Pac)
`
`i|
`
`Y.
`
`Miltenyi Ex. 1037 Page 1
`
`Transposition via Reverse Transcription
`
`eitttaes 40 Number 3
`
`March 1985
`
`Miltenyi Ex. 1037 Page 1
`
`Miltenyi Ex. 1037 Page 1
`
`

`

`Cell makes science easier
`
`One sure way to save you from a tired mind,
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`
`Miltenyi Ex. 1037 Page 2
`
`

`

`ell
`
`Editor
`Benjamin Lewin
`
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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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`Cell is published monthly from January to November and twice monthly in December by The MIT Press, Cambridge, Mas(cid:173)
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`
`Miltenyi Ex. 1037 Page 3
`
`

`

`Volume 40 Number 3 March 1985
`
`Cell
`
`commentary
`Wider Sharing of Materials and Methods
`
`N. R. Cozzarelli
`
`Minireviews
`
`Molecular Organization of the AIDS Retrovirus
`Retroviruses and Retrotransposons: The Role of
`Reverse Transcription in Shaping the Eukaryotic
`Genome
`Apurinic Sites as Mutagenlc Intermediates
`Altering Gene Expression with 5-Azacytidine
`
`Book Reviews
`How Viruses Work
`More on Hormones and Genes
`A Diversity of Monoclonal Antibodies
`Books Received
`
`Articles
`
`Ty Elements Transpose through an RNA
`Intermediate
`
`Isolation and Sequence of a cDNA Encoding the
`Major Structural Protein of Peripheral Myelin
`
`Muscle-Specific Expression of a Gene Affecting
`Acetylcholinesterase in the Nematode
`Caenorhabditis elegans
`
`Insertion Mutagenesis to Increase Secondary
`Structure within the 5' Noncoding Region of a
`Eukaryotic mRNA Reduces Translational Efficiency
`
`Control of ColE1 Plasmid Replication: Initial
`Interaction of RNA I and the Primer Transcript Is
`Reversible
`
`Rous Sarcoma Virus Encodes a Transcriptional
`Activator
`
`Monoclonal Antibodies NORM-1 and NORM-2
`Induce More Normal Behavior of Tumor Cells In
`Vitro and Reduce Tumor Growth In Vivo
`
`A. B. Rabson and M. A. Martin
`D. Baltimore
`
`L.A. Loeb
`
`P.A. Jones
`
`M. S. Hirsch
`
`M. Beato
`
`C. J. Barnstable
`
`J. D. Boeke, D. J. Garfinkel, C. A. Styles,
`and G. R. Fink
`
`G. Lemke and R. Axel
`
`R. K. Herman and C. K. Kari
`
`475-476
`
`477-480
`
`481-482
`
`483-484
`
`485-486
`
`487-488
`
`488-489
`
`489-490
`
`490
`
`491-500
`
`501-508
`
`509-514
`
`J. Pelletier and N. Sonenberg
`
`515-526
`
`J. Tomizawa
`
`S. Broome and W. Gilbert
`
`H. P. Vollmers, B. A. Imhof, I. Wieland,
`A. Hiesel, and W. Birchmeier
`
`527-535
`
`537-546
`
`547-557
`
`(continued)
`
`Miltenyi Ex. 1037 Page 4
`
`

