Volume 5 Number 9 September 1978
`
`Nucleic Acids Research
`
`Characterization of an immunoglobulin cDNA clone containing the variable and constant regions for
`the MOPC 2| kappa light chain
`
`
`Michael D.Stratheaml, Gary Ehstratheaml, Patricia Akopiantzl, Alvin Y.Liul, Gary V.Paddock1,
`and Winston Salser 1'2
`
`Molecular Biology Institute 2, and Department of Biology 1. University of California, Los Angeles,
`CA 90024, USA
`
`Received 14 July 1978
`
`ABSTRACT
`
`restriction endonuclease
`and
`Nucleotide sequence analysis
`mapping
`have
`been
`used
`to characterize
`a
`cDNA copy
`of
`immunoglobulin MOPC 21 Kappa mRNA cloned in the bacterial plasmid
`pMB9. Three regions of the inserted cDNA of plasmid pL21-1
`have
`been sequenced and match the known protein sequence at amino acid
`residues
`1-24,
`128-138
`and
`171-179.
`with these sequences to
`provide absolute correlations between the restriction map and the
`structural gene sequence it has been possible to exactly deduce
`the positions of all 11 of the insert restriction sites mapped
`within the structural gene.
`The
`pL21-1
`insert
`contains
`the
`complete variable and constant regions as well as parts of the 3'
`untranslated and polypeptide leader coding sequences.
`
`
`INTRODUCTION
`
`Wall, Gilmore—Hebert, Higuchi, Komaromy, Paddock, and Salser
`
`the synthesis of immunoglobulin gene copies
`have described
`(1)
`from MOPC 21 mRNA and the construction of clones by the technique
`
`of Higuchi, Paddock, Wall, and Salser
`
`(2).
`
`The
`
`five plasmids
`
`to pL21-5 generated were partially characterized by
`pL21-1
`DNA-RNA hybridization studies (1). Here we present data giving a
`
`precise restriction map of pL21-1,
`
`the
`
`largest of
`
`the
`
`cloned
`
`Kappa chain immunoglobulin gene copies (1). Several nucleic acid
`sequences which
`unambiguously show the relationship of this map
`with the known amino acid sequence
`are
`introduced.
`This will
`
`for the cloning of the
`for gene mapping,
`probes
`provide useful
`corresponding chromosomal sequences and for the eventual analysis
`of the nucleotide sequences of the
`gene
`as
`it occurs
`in the
`chromosome.
`We were
`also guided
`by the need to definitively
`
`by
`characterize probes for the mapping of the transcription unit
`__:a
`
`0 Information Retrieval Limited 1 Falconberg Court London W‘lV5FG England
`
`3101
`
`Genzyme Ex. 1034, pg 888
`
`Genzyme Ex. 1034, pg 888
`
`

`
`Nucleic Acids Research o
`
`the
`
`UV
`
`resistance mapping technique of Hacket and Sauerbier
`
`(3)
`
`and Sauerbier (4).
`
`
`MATERIALS AND METHODS
`
`Cloning of pL21-1 and Preparation of Plasmid DNA
`The
`procedure by which the plasmid pL21-1 was cloned in the
`
`plasmid
`
`pMB9
`
`has
`
`been described earlier
`
`(1)
`
`and
`
`used
`
`the
`
`techniques of Higuchi et al.
`
`(2)
`
`(see also Salser, reference 5,
`
`for review).
`
`The plasmid was separated from the chromosomal
`
`DNA
`
`as described by Padayatty, Cummings, Manske, Higuchi,
`and mRNAs
`Woo and salser (6).
`
`Restriction Enzymes
`restriction endonucleases
`using
`cleaved
`The
`plasmid was
`Alu I, Hae III, Hinc II, Hind III, Hpa II, and Taq
`I.
`Fragment
`
`sizes were determined
`
`by
`
`comparison
`
`to a digest of the ¢x174
`
`bacteriophage genome cleaved by either Alu I or Hae III
`
`to
`
`give
`
`size (Sanger, Air, Barrell, Brown, Coulson,
`known
`fragments of
`reference 7).
