286
`
`
`
`Fig. 3 Double labelling of normal blood B lymphocytes for
`surface immunoglobulin and vimentin filaments. Cells were first
`stained with rhodamine-conjugated F(ab’)2 fragments of rabbit
`IgG to pt chain, washed and allowed to cap. The cells were then
`fixed and stained for vimentin as described in Fig.
`1
`legend.
`Controls with fluoresceinated IgM having no anti—IF reactivity
`were negative. :1, Staining for vimentin; the arrow indicates polar
`redistribution of vimentin in a B cell with redistributed surface
`immunoglobulin; b, same field of view showing surface staining
`with rhodamine-conjugated alltl-p. antibodies.
`
`Nature Vol. 298 15 July 1982
`
`Recombinant plasmids were constructed which contained, in
`different positions, the functional K-chain gene of myeloma T,
`called T1 (refs 6, 7), or the aberrantly joined, but expressed,
`MPC 11 Li gene“'1° (see Fig. 1). Four of the plasmids had the
`K-chain genes inserted in the direction of late,
`two in the
`direction of early SV40 transcription. The coding region of the
`T1 gene is located close [31 base pairs (bp)] to the SV40
`sequences with their promoters in pTKpn6 alone; in the other
`plasmids there are 0.8 and 3.0 kilobases (kb) of mouse DNA
`(with the putative promoters) upstream of the T1 and the
`MPC-11 Lf gene, respectively.
`In pTKpn6-transfected CV1 cells (transfection was done as
`described in ref. 11), a distinct transcript was detected having
`the size of the light chain mRNA from myeloma T, indicating
`correct splicing of the primary transcript (Fig. 2). Standardized
`blot hybridization experiments (not shown) indicated that the
`content of specific l<-chain mRNA in total cellular RNA of
`pTKpn6-transfected CV1 cells was 30-40% that of myeloma
`T cells. Transfection of CV1 cells with the other plasmids led
`to patterns lacking the distinct transcript band (see Fig. 2;
`similar patterns were obtained with pTKpn1,
`the MPC 11
`plasmids, and with pBR322). From DNase and RNase diges-
`tions,
`the hybridizing material consists mainly of partially
`degraded plasmid DNA, but it is possible that it also contains
`Cu A7.0kb
`
`mechanical integration of various cytoplasmic organellesl. The
`very close relationship between the redistribution of surface
`molecules and that of lymphocyte vimentin suggests a novel
`function for intermediate filaments.
`This work was supported by grant 81.1029 from INSERM.
`Received 22 February; accepted 13 May 1982.
`1. Lazarides, E. Nature 283. 249-256 (1980).
`2. Dellagi, K., Brouet. J. C... Perreau. J. & Paulin, D. Proc. natn. Acad. Sci. U.S.A.
`4464150 (1982).
`3. Gabhiani. G.. Chaponnier, C., Zumbe, A. & Vassalli. P. Nature 269, 697—698 (1977).
`P
`Schreiner, G. F., Fumiwara. K., Pollard, T. D. & Unanue, E. R. J. exp. Med. 145,
`1393-1398 (1977).
`Unanue, E. R. & Karnovsky, M. J. J. exp. Med. 140, 12074220 (1974).
`Schreiner, G. F. & Unanue, E. R. Adv. Immun. 24, 37-165 (1976).
`L001’. F. Adv. Immun. 30, 1-120 (1980).
`Bourguignon, L. Y. W., Nagpal, M. L. & Hsing, Y. C. J. Cell Biol. 91, 889-894 (1981).
`Schliwa, M. & Van Blerkom, 1.1. Cell Biol. 90, 222—23S (1981).
`Daley, R. A. & Eckert, B. S. J. Cell Elol. 91, 239 (1981).
`Braun, I. & Unanue, E. R.
`in B Lymphocytes in the Immune Response: Functional,
`Developmental, and Interactive Properties Vol. 15 (eds Klinman, N., Mosier, D. E.,
`Seller. 1. & Vitetta, E. S.) 169-175 (Elsevier, Amsterdam. 1981).
`
`
`'19.
`
`."'.°.‘°.°°.".°‘."'
`
`_...
