HU200366B - Process for producing modified hygromycin resistance gene - Google Patents

Abstract

(1) DNA encoding the last 338, 339 or 340 amino acids of hygromycin B phosphotransferase either alone or in translational reading phase with a transcriptional and translational activator sequence-contg. gene or protein of a gene is new. (2) Recombinant DNA cloning vector comprising the DNA defined above is new. (3) Plasmids pII 141, 143 and 207 are new. (4) Transformant comprising the vector defined above is new.

Description

FIELD OF THE INVENTION The present invention relates to a modified gene that determines resistance to hygromycin B antibiotic. The invention also relates to the preparation of vectors and transformants containing the aforementioned DNA.

British Patent Publication No. 2,100,738 discloses the starting materials, including plasmid pKC222, and the DNA fragment that determines resistance to the hygromycin B antibiotic used in the methods described herein.

However, the above specification does not disclose the use of the modified gene which is the subject of the present invention for the production of fused genes for selection.

Gene fusion with a dominant selectable marker is a suitable method for isolating transcriptional or translational activation sequences and thus for expressing a dominant selectable genetic signal in foreign cells. Because many organisms are sensitive to the aminoglycoside-type hygromycin B, Ahmad et al., Antimicrob. Agents Chemother., 18, 789 (1980); Mann et al., Antibiot. and Chemother, 3, 1279 (1953); Pettinger et al., Antibiot. and Chemother., 3, 1268 (1953); and Singh et al., Naturre, 277, 146 (1979)], the modified gene for resistance to hygromycin B is a valuable dominant, selectable genetic mammal in various host cell systems.

It is of interest to the present invention that the last 338, 339 or 340 amino acids of hygromycin B phosphotransferase, in their natural sequence, describe all amino acids of the enzyme hygromocin B phosphotransferase except the first and second or first, second and third amino acids of the N-terminal end of the naturally occurring enzyme molecule. 40

Below we define certain terms.

A recombinant DNA cloning vector is an autonomously replicating molecule, such as, but not limited to, a plasmid to which one or more additional DNA segments are linked or linked to a DNA molecule.

Transformation: introduction of DNA into a host host cell, followed by a change in the geno5 type and a change in the host cell. Functional polypeptide: a recoverable, biologically active, fully foreign polypeptide or precursor, a recoverable, biologically active, polypeptide consisting of 10 or all of a portion of a homologous polypeptide, or a recoverable, biologically inactive, fusion polypeptide, and a biologically inactivating homologous polypeptide which is specifically cleaved.

Fused gene product: a recoverable, foreign-derived polypeptide that is linked to a portion or all of a homologous polypeptide.

Structural gene: a functional polypeptide-defining DNA that does not contain transcriptional and translational activation sequences.

FIELD OF THE INVENTION The present invention relates to a plasmid comprising a gene containing a transcriptional and translational activation sequence, or a portion thereof, comprising the DNA sequence defining the last 338 amino acids of the hygromycin B phosphotransferase enzyme alone or in a translation reading phase.

To construct the plasmid, a 1.45 30 kb Eco RI restriction fragment of plasmid pKC222 is linked to the Eco RI digested form of pIT122 to give plasmid pIT123. The invention also relates to transformants containing the above DNA.

More particularly, the DNA of the present invention comprises the gene defining the translational activation sequence, or the DNA sequence of a portion of a gene. The present invention also relates to recombinant DNA cloning vectors and transformants containing the aforementioned DNA ribonucleic acid.

More particularly, the DNA sequence of the present invention has the following DNA sequence:

Rm 1

Rm LYS

R ² n 1 CCT 1 1 1 GAA 1 1 1 CTC 1 1 1 ACC 1 1 1 GCG 1 1 1 ACG 1 I 1 1 R ³ n LYS 1 1 1 GGA PRO 1 1 1 CTT GLU 1 1 1 GAG LEU 1 1 1 TGG THR i 1 I CGC ALA 1 f 1 TGC THR ATC 1 1 I GAA 1 1 1 AAG 1 1 f TTC 1 1 1 GAC 1 1 i AGC 1 1 1 GTC 1 1 1 1 1 I BROAD 1LE 1 1 I CTT GLU 1 1 1 TTC LYS 1 1 1 AAG PHE 1 1 1 CTG ASP 1 1 1 TCG SER 1 I 1 CAG VAL TCG i 1 1 GAG 1 1 1 GGC 1 1 1 GAA 1 1 t GAA 1 1 1 TCT i 1 1 CGT 1 1 1 I i I AGC SER 1 1 1 CTC GLU 1 I 1 CCG GLY 1 1 1 CTT GLU 1 i 1 CTT GLU I 1 1 AGA SER 1 1 1 GCA ARG

TCT 1 1 1 AGA SER GTC 1 1 1 CAG WITH GAG 1 1 1 CTC GLU AAG 1 1 1 TTC LYS TTT 1 1 1 AAA PHE CTG 1 1 1 GAC LEU TCC i t 1 GAC t 1 1 CTG ATG t 1 1 CAG 1 1 1 CTC I 1 1 1 1 1 AGG <1 1 CTG 1 1 1 GAC 1 1 1 TAC 1 1 1 GTC 1 1 1 GAG SER ASP LEU MET GLN LEU GCT 1 hr 1 TTC i 1 1 AGC 1 1 1 TTC 1 1 1 DAM 1 1 1 GTA I 1 1 CGA 1 1 1 AAG 1 1 1 TCG I 1 1 AAG 1 1 I CTA i 1 1 CAT ALA PHE SER PHE ASP WITH

