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`
`340
`
`LETTERS TO NATURE
`
`NATURE VOL. 302 24 MARCH 1983
`
`Table 3 Expression of Lyt 1.1 and Lyt 2.1 antigens by FACS analysis
`
`Control mice
`VAA-fed mice
`Student's r-test
`
`% Lymph node cells expressing
`Lyt 1.1
`Lyt 2.1
`24.4± 1.4
`83.6± 1.1
`24.2±0.7
`90.1±0.6
`Non significant
`P<0.001
`difference
`
`Immunofluorescently stained CBA lymph node cells were analysed
`using FACS-11 (Becton-Dickinson) as described previously30
`3
`\ but
`'
`with the argon ion laser set at 300m W, 488 nm and the photomultiplier
`tube (EM! 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 1 04 lymph node cells. The fluorescein isothiocyanate(cid:173)
`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 cells30
`• The
`values in the table were calculated after subtracting the count for
`immunoglobulin-positive cells and represent the mean ±s.e. calculated
`from· seven determinations. These results have been repeated in two
`subsequent experiments.
`
`Our data are consistent with recent studies of Loveland and
`21 who have clearly shown that Lyt 1 + T cells alone
`McKenzie20
`'
`(depleted of Lyt 2+ T cells) were sufficient to restore immune
`responses generated against H-2 and non-H-2 alloantigens in
`A TXBM (adult thymectomized, irradiated and bone-marrow
`reconstituted) mice. Our data also corroborate their view21 that
`Lyt 1 + ce::lls 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 hypothesis17 that the
`anti-cancer action of vitamin A acetate 17
`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'h 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.
`
`R~::ceived 1 December 1982: accepted 21 January 1983.
`
`1. Moore, T. in The Vitamins (eds Sebrell, W. H. & Harris, R. S.) 245-266 (Academic, New
`York. 1967).
`2. Goodman, D. S. Fedn Proc. 38, 2501-2503 (1979).
`3. Bollag, W. in Retinoids, Advances in Basic Research and Therapy (eds Orfanos, C E. et
`al.) 5-11 (Springer, Berlin, 1981).
`4. Sporn, M. B. in Retinoids, Advances in Basic Research and Therap}' (eds Orfanos, C. E.
`e' a/.) 73-76 (Springer, Berlin, 1981).
`5. Pawson, B. A. Ann. N. Y. Acad. Sci. 359, 1-8 {1981).
`6. Dresser, D. W. Naru" 217,527-529 {1968).
`7. Floersheim, G. L. & Bollag. W. Transplantlltion 1S, 564-567 (1972) ..
`8. Jurin, M. & Tannock,l. F. Immunology 23,283-287 {1972).
`9. Cohen, B. E. & Cohen, I. K. J. Immrm. 111, 1376-1380 (1973).
`10. B1alcck, J. E. & Gifford, G. E. Proc. nam. Acad. Sci. U.S.A. 74, 5382-5386 {1977).
`11. Dennen, G. & Lotan, R. Eur. I. Immun. 8, 23-29 (1978).
`i2. Dennert, G., Crowley, C., Kouba, J. & Lotan, R.I. nam. Cancer fnst. 6:Z, 89-94 (1979).
`13. Goldfarb, R. H. & Herbennan, R. B. J. Immun. 126,2129-2135 (1981).
`14. Abb, J., Abb, H. & Deinhardt, F. Immunophdrmacolog)' 4, 303-310 (1982).
`15. Sporn, M. B. & Newton, D. L. Fedn Proc. 33,2528-2534 (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. Mednwar, P. B., Hunt, R. & Mertin, J. Proc. R. Soc. Lend. 8206, 265-280 {1979).
`19. Malkovsky, M, er al. J. Immun. 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. Transplantarion 33, 217-221 ( 1982).
`22. Moon, R. C., Grubbs, C. J. & Sporn, M. B. Cancer Res. 36, 2626-2630 (1976).
`23. Malkovsk}•, M., Asherson, C. L., Stockinger, B. & \l/atkins, M. C. Namre 300, 652-655
`{1982).
`24. Hughes, W. L. ez al. Fedn Proc. 23,640-648 (1964).
`25. Julius, M. H., Simpson, E. & Herzenberg. L.A. Eur. 1. Immtm. 3, 645-649 {1973).
`26. Mage, M.G., McHugh, L. L. & Rothstein, T. L. J. Immrm. Merh. 15, 47-56 {1977).
`27. Mage, M. e1 al. Eur. I. Immun.ll, 22&-235 {1981).
