Amgen Inc. v. F. Hoffmann-LaRoche LTD et al

Filing 810

DECLARATION re #808 MOTION in Limine To Preclude Amgen Inc. From Making Assertions That Contradict Statements Made in Specifications of Patents-In-Suit (By Kregg T. Brooks) by F. Hoffmann-LaRoche LTD, Roche Diagnostics GmbH, Hoffmann LaRoche Inc.. (Attachments: #1 Exhibit A#2 Exhibit B, Part 1#3 Exhibit B, Part 2#4 Exhibit B, Part 3)(Brooks, Kregg)

Case 1:05-cv-12237-WGY

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Amgen Inc. v. F. Hoffmann-LaRoche LTD et al

Doc. 810 Att. 3

U S . Patent

Aug. 15, 1995

Sheet 8 of 27

Dockets.Justia.com

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ATTGGCCMGAGGTGGCTGGGTTCMGGACCGGCGACTTGTCMGGACCCCGGAAGGGGGAGGGGGGTGGG
GCAOCCTCCACGTGCCGCGGGGACTTGGGGGAGTTCTTGGGGATGGCWCCTGGCCTGTTGAGGGGCA

TTGCACACGCACAGATCMTMGCCAGAGGCAGCACCTGAGTGCTTGCATGGTTGGGACAGGMGGACGAG

CTGGGGCAGAGACGTGGGGATGAAGGAAGCTGTCCTTCCACAGCCACCCTTCTCCCCCCCCGCCTGACTCT
-23
-2 0

CAGCCTGGCTATCTGTTCTAO

Glu Cys Pro Ala Trp Leu Trp Leu Leu Leu Ber Leu AA TGT CCT GCC TGG CTG TOG CTT CTC CTG TCC CTG

-10 -1 +I Leu Ser Leu Pro Leu Gly Leu Pro Val Leu Gly Ala Pro Pro Arg Leu Ile Cys CTG TCG CTC CCT CTG sac CTC CCA GTC CTG GGC GCC CCA CCA c ~ c CTC ATC TGT

10 20 * Asp Ber Arg Val Leu Glu Arg Tyr Leu Leu Glu Ala Lys Glu Ala Glu Asn Ile GAC AGC CGA GTC CTG GAG AGO TAC CTC TTG GAG GCC AAG GAG GCC GAG AAT ATC
26

Thr ACG GTGAGACCCCTTCCCCAGCACATTCCACAGAACTCACGCTCAGGGCTTCAGGGMCTCCTCCCAGAT
CCAGGMCCTGGCACTTGGTTTGGGGTGGAGTTGGGAAGCTAGACACTGCCCCCCTACATAAGMTMGTC

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TGGTGGCCCCAAACCATACCTGAAACTAGGCMGGAGCAAAGCCAGCAGATCCTACGCCTGTGGGCCAGGG

27

30

Thr Gly Cys Ala Glu
CCAGAGCCTTCAGGGACCCTTGACTCCCCGGGCTGTGTGCATTTCAG

ACG GGC TOT GCT GAA

*
50
55

40

His cys 8er Leu Asn Glu Asn I l e Thr Val Pro Asp Thr &ys Val Asn Phe Tyr CAC TGC AQC TTG AAT GAG AAT ATC ACT GTC CCA GAC ACC AAA GTT AAT TTC TAT
Ala Trp Lys Arg Wet Glu GCC TGG AAQ AGG ATG GAG GTGAGTTCCTTTTTTTTTTTTTTTCCTTTCTTTTGGAGAATCTCATT
TGCGAGCCTGATTTTGGATGAAAGGGAGMTGATCGGGGGAAAGGTAAAATGGAGCAGCAGAGATGAGGCT

GTGMGTGGTGCATGGTGGTAGTCCCAGATATTTGGAAGGCTGAGGCGOGAGGATCGCTTGAGCCCAGGAA

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FIG. 6D

CACTCATTCATTCATTCATTCATTCAACMGTCTTATTGCATACCTTCTGTTTGCTCAGCTTGGTGCTTGG
GGCTGCTGAGGGGCAGGAGGGAQAGGGTGACATGGGTCAGCTCGACTCCCAGAGTCCACTCCCTGTAG
56 60 70

Val Gly Gln Gln Ala Val Glu Val Trp Gln Gly Leu Ala Leu Leu Ser Glu Ala GTC 000 CAG CAG GCC GTA GAA GTC TGG CAG GGC CTG GCC CTG CTG TCG O M GCT
80