`Hae
`Fiddes, Hutchison, Slocombe, and Smith,
`III,
`Hinc
`
`II, Hind
`
`III,
`
`and Hpa II were purchased from New England
`
`Biolabs (NEB), Alu I
`
`I was prepared by S.
`developed by M. Komaromy.
`
`from Bethesda Research Laboratories, and Taq
`Hendrich
`an
`
`using
`
`unpublished
`
`technique
`
`Reaction Conditions
`
`Except
`
`in the case of Taq I, preparatory enzyme digests were
`
`carried out
`
`for two hours at 37°C in 6 mM Tris-Hcl pH 7.5,
`
`6 mM
`
`(for Hae III); the preceding
`6 mM 2—mercaptoethano1 (2-ME)
`MgC12,
`plus 100 ug/ml gelatin (Alu I); 10 mM Tris-HC1,
`pH 7.9,
`7
`mM
`Mgclz,
`1.0 mM dithiothreitol (DTT), 60 mM NaC1 (Hinc II and Hind
`III); 10 mM Tris-HC1, pH 7.4,
`6 mM KCl, 10 mM MgC12, 1.0 mM DTT,
`Bovine
`Digestion
`
`and
`
`100
`
`ug/ml
`
`with Taq I was carried out
`
`for
`
`Serum Albumin (BSA)
`two
`hours
`
`(Hpa II).
`
`at
`
`62°C
`
`in
`
`10
`
`mM
`
`Tris-HC1
`
`pH 8.4,
`
`6 mM 2-ME and 100 ug/ml gelatin.
`mM MgC12,
`6
`one
`hour
`in
`Restriction mapping reactions were carried out for
`the conditions above. All double digests were carried out
`
`in Alu
`
`I buffer. Digestion with Taq I was carried out for two hours at
`0
`
`52 c in 10 mM Tris—HCl.
`
`
`3102
`
`Genzyme Ex . 1034, pg 889
`
`Genzyme Ex. 1034, pg 889
`
`

`
`Nucleic Acids Research
`
`32? Labeling and Seguence Analysis
`The 5'
`terminal phosphate was removed by bacterial
`
`alkaline
`
`in 10 mM Tris-HC1,
`phosphatase (Worthington Biochemical, grade f)
`pH 9.0,
`for
`30 min.
`at 37 C.
`The reaction mixture was then
`
`to
`ether
`twice phenol extracted and twice rinsed with diethyl
`inhibit
`any
`remaining
`activity. Gamma-329 ATP was synthesized
`from 32sP (ICN Pharmaceuticals) by
`the procedure of Maxam and
`Gilbert
`(8).
`Gamma-32? was
`transferred to
`the 5'
`terminal
`
`T4 polynucleotide
`using
`phosphate of the restriction fragment
`kinase
`(PL Biochemicals)
`(8).
`The fragments were then either
`
`strand separated or
`
`cleaved
`
`with
`
`a
`
`second
`
`restriction
`
`endonuclease,
`
`to give
`
`singly labeled fragments which were then
`
`sequenced using the partial
`Gilbert (8).
`
`cleavages described by Maxam and
`
`Gel Electrophoresis
`Several
`acrylamide slab gel
`
`types were used, depending upon
`
`the restriction fragment sizes:
`
`6%
`
`acrvlamide
`
`(Eastman Kodak)
`
`with
`
`6
`
`or
`
`12% glycerol for smaller fragments (<500 base pairs)
`
`and 8% acrylamide for larger
`
`fragments.
`
`DNA was
`
`eluted
`
`from
`
`these preparatory gels using 0.5 M NaAc, 10 mM MgCl2, 0.1% SDS,
`and 0.1 mM EDTA. Twenty percent acrylamide 7 M urea
`slab gels
`
`were used for the sequence analysis.
`
`RESULTS
`
`The
`
`results
`
`in
`
`the previous paper have indicated that the
`
`plasmid pL2l-1 contains both variable and constant
`
`regions
`
`from
`
`MOPC
`
`21 immunoglobulin mRNA (1).