HU 200366 Β

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rm

LYS

GGA 1 1 1 GGG 1 1 1 CGT 1 1 1 GGA 1 1 1 TAT 1 1 1 GTC 1 1 1 CTG 1 1 1 CGG 1 1 1 GTA 1 1 1 AAT 1 1 I AGC 1 1 1 TGC 1 t i GCC 1 1 1 1 1 1 CCT 1 1 1 ccc 1 1 1 GCA 1 1 1 CCT 1 1 1 OVER THE i 1 1 CAG 1 1 1 GAC 1 1 1 GCC 1 1 1 CAT 1 1 1 TTA 1 1 1 TCG 1 1 1 ACG 1 1 1 CGG GLY GLY ARG GLY TYR WITH LEU ARG WITH ASN SER CYS ALA DAM 1 1 1 GGT 1 1 1 TTC 1 1 1 TAC 1 1 1 AAA 1 1 1 DAM 1 1 1 CGT 1 1 1 TAT 1 1 1 GTT 1 1 1 TAT 1 1 1 CGG 1 1 1 CAC 1 1 1 TTT 1 1 1 1 1 1 CTA 1 1 1 CCA 1 1 1 AAG 1 1 1 ATG 1 1 1 TTT 1 1 1 CTA 1 1 1 GCA 1 1 1 ATA 1 1 1 CAA 1 1 1 ATA 1 1 1 GCC 1 1 1 GTG 1 I 1 AAA ASP GLY PHE TYR LYS ASP ARG TYR WITH TYR ARG HIS PHE GCA 1 1 1 TCG 1 1 1 GCC 1 1 1 GCG 1 1 1 CTC 1 1 1 CCG 1 1 1 ATT 1 1 1 CCG 1 1 1 GAA 1 1 1 GTG 1 1 1 CTT 1 1 1 GAC 1 1 1 ATT 1 1 1 1 1 1 CGT 1 ι 1 AGC 1 1 1 CGG 1 1 1 CGC 1 1 1 GAG 1 1 1 GGC 1 1 1 TAA 1 1 1 GGC 1 1 1 CTT 1 1 1 CAC 1 1 1 GAA 1 1 I CTG 1 1 1 TAA ALA SER ALA ALA LEU PRO ILE PRO GLU WITH LEU ASP ILE GGG 1 1 1 GAA 1 1 1 TTC t 1 1 AGC 1 1 1 GAG 1 1 1 AGC 1 1 1 CTG 1 1 1 ACC 1 1 1 TAT 1 1 1 TGC 1 1 1 ATC 1 1 i TCC 1 1 1 CGC 1 1 1 1 1 I ccc 1 1 1 CTT 1 1 1 AAG 1 1 1 TCG 1 1 1 CTC 1 1 1 TCG 1 1 1 GAC 1 1 1 TGG 1 1 1 ATA 1 1 1 ACG I 1 1 TAG 1 1 1 AGG 1 1 1 GCG GLY GLU PHE SER GLU SER LEU THR TYR CYS ILE SER ARG CGT 1 1 1 GCA 1 1 1 CAG 1 1 1 GGT 1 1 1 GTC 1 1 1 ACG 1 1 1 TTG 1 1 1 CAA 1 1 1 GAC 1 1 1 CTG 1 1 1 CCT 1 1 1 GAA 1 1 1 ACC 1 1 1 1 1 1 GCA 1 1 1 CGT 1 1 1 GTC 1 1 ι CCA 1 1 1 CAG 1 1 1 TGC 1 1 1 AAC 1 1 1 GTT 1 1 1 CTG 1 1 1 GAC 1 1 1 GGA 1 1 1 CTT 1 1 1 TGG ARG ALA GLN GLY WITH THR LEU GLN ASP LEU PRO GLU THR GAA 1 1 1 CTG 1 1 1 CCC 1 1 1 GCT 1 1 1 GTT 1 I 1 CTG 1 1 1 CAG 1 1 1 CCG 1 1 1 GTC 1 1 1 GCG 1 1 1 GAG t 1 1 GCC 1 1 1 ATG 1 1 1 1 1 1 CTT 1 1 1 GAC 1 1 1 GGG 1 1 1 CGA 1 I 1 CAA 1 1 i GAC 1 1 1 GTC 1 1 1 GGC 1 ι 1 CAG 1 1 1 CGC 1 1 1 CTC 1 1 1 CGG 1 1 1 TAC GLU LEU PRO ALA WITH LEU GLN PRO WITH ALA GLU ALA MET DAM 1 1 1 GCG 1 1 1 ATC 1 1 t GCT 1 1 1 GCG 1 1 1 GCC 1 1 1 DAM 1 1 1 CTT 1 1 1 AGC 1 1 1 CAG 1 I 1 ACG 1 1 1 AGC 1 1 1 GGG 1 1 1 I 1 i CTA 1 1 1 CGC 1 1 1 BROAD 1 1 1 CGA l l 1 CGC 1 1 1 CGG 1 1 1 CTA 1 1 1 GAA 1 1 1 TCG 1 i 1 GTC 1 1 1 TGC 1 1 1 TCG 1 1 1 CCC ASP ALA ILE ALA ALA ALA ASP LEU SER GLN THR SER GLY TTC 1 1 1 GGC 1 1 1 CCA 1 1 1 TTC 1 1 1 GGA 1 1 1 CCG 1 1 1 CAA 1 1 1 GGA 1 1 1 ATC 1 1 1 GGT 1 1 1 CAA 1 1 1 TAC 1 1 1 ACT 1 1 1 1 1 1 AAG 1 1 1 CCG 1 1 1 GGT 1 1 1 AAG 1 1 1 CCT 1 1 1 GGC 1 I 1 GTT III CCT 1 1 1 TAG 1 1 1 CCA 1 1 1 GTT 1 1 1 ATG 1 1 1 TGA PHE GLY PRO PHE GLY PRO GLN GLY ILE GLY GLN TYR THR ACA TGG CGT DAM TTC OVER THE TGC GCG ATT GCT DAM CCC CAT 1 1 1 f 1 1 1 1 1 1 1 1 1 1 1 1 1 t 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 TGT ACC GCA CTA AAG TAT ACG CGC TAA CGA CTA GGG GTA THR TRP ARG ASP PHE ILE CYS ALA ILE ALA ASP PRO HIS GTG 1 1 1 TAT 1 1 i CAC I 1 1 TGG 1 1 1 CAA 1 1 1 ACT 1 1 1 GTG 1 1 1 ATG 1 1 1 GAC 1 1 1 GAC 1 1 1 ACC 1 1 1 GTC 1 1 1 AGT 1 1 t 1 1 1 CAC ι 1 1 OVER THE 1 1 1 GTG 1 1 1 ACC 1 1 1 GTT 1 1 1 TGA I 1 1 CAC 1 1 TAC 1 1 1 CTG 1 1 1 CTG 1 1 1 TGG 1 1 1 CAG 1 1 1 TCA WITH TYR HIS TRP GLN THR WITH MET ASP ASP THR WITH SER GCG 1 1 1 TCC 1 1 1 GTC 1 1 1 GCG 1 1 1 CAG 1 1 1 GCT I 1 1 CTC 1 1 1 DAM 1 1 1 GAG 1 1 1 CTG 1 1 1 ATG 1 1 1 CTT 1 1 1 TGG I 1 1 1 i 1 CGC 1 1 1 AGG 1 1 1 CAG 1 1 1 CGC 1 1 1 GTC 1 I 1 CGA 1 I 1 GAG 1 1 1 CTA 1 1 1 CTC 1 1 1 GAC I 1 1 TAC 1 1 1 GAA 1 1 I ACC ALA SER WITH ALA GLN ALA LEU ASP GLU LEU MET LEU TRP GCC 1 1 1 GAG ι i 1 GAC 1 1 1 TGC III CCC 1 1 1 GAA 1 1 1 GTC 1 1 1 CGG 1 1 1 CAC 1 1 1 CTC 1 1 1 GTG I 1 1 CAC 1 1 1 GCG 1 1 1 Ι 1 1 CGG 1 I 1 CTC 1 1 1 CTG 1 1 1 ACG 1 1 1 GGG 1 1 I CTT 1 1 1 CAG 1 1 i GCC 1 1 1 GTG 1 1 1 GAG I 1 1 CAC 1 1 1 GTG 1 1 1 CGC ALA GLU ASP CYS PRO GLU WITH ARG HIS LEU WITH HIS ALA