`

`Organelle, Bead, and Mlcrotubule Translocations
`Promoted by Soluble Factors from the Squid Giant
`Axon
`
`Rapid Changes in Specificity within Single Clones
`of Cytolytlc Effector Cells
`
`Stimulation of the T3-T Cell Receptor Complex
`Induces a Membrane-Potential-Sensitive Calcium
`Influx
`
`The T Cell Differentiation Antigen Leu-2/TB Is
`Homologous to lmmunoglobulin and T Cell
`Receptor Variable Regions
`
`The Drosophila EGF Receptor Gene Homolog:
`Conservation of Both Hormone Binding and Kinase
`Domains
`
`Protein Phosphorylation at Tyrosine Is Induced by
`the v-erbB Gene Product In Vivo and In Vitro
`
`R. D. Vale, B. J. Schnapp, T. S. Reese,
`and M. P. Sheetz
`
`559-569
`
`J. Reimann and R. G. Miller
`
`H. C. Oettgen, C. Terhorst, L. C. Cantley,
`and P. M. Rosoff
`
`571-581
`
`583-590
`
`V. P. Sukhatme, K. C. Sizer, A. C. Vollmer,
`T. Hunkapiller, and J. R. Parnes
`
`591-597
`
`E. Livneh, L. Glazer, D. Segal,
`J. Schlessinger, and B.-Z. Shilo
`
`599-607
`
`T. Gilmore, J. E. DeClue, and G. S. Martin
`
`609-618
`
`Antibodies against a Synthetic Peptide as a Probe
`for the Kinase Activity of the Avian EGF Receptor
`and v-erbB Protein
`
`R. M. Kris, I. Lax, W. Gullick,
`M. D. Waterfield, A. Ullrich, M. Fridkin,
`and J. Schlessinger
`
`Influenza Virus M2 Protein Is an Integral Membrane
`Protein Expressed on the Infected-Cell Surface
`
`R. A. Lamb, S. L. Zebedee,
`and C. D. Richardson
`
`619-625
`
`627-633
`
`Vesicles and Clsternae in the Trans Golgi Apparatus
`of Human Fibroblasts Are Acidic Compartments
`
`Requirement for Metalloendoprotease in Exocytosis:
`Evidence In Mast Cells and Adrenal Chromaffin
`Cells
`
`The Hierarchy of Requirements for an Elevated
`Intracellular pH during Early Development of Sea
`Urchin Embryos
`
`Identification of the Sequence Responsible for the
`Nuclear Accumulation of the Influenza Virus
`Nucleoproteln in Xenopus Oocytes
`
`Enzymatic Cross-Linking of lnvolucrin and Other
`Proteins by Keratinocyte Particulates In Vitro
`
`Keratinocyte-Specific Transglutaminase of Cultured
`Human Epidermal Cells: Relation to Cross-Linked
`Envelope Formation and Terminal Differentiation
`
`Monoclonal Antibody to a Membrane Glycoprotein
`Inhibits the Acrosome Reaction and Associated
`Ca2• and W Fluxes of Sea Urchin Sperm
`
`R. G. W. Anderson and R. K. Pathak
`
`635-643
`
`D. I. Mundy and W. J. Strittmatter
`
`645-656
`
`F. Dube, T. Schmidt, C. H. Johnson,
`and D. Epel
`
`657-666
`
`J. Davey, N. J. Dimmock, and A. Colman
`
`667-675
`
`M. Simon and H. Green
`
`S. M. Thacher and R. H. Rice
`
`J. S. Trimmer, I. S. Trowbridge,
`and V. D. Vacquier
`
`677-683
`
`685-695
`
`697-703
`
`(continued)
`
`Miltenyi Ex. 1037 Page 5
`
`

`

`Adenovirus E1a Proteins Repress Transcription from
`the SV40 Early Promoter
`
`A. Velcich and E. Ziff
`
`Transcripts and the Putative RNA Pregenome of
`Duck Hepatitis B Virus: Implications for Reverse
`Transcription
`
`M. Buscher, W. Reiser, H. Will,
`and H. Schaller
`
`705-716
`
`717-724
`
`Positions Available
`
`Announcements
`
`Directory of Advertisers
`
`The cover shows yeast colonies transformed with
`plasmids carrying the transposon Ty (upper panel),
`which forms small, sectioned colonies. Control plas(cid:173)
`mids, lacking Ty, make large, smooth colonies (lower
`panel). For details, see the article by Boeke et al. in
`this issue.
`
`Miltenyi Ex. 1037 Page 6
`
`