Val Leu Arg Gly Gln Ala Leu Leu Val Asn Ser Ser Gln Pro Trp Glu Pro Leu GTC CTG caa GGC CAG GCC CTG TTG GTC MC TCT TCC CAG cca TGG GAG ccc CTG
100

*

90

Gln Leu His Val Asp Lys Ala Val Ser Gly Leu Arg Ser Leu Thr Thr Leu Leu CAG CTG CAT GTG GAT AAA GCC GTC AGT GGC CTT CGC AGC CTC ACC ACT CTG CTT
110 115

Arg Ala Leu Gly Ala Gln CGG GCT CTG GGA GCC CAG GTGAGTAGGAGCGGACACTTCTGCTTGCCCTTTCTGTMGAAGGGGA
GMGGGTCTTGCTMGGAGTACAGGMCTGTCCGTATTCCTTCCCTTTCTGTGGCACTGCAGCGACCTCCT
116 12 0

GTTTTCTCCTTGGCAG

Lys Glu Ala Ile Ber Pro Pro Asp Ala Ala Ber Ala Ala MG GAA acc ATC TCC CCT CCA GAT GCG GCC TCA OCT GCT

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FIG. 6E

130

140

Pro Leu Arg Thr Ile Thr Ala Asp Thr Phe Arg Lys Leu Phe Arg Val Tyr Ber CCA CTC CGA ACA ATC ACT GCT GAC ACT TTC CGC AAA CTC TTC CGA GTC TAC TCC
150 160

Asn Phe Leu Arg Gly Lys Leu Lys Leu Tyr Thr Gly Glu Ala Cys Arg Thr Gly MT TTC CTC coa GGA MG CTG MG CTG TAC ACA GGG GAG GCC TGC AGG ACA GGG
166

Asp Arq OP GAC AGA TGA CCAGOTGTGTCCACCTGGGCATATCCACCACCTCCCTCACCMCATTGCTTGTGCCACA
CCCTCCCCCGCCACTCCTGAACCCCGTCGAGGGGCTCTCAGCTCAQCGCCAQCCTGTCCCATGGACACTCC

AGTGCCAGCMTGACATCTCAOGGGCCAGAGGMCTGTCCAGAGAGCMCTCTGAGATCTMOGATGTCAC
AGGGCCMCTTGMGGGCCCAGAGCAGGMGCATTCAGAGAGCAGCTTTAAACTCAOGGACAGAGCCATGC

TGGGMGACGCCTGAGCTCACTCGGCACCCTGCAAAATTTGATGCCAGGACACGCTTTGGAGGCGATTTAC

CTGTTTTCGCACCTACCATCAGOOACAGGATGACCTGGAGMCTTAGGTGGCMGCTGTGACTTCTCCAGG

AXGATXGQGGCTGGCCTCTGGCTCTCATGGQGTCCMGTTTTGTGTATTCTCMCCTATTGACAGACTGM

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U.S. Patent

Aug. 15, 1995

Sheet 13 of 27

FIG. 7

xb81

-1 1 MetAla

CTAG AAACCATGAG GGTAATAAAA TAATGGCTCC GCCGCGTCTG TTTGGTACTC CCATTATTTT ATTACCGAGG CGGCGCAGAC ATCTGCGACT CGAGAGTTCT GGAACGTTAC CTGCTGGAAG CTAAAGAAGC TAGACGCTGA GCTCTCAAGA CCTTGCAATG GACGACCTTC GATTTCTTCG TGAAAACATC ACCACTGGTT GTGCTGAACA CTGTTCTTTG AACGAAAACA ACTTTTGTAG TGGTGACCAA CACGACTTGT GACAAGAAAC TTGCTTTTGT TTACGGTACC AGACACCAAG GTTAACTTCT ACGCTTGGAA ACGTATGGAA AATGCCATGG TCTGTGGTTC CAATTGAAGA TGCGAACCTT TGCATACCTT GTTGGTCAAC AAGCAGTTGA AGTTTGGCAG GGTCTGGCAC TGCTGAGCGA CAACCAGTTG TTCGTCAACT TCAAACCGTC CCAGACCGTG ACGACTCGCT GGCTGTACTG CGTGGCCAGG CACTGCTGGT AAACTCCTCT CAGCCGTGGG CCGACATGAC GCACCGGTCC GTGACGACCA TTTGAGGAGA GTCGGCACCC AACCGCTGCA GCTGCATGTT GACAAAGCAG TATCTGGCCT GAGATCTCTG TTGGCGACGT CGACGTACAA CTGTTTCGTC ATAGACCGGA CTCTAGAGAC ACTACTCTGC TGCGTGCTCT GGGTGCACAG AAAGAGGCTA TCTCTCCGCC TGATGAGACG ACGCACGAGA CCCACGTGTC TTTCTCCGAT AGAGAGGCGG GGATGCTGCA TCTGCTGCAC CGCTGCGTAC CATCACTGCT GATACCTTCC CCTACGACGT AGACGACGTG GCGACGCATG GTAGTGACGA CTATGGAAOG GCAAACTGTT TCGTGTATAC TCTAACTTCC TGCGTGGTAA ACTGAAACTG CGTTTGACAA AGCACATATG AGATTGAAGG ACGCACCATT TGACTTTGAC
8alI -