HU 200366 Β

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rm

LYS

DAM 1 1 1 TTC 1 1 1 GGC 1 1 1 TCC 1 1 1 AAC 1 1 1 AAT 1 1 1 GTC 1 1 1 CTG 1 1 1 ACG 1 1 1 GAC 1 1 1 AAT 1 1 1 GGC 1 1 1 CGC 1 1 1 1 1 1 CTA 1 1 1 AAG 1 1 1 CCG i 1 1 AGG 1 1 1 TTG 1 1 1 TTA I 1 1 CAG 1 to 1 GAC 1 1 1 TGC 1 1 1 CTG 1 1 1 TTA 1 1 1 CCG 1 1 1 GCG ASP PHE GLY SER ASN ASN WITH LEU THR ASP ASN GLY ARG OVER THE 1 1 1 ACA 1 1 1 GCG 1 1 1 GTC 1 1 1 ATT 1 f 1 GAC 1 1 1 TGG 1 1 1 AGC 1 1 1 GAG 1 1 1 GCG 1 i 1 ATG 1 1 1 TTC 1 1 1 GGG t 1 1 1 1 1 TAT 1 l 1 TGT 1 1 1 CGC 1 1 1 CAG 1 1 1 TAA 1 1 1 CTG 1 1 1 ACC 1 i 1 TCG 1 1 1 CTC 1 1 1 CGC 1 1 1 TAC 1 1 1 AAG ι 1 1 ccc ILE THR ALA WITH ILE ASP TRP SER GLU ALA MET PHE GLY DAM 1 1 1 TCC 1 1 1 CAA 1 1 1 TAC 1 1 1 GAG 1 1 1 GTC 1 1 1 GCC 1 1 1 AAC 1 1 1 ATC 1 1 1 TTC 1 1 1 TTC 1 1 1 TGG 1 1 1 AGG 1 1 1 1 i 1 CTA 1 1 1 AGG 1 1 1 GTT 1 1 1 ATG 1 1 1 CTC 1 1 ι CAG 1 f 1 CGG 1 1 1 TTG 1 1 1 TAG 1 I 1 AAG 1 1 1 AAG I 1 1 ACC 1 1 1 TCC ASP SER GLN TYR GLU WITH ALA ASN ILE PHE PHE TRP ARG CCG 1 1 1 TGG 1 1 1 TTG 1 1 1 GCT 1 1 1 TGT III ATG 1 1 1 GAG 1 1 1 CAG 1 1 1 CAG 1 1 1 ACG 1 1 1 CGC 1 i 1 TAC 1 1 1 TTC 1 1 1 1 1 1 GGC 1 1 1 ACC 1 hr 1 AAC 1 1 1 CGA 1 1 1 ACA 1 1 1 TAC 1 1 1 CTC 1 1 1 GTC 1 1 1 GTC 1 1 1 TGC 1 1 1 GCG 1 i 1 ATG 1 1 1 AAG PRO TRP LEU ALA CYS MET GLU GLN GLN THR ARG TYR PHE GAG 1 1 1 CGG 1 1 1 AGG 1 1 1 CAT 1 1 1 CCG 1 1 1 GAG 1 1 1 CTT 1 1 1 GCA 1 1 1 GGA 1 1 1 TCG 1 1 1 CCG 1 1 1 CGG 1 1 1 CTC 1 1 1 1 1 ι CTC 1 ι 1 GCC 1 1 1 TCC 1 1 1 GTA 1 1 I GGC f 1 1 CTC 1 1 1 GAA 1 1 1 CGT 1 1 1 CCT 1 1 1 AGC 1 1 1 GGC 1 1 1 GCC 1 1 1 GAG GLU ARG ARG HIS PRO GLU LEU ALA GLY SER PRO ARG LEU CGG 1 1 1 GCG 1 1 1 TAT 1 1 1 ATG 1 1 1 CTC 1 1 1 CGC 1 1 1 ATT 1 1 1 GGT i 1 1 CTT 1 1 1 GAC 1 II CAA 1 1 1 CTC 1 1 1 TAT 1 1 1 1 1 1 GCC 1 1 1 CGC 1 1 I OVER THE 1 1 1 TAC 1 I 1 GAG 1 1 1 GCG 1 1 1 TAA 1 1 1 CCA 1 1 1 GAA 1 1 ι CTG 1 1 1 GTT 1 1 1 GAG 1 1 1 ATA ARG ALA TYR MET LEU ARG ILE GLY LEU ASP GLN LEU TYR CAG 1 1 1 AGC 1 1 1 TTG 1 1 1 GTT 1 1 1 GAC 1 1 1 GGC 1 1 1 AAT 1 1 1 TTC 1 1 1 DAM 1 1 1 DAM 1 1 1 GCA 1 hr 1 GCT 1 1 1 TGG t 1 1 1 1 1 GTC 1 1 1 TCG 1 1 1 AAC 1 1 1 CAA 1 1 1 CTG I 1 1 CCG 1 1 1 TTA 1 1 1 AAG ι ι ι CTA 1 1 1 CTA 1 1 i CGT 1 1 I CGA 1 1 1 ACC GLN SER LEU WITH ASP GLY ASN PHE ASP ASP ALA ALA TRP GCG 1 1 1 CAG 1 1 1 GGT 1 1 1 CGA 1 1 1 TGC 1 1 1 GAC 1 1 1 GCA 1 1 1 ATC 1 1 i GTC 1 1 1 CGA 1 1 1 TCC 1 1 1 GGA 1 1 1 GCC 1 1 ί 1 1 1 CGC 1 ι 1 GTC 1 1 1 CCA 1 I 1 GCT 1 1 1 ACG 1 1 1 CTG I I 1 CGT 1 1 1 TAG 1 1 1 CAG 1 ι 1 GCT 1 I 1 AGG I 1 I CCT 1 1 1 CGG ALA GLN GLY ARG CYS ASP ALA ILE WITH ARG SER GLY ALA GGG 1 1 1 ACT 1 1 1 GTC 1 1 i GGG 1 1 1 CGT 1 1 1 ACA 1 1 1 CAA 1 1 i ATC 1 1 1 GCC 1 1 1 CGC 1 1 1 AGA 1 1 1 AGC 1 1 1 GCG I 1 1 1 1 1 ccc 1 1 1 TGA i 1 1 CAG 1 1 I CCC 1 1 1 GCA 1 1 1 TGT 1 1 1 GTT 1 1 1 TAG 1 1 1 CGG 1 1 1 GCG 1 1 1 TCT 1 f 1 TCG ι 1 i CGC GLY THR WITH GLY ARG THR GLN ILE ALA ARG ARG SER ALA GCC 1 1 1 GTC 1 1 1 TGG 1 1 1 ACC 1 1 1 DAM 1 1 1 GGC 1 I 1 TGT 1 1 1 GTA 1 1 1 GAA 1 1 1 GTA 1 1 1 CTC 1 1 1 GCC I 1 1 DAM 1 1 1 1 ι 1 CGG 1 1 1 CAG 1 1 1 ACC 1 1 1 TGG 1 1 1 CTA 1 I i CCG 1 1 1 ACA 1 1 1 CAT 1 I I CTT 1 1 1 CAT 1 1 1 GAG 1 1 1 CGG 1 1 1 CTA ALA WITH TRP THR ASP GLY CYS WITH GLU WITH LEU ALA ASP AGT 1 1 1 GGA i i 1 AAC 1 1 1 CGA 1 1 1 CGC 1 1 1 CCC 1 I 1 AGC 1 1 1 ACT 1 1 1 CGT t 1 1 CCG 1 1 1 AGG 1 1 1 GCA I 1 1 AAG 1 1 1 1 1 i TCA 1 1 1 CCT 1 1 1 TTG 1 1 1 GCT 1 1 I GCG 1 1 1 GGG 1 1 1 TCG 1 1 1 TGA 1 1 1 GCA 1 1 1 GGC 1 1 1 TCC 1 1 1 CGT 1 1 1 TTC SER GLY ASN ARG ARG PRO SER THR ARG PRO Arg ALA LYS