`
`The sequences inserted in this
`
`plasmid have now been
`
`characterized by
`
`sequence
`
`analysis
`
`and
`
`mapped with restriction enzymes.
`
`variable Region Nucleotide Seguence of pL2l-1
`Cleavage
`of
`the
`plasmid
`pL2l-1 with
`
`endonuclease Alu I
`
`yielded four fragments 291, 245, 55 and 53
`
`base pairs
`
`in size
`
`which did
`
`not
`
`co-migrate with the fragments of parent plasmid
`
`pMB9, cut with the same enzyme. We
`
`suspected
`
`the
`
`presence of
`
`additional "hidden" Alu I bands since the sum of the sizes of the
`
`
`
`M03
`
`Genzyme Ex. 1034, pg 890
`
`Genzyme Ex. 1034, pg 890
`
`

`
`Nucleic Acids Research
`
`four
`
`fragments was
`
`less
`
`than the size of the entire insert as
`
`restriction mapping
`estimated from other data (1). Our further
`showed
`three
`additional
`insert fragments co-migrating with pMB9
`
`The 245 base
`fragments (see Results, Section C, and Figure 1-A).
`pair fragment was eluted,
`labeled at its 5'
`terminus with
`32?,
`and cleaved with Hae III to give two fragments whose lengths were
`
`85
`
`and
`
`160 base pairs.
`
`Nucleotide sequencing reactions were
`
`77
`sequence of
`A
`fragment.
`pair
`carried out on the 85 base
`nucleotides could be read from the autoradiograph of the sequence
`
`gel
`
`(Figure
`
`2-A).
`
`This
`
`sequence corresponds exactly to amino
`Figure !-A
`
`
`
`(|"7)
`
`uI.:s-r.u:r
`
`1' or
`
`
`
`(Ill)
`
`
`
`All:
`
`I
`
`vn..n- I-II
`
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`
`1
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`u
`I.
`.'filI Ila HM. MK:
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`:\TI' I-'|'A NIH :\l|
`I111: A-H:
`man u.- \'.u um. um u.T..' -.‘.r I'r:I-I—.\-1 -..-r -1.» -.-.-u- an -..-r ul tn. ..|.. an: run 11:: I.»-a nu In I.-In fia-r‘.."F
`I
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`II
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`in
`
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`
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`Anlnu .\. Id Suqun.-nu:
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`
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`
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`
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`
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`
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`
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`‘Ell H-.'
`
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`
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`
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`
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`I-‘I
`II.’ III |1'-
`I1’- Uh I11 I1! I" II0 IIII
`
`'[
`
`.
`the area
`in
`Figure 1-A shows a restriction endonuclease map of EL21—1
`containing and
`surrounding the
`insert.
`The orients ion of the translated
`(variable
`constant and 5
`pol peptide leader) and 3 untranslated (UT) and 5
`untranslated are shown above t e restriction map.
`Restriction enzyme
`sites
`are indicated, and the distances (in base pairs
`between restriction sites are
`shown
`o
`horizontal numbers. Vertical numbers in parenthesis indicate amino
`acid residue (9).
`The oly A/T tails
`(60
`base pairs
`are
`expected from
`insertion technique
`E
`but
`has
`not been confirmed} are shown to mark the
`insertion sites.
`The nucleotide sequences obtained are
`indicated by
`lines.
`Figure
`1-3
`shows
`three
`sequences of
`7?.
`H and 27 nucleotides derived by
`nucleotide sequence
`analysis
`of
`the
`autora iogra he
`shown
`in
`F1 ure
`2.
`ggquence 1 is from the Alu I 2&5 base pair fragment
`-terminally iabe ed with
`P,
`then
`cleaved with Hae III. Sequences 2 and 3 are complementary strands
`from the labeled 180 base pair fragment of
`a Hinc II di eat.
`Sequence
`1
`provided
`1? nucleotides, while
`sequence
`2
`from the
`sat-running coding
`strand, yielded 3H and the slow-running non-codin? strand 2‘.