`28. Zembala, M. A., Asherson, G. L., James, B. M. B., Stein, V. E. & Watkins, M. C. I.
`Immun.129, 1823-1829 {1982).
`29. Gorini, G., Medgyesi, G. A. & Doria, G. J. Immun. 103, 1132-1142 (1969).
`30. Abehsira, 0., Edwards, A. & Simpson, E. Eur. I. Immun. 11, 275-281 (1981).
`31. Simon, M. M. el al. Eur. J. Immun. 11,246-250 (!9R1).
`
`0028-0836/83/120340-03$0' nn
`
`-
`
`Transfer of 21 cloned imm.u.:noglobu.lin
`light-chain gene to
`mutant h.ybridoma cells
`restores specific an.tibod.y production
`
`Atsuo Och!, Robert G. Hawley,
`Marc .J. Shulman* & Nobumichl Ho:mmi
`
`Ontario Cancer Institute and Department of Medical Biophysics,
`University of Toronto, 500 Sherbourne Street, Toronto,
`Canada M4X 1K9
`*Rheumatic Disease Unit, Wellesley Hospital, and
`Department of Medical Biophysics, University of Toronto,
`Toronto, Canadli M4Y 113
`
`The expression of immunoglollnilln (llg) genes is regulated at
`several lewels. For example, although ut-cluun production
`requires a DNA rearrangement that juxtaposes variable and
`joining segments', thds reiD!l'rangement is not sufficlemt for 1t •
`chain gene expres;ion; that is, some ceil types G!o not permit
`immunogiobuiin productionl-4. The mechanisms responsible
`for the repletion of ~he expression-of relm'anged immuno(cid:173)
`globulin genes are poorly understood. The tecluniqne of modify(cid:173)
`hug cloned genes in vitro and transferring the modified genes
`ao cells in cwmre provides a tool for ideniliying the smmctural
`fearures required for gene expression. To amalyse immuno(cid:173)
`glollmfua genes in this manner, however, it ns mt necessary to
`use, es recipients, cells that mom11ally permit immumogDobulin
`production. We report here that 111 cDoned :c-chain gene is
`expressed in immunoglobulim-producing hybridoma cells. Fur(cid:173)
`thermore, the product of the tran~ferred K -chain gene is capable
`of res~orillllg specific antibody p~cdnctiollll to the trlllnsformed
`celi.s.
`The hybridoma Sp603 produces IgM(K) specific for the hap(cid:173)
`ten 2,4,6-trinitrophenyl (TNP) 5
`• The rearranged gene encoding
`the TNP-specific K chain (KTNp) has been cloned . As recipient
`cells, we used the mutant cell line igk-14 which was derived
`from Sp603 and does not produce the KTNP chain. As shown
`in Fig. 3, the KTNP gene is apparently deleted from the igk-14
`cell line. Because the igk-14 cells still produce the TNP-specific
`IL heavy chain, it would be expected that the expression of the
`KTNP gene in these cells would restore the production of TNP(cid:173)
`specific IgM. To select for cells that have taken up the KTNP
`gene, the cloned KTNP gene, designated TKl, was inserted into
`the plasmid pSV2-neo, which contains a bacterial phos(cid:173)
`photransferase gene (neo) and confers resistance to the amino(cid:173)
`glycoside antibiotic G418 (ref. 7). In the present study, we have
`used two recombinant plasmids: in the plasmid pT-TKl, 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-TKl, the orientation of the gene is
`reversed (Fig. 1).
`To transfer the DNA, bacteria harbouring the pT-TKl and
`pR-TKl plasmids were converted to protoplasts and fused with
`igk-14, after the method of Schaffner". G418-resistant transfor(cid:173)
`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 10-4 to 10-5 per input igk-14 cell, and was comparable for
`the pT-Td, pR-TKl 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
`transform ants from pT- TKl and about 30% of the transfor(cid:173)
`mants from pR- TKl 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(cid:173)
`ing dilution and studied further. The production of the KTNP
`chain has been assayed by TNP-specific haemagglutination and
`
`© 1983 Macmillan Journals Ltd
`Sanofi/Regeneron Ex. 1 021 , pg 595
`
`pi
`a!