`
`Expression of mouse
`immunoglobulin genes in monkey cells
`
`Falko G. Falkner & Hans G. Zachau
`
`Institut fur Physiologische Chemie, Physikalische Biochemie und
`Zellbiologie der Universitiit Miinchen, 8000 Miinchen 2, FRG
`
`The generation of functional immunoglobulin light-chain genes
`from variable (V), joining (J), and constant (C) gene segments
`has been elucidated recently”. The structural features which
`regulate the expression of immunoglobulin genes, however,
`have not been well defined. To further our understanding of
`immunoglobulin gene expression, African green monkey cells
`(CV1), HeLa cells, and mouse L cells were transfected with
`K-chain gene-containing plasmids. We report here that efficient
`transient expression occurred in the CV1 and HeLa systems
`when the gene was placed, in a recombinant plasmid, under
`simian virus 40 (SV40) promoter control. This is the first report
`of immunoglobulin gene expression in a eukaryotic gene trans-
`fer system. No significant expression of the K-chain genes under
`the control of their own promoters was detected, however, in
`systems in which, for example, the B-globin gene is expressed
`from its own promoter”. Possible reasons for this difference
`are discussed.
`
`0O28~0836/82/280286—()3$O1.00
`
`
`
`5’
`the
`pTKpn6,
`plasmids.
`recombinant
`of
`1 Construction
`Fig.
`EcoRI/BamHI fragment (5.5 kb) of fragment Tl (ref. 6) was ligated to the
`large EcoRI/Baml-II fragment of pBR322; the resulting plasmid pT1 was
`cleaved with Kprll, treated with alkaline phosphatase, and ligated with
`Kpnl-cleaved SV40 DNA; the clone having the opposite SV40 orientation
`is called pTKpn1. pTEco5, a plasmid (donated by U. Hiinggi) consisting
`of the large EcoRI/BamHI fragments of pBR322 and SV40 DNA cleaved
`with EcoRI, treated with phosphatase, and ligated to the 14—kb EcoRI
`fragment T1. pTPvu3, EcoRI linkers (Collaborative Research) were added
`to Pvul1—cleaved SV40 DNA; the origin-containing fragment (isolated by
`electrophoresis on a low-melting point agarose gel) was cloned into the
`EcaRI site of pT1. pTHind2, the Hindlll origin fragment of SV40 was
`ligated with Hindlll-cleaved pBR322 DNA and the clone having the EcoRI
`site close to the origin was chosen (pHind ori); the EcoRI fragment T1 was
`introduced into this site. pMPC—Hind1, the MPC-11 Lf gene”, cloned as
`a 6.5-kb Sstl fragment” in AgtWES (donated by H. Schnell), was isolated
`as a 10-kb Ec0RI fragment and ligated with EcoRI—cleaved, phosphatase-
`treated pHind ori DNA. pMPC-Baml, the TK gene plasmid“ (donated
`by C. Weissmann) was partially cleaved with BamHI; full—length linear
`molecules were isolated, phosphatase-treated, and ligated with BamHI—
`cleaved SV40 DNA. One of the orientational isomers was chosen and
`cleaved at the indicated Sstl site into which the 6.5-kb MPC 11 Lt fragment
`was ligated (other orientational isomer pMPC»Bam20). The structure of
`all recombinants was verified by restriction analysis. Enzymes were prepared
`in our laboratory (EcoRI,
`ligase, BamHI, Hindlll) or obtained from
`Boehringer—Mannheim (phosphatase) and BRL (Kprll, Pvull, Sstl). I ,
`lmmunoglobulin gene fragments, coding and n0n~coding regions, respec-
`tively; ——, SV40; — —, TK plasmid“, only in pMPC-Sst1; —, pBR322
`and, in pMPC~Hind1, also /\ phage. B, Baml-II; E, EcoRI; E(P), Pvul site
`Mylan v. Genentech
`conve1‘tadyiIi2t‘c>filnvE‘c@IesIiieéIliltélé
`III; K, Kpnl; S, Sstl.