`

`Cell, Vol. 40, 591-597, March 1985, Copyright © 1985 by MIT
`
`0092-8674/85/030591-07 $02.00/0
`
`The T Cell Differentiation Antigen Leu-2/TS
`Is Homologous to lmmunoglobulin and
`T Cell Receptor Variable Regions
`
`Vikas P. Sukhatme;t Kurt C. Sizer,*
`Amy C. Vollmer,• Tim Hunkapiller,t and
`Jane R. Parnes•
`• Division of Immunology
`Department of Medicine
`Stanford University Medical Center
`Stanford, California 94305
`i Division of Biology
`California Institute of Technology
`Pasadena, California 91125
`
`Summary
`
`Leu-2/TB is a cell surface glycoprotein expressed by
`most cytotoxic and suppressor T lymphocytes. Its ex(cid:173)
`pression on T cells correlates best with recognition of
`class I major histocompatibility complex antigens,
`and it has been postulated to be a receptor for these
`proteins. We have determined the complete primary
`structure of Leu-2/TB from the nucleotide sequence of
`its cDNA. The protein contains a classical signal pep(cid:173)
`tide, two external domains, a hydrophobic transmem(cid:173)
`brane region, and a cytoplasmic tail. The N-terminal
`domain of the protein has striking homology to varia(cid:173)
`ble regions of immunoglobulins and the T cell recep(cid:173)
`tor. The membrane-proximal domain appears to be a
`hinge-like region similar to that of immunoglobulin
`heavy chains. The superfamily of immunologically im(cid:173)
`portant surface molecules can now be extended to in(cid:173)
`clude Leu-2/TB.
`
`Introduction
`
`The Leu-2/TS T cell differentiation antigen is a cell surface
`glycoprotein expressed by distinct subsets of human T
`lymphocytes. It is the analog and presumed homolog of
`mouse Lyt-2,3 (Reinherz and Schlossman, 1980; Ledbet(cid:173)
`ter et al., 1981). Traditionally, these differentiation antigens
`have been considered markers of T cell function: cytotox(cid:173)
`ic and suppressor cells expressing Leu-2/TS (Lyt-2,3 in
`mouse) and helper/inducer cells expressing the alterna(cid:173)
`tive antigen Leu-3/T4 (L3T4 in mouse). However, recent
`data from many laboratories have indicated that expres(cid:173)
`sion of Leu-2/TS or Leu-3/T4 correlates not so much with
`function as with recognition by T cells of class I (HLA-A,
`B, C) or class II (HLA-DP, DQ, DR) major histocompatibility
`complex (MHC) antigens, respectively (Engleman et al. ,
`1981; Krensky et al., 1982a, 1982b; Meuer et al., 1982).
`Leu-2/TS is thought to be involved in the recognition by
`cytotox ic T cells of their targets. Monoclonal antibodies
`directed against Leu-2/TS inhibit cytotoxicity by most Leu-
`2/TS• killer T cells (Meuer et al., 1982; Spits et al., 1982;
`Reinherz et al., 1983). This block is at the step of con-
`
`t Prese nt address: Department of Medicine, University of Chicago,
`Chic ago, Illinois 60637.
`
`jugate formation between the cytotoxic cell and the target
`cell, rather than at the later killing steps (Tsoukas et al.,
`1982; Landegren et al., 1982; Moretta et al. , 1984). It has
`been suggested that the function of Leu-2/TS (or Lyt-2,3)
`is to increase the avidity of, or stabilize the interaction be(cid:173)
`tween, the T cell and its target (Meuer et al., 1982; Mac(cid:173)
`Donald et al., 1982; Moretta et al. , 1984), perhaps by bind(cid:173)
`ing to nonpolymorphic regions of class I MHC molecules
`(Krensky et al., 1982a; Meuer et al., 1982; Ball and
`Stastny, 1982; Spits et al., 1982; Biddison et al., 1982; En(cid:173)
`gleman et al., 1983).
`On peripheral T cells the Leu-2/TS protein consists of
`dimers and higher multimers of a 32-34 kd glycosylated
`subunit, linked together by disulfide bridges (Ledbetter et
`al., 1981 ; Snow et al., 1983). On thymocytes, in addition
`to homodimers of this polypeptide, a 46 kd subunit is disul(cid:173)
`fide linked to the smaller chain in tetramers and higher
`multimers (Ledbetter et al. , 1981; Snow and Terhorst,
`1983). This larger subunit does not bear the TS deter(cid:173)
`minant, is unrelated to the smaller subunit by peptide
`mapping, and is presumably not required for the function
`of the molecule on mature T cells (Snow and Terhorst,
`1983). Snow et al. (1984) have recently sequenced 25
`N-terminal residues of Leu-2/TS and found no significant
`homology to any other proteins. Determination of the com(cid:173)
`plete primary structure of Leu-2/TS would be of enormous
`value not only for evolutionary comparisons but also for
`further studies of the function of the protein in T cell recog(cid:173)
`nition . To this end we have recently isolated cDNA clones
`that encode Leu-2/TS (Kavathas et al., 1984). We have
`now determined the complete amino acid sequence of the
`Leu-2/TS protein from the nucleotide sequence of a cDNA
`clone. We find that the protein has a classical signal pep(cid:173)
`tide, two external domains, a transmembrane region, and
`an intracytoplasmic tail. One of the external domains has
`striking sequence homology to variable regions of other
`immunological recognition molecules. The results sug(cid:173)
`gest a common evolutionary origin of Leu-2/TS, immuno(cid:173)
`globulin, and T cell receptor genes.
`
`Results
`
`Nucleotide Sequence of Leu-2/TB cDNA
`The nucleotide sequence of Leu-2/TS was determined
`from a single cDNA clone, pL2-M, containing a 2 kb insert,
`although large portions were confirmed on pdditional
`clones. Figure 1 shows a restriction map of the insert of
`clone pl2-M, indicating the enzyme sites used for sub(cid:173)
`cloning into M13 vectors. The nucleotide sequence of this
`clone is presented in Figure 2. There are two long open
`reading frames of similar sizes at the 5' end of this clone.
`One of these extends from the beginning of the clone
`through nucleotide 632. There are no methionines in this
`frame, so if it represents a translated protein, our clone is
`missing the initiation codon. The second open reading
`frame begins at nucleotide 1 and extends through nucleo(cid:173)
`tide 792. This latter frame encodes Leu-2/TS, as deter-
`
`Miltenyi Ex. 1037 Page 7
`
`