`(I
`cl
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`th
`gt
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`ol
`di
`cl
`fe
`ol
`th
`V(
`
`tr
`pt
`th
`D
`gt
`R
`T
`th
`T:
`th
`cc
`, pc
`in
`
`Merck Ex. 1021, pg 621
`
`

`
`!'!_NA~TU!}!!'R~Ec__:v~o~L"._1!3ogzJ2~4~M~A~RS:CH!!_!t2!98~3----------[LETTflTEEFR~S;i-01'i/\fURE
`
`341
`
`HI
`
`a
`
`b
`
`c
`
`d
`
`e
`
`f
`
`pBR 322 ori
`
`SV40on
`Fig. 1 Structure of TK 1 transducing plasmids. The TKl fragment
`(9.6 kb) 6 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-TKl (TKl inserted in
`tandem with the SV40 early promoter) and pR-Tk 1 (Tiel orienta(cid:173)
`tion reversed with respect to the SV40 early promoter). The
`directions of transcription of the KTNP gene and SV 40 early region
`are indicated by arrows.
`
`~
`!
`
`:i
`!
`
`.J
`
`plaque formation (Table 1). Synthesis of the KrNP 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 KTNP protein. Low level production of the KTNp
`chain by R11L3 can be detected both as low titre haemaggluti(cid:173)
`nation and as a weak band on SDS-PAGE. In general, the
`intensities of the bands corresponding to the KTNP chain are
`roughly proportional to the TNP-specific haemagglutination
`titres.
`To analyse the KTNP genes in the transformed cell lines,
`transformant DNA was digested with the restriction endonu(cid:173)
`clease BamHI, fractionated by agarose gel electrophoresis and
`transferred to nitrocellulose. The blot was then probed with a
`eDNA clone of the K constant-region gene segiLent (Fig. 3).
`Previous work has shown that this probe detects three K-chain
`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 KTNP
`gene6
`, 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(cid:173)
`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
`9
`10
`vectors7
`,
`'
`'
`We can estimate the copy number of the I<TNP gene in the
`transformants by comparing the intensity of the band corres(cid:173)
`ponding to the KTNP gene in DNA from the transformants with
`the intensity of the band corresponding to the KTNP gene in the
`DNA of Sp603 which apparently contains one copy of the KTNP
`gene per cell6
`• By this criterion, the transformants T3L2 and
`R31L4 have multiple copies. of the KTNP gene per cell, while
`T1L2 and Rl1L3 contain about 1 copy per cell. It is interesting
`that the transformant R31L4 makes more KTNP chain than does
`T3L2, although T3L2 has more copies of the Krx.n gene. Fur(cid:173)
`thermore, R31L4 makes about 10-fold less KTNP chain per gene
`copy than does the wild-type hybridoma, although it should be
`pointed out that we do not knew if all copies or the KTNP gene
`in R31L4 function equally efficiently. This variability in gene
`
`Identification of KTNrchain production in hybridoma cell
`Fig. 2
`lines and pT-TKl and pR-TKl transformants. Lane a, igk-14;
`lane b, T1L2; lane c, T3L2; lane d, R11L3; lane e, R31L4; lane
`{, Sp603. Secreted immunoglobulin was radiolabelled by incubat(cid:173)
`ing cells for 18 h in leucine-free Dulbecco's modified Eagle's
`medium containing 14C-leucine (5 ~-t-Ci ml- 1
`) and dialysed fetal
`calf serum (5%). Immunoglobulin was reduced and culture super-
`natants analysed by SDS-PAGE as described previousll.
`
`Tallie 1 Secretion of TNP-specific lgM by G418-resistant.
`transform ants
`
`Vector
`pT-TKl
`
`Cell lines
`Sp603
`igk-14
`Transformants
`TlL2
`T3L2
`T4Ll
`Tl2e
`Tl?Ll
`Tl9L1
`T21L2
`R2L6
`R9L3
`R10L18
`R11L3
`R20Ll
`R22Ll
`R31L4
`
`Haemagglutination TNP plaque
`titre
`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-Tid or pR-TKl plasmids. The methods for increaSing
`the plasmid copy number and making protoplasts have been described
`• About 1010 protoplasts were collected by centrifugation.
`elsewhere14
`107 igk-14 ce!Is, grown as described previousll, 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(cid:173)
`ene glycol 1000 (PEG), as described by Taniguchi and Miller for the
`production of T-cell hybridomas 15
`• 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% ietal 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 1 mg ml- 1 of G418 (a gift of
`Schering Corporation). The transformants resistant to 0418 were
`screened for the production of TNP-specific IgM by testing for TNP(cid:173)
`spedfic piaque 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 elsewhere 5 The sensitivity of the assay was enhanced by
`including monoclonal rat anti-t-L antibody C2-23, as described else(cid:173)
`where (M. Potash er a/., in prepara!ion). Plaquing was done on
`-104 cells, alsc as described previously'.