GAA R ⁴ III I CTT R ⁵ , GLU

HU 200366 Β where

The deoxyadenyl,

G deoxyguananidyl,

C deoxy-citizil,

T is thymidyl,

The R and R ² lysine defining triplets, independently of one another,

R ¹ and R are deoxyribonucleotide triplets wherein the nitrogenous base is complementary to the corresponding R and R ² base, m and n are 0 or 1 with the proviso that when n = 0, m = 0, and, if m = 1, then n = 1,

The R ⁴ translational stop codon deoxyribonucleotide triplet, and

R ^{5 is} a triplet of deoxyribonucleotides in which the nitrogen-containing base is complementary to its corresponding R ⁴ base.

The amino acids defined by the above DNA are indicated by the following abbreviations:

MET = methionine,

LYS = lysine,

PRO = proline,

GLU = glutamic acid,

LEU = leucine,

THR = threonine,

ALA = alanine,

SER = serine,

VAL = Choice,

PHE = phenylalanine,

ILE = isoleucine,

GLY = glycine,

ASP = aspartic acid,

GLN = glutamine,

ARG = arginine,

CYS = cysteine,

TRP = tryptophan,

ASN = asparagine,

HIS = histidine and

TYR = tyrosine.

The DNA of the present invention wherein:

R, R ¹ , R ² , R ³ , R ⁴ and R ⁵ corresponding to the genetic code (Watson, JD, Molecular, Biology of the Gene, WA Benjamin Inc., Menlo Park, Califomia, 1976) can be synthesized in a known manner phospho triester method, from fully protected trideoxyribonucleotide building blocks. Dyen synthetic methods are well known in the art and can be carried out by Itakura et al., Science, 1987, 1056 (1977), Crea et al., Proc. Nat Acad. Sci., USA, 75, 5765 (1978). It will be appreciated by those skilled in the art that the amino acids defined by the DNA sequence described above may also be made with other DNA sequences. These DNA sequences can be used due to the degeneracy of the genetic code and thus fall within the scope of the invention.