`The data
`for
`sequence
`one was
`ambi uous
`in
`two positions
`labeled N
`in Pi are 2-A and
`described in the text.
`ata from the
`related plasmid ptal-5 S
`.
`Clarke
`personal
`communication]
`suggests
`that
`there may
`be
`an
`add tional Alu i
`ragment about 30 to in size extendins to the right from ser 205. This
`small
`fragment was apparently missed in our experiments.
`
`
`3104
`
`Genzyme Ex. 1034, pg 891
`
`Genzyme Ex. 1034, pg 891
`
`

`
`Nucleic Acids Research
`
`acid residues 1-24 of the protein sequence (Svasti and Milstein,
`
`reference
`
`9)
`
`and
`
`in
`
`addition includes five of the nucleotides
`
`coding
`
`for
`
`the
`
`pre-polypeptide
`
`leader.
`
`This
`
`experiment
`
`unambiguously
`
`locates
`
`one of the Hae III sites at Ala 25 in the
`
`an
`that
`sequence coding for the variable region and establishes
`Alu
`I
`site occurs a few nucleotides prior to the first codon of
`
`Two nucleotides at
`the variable region.
`difficult
`to
`interpret
`from
`the
`
`amino
`ladder
`
`acid 15
`sequence
`
`proved
`gel
`
`autoradiograph and these two nucleotides
`
`are
`
`left
`
`unassigned.
`
`These problematic nucleotides may be due to poor resolution of
`
`the gel
`
`in this region but it is also possible that the bases may
`
`have been deleted during the
`
`cloning
`
`procedures or
`
`subsequent
`
`propagation of the plasmid.
`
`Constant Region Nucleotide sequences of pL2l-l
`A Hinc
`II
`cleavage was carried out
`
`on pL2l-l,
`
`three
`
`fragments were
`
`observed which were
`
`not present
`
`in parallel
`
`one of these fragments 170
`digests of
`the parent plasmid pMB9.
`its 5'
`terminues, strand
`base pairs in size was labeled at
`from the
`complementary
`separated and sequenced.
`Two sequences
`strands
`‘were deduced from the autoradiograph shown in Figure 2-8
`
`and
`
`are
`
`shown
`
`in Figure 1-8.
`
`From the
`
`slow strand,
`
`27
`
`nucleotides
`
`(coding
`
`for
`
`amino.acids 171 to 179 in the constant
`
`region) were read, while the fast
`
`strand gave
`
`a 34-nucleotide
`
`sequence
`
`(coding
`
`for
`
`amino
`
`acids
`
`128
`
`to 138 in the constant
`
`region). This experiment unambiguously locates Hinc II
`sites at codons 125 and 181 in the constant region.
`
`cleavage
`
`gegtriction Mapping of pL2l-l
`As described below, our restriction mapping indicates that
`
`in pL2l-l
`
`the
`
`sequence
`
`coding
`
`for
`
`the
`
`entire variable and
`
`constant
`
`regions
`
`have
`
`been
`
`inserted into the Eco RI cleaved
`
`The logic used in constructing the map of pL2l-l
`plasmid pMB9.
`will
`be
`summarized briefly below but we will not attempt to
`describe all of the data since many features
`of
`the map
`are
`
`be
`cannot
`independently verified by a variety of data which
`detailed in
`the space available. Our analysis was aided by the
`
`
`3105
`
`Genzyme Ex. 1034, pg 892
`
`Genzyme Ex. 1034, pg 892
`
`

`
`Nucleic Acids Research
`
`AJC. An: 5).
`
`g
`
`Q
`7
`
`- . 9 A-in A:-n on p
`“
`"
`‘
`.
`3
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`
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`
`C
`1’
`'
`'
`0
`
`D
`.
`
`'
`'
`on
`
`s
`
`?
`E
`' .
`.
`51
`3
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`
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`‘Q
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`o
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`
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`
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`
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`
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`
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`
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`
`_
`
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`.