`
`. - - - - - - - - - - - - - ·------------
`-
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`--
`
`Sqpp'i'Bt£Q@QWQQ Ex 1Q?1 QQ §ftft
`
`- i
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`cs,
`
`1tedl at
`luction
`le and
`for IIC·
`permit
`msible
`munno-
`todify-
`genes
`BCWraJ
`IIUUIIIO•
`1acy to
`obulin
`ene is
`i. Fur-
`!pable
`ormed
`
`e hap-
`::oding
`:ipient
`erived
`;hown
`gk-14
`Jecific
`of the
`TNP-
`: KTNP
`d into
`phos-
`mino-
`:have
`, TKl
`be in
`SV40
`,ne is
`
`r and
`I with
`tsfor-
`ogend
`from
`lefor
`stant
`ecific
`stant
`sf or-
`;t, of
`ector
`
`imit-
`KTNp
`1 and
`
`als Ltd
`
`Merck Ex. 1021, pg 622
`
`

`
`~34~2 ----------------------------------iL£F',I,I~E~RS~TfO~N~A~JnURE----------------~N~A~~~~vo~L~-~30=2~2~4~MAR~c~H~1~9s=3
`
`N.
`
`a
`
`b
`
`c
`
`d
`
`e
`
`.r
`J
`
`9.6-
`
`5.9-
`5.4-
`
`Fig. 3 Blot hybridization of DNAs from hybridoma cell lines
`and pT-TKl and pR-TKl transformants. Lane a,.igk-14; lane b,
`T1L2; lane c, T3L2; lane d, R11L3; lane e, R31L4; lane{, Sp603.
`BamHI-digested DNA samples (20 f.Lg) were electrophoresed
`through a 1% agarose gel at 2 V em -I for 40 h. After transfer to
`· nitrocellulose 16
`, the blot was hybridized with a 32P-labelled eDNA
`clone of the K constant-region gene segment (pL21-5J provided
`by R. Wall) by a method described elsewhere .
`
`expression raises the question of whether all the regulatory
`elements of the normal KTNP gene are present or functioning
`on the cloned fragment. The messenger RNA cap sites have
`been identified for several immunoglobulin K -chain genes 11
`. In
`all cases, the cap site seems to be within 30 base pairs (bp) of
`the initiation codon. As the TKl fragment has 5 kb of DNA
`upstream of the initiation codon, it is likely that the DNA
`fragment carries the KTNP gene promoter. Similarly, the poly(A)
`addition site is estimated to lie 211 bp downstream of the
`constant-region gene segment12
`, 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 KTNP gene in the various transformants.
`Falkner and Zachau have studied the transient expression of
`mouse immunobulin K-chain genes in African green monkey
`cells (CVl), HeLa cells and mouse L cells 13
`• In the case of the
`monkey CVl 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(cid:173)
`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 Societ;
`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 17 and Oi et al. 18 have
`:ecently reported the expression of cloned K light chain genes
`m transformed lymphoid cells.
`
`Received 17 November 1982: accepted 19 January 1983.
`I. Seidman, J. G., Max, E. E. & Leder, P. Narure 280,370-375 (1979).
`2. Parslow, T. G. & Granner, D. K. Nature 299,449-451 (1982).
`3. Coffino, P., Knowles, B., Nathenson, S. G. & Scharff, M.D. Nature new Biol. 231,87-90
`(1971).
`4. Ar-Rmohdi, A., Tan. K. B. & Croce, C. M. Somatic Celf Genet. B, 151-161 (1982).
`5. KOhler, G. & Shulman, M. I. Eur. J. Irnmun. 10, 467-476 (1980).
`6. Haw\ey, R. G., Shulman, M. J., Murialdo, H., Gibson, D. M. & Hozumi, N. Proc. narn.
`Acad. Sci. U.S.A. 79,7425-7429 (1982).
`7. Southern, P. J. & Berg, P. J. molec. appl. Genet. 1, 327-341 {1982).
`8. Schaffner, W. Proc. r~arn. Acad. Sci. U.S.A. 77, 2163-2167 (1980).
`9. Mulligan, R. C. & Berg. P. Proc. narn. Acad. Sci. U.S.A •. 78,2072-2076 (1981).
`10. Canaani, D. & Berg. P. Proc. natn. Acad. Sci. U.S.A. 79,5166-5170 t1982).