TATACTGGCG AAGCATGCCG TACTGGTGAC CGCTAATAG ATATGACCGC TTCGTACGGC ATGACCACTG GCGATTATCA GCT

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Sheet 14 of 27

FIG. 8
Hind111 AGCTTGGATA
-1 +1 AlcgAla AAAGAGCTCC ACCAAGATTG ATCTGTGACT CGAGAGTTTT ACCTAT TTTCTCGAGG TGGTTCTAAC TAGACACTGA GCTCTCAAAA

GGAAAGATAC TTGTTGGAAG CTAAAGAAGC TGAAAACATC ACCACTGGTT CCTTTCTATG AACAACCTTC GATTTCTTCG ACTTTTGTAG TGGTGACCAA GTGCTGAACA CTGTTCTTTG AACGAAAACA TTACGGTACC AGACACCAAG CACGACTTGT GACAAGAAAC TTGCTTTTGT AATGCCATGG TCTGTGGTTC GTTMCTTCT ACGCTTGGAA ACGTATGGM GTTGGTCAAC AAGCTGTTGA CAATTGMGA TGCGAACCTT TGCATACCTT CAACCAGTTG TTCGACAACT AGTTTGGCAA GGTTTGGCCT TGTTATCTGA AGCTGTTTTG AGAGGTCAAG TCAAACCGTT CCAAACCGGA ACAATAGACT TCGACAAAAC TCTCCAGTTC CCTTGTTGGT TAACTCTTCT CAACCATGGG AACCATTGCA ATTGCACGTC GGAACAACCA ATTGAGAAGA GTTGGTACCC TTGGTAACGT TAACGTGCAG GATAAAGCCG TCTCTGGTTT GAGATCTTTG ACTACTTTGT TGAGAGCTTT CTATTTCGGC AGAGACCAAA CTCTAGAAAC TGATGAAACA ACTCTCGAAA GGGTGCTCAA AAGGAAGCCA TTTCCCCACC AGACGCTGCT TCTGCCGCTC CCCACGAGTT TTCCTTCGGT AAAGGGGTGG TCTGCGACGA AGACGGCGAG CATTGAGAAC CATCACTGCT GATACCTTCA GAAAGTTATT CAGAGTTTAC GTAACTCTTG GTAGTGACGA CTATGGAAGT CTTTCAATAA GTCTCAAATG TCCAACTTCT TGAGAGGTM ATTGAAGTTG TACACCGGTG AAGCCTGTAG AGGTTGAAGA ACTCTCCATT TAACTTCAAC ATGTGGCCAC TTCGGACATC AACTGGTGAC AGATAAGCCC GACTGATAAC AACAGTGTAG TTGACCACTG TCTATTCGGG CTGACTATTG TTGTCACATC