`
`—
`
`.
`
`.
`
`.
`
`.
`‘O
`.
`
`a
`
`II‘.
`
`Figure 2-h shows the autoradio raph of a ladder seguencing gel and the 77
`nucleotide sequence derived from t.
`The 235 base pa r restriction fragment
`labeled at its nlu I terminal 5
`phosphate was
`cleaved with Hae
`III
`(see
`text).
`The
`85
`base pair fragment was divided into six fractions, and each
`fraction was chemicall
`cleaved at one of the four bases.
`The products were
`resolved by aleetrop oresis
`on
`a
`201 denaturing acrglamide gel.
`The six
`reactions were divided
`into three portions
`and
`cede
`onto
`the gel at
`different intervals.
`Ehe reaction conditions used for each cleavage are
`iven
`bx Haxam and Gilbert 3) under the Following titles: A>C, "alternative 3 rung
`a enine, weak
`cytosine
`c1aava§e';
`A>G.
`strong adenine‘
`weak
`guanine
`cleavages“:
`G>Ai
`“strong guan ne.
`ueak
`adenine cleave e ; 6. "alternative
`guanine reaction ; C, "cleave e at cytosine"; and T+C,
`"o esvage
`of
`th ins
`and
`cytosine.“
`Brackets
`s
`H the regions in common among the three se a of
`tracts. Electra horesis is from to
`to bottom. Nucleotides
`coding for
`two
`bases
`or val 1
`are not diseerneb e
`being too closely s aced for one set of
`tracks and cannot be read by either other set of
`tracks.
`a the
`C
`reactions
`were weak.
`data use discerned from the A>C. C and T+C reactions. Figure 2-B
`ahous autoradiographs of ladder sequencing gels and the
`3H nucleotides
`read
`from the coding strand and 27 nucleotide read from the non—codin3 strand.
`The
`158
`base pair fra
`ent was labeled at its Hinc 1% terminal 5
`hosphates and
`was strand-separate
`(see text}. The fast strand codin§ strand
`was
`broken
`into six reactions [Figure 2-» legend} and run in the E rst six tracks, while
`the other six tracks came from the 51:
`reactions
`on
`the
`slow (non-codin§)
`strand.
`The
`fourth nucleotide from the 5
`end in the non—coding strand s
`split due to a cracking of the gel.
`
`
`3106
`
`Genzyme Ex. 1034, pg 893
`
`Genzyme Ex. 1034, pg 893
`
`

`
`Nucleic Acids Research
`
`0 °>A Am A)‘:
`
`..cA..aoA..a..caVAcT:a
`
`7 c
`
`.9
`
`8.........o..mm2..Il.o...t
`
`Du
`
`NDN- CDDINO
`
`3107
`
`Genzyme Ex. 1034, pg 894
`
`Genzyme Ex. 1034, pg 894
`
`

`
`Nucleic Acids Research
`
`availability of a complete restriction map determined
`
`by
`
`John
`
`Rogers
`
`for the sequences of the parent plasmid pMB9 lying within
`
`the Tag fragment containing the Eco RI site (Rogers, Clarke
`
`and
`
`Salser, manuscript
`
`in preparation,
`
`reference 10).
`
`our data
`
`confirms the findings of Rogers in this
`
`region.
`
`In order
`
`to
`
`identify those
`
`restriction fragments
`
`carrying portions of the
`
`insert, parallel digestions were carried out on
`
`pL21—1
`
`and
`
`the
`
`parent
`
`plasmid
`
`pMB9.
`
`Those fragments found only in the pL21—1
`
`digest were judged to carry portions of the
`
`insert,
`
`along with
`
`and plasmid sequences. As a supplementary
`possible A/T joints
`approach, we isolated quantities of a Tag I restriction fragment
`
`which
`
`contains
`
`the entire insert sequence and then digested it
`
`with one or
`
`two
`
`additional
`
`enzymes.
`
`This method gives much
`
`clearer
`
`results
`
`since purification of
`
`the Tag
`
`I
`
`fragment
`
`eliminates 90% of
`
`the
`
`plasmid
`
`sequences.