By proper digestion of plasmid pKC222, the DNA molecule described above, where m = 0, R ^{4 is} TAG and R ^{5 is} ATC, is prepared. The Hph1 restriction site at the start of the coding region for the hygromycin B phosphotransferase gene in pKC222 is well suited for this purpose. so the

Following HphI-PstI digestion, plasmid pKC222 yields the hygromycin B phosphotransferase gene, consisting of 325 base pairs (plus the single-stranded end), which defines the hygromycin B phosphotransferase polypeptide 4-112. amino acid A is cleaved with the restriction enzyme Hphl 3 'to the left side, then bound to the fragment by a BamHI molecular linker, cleaved with EcoRI and then ligated to the BamHIEcoRI digested fragment of plasmid pBR322. The resulting plasmid is designated pIT122 and contains a portion of the hygromycin B phosphotransferase gene and can be used as starting material.

Hygromycin B phosphotransferase enzyme 113-341. amino acid residues of plasmid pKC222, which is inserted into the properly cleaved plasmid ρΓΓ122. The resulting plasmid is designated pIT123, which contains the entire structural gene portion of the hygromycin B phosphotransferase except for the first 9 nucleotide pairs where the BamHI linker is located. The gene thus produced describes a hygromycin B phosphotransferase that has lost the first 3 amino acids relative to the polypeptide defined by the natural gene. A restriction map of plasmid ρΓΓ123 is provided in Figure 1 of the accompanying drawings.

The DNA of the present invention is obtained from plasmid pKC222, the plasmid is 6.8 kb, and is constructed by plasmid pKC203.

The 2.75 kb SalI-BglII fragment was ligated to the 4.1 kb SalI-BglII fragment of plasmid pKC7. Plasmid pKC2O3 is 15 kb in size and can be easily isolated from the E coli · JR225 strain found in the American Type Culture Collection, Rockville, Maryland, USA. The strain can be ordered as a source of plasmid pKC203 under ATCC No. 31912. Plasmid pKC7 is known in the art (ATCC 37084) and may be prepared according to the method of Rao and Rogers (Gene, 1979, 7, 79). A restriction winter image of plasmids pKC222 and pKC203 is shown in Figures 1 and 2.

The deoxyribonucleic acid of the invention may be used as a dominant selectable primer when linked to a gene or a portion of a gene comprising a transcriptional and translational activation sequence in a translationally read phase. The number of amino acids encoded by the gene or portion thereof is not critical to the process of the invention. In the case of a bacterial gene, fusion is accomplished by linking the incomplete aph (4) plasmid pIT123 gene to pUC7. Plasmid pUC7 is readily available and prepared according to Vieira and Messing, Gene, 9, 259 (1982), contains a portion of the E. coli lac Z gene and contains a single BamHI restriction site to the right of the lac operator and translation initiation signals. At the BamHI site, the reading phase of the lac Z gene fragment corresponds to that of the truncated aph (4) gene of plasmid pIT123. Thus, when the two genes are joined at the BamHI site such that plasmid ρΓΓ123

The 1.3 kb BamHI-Bgin fragment was ligated into the BamHI site of plasmid pUC7 to obtain a hybrid gene that conferred resistance to hygromycin B. The above illustrated composition is the first 20 amino acids 5 of the lac Z gene

HU 200366 Β consists of a dominant coding sequence fused to the truncated aph (4) gene. A restriction map of the pIT144 plasmid containing the resulting gene is shown in Figure 2 attached. A similar plasmid, designated pKC307, was constructed by ligating the blunt-ended Hphl fragment of plasmid pIT104 to the HincII-digested blunt-end fragment of pUC8. The latter plasmid is similar to pUC7 and is readily available or prepared according to the method of Vieira and Messing, 1982 (supra).

The deoxyribonucleic acid of the invention may also be linked to eukaryotic genes or gene fragments, such as the yeast heat shock gene (YG101) reported by Ingolia et al., Mol. and Cellular Biol., 2, 1388 (1982)]. The mobilization of the transcriptional and translational activation sequence of YG101 on the 750 bp BamHI-BglII fragment of plasmid ρΓΓ118 makes fusion particularly advantageous. by constructing plasmid pMC1587 in place of BamHI. The 1.3 kb BamHI-BglII fragment of plasmid pIT123 is ligated into the bamfunctional plasmid designated pIT208 to replace the BamHI site of pIT207. Plasmid pIT208 is selectable in E. coli cells and confers resistance to the hygromycin B antibiotic in yeast and thus illustrates the method of the invention. The restriction map of plasmid pIT208 is presented in Figure 3 below.

The heat shock gene YG100 (Ingolia et al. (1982), supra) may similarly be used to produce preferred translational fusions. A 1 kb BamHI-BglII fragment containing the transcriptional and translational activation sequence of plasmid pIT120 is ligated into the BamHI-digested plasmid pIT213. The latter plasmid contains the known plasmid pRB5, a kanamycin resistance determining gene, and the 1.3 kb BamHI-BglII fragment derived from plasmid pIT123 containing the truncated hygromycin B resistance gene. The source of the hsc100 transcriptional and translational activation sequence pIT120 is readily isolated from E. coli K12 JA221 / pIT120 cells. The strain can be found in the Northern Region Research Laboratory, Peoria, Illinois, USA, a permanent strain collection. The strain can be used as a source of the plasmid and can be ordered under the accession number NRRL B-15603.

Following ligation of pIT120 and pIT213 fragments as described above, a bifunctional plasmid designated pIT217 is obtained. Plasmid pIT217 confers selective resistance to hygromycin B antibiotic in E. coli cells in yeast and thus further illustrates the method of the invention.

One skilled in the art will recognize that the BamHI-digested pIT213 fragment and the 750-base BamHI-BglII fragment of plasmid pIT118, after ligation, also result in a similar fusion product that is within the scope of the invention. The resulting plasmid, designated pIT215, is selectable in E. coli cells and confers resistance to hygromycin B antibiotic in yeast.

