursor‘, the signal sequence of the OT-Nplprecursor is
`immediately followed by the nonapeptide hormone which is
`connected to neurophysin l by a Gly-=Lys-Arg sequence. A
`striking feature of the nucleic acid sequence is the 197-nnclem
`tide long perfect homology with the AVP-Npfl precursor
`mRNA sequence encoding the conserved middle part of
`neurophysins I and ll.
`A cDNA library of bovine hypothalamic mRNA‘ was
`screened for plasmids containing the mRNA sequence for the
`OT-Npl precursor. Bovine neurophysins I and II show nearly
`80% homology in their amino acid sequences,
`including a
`complete homology between amino acids 10 and 74, so con-
`siderable cross-hybridization was expected between the mRNA
`sequences for the AVP-NpII precursor and the OT-Npl pre-
`cursor.
`
`The cloned cDNA encoding the AVP—NpII precursor‘ was
`therefore chosen as a hybridization probe for in situ colony
`screening9. Out of 5,000 recombinants, 63 clones gave a positive
`hybridization signal. Restriction analysis of
`the plasmids
`revealed two groups of 47 and 16 members containing different
`types of CDNA inserts (see Fig. 1 for examples). The first group
`represented cDNA sequences specific for the AVP-NpII pre-
`cursor‘. Sequence analysis of members of the second group
`(Fig. 1) showed that these contained OT—NpI precursor-specific
`sequences.
`Figure 2 shows the nucleotide sequences of the cloned OT-
`Npl precursor CDNA and of the previously described AVP—
`NpII precursor CDNA‘ plus the predicted amino acid sequences.
`
`1 Present addresses: MIT Center for Cancer Research, 77 Massachusetts Avenue,
`Cambridge, Massachusetts 02138. USA (H.L.); University of Southern California, School of
`Medicine, Department of Microbiology. 2025 Zonal Avenue, Los Angeles, California
`90033. USA (M.G.).
`
`
`
`.1’_._..-4;-._.
`
`
`
`Fig. 3 Blot hybridization of DNAs from hybridoma cell lines
`and p'l'—TK1 and pR-TK1 transformants. Lane a,.igk-14; lane b,
`T1L2; lane c, T3L2; lane d, R11L3; lane e, R31L/l; lane f, Sp603.
`BamHI-digested DNA samples (20 ug) were electrophoresed
`through a 1% agarose gel at 2 V cm" for 40 h. After transfer to
`nitrocellulose”, the blot was hybridized with a 321’-labelled cDNA
`clone of the K constant-region gene segment (pL21-5 provided
`by R. Wall) by a method described elsewhere‘.
`
`'
`
`expression raises the question of whether all the regulatory
`elements of the normal K-1-Np gene are present or functioning
`on the cloned fragment. The messenger RNA cap sites have
`been identified for several immunoglobulin K-chain genes“. In
`all cases, the cap site seems to be within 30 base pairs (bp) of
`the initiation codon. As the TK1 fragment has 5 kb of DNA
`upstream of the initiation codon,
`it is likely that the DNA
`fragment carries the Kjwp gene promoter. Similarly, the poly(A)
`addition site is estimated to lie 211 bp downstream of the
`constant-region gene segment”, which is well within the 1.2 kb
`of downstream flanking DNA. Experiments are in progress to
`determine the molecular basis for the differences in the level
`of expression of the Km; gene in the various transformants.
`Falkner and Zachau have studied the transient expression of
`mouse immunobulin K-chain genes in African green monkey
`cells (CV1), HeLa cells and mouse L cells”. In the case of the
`monkey CV1 cells and the HeLa cells, the K-chain gene was
`expressed only when transcription of the gene was placed under
`the’ direct control of an SV40 promoter after removal of the
`putative K -chain gene promoter region. In the case of the mouse
`L cells, the K-chain gene was not expressed. There are many
`structural differences between the vectors used by Falkner and
`Zachau and those used here. On the other hand, differences in
`the cellular environments might underlie the different require-
`ments for K-(chain gene expression. It should be possible to
`resolve these differences by using the gene transfer .system
`described here on cells of different types.
`'
`This work was supported by grants from the MRC of Canada,
`the National Cancer Institute of Canada, the Arthritis Society
`of Canada and the Allstate Foundation. A.O. was supported
`by a research fellowship of the National Cancer Institute of
`Canada. R.G.H. was supported by a studentship of the MRC
`of Canada.
`Note added in proof: Rice and Baltimore” and Oi er al.” have
`recently reported the expression of cloned K light chain genes
`in transformed lymphoid cells.
`Received 17 November 1982; accepted 19 January 1983.
`Seidman, J. (3.. Max, E. E. & Leder, P. Nature 280. 370-375 (1979).
`. Parslow. T. G. & Granner, D. K. Nature 299, 449-451 (1982).
`Cofiino, P., Knowles. B., Natl-ienson, S. G. & Scharfi, M. D. Nature new Biol. 231, 87-90
`(1971).
`Ar—Rushdi. A.. Tan. K. B. & Croce. C. M. Somatic Cell Genet. 8, 151-161 (1982).
`Kfihler, G. & Shulman, M. J. Eur. J. Immun. 10, 467-476 (1980).
`. I-Iawley, R. G., Shulman, M. 1., Murialdo, I-I.. Gibson, D. M. & Hozumi. N. Prac. nam.
`Acmi. Sci. U.S.A. 79, 7425-7429 (1982).
`Southern. P. J. & Berg, 1’. J. malec. appl. Genet. 1, 327-341 (1982).
`Schaffner, W. Prac. nam. Acad. Sci. U.S.A. 77, 2163-2167 (1980).
`Mulligan, R. C. & Berg. P. Proc. nam. Acad. Sci. U.S.A. 78. 2072-2076 (1981).
`. Canaani. D. & Berg. P. Prac. nam. Acad. Sci. U.S.A. 79, 5166-5170 (1982).
`
`
`
`
`
`C)09“."¢".V'.“5‘""?"
`
`0028.-0836/83/120342—03$01.00
`
`Genzyme Ex. 1021, pg 600
`
`‘
`
`'
`
`' © 19s3'Macmiuan Journals Ltd
`
`Genzyme Ex. 1021, pg 600