`
`As
`
`a
`
`result,
`
`the
`
`subsequent digests only rarely contain overlapping bands and are
`
`easily interpreted.
`
`The
`
`fragments
`
`from Hpa II, Hae III, and Hinc II gave two,
`
`three, and three new insert fragments,
`
`respectively.
`
`Secondary
`
`digestions with Hind III were used to help orient these fragments
`
`the map. There is only one Hind III site in pL21—1
`properly in
`and it is located in the plasmid sequence 310 base pairs
`to
`the
`
`left of
`
`the
`
`Eco RI insertion point (Maniatis, Kee, Bfstratidis
`
`and Kafatos, reference 11). Digestion of the 760
`
`base
`
`pair
`
`Hpa
`
`II
`
`fragment with Hind III yielded fragments of 655 and 105
`
`Since there is a known Hpa II site 105 nucleotides
`base pairs.
`to the
`left of the Hind III site this established that the 760
`
`nucleotide fragment contained the Left A/T joint and extended 280
`nucleotides into the insert
`(these distances include
`the
`length
`
`the A/T joint which would be about 30-100 nucleotides in size
`of
`according to the
`techniques
`used
`in constructing
`the
`clones
`
`the 710 base pair Hae III fragment was
`(reference 1). Similarly,
`cleaved by Hind
`III
`to yield 575 and 135 base pair fragments.
`
`This enabled us to place a Hae III site within the
`
`insert
`
`200
`
`nucleotides
`
`from the start of the left A/T joint. Digestion of
`
`the 512
`
`base pair Hae
`
`III
`
`fragment with
`
`Hpa II
`
`yielded
`
`fragments
`
`of
`
`437 and 80 base pairs. This established that the
`
`insert.
`the
`512 base pair Hae III fragment lies entirely within
`
`
`3108
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`

`
`Nucleic Acids Research
`
`Therefore.
`
`the
`
`745
`
`base pair Hae III fragment must contain the
`
`rightward A/T joint. Similarly, digestions of the isolated Hinc
`II
`fragments with Hind
`III,
`Hpa II and aae III enabled us to
`
`unambiguously map the relative position of the sites of all
`enzymes.
`
`four
`
`Since there were so many Alu I sites within the
`
`insert
`
`and
`
`flanking
`
`plasmid
`
`sequences,
`
`their mapping proved somewhat more
`
`difficult.
`
`This was
`
`accomplished primarily
`
`by
`
`accurately
`
`determining
`
`the
`
`sizes of all products from Alu I digestions of
`
`the individual Hae III, Hpa II and Taq I bands containing
`
`insert
`
`sequences
`
`and
`
`by digesting the Alu I fragments with the other
`
`been
`have
`sites
`the Alu I
`two of
`In addition,
`enzymes.
`unambiguously placed within the structural gene by the nucleotide
`
`sequence analysis reported above.
`
`The map distances were determined by
`
`a variety of double
`
`digestions.
`
`This
`
`enabled
`
`us
`
`to directly measure
`
`(with few
`
`exceptions)
`
`the distances from each cleavage site to the nearest
`
`neighboring
`
`sites for all other enzymes used. Because the ¢x174
`
`marker digests provide accurate size standards this results in
`precise map.
`
`a
`
`Comparison of Restriction Map with the Known Amino Acid sequence
`We
`then
`used
`the
`amino
`acid
`sequence of MOPC
`21
`
`and
`by Svasti
`chain protein determined
`immunoglobulin light
`Hilstein (9)
`to predict possible endonuclease sites within the
`
`structural gene.
`
`A possible
`
`site predicted by
`
`the protein
`
`sequence was
`
`considered to be in good agreement with the map if
`
`there was no other predicted site within 30 nucleotides.
`
`Such
`
`the precision of the map
`refine
`further
`to
`us
`enabled
`cases
`distances.
`In other cases an observed restriction cleavage could
`
`correspond to two or more possible sites.