`

`Cell
`592
`
`A
`I
`
`5'
`5'UT SP
`
`A R
`I
`I
`
`S
`I
`
`R H
`I I
`
`HR
`II
`
`H
`I
`
`M
`I
`
`3' UT
`
`,.........
`100 bp
`
`H f-1
`
`I I I
`
`R
`
`Figure 1. Restriction Map of Leu-2/TB cDNA Clone pl2-M
`A map of the 2 kb Eco RI insert of cDNA clone pl2-M is shown. The
`enzyme sites indicated are those used for subcloning into M13 vectors
`for DNA sequencing. A, Ava I; S, Sau 3A; R, Asa I; H, Hint I. The artifi(cid:173)
`cial Eco RI sites at the 5' and 3' ends are not labeled. The domains
`of the protein encoded by various segments of the cDNA are demar(cid:173)
`cated. Open bar: 5' and 3' untranslated (UT) regions. Hatched: signal
`peptide (SP). Shaded: external protein domains. Speckled: transmem(cid:173)
`brane region (TM). Crosshatched: cytoplasmic tail (C).
`
`mined by comparison with the N-terminal protein se(cid:173)
`quence (Snow et al., 1984). The first ATG in this frame is
`at nucleotide 88. We believe this to be the initiation codon.
`Accordingly, clone pL2-M contains 115 bp of 5' untrans(cid:173)
`lated region (not all shown), 705 bp of protein coding se(cid:173)
`quence (235 amino acids), and 1182 bp of 3' untranslated
`region. Neither this clone nor another extending 85 bp
`more 3' contains a poly(A) tail. The mRNA represented by
`these clones is approximately 2.5 kb in length (Kavathas
`et al., 1984). Our clones span 2.1 kb, so we are missing
`approximately 400 bp. We presume that most of this is 3'
`untranslated region and poly(A) tail.
`
`Met Ala Leu
`ACTTTCCCCCCTCGGCGCCCCACCGGCTCCCGCGCGCCTCCCCTCGCGCCCGAGCTTCGAGCCAAGCAGCGTCCTGGGGAGCGCGTC ATG GCC TTA
`~ V
`Pro Va l 1h r Ala Leu Leu Leu Pro Leu Ala Leu Leu Leu H1s Ala Ala Arg Pro Ser Gin Phe Arg Val Ser Pro
`CCA GTG • cc GCC TTG CTC CTG CCG CTG GCC TTG CTG CTC CAC GCC GCC AGG CCG AGC CAG TTC CGG GTG TCG CCG
`•
`0
`Leu Asp Ar g Thr Trp Asn Leu G!y Glu Thr Val Glu Le u Lys Cys Gin Val Leu Leu Ser Asn Pro Thr Ser G! y
`CTG GAT CGG ACC TGG AAC CTG GGC GAG ACA GTG GAG CTG AAG TGC CAG GTG CTG CTG TCC AAC CCG ACG TCG GG C
`
`- L
`
`Cys Ser Trp Leu Phe Gin Pro Arg G!y Ala Ala Ala S e r Pro Thr Phe Leu Leu Tyr Leu Ser Gin As n Ly G Pr o
`TGC TCG TGG CTC TTC CAG CCG CGC GGC GCC GCC GCC AGT CCC ACC TTC CTC CTA TAC CTC TCC CAA AAC AAG CC C
`
`Lys Ala Ala G!u GI y Leu Asp Thr Gin Ar g Phe Ser Gly Lys Arg Leu G!y As o Thr Phe Val Leu Thr Leu Ser
`AAG GCG GCC GAG GGG CTG GAC ACC CAG CGG TTC TCG GGC AAG AGG TTG GGG GA L ACC TTC GTC CTC ACC CTG AGC
`,__..,.. H
`0
`Asp Phe Arg Arg Glu Asn Glu Gly Tyr Tyr Phe Cys Ser Ala Leu Ser Asn Ser I I e Met Tyr Phe Ser His Phe
`GAC TTC CGC CGA GAG AAC GAG GGC TAC TAT TTC TGC TCG GCC CTG AGC AAC TCC ATC ATG TAC TTC AGC CAC TTC
`
`Va I Pro Val Phe Leu Pro Ala Ly ,; Pro Thr Thr Thr Pro Ala Pro Arg Pro Pro Thr Pro Ala Pro Thr Ile Ala
`GTG CCG GTC TTC CTG CCA GCG AA 1 CCC ACC ACG ACG CCA GCG CCG CGA CCA CCA ACA CCG GCG CCC ACC ATC GCG
`
`-21
`
`-18
`97
`
`8
`172
`
`33
`247
`
`68
`322
`
`B:)
`397
`
`108
`472
`
`-19
`96
`
`I 7 I
`
`32
`246
`
`57
`321
`
`82
`396
`
`107
`471
`
`132
`546