`IPR2016-00710
`IPR2016—00710 © 1982 Macmillan Journals Ltd
`Genentech Exhibit 2022
`Genentech Exhibit 2022
`
`
`
`
`
`Fig. 3 Double labelling of normal blood B lymphocytes for
`surface immunoglobulin and vimentin filaments. Cells were first
`stained with rhodamine-conjugated F(ab’)2 fragments of rabbit
`IgG to pt chain, washed and allowed to cap. The cells were then
`fixed and stained for vimentin as described in Fig.
`1
`legend.
`Controls with fluoresceinated IgM having no anti—IF reactivity
`were negative. :1, Staining for vimentin; the arrow indicates polar
`redistribution of vimentin in a B cell with redistributed surface
`immunoglobulin; b, same field of view showing surface staining
`with rhodamine-conjugated alltl-p. antibodies.
`
`Nature Vol. 298 15 July 1982
`
`Recombinant plasmids were constructed which contained, in
`different positions, the functional K-chain gene of myeloma T,
`called T1 (refs 6, 7), or the aberrantly joined, but expressed,
`MPC 11 Li gene“'1° (see Fig. 1). Four of the plasmids had the
`K-chain genes inserted in the direction of late,
`two in the
`direction of early SV40 transcription. The coding region of the
`T1 gene is located close [31 base pairs (bp)] to the SV40
`sequences with their promoters in pTKpn6 alone; in the other
`plasmids there are 0.8 and 3.0 kilobases (kb) of mouse DNA
`(with the putative promoters) upstream of the T1 and the
`MPC-11 Lf gene, respectively.
`In pTKpn6-transfected CV1 cells (transfection was done as
`described in ref. 11), a distinct transcript was detected having
`the size of the light chain mRNA from myeloma T, indicating
`correct splicing of the primary transcript (Fig. 2). Standardized
`blot hybridization experiments (not shown) indicated that the
`content of specific l<-chain mRNA in total cellular RNA of
`pTKpn6-transfected CV1 cells was 30-40% that of myeloma
`T cells. Transfection of CV1 cells with the other plasmids led
`to patterns lacking the distinct transcript band (see Fig. 2;
`similar patterns were obtained with pTKpn1,
`the MPC 11
`plasmids, and with pBR322). From DNase and RNase diges-
`tions,
`the hybridizing material consists mainly of partially
`degraded plasmid DNA, but it is possible that it also contains
`Cu A7.0kb
`
`mechanical integration of various cytoplasmic organellesl. The
`very close relationship between the redistribution of surface
`molecules and that of lymphocyte vimentin suggests a novel
`function for intermediate filaments.
`This work was supported by grant 81.1029 from INSERM.
`Received 22 February; accepted 13 May 1982.
`1. Lazarides, E. Nature 283. 249-256 (1980).
`2. Dellagi, K., Brouet. J. C... Perreau. J. & Paulin, D. Proc. natn. Acad. Sci. U.S.A.
`4464150 (1982).
`3. Gabhiani. G.. Chaponnier, C., Zumbe, A. & Vassalli. P. Nature 269, 697—698 (1977).
`P
`Schreiner, G. F., Fumiwara. K., Pollard, T. D. & Unanue, E. R. J. exp. Med. 145,
`1393-1398 (1977).
`Unanue, E. R. & Karnovsky, M. J. J. exp. Med. 140, 12074220 (1974).
`Schreiner, G. F. & Unanue, E. R. Adv. Immun. 24, 37-165 (1976).
`L001’. F. Adv. Immun. 30, 1-120 (1980).
`Bourguignon, L. Y. W., Nagpal, M. L. & Hsing, Y. C. J. Cell Biol. 91, 889-894 (1981).
`Schliwa, M. & Van Blerkom, 1.1. Cell Biol. 90, 222—23S (1981).
`Daley, R. A. & Eckert, B. S. J. Cell Elol. 91, 239 (1981).
`Braun, I. & Unanue, E. R.
`in B Lymphocytes in the Immune Response: Functional,
`Developmental, and Interactive Properties Vol. 15 (eds Klinman, N., Mosier, D. E.,
`Seller. 1. & Vitetta, E. S.) 169-175 (Elsevier, Amsterdam. 1981).
`
`
`'19.
`
`."'.°.‘°.°°.".°‘."'
`
`_...