SalI ATGTMCAAA G TACATTGTTT CAGCT

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FIG. 9

Human Monkey

-2 0 -10 [phone] MDVfl[ECPA~~LLSLL8LPLGLPVLQAPPRLICDBROtERYLLEAKEA~NITTGCAEHCSLNENITVPDTK

**************** ******** .........................
50 60 70 80

* ** *
100

*******a*****

MGVBECPA~OltLLSLV8LPLOLPVPGAPPRLICDSRVLERYLL~EAE~MGCSESCSLNENI~DTK
90 110

Human Monkey

*a*** ****a* VNFYAIOI[RMEV(3QQAVmQaWtSEAVLRGQAG-E

~Y~EvoQQAVEVllQGLAfrtSEAVLRGQALLVNS8QPWEPLQLHVD1IAVSQLRS~TTLLRALGAQKE

..................................
12 0
13 0

*

*** ***** *****+**** *

1 40

150

160

Human Monkey

AIBPPDAABAAPLRTITADTBRKLFRWBNFLRGKLKLYTGEACRTGDR

*** **+************** .......................

***

AISLPDAABAAPLRTITADTFCKLFRVYSNFLRGKLKLYTGEACRRGDR

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Sheet 16 of 27

MTTCTAGAAACCATGAGGGTAATAAAATA

CCATTATTTTATTACCCTCATGGTTTCTAG ATGGCTCCGCCGCGTCTGATCTGCGAC

CTCGAGTCGCAGATCAGACGCGGCGGAG TCGAGAGTTCTGGAACGTTACCTGCTG

CTTCCAGCAGGTAACGTTCCAGAACT
GAAGCTAAAGAAGCTGAAAACATC

GTGGTGATGTTTTCAGCTTCTTTAG
ACCACTGGTTGTGCTGAACACTGTTC
CAAAGAACAGTGTTCAGCACAACCA

TTTGAACGAAAACATTACGGTACCG
GATCCGGTACCGTAATGTTTTCGTT

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Sheet 17 of 27

FIG. 11

AATTCTAG AAACCATOAG

XbaI EcoRI

1

3

GGTAATAAAA GATC TTTGGTACTC CCATTATTTT

GCCGCGTCTG CGGCGCAGAC
4 -

2
GGAACGTTAC CCTTGCAATG
6 -

CTAAAOAAGC GATTTCTTCG

GTGCTGAACA CTGTT
8
CACGACTTGT

9

10

11 MGEC CAAA TTGCTTrPGT

-

=I 1 TTACGGTACC G AATGCCATGG CCTAG
12 -

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Sheet 18 of 27

FIG. 12

AATTCGGTACCAGACACCAAGGT GTTAACCTTGGTGTCTGGTACCG

TAACTTCTACGCTTGGAAACGTAT

TTCCATACGTTTCCAAGCGTAGAA GGAAGTTGGTCAACAAGCAGTTGAAGT

CCAAACTTCAACTGCTTGTTGACCAAC
TTGGCAGGGTCTGGCACTGCTGAGCG

GCCTCGCTCAGCAGTGCCAGACCCTG
AGGCTGTACTGCGTGGCCAGGCA GCAGTGCCTGGCCACGCAGTACA
CTGCTGGTAAACTCCTCTCAGCCGT

TTCCCACGGCTGAGAGGAGTTTACCA

GGGAACCGCTGCAGCTGCATGTTGAC
GCTTTGTCAACATGCAGCTGCAGCGG

AAAGCAGTATCTGGCCTGAGATCTG GATCCAGATCTCAGGCCAGATACT

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EaoRI -

Kml 1 A ATTCGGTACC AGACACCMG G GCCATGO TCTOTQGTTC

ACGCTTGGAA ACGTA
TGCGAACCTT

3

2

3

GTTGGTCLC MGCAGTTGA CMCCACTTQ TTCOTCMCT 6 9

5

AG

8

TGCTGAGC& ACGACTCGCT

CGTGGCCAGG E ~ + ~ A T G A QCACCGGTCC C 10 GGCTGTACTG

CAGCCO* GTCWCACCC

CCGCT~CA OCTGCATGTT GA&

15 BgtllIII BamHI A A G TATCGCCT ~~ GAGATCTG GCOACGT COACGTACM CTGTTTC C ATAGACCGOA CTCTAGACCTAC 13

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Sheet 20 of 27

FIG. 14

GATCCAGATCTCTGACTACTCTGC

ACGCAGCAGAGTAGTCAGAGATCTG
TGCGTGCTCTGGGTGCACAGAAAGAGG GATAGCCTCTTTCTGTGCACCCAGAGC

CTATCTCTCCGCCGGATGCTGCATCT
CAGCAGATGCAGCATCCGGCGGAGA

GCTGCACCGCTGCGTACCATCACTG

ATCAGCAGTGATGGTACGCAGCGGTG CTGATACCTTCCGCAAACTGTTTCG ATACACGAAACAGTTTGCGGAAGGT

TGTATACTCTAACTTCCTGCGTGGTA
CAGTTTACCACGCAGGAAGTTAGAGT

AACTGAAACTGTATACTGGCGAAGC

GGCATGCTTCGCCAGTATACAGTTT ATGCCGTACTGGTGACCGCTAATAG TCGACTATTAGCGGTCACCAGTAC

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A U ~ 15, .