`
`157
`621
`
`182
`696
`
`207
`771
`
`214
`863
`
`963
`
`1063
`
`1163
`
`1263
`
`1363
`
`1d63
`
`!563
`
`!663
`
`1763
`
`!863
`
`1963
`
`1975
`
`133
`547
`
`!58
`622
`
`183
`697
`
`208
`772
`
`864
`
`964
`
`1064
`
`1164
`
`Ser Gin Pro Leu Ser Leu Ar g Pro Glu Ala Cys Arg Pro Ala Ai d G!y Gly Ala Val His Thr Arg G!y Leu Asp
`TCG CAG CCC CTG TCC CTG CGC CCA GAG GCG TGC CGG CCA GCG GC G GGG GGC GCA GTG CAC ACG AGG GGG CTG GAC
`,__..,.. TM
`I I e Thr
`Phe Ala Cys Asp II e Tyr Ile Trp Ala Pro Leu Ala G!y Thr Cys G!y Va I Leu Leu Leu Ser Leu Val
`TTC GCC TGT GAT ATC TAC ATC TGG GCG CCC CTG GCC GGG ACT TGT GGG GTC CTT ere CTG TCA CTG GTT ATC ACC
`,____,..CJ
`1s Arg Asn Arg Arg Arg Val Cys Lys Cys Pro Arg Pro Val Val Lys Ser GI y As P Lys Pro
`Leu Tyr
`Cys Asn
`CTT TAC TGC AAC CAC AGG AAC CGA AGA CGT GTT TGC AAA TGT CCC CGG CCT GTG GTC AAA TC G GGA GAC AAG CCC
`....... 31UT
`Ser Leu Ser Ala Arg Tyr Val Trm
`AGC CTT TC G GCG AGA TAC GTC TAA CCCTGTGCAACAGCCACTACATTACTTCAAACTGAGATCCTTCCTTTTGAGGGAGCAAGTCCTTCCCT
`TTCATTTTTTCCAGTCTTCCTCCCTGTGTATTCATTTTCATGATTATTATTTTAGTGGGGGCGGGGTGGGAAAGATTACTTTTTCTTTATGTGTTTGACG
`GGAAACAAAACTAGGTAAAATCTACAGTACACCACAAGGGTCACAATACTGTTGTGCGCACATCGCGGTAGGGCGTGGAAAGGGGCAGGCCAGAGCTACC
`CGCAGAGTTCTCAGAATCATGCTGAGAGAGCTGGAGGCACCCATGCCATCTCAACCTCTTCCCCGCCCGTTTTACAAAGGGGGAGGCTAAAGCCCAGAGA
`CAGCTTGATCAAAGGCACACAGCAAGTCAGGGTTGGAGCAGTAGCTGGAGGGACCTTGTCTCCCAGCTCAGGGCTCTTTCCTCCACACCATTCAGGTCTT
`TCTTTCCGAGGCCCCTGTCTCAGGGTGAGGTGCTTGAGTCTCCAACGGCAAGGGAACAAGTACTTCTTGATACCTGGGATACTGTGCCCAGAGCCTCGAG
`1264
`1364 GAGGTAATGAATTAAAGAAGAGAACTGCCTTTGGCAGAGTTCTATAATGTAAACAATATCAGACTTTTTTTTTTTATAATCAAGCCTAAAATTGTATAGA
`CCTAAAATAAAATGAAGTGGTGAGCTTAACCCTGGAAAATGAATCCCTCTATCTCTAAAGAAAATCTCTGTGAAACCCCTACGTGGAGGCGGAATTGCTC
`TC CCAGCCCTTGCATTGCAGAGGGGCCCA TGAA AGAGGACAGGCTACCCC TTT ACAAATAGAATTTGAGCATCAGTGAGGTTAAACTAAGGCCCTCTTGA
`ATCTCTGAATTTGAGATACAAACATGTTCCTGGGATCACTGATGACTTTTTATACTTTGTAAAGACAATTGTTGGAGAGCCCCTCACACAGCCCTGGCCT
`CCGCTCAACTAGCAGATACAGGGATGAGGCAGACCTGACTClCTTAAGGAGGCTGAGAGCCCAAACTGCTGTCCCAAACATGCACTTCCTTGCTTAAGGT
`ATGGTACAAGCAATGCCTGCCCATTGGAGAGAAAAAACTTAAGTAGATAAGGAAATAAGAACCACTCATAATTCTTCACC TT AGGAATAATCTCCTGTTA
`
`1464
`
`1564
`
`1664
`
`I 764
`
`1864
`
`1964
`
`ATATGGTGT ACA
`
`Figure 2. Nucleotide Sequence and Deduced Amino Acid Sequence of the Insert of Leu-2/TB cDNA Clone pL2-M
`Horizontal arrows denote the beginning of the predicted protein domains. Open circles are placed above the two cysteines in the V-like domain that
`are conserved in immunoglobulin and T cell receptor V regions. A closed circle is above the potential site for N-linked glycosylation. A horizontal
`bar is over the 21 amino acid sequence homologous to the mouse fgA hinge. Numbers in the left and right margins, respectively, refer to the first
`and last nucleotides or amino acids in each line. Abbreviations: L, leader (signal peptide); V, variable region-like domain; H, hinge; TM, transmem·
`brane region; Cy, cytoplasmic tail; 3'UT, 3' untranslated region.
`
`Miltenyi Ex. 1037 Page 8
`
`