We can also produce other products using different genes. For example, the eukaryotic phosphoglycerate kinase gene (PGK) can be linked to the deoxyribonucleic acid encoding the truncated hygromycin B resistance of the present invention by linking the 230 bp HI fragment of plasmid Bam HI with the 230 bp fragment of the ρΓΓ143 transcriptional and translational activation sequence. The PGK transcriptional and translational activation sequence was isolated from pIT143, the plasmid was prepared by treating the 958 base pair Clal-HincII fragment of plasmid pIT141 with the restriction enzyme MboII, removing the resulting single-stranded fragment with the DNA polymerase K BamHI linkers having the sequence SEQ ID NO: 3 are ligated and the fragment containing the linker is ligated to the BamHI fragment of pUC8. Plasmid pIT141 contains the entire PGK gene, which is used to construct plasmid pIT143. Plasmid pIT141 can be readily isolated from E. coli K12 JA221 / pIT141 cells, as found in the Northern Region Research Laboratory, Peoria, Illinois, USA. The strain can be ordered as the source of the plasmid under the accession number NRRL B-15602.

One skilled in the art will recognize that a large number of genes or gene portions can be replaced by the illustrative bacterial lacZ and eukaryotic PGK, YG100 and YG101 genes. The number of amino acids determined by the genes or gene portions is not considered to be significant for the purposes of the present invention. It can be used as a gene, for example

1) E. coli genes, such as the trpE or lipoprotein gene;

2) Saccharomyces cerevisiae genes, such as the α-factor gene;

3) Bacillus genes, such as the α-amylase gene or the spoVG gene;

4) a Streptomyces gene, such as the thiostrepton resistance, neoncin resistance, or viomycin resistance gene;

5) viral or bacteriophage genes such as those described by the lambdaPL or lambdaPR promoters;

6) mammalian genes such as thymidine kinase or the dihydrofolate reductase gene; furthermore

7) plant genes such as octopine synthetase or nopaline synthase genes.

The above genes may be truncated by treatment with appropriate restriction enzyme and / or Bal31 nuclease and then supplemented with molecular linkers, depending on the options available and the desired fusion. Molecular linkers can be obtained from the market or, if special or unusual gene fusion is to be made, according to the judgment of Judakura et al., 1977, supra; Crea et al., 1978, supra]. Particularly advantageous fusion is the method of ligating the pIT123 plasmid containing the aph (4) gene to the 4.5 kb BamHI-BglII fragment of plasmid ρΙΑ7Δ4Δ1. The latter fragment contains the transcriptional and translational activation sequence and also contains the 15 amino acid coding region of the bacterial trp LE 'gene. As a result of this ligation, the 5.8 kb plasmid pIT125 is obtained

HU 200366 Β

One skilled in the art will recognize that other truncated genes, with or without molecular linkers, may be fused to the truncated aph (4) gene of the present invention, in which case the plasmids of the invention will also be obtained.

The vectors containing the DNA of the present invention can be used in any hygromycin B antibiotic-sensitive host cell, provided that

1) the vector replicates in the host cell or is integrated into the host cell chromosome;

2) the gene fused to the truncated aph (4) gene is expressed in the host cell; and 3) the host cell is transformable.

Suitable host cells include, for example, the following 10 cells of K12 HB101, E. coli K12 RR1, Streptomyces, Streptomyces ambofaciens, Bacillus, Bacillus subtilis, Saccharomyces cerevisiae, mammalian cells, and plant cells, especially Angiospermous cells.

One skilled in the art will recognize that other host cells can be transformed with the vectors containing the DNA of the invention, and that transformation is within the scope of the invention.

All embodiments of the process of the invention are preferred. However, some of the DNA sequences, cloning vectors and transformants of the invention are even more preferred. A preferred DNA sequence is, for example, a