1995

Sheet 21 of 27

FIG. 15

BamBI -

BqlII GA TCCAGATCTCTG GTCTAGAGAC
1
3

ACTAC~TGC TGATGAGACG

ZGGTGCACAG AAAGAG CCCACGTGTC

2
L

4
T

GGATGCTGCA TC CCTACGACGT

CGCTGCGTAC CATCACT GCGACGCATG

6

8

GCAMCTGTT T C ~ G T A T A C TGCGTGGT CGTTTGACM A G C A C A T ~ GAGATTGMGG

TCTAACTTCC
1s

11

10
TATACTGGCG ATATGACCGC

12

W

I

14

TACTGGTGAC CGCTMTAG ATGACCACTG GCGATTATC

AGCT

16

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Sheet 22 of 27

FIG. 16

AATTCAAGCTTGGATAAAAGAGCT GTGGAGCTCTTTTATCCAAGCTTG CCACCAAGATTGATCTGTGACTC TCTCGAGTCACAGATCAATCTTG GAGAGTTTTGGAAAGATACTTGTTG
CTTCCAACAAGTATCTTTCCAAAAC

GMGCTAAAGAAGCTGAAAACATC GTGGTGATGTTTTCAGCTTCTTTAG
ACCACTGGTTGTGCTGAACACTGTTC

CAAAGAACAGTGTTCAGCACAACCA

TTTGAACGAAAACATTACGGTACCG
GATCCGGTACCGTAATGTTTTCGTT

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Sheet 23 of 27

ECORI -

Hind111 A AATTCA AGCTTGGATA G TTCGAACCTAT
2 -

ow-GATAC
CCTTTCTATG

5

7

6

8 -

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Sheet 24 of 27

FIG. 18

AATTCGGTACCAGACACCAAGGT GTTAACCTTGGTGTCTGGTACCG TMCTTCTACGCTTGGAAACGTAT TTCCATACGTTTCCAAGCGTAGM

GGAAGTTGGTCAACAAGCAGTTGMGT
CCAAACTTCAACTGCTTGTTGACCAAC

TTGGCMGGTTTGGCCTTGTTATCTG
GCTTCAGATMCAAGGCCAAACCTTG AAGCTGTTTTGAGAGGTGMGCCT
AACAAGGCTTGACCTCTCAAAACA

TGTTGGTTMCTCTTCTCAACCATGGG
TGGTTCCCATGGTTGAGMGAGTTMCC

MCCATTGCAATTGCACGTCGAT CTTTATCGACGTGCAATTGCAA

AAAGCCGTCTCTGGTTTGAGATCTG
GATCCAGATCTCAAACCAGAGACGG

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Sheet 25 of 27

FIG. 19

EcoRI -

1
2 -

A ATTCGGT~CCAGACACCAAG GCCATGG TCTGTGGTTC

5

GTTGGTLULGCTGTTGA CAACCAGTTG TTCGACAACT

7
9 8 -

6

GG~TTGGCCTTGTTATCT CCAAACCGGA

10

AGAGGTCAAG TCTCCAGTTC

15 mI I 1 GCCG GTCTGGTTT GAGATCTG CTATTT GC AGAGACCAAA CTCTAGACCTA G G* A 16

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Sheet 26 of 27

FIG. 20
GATCCAGATCTTTGACTACTTTGTT TCTCAACAAAGTAGTCAAAGATCTG GAGAGCTTTGGGTGCTCAAAAGGAAG
ATGGCTTCCTTTTGAGCACCCAAAGC

CCATTTCCCCACCAGACGCTGCTT
GCAGAAGCAGCGTCTGGTGGGGAA

CTGCCGCTCCATTGAGAACCATC CAGTGATGGTTCTCAATGGAOCG ACTGCTGATACCTTCAGAAAGTT GAATAACTTTCTGAAGGTATCAG ATTCAGAGTTTACTCCAACTTCT CTCAAGAAGTTGGAGTAAACTCT
TGAGAGGTAAATTGAAGTTGTACAC

ACCGGTGTACAACTTCAATTTACCT
CGGTGAAGCCTGTAGAACTGGT CTGTCACCAGTTCTACAGGCTTC GACAGATAAGCCCGACTGATAA GTTGTTATCAGTCGGGCTTAT CAACAGTGTAGATGTAACAAAG TCGACTTTGTTACATCTACACT