`

`Leu-2/T8 cDNA Sequence
`593
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`
`Figure 3. Comparison of the Amino Acid Sequence of the N-Terminal Domain of Leu-2/T8 with lmmunoglobulin and T Cell Receptor Variable Region
`Seq uences
`The Leu-2/T8 sequ ence is the first 96 am ino acids of the mature protein. Simi larities to Leu-21T8 are denoted by a dot (.); gaps in the alignments
`are indicated by blank spaces. lmmunoglobulin lambda, kappa, and heavy chain V regions are denoted V1, V,, and VH, T cell recepto r a and fl cha in
`V regions are denoted V0 and Vp- The sequ ence alignments were derived by comparing all members of a given class of V regions (e.g. kappa,
`lambda) against one another and Leu-21T8 simultaneously to achieve the best alignment for a group of sequences, with attention paid to alignment
`of certain key co nserved res idues. All of the immunog lobul in V sequences in the Dayhoff bank we re utilized , as were all published and some unpub(cid:173)
`lished T cell receptor sequences (L. E Hood, personal co mmun ication)_ The sequences we re from the following sources : human Ha V,-I (S hinoda
`et al., 1970) ; human Meg V,-II (Fett and Deutsch. 1974); mouse U61 V, (Vrana et al , 1979); human Len V, (Schneider and Hilschmann . 1975); mouse
`VH (Capra and Nisonoff, 1979); rat IR2 VH (Hellman et al. , 1982); mouse TT11 V0 (Chien et al. , 1984); human TY35 Vp (Yanagi et al, 1984).
`
`Predicted Amino Acid Sequence of Leu-2/TB
`The predicted amino acid sequence of Leu-2/T8 is shown
`in Figure 2. The protein begins with a 21 amino acid
`hydrophobic leader sequence, presumably representing
`a signal peptide that is cleaved off as the protein passes
`into the endoplasmic reticulum . The indicated start of the
`mature processed protein is based upon comparison with
`the published N-terminal protein sequence (Snow et al.,
`1984). Our N-terminal sequence differs from the latter in
`only two places. We predict a glutamine at residue 2, while
`the protein sequence indicates glutamic acid at this posi(cid:173)
`tion . As the cDNA was sequenced multiple times on both
`strands, we believe that either this residue became deami(cid:173)
`dated during the protein purification or there was an error
`made by reverse transcriptase during the cDNA cloning.
`Snow et al. (1984) placed a gap of one amino acid between
`the cysteine at residue 22 and the two leucines at what
`were considered residues 24 and 25. We predict two
`amino acids in this region : glutamine at residue 23 and va(cid:173)
`line at 24. This discrepancy is likely to be a protein se(cid:173)
`quencing error, since it is at the very end of the deter(cid:173)
`mined sequence. However, it is possible that either or both
`of these discrepancies could be the result of polymor(cid:173)
`ph ism .
`The mature Leu-2/T8 protein consists of 214 amino
`acids. A 24 amino acid hydrophobic segment begins at
`position 162. We believe this to represent a transmem(cid:173)
`bra ne region . The following 29 amino acid C-terminal seg(cid:173)
`ment (residues 186-214) is highly charged (ten basic
`res idues and only one acidic residue) and is presumably
`an intracytoplasmic tail. Our sequence predicts an ungly(cid:173)
`cosylated mature protein chain of 23,554 daltons as com(cid:173)
`pared to the described 32,000-34,000 dalton glycosylated