`
`For instance, Hinc
`
`11
`
`sites
`
`are possible
`
`at
`
`amino
`
`acid
`
`residues 176. 178 and 180.
`
`Usually,
`the nucleotide sequence data we present
`ruled out all
`but
`one possibility.
`only one
`ambiguity remains:
`the Hpa 11
`site, shown on the restriction map (Figure 1-A) at amino acid 53.
`A possible cleavage site for this enzyme is also found
`at
`amino
`acid
`56
`(7 base pairs away). We have no definitive information
`' for the correct placement in this case.
`
`3109
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`

`
`Nucleic Acids Research
`
`
`orscussrgg
`
`is
`it
`that
`shown
`Sequence analysis of plasmid pL21-1 has
`indeed
`the
`Kappa light chain immunoglobin message of HOPC 21.
`
`Restriction mapping carried out on pL2l—l shows that
`
`the
`
`insert
`
`includes
`
`sequences
`
`coding for
`
`the entire amino acid sequence of
`
`the variable and constant regions. Analysis of
`
`two
`
`restriction
`
`from the plasmid pL2l-1 has given sequences of 77, 34
`fragments
`and 27 nucleotides. These three
`sequences of nucleotides
`are
`
`consistent with
`
`the
`
`known
`
`amino acid sequences (reference 9).
`
`Restriction sites and sequences obtained from another plasmid
`
`in
`
`this
`
`series
`
`(pL21-5, Clarke and Salser, reference 12)
`
`show that
`
`this cloned cDNA lies entirely within that of pL21-1 and contains
`
`the complete constant region as well as portions of the variable
`and 3' untranslated regions.
`
`Seidman, Edgell, and Leder
`
`(13)
`
`have
`
`cloned Kappa
`
`light
`
`chain messenger
`
`sequences
`
`from MOPC 149 and have determined a
`
`nucleotide sequence coding for amino acid residues 173 to
`
`196 in
`
`the constant region. All Kappa light chains are
`
`known
`
`to
`
`have
`
`identical
`
`amino
`
`acid
`
`sequences
`
`in
`
`their
`
`constant
`
`regions.
`
`also expect
`According to current models of V-C joining we would
`them to have an identical nucleotide sequence in this region.
`As
`
`from this, we find identical nucleotide sequences in a
`predicted
`2l-nucleotide region where the data overlap (Figure 3).
`
`Milstein, Brownlee, Cheng, Hamlyn, Proudfoot and Rabbitts
`
`(14) have carried out sequence analysis of MOPC 21
`method
`of
`T1
`Libonuclease
`digestion and
`
`the
`by
`mRNA
`two-dimensional
`
`fractionation. Our sequence data agrees with
`
`theirs with
`
`two
`
`exceptions.
`
`They represented the codon at amino acid 171 as AGC,
`
`Figure 3
`.11.
`.('-C .\CC CTC ACO... Mllstein at
`:\CC TAC ACC ATG M}.
`...’\ AG;
`HUPC 2|
`A.
`. .A A01‘ ACC TAC AGC ATL AGL /mc :\CC CTC
`This Paper
`HDPC 21
`H.
`TAC AGC ATG ACC AGC ACC CTC ACG
`‘Ieldn.-In er nl ,
`HUPC [/09
`(J.
`D. Protein Sequence Asp Set Thr Tyr Ser Net Set Set Thr Leu Thr
`I75
`180
`170
`
`I977
`IFHB
`
`I"
`
`E‘;
`nd
`21
`or the nucleotide sequences from HOPE
`Figure 3 shows the overla
`MOPC 1H9 (13, 1a).
`Shown are the coding sequences for amino acids 171 to 1 1.
`A
`shows
`the nucleotide
`sequence
`from MDPC
`21 Ilfli. Row B-shows the
`Row
`sequence from HOPC 21. this paper, and Row C shows the se uence from HOPC
`1H9
`(13).
`Row B shows the known amino acid sequence (10). w th the number of the
`amino acid residues indicated below.