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Sheet 27 of 27

FIG. 21

1

GATC

CAGATCTTTGACTACTTTGT
GTCTAGAAAC TGATGAAACA
2 -

-1 1

1

3

GGGT~CTCAA AAGGAA CA CCCACGAGTT TTCCTTCGGT
4 -

TTTCCCCACC

5

AGACGCTGCT OGGGTGG TCTGCGACGA
6 -

13

15

TCCAACTTCT AGGTTGAAGA

TACA

GGTG A A ~ C T G T A G TTCGGACATC

GACTGAT

8alI ATGTAACAAA G TACATTGTTT CAGCT
20 -

-

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L

PRODUCI'ION OF RECOMBINANT ERYTHROPOIETIN
This is a continuation of my co-pending U.S. patent 5 application Ser. No. 675,298, filed Nov. 30, 1984 and issued as U.S. Pat. No. 4,703,008 on Oct. 27, 1987, which was a continuation-in-part of my copending U.S. patent application Ser. No. 561,024, filed Dec. 13, 1983, now abandoned, and a continuation-in-part of Ser. No. 10 582,185, filed Feb. 21, 1984, now abandoned, and a continuation-in-part of Ser. No. 655,841, filed Sep. 28, 1984 now abandoned. BACKGROUND 15 The present invention relates generally to the manipulation of genetic materials and, more particularly, to recombinant procedures making possible the producpossessing part or all of the primary tion of structural conformation and/or one or more of the 20 biological properties of naturally-occurring erythropoietin. A. Manipulation Of Genetic Materials Genetic materials may be broadly defined as those 25 chemical substances which program for and guide the manufacture of constituents of cells and viruses and direct the responses of cells and viruses. A long chain polymeric substance known as deoxyribonucleic acid (DNA) comprises the genetic material of all living cells 30 and viruses except for certain viruses which are programmed by ribonucleic acids (RNA). The repeating units in DNA polymers are four different nucleotides, each of which consists of either a purine (adenine or guanine) or a pyrimidine (thymine or cytosine) bound to 35 a deoxyribose sugar to which a phosphate group is attached. Attachment of nucleotides in linear polymeric form is by means of fusion of the 5' phosphate of one nucleotide to the 3' hydroxyl group of another. Functional DNA occurs in the form of stable double 40 stranded associations of single strands of nucleotides (known as deoxyoligonucleotides), which associations occur by means of hydrogen bonding between purine and pyrimidine bases [i.e., "complementary" associations existing either between adenine (A) and thymine 45 (T) or guanine (G) and cytosine (C)]. By convention, nucleotides are referred to by the names of their constituent purine or pyrimidine bases, and the complementary associations of nucleotides in double stranded DNA (i.e., A-T and G-C) are referred to as "base 50 pairs". Ribonucleic acid is a polynucleotide comprising adenine, guanine, cytosine and uracil (U), rather than thymine, bound to ribose and a phosphate group. Most briefly put, the programming function of DNA is generally effected through a process wherein specific 55 DNA nucleotide sequences (genes) are "transcribed into relatively unstable messenger RNA (mRNA) polymers. The mRNA, in turn, serves as a template for the formation of structural, regulatory and catalytic proteins from amino acids. This mRNA "translation" process 60 involves the operations of small RNA strands (tRNA) which transport and align individual amino acids along the mRNA strand to allow for formation of polypeptides in proper amino acid sequences. The mRNA "message" derived from DNA and providing the basis for 65 the tRNA supply and orientation of any given one of the twenty amino acids for polypeptide "expression", is in the form of triplet "codons" -sequential groupings