`subunit size (Ledbetter et al., 1981; Snow and Terhorst,
`1983). However, when Snow et al. (1984) treated the iso(cid:173)
`lated protein with trifluoromethane sulfonic acid (TFMS) to
`remove both N-linked and O-linked carbohydrate, the size
`was reduced only 1.5-2 kd. The reasons for this discrep(cid:173)
`ancy are unclear, but most likely relate to incomplete
`cleavage of carbohydrates from the protein with TFMS.
`Although our sequence predicts one potential site for
`N-l inked glycosylation (Arg-X-Thr or Arg-X-Ser) , at position
`28, it may not be used, since experiments with endoglyco(cid:173)
`sidase F suggest that there is no N-linked glycosylation
`(Snow and Terhorst, 1983).
`
`Leu-2/TB Is Homologous to lmmunoglobulin and
`T Cell Receptor Variable Regions
`We searched the Dayhoff protein sequence data bank for
`homologies of the predicted Leu-2/T8 protein sequence to
`other proteins. The greatest amount of homology found
`was to immunoglobulin light chain variable (V) regions,
`with less but still significant homology to immunoglobulin
`heavy chain V regions (VH) and V regions of the er and {3
`chains of the T cell receptor. Comparisons of the Leu-2/T8
`protein sequence with examples of immunoglobul in and
`T cell receptor V region sequences are illustrated in Fig(cid:173)
`ure 3. The segment of Leu-2/T8 that is homologous to a
`V region is at the N terminus and extends to amino acid
`96. For the V regions the homology to Leu-2/T8 stops just
`before the D (diversity) segment (if one is present) or the
`J (joining) segment. With the alignment shown the homol(cid:173)
`ogy is on the order of 30%-35% to lambda and kappa V
`regions, 20%-22% to VH, and 24% to T cell receptor er
`and {3 chain V regions. The homology increases substan(cid:173)
`tially (e.g. up to 56% for lambda or 58% for kappa) if one
`includes conservative amino acid substitutions. As shown
`in Figure 3, there are seven amino acids that are con(cid:173)
`served in all of these sequences. These include the two
`cysteine residues involved in the classical intrachain di(cid:173)
`sulfide loop of V regions (positions 22 and 94 for Leu-2/T8)
`and the tryptophan at position 35, which is thought to be
`important for the proper folding of immunoglobulin do(cid:173)
`mains. Leu-2/T8 contains most of the residues of light
`chain V regions that are used for association with heavy
`chains (Poljak et al., 1975), as well as many of the V region
`contact residue s in light chain dimers (Davies et al. , 1975).
`We therefore predict that the two V-like domains in Leu-
`2/T8 dimers are associated with one another (noncova(cid:173)
`lently), and probably form a single binding site for a pre(cid:173)
`sumed ligand . As might be expected, the homology of
`Leu-2/T8 to V regions is predominantly to framework
`rather than hypervariable regions. The level of homology
`does not vary substantially across mammalian species.
`In contrast to the findings for the N-terminal external do(cid:173)
`main, the 65 amino acid membrane-proximal domain of
`Leu-2/T8 bears no strong resemblance to constant region
`domains of immunoglobulins or the T cell receptor and is
`not homologous to other known proteins. However, a
`stretch of 21 amino acids in the center of this domain
`(residues 120- 140) is homologous to the hinge region of
`
`Miltenyi Ex. 1037 Page 9
`
`