`
`0028 ·0836/83/120342-03$01.00
`
`11. Kelley, D. E., Coleclough, C. & Perry, R. P. Cell29, 681-689 {1982).
`12. Hamlyn, P. H., Brownlee, G. G., Cheng, C. C., Gait, M. J. & Milstein •. c. Cell 15,
`1067-1075 (1978).
`13. Falkner, F. G. & Zachau, H. G. Natu'< 298,286-288 (1982).
`14. Sandri-Goldin, R. M., Goldin, A. L., Levine, M. & Glorioso, I. C. Malec. cell. Bioi. 1,
`743-752 (1981).
`15. Taniguchi, M. & Miller, J. F. A. P. J. exp. Med. 148,373-382 (1978).
`16. Southern, E. M. J. mofec. Biol. 97,503-517 (1975).
`17. Rice, D. & Baltimore, D. Proc. narn. Acad. Sci. U.S.A. 79,7862-7865 11982).
`18, Oi, V. T., Morrison, S. L., Herzenberg, L. A. & Berg, P. Proc. narn. Acad. Sci. U.S.A.
`80,825-829 (1983).
`
`Deduced amino add sequence
`from. the bovine
`oxytocin~neurophysin I
`precursor cD N A
`H. Land*:!:, M. Grez*:l:, §.Ruppert*, H. §dnmalet,
`M. R.ehbeint, D. Richtert & G. Schutz*
`• Institute of Cell and Tumor Biology, German Cancer Research
`Center, Im Neuenheimer Feld 280, D-6900 Heidelberg, FRG
`t Institut fiir Physiologische Chemie, Abteilung Zellbiochemie,
`Universitat Hamburg, UKE, 2000 Hamburg 20, FRG
`
`The nonapeptide hormone o~ocin-like arginine-vasopressin
`(A VP)1
`-4 is synthesized as part of a larger preclll'!lor polypep(cid:173)
`tide. The precursor also includes the neuoplnysin molecule with
`which the hoH"mone is associated in the neurosecretory graneles
`of the hypotha.Damo-pitoitary tract. A protein oE molecular
`weight (M,)- 20,000 has been isolated from supll'lloptic nuclei
`of rat hypothalami which, after tryptic cleavage, released a
`neurophysin-like molecule of M,-10,000 and an o!igopeptide
`related to oxytocin5
`• This res111lt was complemented by in vitro
`translation of bovine hypothalamic onRNA &.;~. Among the
`primary
`translation products a
`single polypeptide of
`M, -16,500 was shown to contain antigenic i!l!ellerminants rec(cid:173)
`ognized by specific antisera against bovine nenrophysin I and
`oxytocin. Here we report the IUJI!ino mcid seqmence of the bovine
`oxytocin-neurophysin I (OT -Npl) precursor which was derived
`from sequence analysis of the cloned eDNA. As is ~he case for
`the bovine surginine-vasopressin-nemrophysm II (A VP-Npll)
`precursor4
`, the signllll sequence of the 01'-Npl precu.rsor is
`immediately followed by the nonapeptide hormone which is
`co111nected to neurophysi.n I by a Giy-Lys-Arg seqeence. A
`striking featWire of the DWicleic 111cid sequence is the 197 -nncleo·
`tide long perfect homology with the AVP-Npll premrsor
`mRNA seqWience encoding tbe conserved middle part of
`ne111rophysins I ami II.
`A eDNA library of bovine hypothalamic mRNA 4 was
`screened for plasmids containing the mRNA sequence for the
`OT-Npi 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(cid:173)
`siderable cross-hybridization was expected between the mRNA
`sequences for the AVP-Npll precursor and the OT-Npl pre(cid:173)
`cursor.
`The cloned eDNA encoding the AVP-Np!I 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 4 7 and 16 members containing different
`types of eDNA inserts (see Fig. 1 for examples). The first group
`represented eDNA sequences specific for the A VP-Npii pre(cid:173)
`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(cid:173)
`Npi precursor c:QNA and of the previously described A VP(cid:173)
`Np!I precursor eDNA 4 plus the predicted amino acid sequences.
`
`t Present addresses: MIT Center for Cancer Research, 77 M2ssachusetts Avenue,
`Cambridge, MassachusetlS 02138, USA (H.L.); Universiry of Southern California. School of
`Medicine, Department of Microbiology, 2025 Zonal Avenue, Los Angeles, California
`90033, USA (M.G.).
`
`· © 1983.Macmillan Journals Ltd
`Sanofi/Regeneron Ex. 1 021 , pg 597
`
`Merck Ex. 1021, pg 623