two: E. coli. E. coli K12, E. coli K12 JA221, E. Mense. The base sequence of the self is a following: CCT 1 1 1 GAA 1 1 1 CTC 1 1 1 ACC 1 1 1 GCG 1 1 1 ACG 1 1 1 TCT <1 1 GTC 1 1 i GAG 1 1 1 AAG 1 1 1 1 1 1 GGA i 1 1 CTT 1 1 1 GAG 1 1 i TGG 1 1 1 CGC 1 1 1 TGC I 1 1 AGA 1 1 i CAG 1 1 1 CTC 1 1 1 TTC TTT I I I CTG 1 1 1 ATC 1 1 1 GAA 1 hr 1 AAG 1 1 1 TTC 1 1 1 GAC i 1 i AGC 1 1 1 GTC 1 1 1 TCC ili GAC 1 1 1 CTG I 1 1 ATG 1 1 1 1 1 1 AAA 1 1 1 GAC 1 1 1 BROAD 1 hr 1 CTT 1 I 1 TTC I 1 1 AAG 1 1 1 CTG 1 1 i TCG 1 1 ι CAG 1 ι I AGG 1 1 1 CTG 1 L 1 GAC 1 1 I TAC CAG 1 1 1 CTC 1 1 1 TCG 1 1 1 GAG 1 1 1 GGC 1 1 1 GAA 1 1 1 GAA 1 1 1 TCT 1 1 1 CGT 1 1 1 GCT 1 1 1 TTC 1 1 1 AGC 1 1 i TTC 1 1 1 1 II GTC 1 1 1 GAG 1 1 1 AGC ι ι ι CTC 1 1 1 CCG 1 1 1 CTT 1 1 1 CTT 1 1 1 AGA ι 1 1 GCA ι 1 1 CGA 1 1 ι AAG 1 1 ι TCG 1 1 I AAG DAM 1 1 1 GTA 1 1 1 GGA 1 1 1 GGG 1 1 1 CGT 1 1 1 GGA 1 1 1 TAT 1 1 1 GTC 1 i t CTG 1 I i CGG 1 1 1 GTA t 1 1 AAT <1 i AGC 1 1 1 1 1 1 CTA 1 1 1 CAT 1 1 t CCT 1 1 1 CCC 1 1 1 GCA 1 1 1 CCT 1 1 1 ATA 1 1 I CAG 1 1 1 GAC 1 1 I GCC 1 1 1 CAT I 1 1 TTA 1 1 1 TCG TGC 1 1 1 GCC 1 1 1 DAM 1) 1 GGT 1 1 1 TTC 1 1 1 TAC 1 1 1 AAA 1 i 1 DAM i 1 1 CGT 1 1 1 TAT 1 1 1 GTT 1 1 1 TAT 1 1 1 CGG t! 1 1 1 1 ACG i 1 i CGG 1! 1 CTA 1 1 1 CCA 1 1 1 AAG 1 1 1 ATG 1 1 ι TTT 1 1 1 CTA 1 1 1 GCA 1 1 1 OVER THE 1 1 1 CAA 1 1 1 OVER THE 1 i 1 GCC CAC 1 1 1 TTT 1 1 t GCA 1 1 i TCG 1 1 1 GCC 1 1 1 GCG 1 1 1 CTC 1 1 1 CCG 1 1 1 ATT 1 1 1 CCG 1 1 1 GAA 1 hr 1 GTG 1 1 1 CTT 1 1 1 1 1 1 GTG 1 1 I AAA 1 I 1 CGT 1 1 1 AGC 1 1 1 CGG 1 1 1 CGC 1 1 1 GAG 1 1 1 GGC 1 1 1 TAA 1 1 1 GGC 1 1 ι CTT I 1 1 CAC 1 1 1 GAA GAC 1 1 1 ATT 1 1 t GGG 1 1 1 GAA 1 1 1 TTC 1 1 1 AGC 1 1 1 GAG 1 1 1 AGC 1 1 1 CTG 1 l 1 ACC 1 1 1 TAT 1 1 1 TGC 1 1 1 ATC 1 1 1 1 1 1 CTG 1 1 1 TAA 1 1 1 CCC 1 1 1 CTT 1 1 1 AAG 1 1 1 TCG 1 1 1 CTC 1 1 1 TCG 1 1 1 GAC 1 1 1 TGG 1 1 1 ATA 1 1 1 ACG 1 1 1 TAG TCC 1 1 t CGC 1 1 1 CGT 1 1 1 GCA 1 t t CAG 1 1 1 GGT 1 hr 1 GTC 1 1 1 ACG 1 1 1 TTG 1 1 1 CAA 1 1 1 GAC 1 1 1 CTG i 1 1 CCT 1 1 1 1 1 1 AGG 1 1 1 GCG 1 1 1 GCA 1 1 1 CGT 1 1 1 GTC 1 ι 1 CCA 1 1 1 CAG 1 1 1 TGC 1 1 1 AAC 1 1 1 GTT i 1 1 CTG i 1 I GAC 1 1 ι GGA GAA 1 i 1 ACC 1 1 t GAA 1 1 1 CTG 1 1 1 CCC 1 1 1 GCT j 1 1 GTT 1 1 1 CTG 1 1 1 CAG 1 1 1 CCG t 1 1 GTC 1 1 1 GCG 1 1 1 GAG 1 1 1 1 1 1 CTT 1 1 1 TGG i 1 1 CTT 1 1 1 GAC 1 1 1 GGG I 1 1 CGA 1 1 1 CAA f 1 1 GAC t i 1 GTC 1 1 1 GGC 1 ι 1 CAG 1 1 1 CGC i 1 i CTC GCC 1 1 t ATG 1 1 1 DAM 1 1 1 GCG 1 1 1 ATC GCT 1 i 1 GCG 1 1 i GCC 1 1 1 DAM 1 1 1 CTT 1 1 1 AGC 1 1 1 CAG 1 1 1 ACG 1 1 1 1 1 1 CGG 1 1 1 TAC 1 1 1 CTA 1 1 1 CGC I 1 1 BROAD 1 1 1 CGA 1 1 1 CGC 1 1 1 CGG 1 1 1 CTA 1 1 1 GAA 1 1 1 TCG 1 1 1 GTC 1 1 1 TGC AGC 1 1 1 GGG 1 1 1 TTC 1 1 1 GGC 1 1 1 CCA 1 1 1 TTC i 1 1 GGA 1 1 1 CCG 1 1 1 CAA t i 1 GGA 1 1 1 ATC 1 i 1 GGT 1 1 1 CAA 1 1 1 1 1 1 TCG 1 1 I ccc 1 1 1 AAG 1 1 1 CCG 1 1 1 GGT l 1 1 AAG 1 1 1 CCT 1 1 1 GGC 1 1 1 GTT 1 1 1 CCT 1 1 1 TAG i 1 1 CCA 1 1 1 GTT TAC 1 1 1 ACT i 1 1 ACA 1 1 1 TGG 1 f 1 CGT 1 1 1 DAM 1 1 1 TTC 1 1 1 OVER THE 1 1 1 TGC 1 1 1 GCG 1 1 1 ATT 1 1 1 GCT 1 1 i DAM 1 1 1 1 1 1 ATG I 1 1 TGA 1 1 1 TGT i 1 1 ACC 1 1 1 GCA 1 i ι CTA 1 1 1 AAG 1 1 1 TAT 1 1 1 ACG 1 1 1 CGC I 1 1 TAA 1 1 1 CGA 1 1 1 CTA