`

`Cell
`594
`
`PA PRPPTPAPTIASQP LSL RP
`Leu-2 / TB Hing e
`** *** **
`*
`* * **
`Mou se CH« Hi ng e PTPPPPITIPSC QP SLSLQRP
`
`Figure 4. Homology of Leu-2fT8 Hinge to Mouse lgA Hinge
`Amino acids 120-140 of Leu-2fT8 are shown. Amino acids 220-240 of
`the mouse a heavy chain (CH. ) are below. The entire CH. hinge
`spans amino acids 218- 240. Homologies are denoted by an asterisk
`('), and gaps, by blank spaces. The CH. hinge sequence is from
`Auffray et al. (1981).
`
`the mouse lgA heavy chain (Figure 4). Although we doubt
`an evolutionary significance of this homology, evidence
`discussed below suggests that the domain containing this
`sequence is a hinge. The remaining protein domains ap(cid:173)
`pear unique to Leu-2/T8.
`
`Structural Predictions
`We have analyzed the Leu-2/T8 sequence using a formula
`similar to that of Kyte and Doolittle (1982) for determining
`the degree of hydrophobicity or hydrophilicity of segments
`of a protein . Such analyses allow one to predict which por(cid:173)
`tions of a protein are exposed, interior, or embedded in a
`membrane. A hydrophobicity plot of the Leu-2/T8 se(cid:173)
`quence is shown in Figure 5. The putative signal peptide
`(residues -21 to -1) and transmembrane domain (resi(cid:173)
`dues 162 to 185) are both demonstrated to be highly hydro(cid:173)
`phobic, while the presumed intracytoplasmic tail (residues
`186 to 214) is very hydrophilic. The V-like N-terminal exter(cid:173)
`nal domain has alternating stretches of hydrophobic and
`hydrophilic residues as seen in globular proteins (Kyte
`and Doolittle, 1982). Furthermore, the plot for this domain
`is virtually superimposable on those for kappa, lambda,
`and heavy chain V regions, except for an extra hydrophilic
`segment in Leu-2/T8 between amino acids 6 and 15 (data
`not shown). These results suggest a great deal of conser(cid:173)
`vation of structure among these protein domains. We fur(cid:173)
`ther analyzed the Leu-2/T8 protein sequence according to
`the parameters of Chou and Fasman (1978) and find that
`the predicted (3-sheet structure of the N-terminal domain
`is the same as that for light chain V regions. In contrast,
`the membrane-proximal external domain does not fit into
`any structure. Therefore this domain, which was shown to
`contain a sequence homologous to the mouse lgA hinge,
`is itself predicted to be a hinge region .
`
`The Leu-2/TS Gene Does Not Rearrange in Cells
`That Express the Protein
`Variable regions of immunoglobulins and the T cell recep(cid:173)
`tor are not expressed as functional receptor molecules un(cid:173)
`til their genes rearrange during the development of B cells
`or T cells, respectively, and become juxtaposed to either
`a J or D and J segments just upstream from a constant re(cid:173)
`gion gene. Since these proteins are homologous to Leu-
`2/T8, it was important to determine whether the Leu-2/T8
`gene also rearranges in T cells that express this protein.
`We have therefore compared Southern blots of DNA from
`human placenta (which does not express Leu-2/T8 protein
`or mRNA) and from a Leu-2/T8-expressing thymoma cell
`line, JM, hybridized to the insert of cDNA clone pL2-M . As
`
`illustrated in Figure 6, no difference could be detected be(cid:173)
`tween the two DNAs with any of the four enzymes used
`(Barn HI, Eco RI, Apa I, and Eco RV). There is also no
`difference seen with Hind Ill (data not shown). Since the
`probe contains the entire coding sequence as well as
`most of the noncoding portions, we conclude that no ma(cid:173)
`jor rearrangements are required for expression of this
`gene. Although we cannot exclude very small rearrange(cid:173)
`ments with either little or no deletion of DNA, immuno(cid:173)
`globulin and T cell receptor type rearrangements would
`have been easily detectable.
`The data shown in Figure 6 also suggest that Leu-2/T8
`is encoded by a single gene. The Southern blots show one
`to three hybridizing bands, depending upon the enzyme.
`Differential hybridization to 5' and 3' probes indicates that
`when multiple bands are present, they represent portions
`of a single gene, and all of the hybridizing fragments can
`be accounted for on a s