`
`Expression of mouse
`immunoglobulin genes in monkey cells
`
`Falko G. Falkner & Hans G. Zachau
`
`Institut fur Physiologische Chemie, Physikalische Biochemie und
`Zellbiologie der Universitiit Miinchen, 8000 Miinchen 2, FRG
`
`The generation of functional immunoglobulin light-chain genes
`from variable (V), joining (J), and constant (C) gene segments
`has been elucidated recently”. The structural features which
`regulate the expression of immunoglobulin genes, however,
`have not been well defined. To further our understanding of
`immunoglobulin gene expression, African green monkey cells
`(CV1), HeLa cells, and mouse L cells were transfected with
`K-chain gene-containing plasmids. We report here that efficient
`transient expression occurred in the CV1 and HeLa systems
`when the gene was placed, in a recombinant plasmid, under
`simian virus 40 (SV40) promoter control. This is the first report
`of immunoglobulin gene expression in a eukaryotic gene trans-
`fer system. No significant expression of the K-chain genes under
`the control of their own promoters was detected, however, in
`systems in which, for example, the B-globin gene is expressed
`from its own promoter”. Possible reasons for this difference
`are discussed.
`
`0O28~0836/82/280286—()3$O1.00
`
`
`
`5’
`the
`pTKpn6,
`plasmids.
`recombinant
`of
`1 Construction
`Fig.
`EcoRI/BamHI fragment (5.5 kb) of fragment Tl (ref. 6) was ligated to the
`large EcoRI/Baml-II fragment of pBR322; the resulting plasmid pT1 was
`cleaved with Kprll, treated with alkaline phosphatase, and ligated with
`Kpnl-cleaved SV40 DNA; the clone having the opposite SV40 orientation
`is called pTKpn1. pTEco5, a plasmid (donated by U. Hiinggi) consisting
`of the large EcoRI/BamHI fragments of pBR322 and SV40 DNA cleaved
`with EcoRI, treated with phosphatase, and ligated to the 14—kb EcoRI
`fragment T1. pTPvu3, EcoRI linkers (Collaborative Research) were added
`to Pvul1—cleaved SV40 DNA; the origin-containing fragment (isolated by
`electrophoresis on a low-melting point agarose gel) was cloned into the
`EcaRI site of pT1. pTHind2, the Hindlll origin fragment of SV40 was
`ligated with Hindlll-cleaved pBR322 DNA and the clone having the EcoRI
`site close to the origin was chosen (pHind ori); the EcoRI fragment T1 was
`introduced into this site. pMPC—Hind1, the MPC-11 Lf gene”, cloned as
`a 6.5-kb Sstl fragment” in AgtWES (donated by H. Schnell), was isolated
`as a 10-kb Ec0RI fragment and ligated with EcoRI—cleaved, phosphatase-
`treated pHind ori DNA. pMPC-Baml, the TK gene plasmid“ (donated
`by C. Weissmann) was partially cleaved with BamHI; full—length linear
`molecules were isolated, phosphatase-treated, and ligated with BamHI—
`cleaved SV40 DNA. One of the orientational isomers was chosen and
`cleaved at the indicated Sstl site into which the 6.5-kb MPC 11 Lt fragment
`was ligated (other orientational isomer pMPC»Bam20). The structure of
`all recombinants was verified by restriction analysis. Enzymes were prepared
`in our laboratory (EcoRI,
`ligase, BamHI, Hindlll) or obtained from
`Boehringer—Mannheim (phosphatase) and BRL (Kprll, Pvull, Sstl). I ,
`lmmunoglobulin gene fragments, coding and n0n~coding regions, respec-
`tively; ——, SV40; — —, TK plasmid“, only in pMPC-Sst1; —, pBR322
`and, in pMPC~Hind1, also /\ phage. B, Baml-II; E, EcoRI; E(P), Pvul site
`Mylan v. Genentech
`conve1‘tadyiIi2t‘c>filnvE‘c@IesIiieéIliltélé
`III; K, Kpnl; S, Sstl.
`IPR2016-00710
`IPR2016—00710 © 1982 Macmillan Journals Ltd
`Genentech Exhibit 2022
`Genentech Exhibit 2022
`
`
`
`
`
`© Nature Publishing Group1982
`
`
`
`III
`
`N22222V2lZ9BJ5J2!yI9l2
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