of three nucleotide bases. In one sense, the formation of a protein is the ultimate form of "expression" of the programmed genetic message provided by the nucleotide sequence of a gene. "Promoter" DNA sequences usually "precede" a gene in a DNA polymer and provide a site for initiation of the transcription into mRNA. "Regulator" DNA sequences, also usually "upstream" of (i.e., preceding) a gene in a given DNA polymer, bind proteins that determine the frequency (or rate) of transcriptional initiation. Collectively referred to as "promoter/regulator" or "control" DNA sequence, these sequences which precede a selected gene (or series of genes) in a functional DNA polymer cooperate to determine whether the transcription (and eventual expression) of a gene will occur. DNA sequences which "follow" a gene in a DNA polymer and provide a signal for termination of the transcription into mRNA are referred to as transcription "terminator" sequences. A focus of microbiological processing for the last decade has been the attempt to manufacture industrially and pharmaceutically significant substances using or-ganishs which either do not initially have coded information concerningthe desired product included in their DNA, or (in the case of mammalian cells in culture) do not ordinarily express a chromosomal gene at appreciable levels. Simply put, a gene that specifies the structure of a desired polypeptide product is either isolated from a "donor" organism or chemically synthesized and then stably introduced into another organism which is preferably a self-replicating unicellular oreanism such as bacteria. veast or mammalian cells . in culture. Once this is done, the existing machinery for gene expression in the "transformed" or "transfected" microbial host cells operates to construct the desired product, using the exogenous DNA as a template for transcription of mRNA which is then translated into a continuous sequence of amino acid residues. The art is rich in patent and literature publications relating to "recombinant DNA" methodologies for the isolation, synthesis, purification and amplification of genetic materials for use in the transformation of selected host organisms. U.S. Pat. No. 4,237,224 to Cohen, et al., for example, relates to transformation of unicellular host organisms with "hybrid" viral or circular plasmid DNA which includes selected exogenous DNA sequences. The procedures of the Cohen, et al. patent first involve manufacture of a transformation vector by enzymatically cleaving vital circular plasmid DNA to form linear DNA strands. Selected foreign ("exogenous"or "heterologous") DNA strands usually including sequences coding for desired product are prepared in linear form through use of similar enzymes. The linear vital or plasmid DNA is incubated with the foreign DNA in the presence of ligating enzymes capable of effecting a restoration process and "hybrid vectors are formed which include the selected exogenous DNA segment "spliced" into the viral or circular DNA plasmid. Transformation of compatible unicellular host organisms with the hybrid vector results in the formation of multiple copies of the exogenous DNA in the host cell population. In some instances, the desired result is simply the amplification of the foreign DNA and the "product" harvested is DNA. Note frequently, the goal of transformation is the expression by the host cells of the exogenous DNA in the form of large scale synthesis of isolatable quantities of commercially significant prow
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3

5,441,868

4

tein or poiypeptide fragments coded for by the foreign DNA. Seealso, e.g., U.S. Pat. Nos. 4,264,731 (to Shine), 4,273,875 (to Manis), 4,293,652 (to Cohen), and European Patent Application 093,619, published Nov. 9, 1983. 5 The development of specific DNA sequences for splicing into DNA vectors is accomplished by a variety of techniques, depending to a great deal on the degree of "foreignness" of the "donor" to the projected host and the size of the polypeptide to be expressed in the 10 host. At the risk of over-simplification, it can be stated that three alternative principal methods can be employed: (1) the "isolation" of double-stranded DNA sequence from the genomic DNA of the donor; (2) the chemical manufacture of a DNA sequence providing a 15 code for a polypeptide of interest; and (3) the in vitro synthesis of a double-stranded DNA sequence by enzymatic "reverse transcription" of mRNA isolated from donor cells. The last-mentioned methods which involve formation of a DNA "complement" of mRNA are gen- 20 erally referred to as "cDNA" methods. Manufacture of DNA sequences is frequently the method of choice when the entire sequence of amino acid residues of the desired polypeptide product is known. DNA manufacturing procedures of co-owned, 25 co-pending U.S. patent application Ser. No. 483,451, by Alton, et al., (filed Apr. 15, 1983 and corresponding to PCT US83/00605, published Nov. 24, 1983 as W083/04053), for example, provide a superior means for accomplishing such highly desirable results as: pro- 30 viding for the presence of alternate codons commonly found in genes which are highly expressed in the host organism selected for expression (e.g., providing yeast or E.coli "preference" codons); avoiding the presence of untranslated "intron" sequences (commonly present 35 in mammalian genomic DNA sequences and mRNA transcripts thereof) which are not readily processed by procaryotic host cells; avoiding expression of undesired "leader" polypeptide sequences commonly coded for by genomic DNA and cDNA sequences but frequently 40 not readily cleaved from the polypeptide of interest by bacterial or yeast host cells; providing for ready insertion of the DNA in convenient expression vectors in association with desired promoter/regulator and terminator sequences; and providing for ready construction 45 of genes coding for polypeptide fragments and analogs of the desired polypeptides. When the entire sequence of amino acid residues of the desired polypeptide is not known, direct manufacture of DNA sequences is not possible and isolation of 5 0 DNA sequences coding for the polypeptide by a cDNA method becomes the method of choice despite the potential drawbacks in ease of assembly of expression vectors capable of providing high levels of microbial expression referred to above. Among the standard pro- 55 cedures for isolating cDNA sequences of interest is the preparation of plasmid-borne cDNA "libraries" derived from reverse transcription of mRNA abundant in donor cells selected as responsible for high level expression of genes (e.g., libraries of cDNA derived from pituitary 60 cells which express relatively large quantities of growth hormone products). Where substantial portions of the polypeptide's amine acid sequence are known, labelled, single-stranded DNA probe sequences duplicating a sequence putatively present in the "target" cDNA may 65 be employed in D N A D N A hybridization procedures carried out on cloned copies of the cDNA which have been denatured to single stranded form. [See, generally,