`It is seen that at Ser 111 a difference
`exists
`between
`the
`two MDPC 21 sequences (see text).
`The figure only shows
`those portions of the sequences from references 13 and 1H whic
`overlap our
`data.
`
`
`3110
`
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`Genzyme Ex. 1034, pg 897
`
`

`
`Nucleic Acids Research
`
`the
`our sequence data indicates an AGT codon. Secondly,
`whereas
`1-3,
`mapping data, coupled with
`the
`sequence data
`in Figure
`clearly place
`a Hinc II cleavage site (GTPyPuAC) at amino acid
`125. This is inconsistent with the sequence
`of
`the
`T1 digest
`fragment
`assigned
`to
`this position by Milstein et al.
`(14),
`which indicates a sequence of ACTAAC rather than GTPyPuAC.
`
`
`ACKNOWLEDGEMENTS
`
`we would like to thank
`
`R.
`
`Hammen
`
`and
`
`S.
`
`Hendrich
`
`for
`
`technical assistance and M. Wynne for secretarial help. Special
`thanks
`go to H. Heindell for providing thoughtful advice. This
`research was supported by American Cancer Society grant VC-240
`
`and PHS grants GM185S6 and HL21831.
`
`REFERENCES
`I) Wall, R., Gilmore-Hebert, M., Higuchi, R., Komaromy, N.,
`Paddock, G., and Salser, N.
`(1978)
`In preparation.
`2) Higuchi, R.,
`Paddock, G., wall, R., and Salser, w.
`Proc. Natl. Acad. Sci.
`USA 73:3l46-3150.
`3) Hacket, P.,
`and Sauerbier, W.
`(1975)
`J. Mol. Biol.
`91:235-256.
`(1976) Advances in Radiation Biolo9Y. Vol.
`4) Sauerbier, W.
`6, Academic Press..
`in Genetic
`sequences
`cDNA
`5) Salser,
`W.
`(1978) Cloning
`Engineering, Chakrabarty, A.M., Ed.,
`CRC Press,
`Palm
`Beach, FL.
`
`(1976)
`
`7)
`
`(1977)
`
`Proc. Natl. Acad.
`
`C.
`
`(1972) Biochem.
`
`Clarke,
`
`P.
`
`and Salser, W.
`
`(1978)
`
`J.
`
`In
`
`6) Padayatty, J., Cummings, I., Manske, C., Higuchi, R., Woo.
`S., and Sa1se1,~W.
`(1978)
`In preparation.
`Sanger, F., Air, G., Barrell, 3., Brown, N., Coulson, A.,
`Piddes, J., Hutchison III, C., slocombe, P., and smith,
`N.,
`(1977) Nature 265:687-695.
`8) Maxam, A.,
`and Gilbert, W.
`Sci.USA 74:560-564.
`9) Svasti,
`J.,
`and Milstein,
`128:427-444.
`10) Rogers,
`J.,
`preparation.
`11) Maniatis, T., Kee, 5., Efstratiadis, A., Kafatos, P.
`Cell 8:163-182.
`12) Clarke, P., and Salser, W.
`13)
`Seidman, J., Edgell, M.,
`27l:582-585.
`G., Cheng, C., Hamlyn, P.,
`Brownlee,
`14) Milstein,
`C.,
`(1976) Mosbacher Colloquium,
`Proudfoot, N., Rabbits, T.
`Gesellschaft
`fur Biologische Chemie 27:75-85.
`15) Present address, G.
`Paddock, Dept.
`of Basic
`and Clinical
`Immunology
`and Microbiology, Medical University of South
`Carolina, Charleston, South Carolina 29401.
`_——i—
`3111
`
`In preparation.
`(1978)
`and Leder,
`P.
`(1978) Nature
`
`(1976)
`
`Genzyme Ex. 1034, pg 898
`
`Genzyme Ex. 1034, pg 898
`
`

`
`Nucleic Acids Research
`
`3112
`
`Genzyme Ex. 1034, pg 899
`
`Genzyme Ex. 1034, pg 899