the disclosure and discussions of the art provlded in U.S. Pat. No. 4,394,443 to Weissman, et al. and the recent demonstrations of the use of long oligonucleotide hybridization probes reported in Wallace, et al., Nuc. Acids Res, 6, pp. 3543-3557 (1979), and Reyes, et al., P.N.A.S. (U.S.A.), 79, pp. 3270-3274 (1982), and Jaye, et al., Nuc. Acids Res., 1 1 , pp. 2325-2335 (1983). See also, U.S. Pat. No. 4,358,535 to Falkow, et al., relating to D N A D N A hybridization procedures in effecting diagnosis; published European Patent Application Nos. 0070685 and 0070687 relating to light-emitting labels on single stranded polynucleotide probes; Davis, et al., "A Manual for Genetic Engineering, Advanced Bacterial Genetics" Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y. (1980) at pp. 55-58 and 174-176, relating to colony and plaque hybridization techniques; and, New England Nuclear (Boston, Mass.) brochures for "Gene Screen" Hybridization Transfer Membrane materials providing instruction manuals for the transfer and hybridization of DNA and RNA, Catalog No. NEF-972.1 Among the more significant recent advances in hybridization procedures for the screening of recombinant clones is the use of labelled mixed synthetic oligonucleotide probes, each of which is potentially the complete complement of a specific DNA sequence in the hybridization sample including a heterogenous mixture of single stranded DNAs or RNAs. These procedures are acknowledged to be especially useful in the detection of cDNA clones derived from sources which provide extremely low amounts of mRNA sequences for the polypeptide of interest. Briefly put, use of stringent hybridization conditions directed toward avoidance of non-specific binding can allow, e.g., for the autoradiographic visualization of a specific cDNA clone upon the event of hybridization of the target DNA to that single probe within the mixture which is its complete complement. See generally, Wallace, et al., Nuc. Acids Res, 9, pp. 879-897 (1981); Suggs, et al. P.N.A.S. (U.S.A.), 78, pp. 6613-6617 (1981); Choo, et al., Nature, 299, pp. 178-180 (1982); Kurachi, et al., P.N.A.S. (U.S.A.), 79, pp. 6461-6484 (1982); Ohkubo, et a. P.N.A.S. (U.S.A.), l, 80, pp. 2196-2200 (1983); and Komblihtt, et al. P.N.A.S. (U.S.A.), 80, pp. 3218-3222 (1983). In general, the mixed probe procedures of Wallace, et al. (1981), supra, have been expanded upon by various workers to the point where reliable results have reportedly been obtained in a cDNA clone isolation using a 32-member mixed "pool" of 16-base-long (16-mer) oligonucleotide probes of uniformly, varying DNA sequences together with a single 11-mer to effect a two-site "positive" confirmation of the presence of cDNA of interest. See, Singer-Sam, et al., P.N.A.S. (U.S.A.), 80, pp. 802-806 (1983). The use of genomic DNA isolates is the least common of the three above-noted methods for developing specific DNA sequences for use in recombinant procedures. This is especially true in the area of recombinant procedures directed to securing microbial expression of mammalian polypeptides and is due, principally to the complexity of mammalian genomic DNA. Thus, while reliable procedures exist for developing phage-borne libraries of genomic DNA of human and other mammalian species origins [See, e.g., Lawn, et al. Cell, 15, pp, 1157-1 174 (1978) relating to procedures for generating a human genomic library commonly referred to as the "Maniatis Library"; Karn, et al., P.N.A.S. (U.S.A.), 77, pp. 5172-5176 (1980) relating to a human genomic li-