HU 200366 Β

CCC 1 1 1 CAT 1 1 1 GTG t t 1 TAT 1 1 1 Ι 1 1 GGG 1 to 1 GTA 1 I 1 CAC 1 1 1 ATA GTC 1 1 i AGT 1 1 1 GCG 1 1 1 TCC 1 1 1 1 ι 1 CAG 1 1 1 TCA 1 1 1 CGC 1 I 1 AGG CTT 1 1 1 TGG 1 1 1 GCC 1 1 i GAG 1 1 1 i 1 I GAA 1 1 l ACC ι ι 1 CGG 1 1 1 CTC CAC 1 1 1 GCG 1 1 1 DAM 1 1 1 TTC 1 1 1 1 1 1 GTG I 1 1 CGC 1 1 1 CTA 1 1 1 AAG AAT GGC CGC OVER THE 1 1 1 1 1 1 1 1 1 1 1 1 TTA CCG GCG TAT ATG 1 1 1 TTC 1 1 I GGG 1 1 1 DAM 1 1 I 1 1 1 TAC 1 1 t AAG 1 1 1 CCC 1 1 1 CTA TTC 1 1 1 TGG 1 1 1 AGG 1 1 t CCG 1 1 1 1 1 1 AAG 1 ι 1 ACC 1 1 1 TCC 1 1 1 GGC CGC 1 I 1 TAC 1 1 1 TTC I 1 1 GAG 1 1 1 1 1 I GCG I 1 1 ATG 1 1 1 AAG 1 I 1 CTC CCG 1 1 t CGG 1 1 1 CTC 1 1 1 CGG 1 1 1 GGC 1 1 1 GCC 1 1 1 GAG I I 1 GCC CAA 1 1 1 CTC 1 1 t TAT 1 1 1 CAG 1 I t 1 1 1 GTT 1 J I GAG 1 1 J OVER THE 1 1 1 GTC GCA 1 I 1 GCT i 1 1 TGG 1 1 J GCG 1 1 1 1 i 1 CGT 1 (1 CGA 1 1 1 ACC 1 ι 1 CGC TCC t 1 t GGA 1 (i GCC 1 I 1 GGG 1 I 1 I 1 ι AGG 1 1 1 CCT 1 1 1 CGG 1 1 1 CCC AGA 1 1 1 AGC i i 1 GCG 1 1 1 GCC 1 1 1 ι 1 1 TCT 1 i 1 TCG 1 1 1 CGC 1 1 1 CGG CTC t 1 1 GCC 1 1 1 DAM 1 1 1 AGT 1 1 1 1 1 1 GAG 1 1 1 CGG 1 1 1 CTA 1 1 1 TCA AGG 1 1 1 GCA 1 1 1 AAG 1 1 1 GAA 1 1 1 1 1 1 TCC 1 1 1 CGT 1 1 1 TTC 1 1 1 CTT

CAC I I I GTG

GTC I I I CAG

GAC I 1 I CTG

GGC I 1 I CCG

ACA I 1 I TGT

TCC I 1 I AGG

TGG I I I ACC

CGG I I I GCC

GCG I I I CGC

AGC I i I TCG

CAG I I I GTC

ACT I I I TGA

CAG

GGA I I I CCT

TAG I I I ATC

TGG I I I ACC

GCG I I I CGC

TGC I I I ACG

TCC I I I AGG

GCG I I I CGC

CAA I I I GTT

TTG I I I AAC

AGG I I I TCC

TAT I I I ATA

TTG I I I AAC

GGT I I I CCA

GTC I I I CAG

TGG I I I ACC

AAC I I I TTG

CAA I I I GTT

CAG I I I GTC

CCC I I I GGG

AAC I I I TTG

GTC I I I CAG

TAC I I I ATG

GCT I I I CGA

CAT I I I GTA

ATG I I I TAC

GTT I I I CAA

CGA I I I GCT

GGG I I I CCC

ACC I I I TGG

CGA I I I GCT

ACT I I I TGA

GCT I I I CGA

GAA I I I CTT

AAT I I I TTA

ATT I I I TAA

GAG I I I CTC

TGT I I I ACA

CCG I I I GGC

CTC I I I GAG

GAC I I I CTG

TGC I I I ACG

CGT I I I GCA

GAT I I I CTA

CGC

I I I

GCG

GTG I I I CAC

CTC I I I GAG

GTC I I I CAG

GTC I I I CAG

GAC I I I CTG

GTC I I I CAG

ATG I 1 I TAC

GAG I I I CTC

CGC I I I GCG

GGC I I I CCG

GAC I I I CTG

ACA 1 I I TGT

GGC I I I CCG

CCC I I I GGG

ATG I I I TAC

GAT I I I CTA

CGG I I I GCC

CTG I I I GAC

TGG I I I ACC

GCC I I I CGG

GAG I I I CTC

CTT I I I GAA

ATT I I I TAA

AAT I I I TTA

GCA I I I CGT

CAA I I I GTT

TGT I I I ACA

AGC I I I TCG

GAC I I I CTG

GAG I I I CTC

CAC I I I GTG

ACG I I I TGC

AGC I I I TCG

AAC I I I TTG

CAG I I I GTC

GCA

I I I

CGT

GGT

I I I

CCA

TTC I I I AAG

ATC I I I TAG

ATC I I I TAG

GTA I I I CAT

ACT I I I TGA

GAC I I I CTG

CTG I I I GAC

CTC I I I GAG

GAC I I I CTG

GAG I I I CTC

ATC I I I TAG

CAG I I I GTC

GGA I I I CCT

CTT I I I GAA

GAT I I I CTA

GTC I I I CAG

GCC

I I I

CGG

GAA

I I I CTT

CGT I I I GCA

ACC I I I TGG

ATG I I I TAC

GTG I I I CAC

AAC I I I TTG

GCG I I I CGC

TTC I I I AAG

ACG I I I TGC

TCG I I I AGC

GAC I I I CTG

GAT I I I CTA

CGA I I I GCT

CGC I I I GCG

GTA

I I I CAT

CCG I I I GGC where:

The deoxyadenyl,

G is deoxyguanidyl, C is deoxysilicyl and T is thymidyl;

preferred plasmids are pIT123, pIT125, pKC307, pIT208, pIT215, pIT217, pIT219 and pIT144;

preferred transformants are E. coli 65 K12 JA221 / pIT123, E. coli K12 JA221 / pIT125, E.

HU 200366 Β coli K12 JA221 / pKC307, E. coli K12 JA221 / pIT208, E. coli K12 JA221 / pIT217, E. coli K12 JA221 / pIT219, E. coli K12 JA221 / pIT219, Saccharomyces cerevisiae / pIT208, Saccharomyces cerevisiae / pIT215, Saccharomyces cerevisiae / pIT217 and Saccharomyces cerevisiae / pIT219.

The DNA of the present invention can be used as selectable markers in both homologous (E. coli) and heterologous (non-E. coli) systems and thus enables the production of selectable carriers for cloning genes in host cells of various nature. The fact that the DNA of the invention confers resistance to the hygromycin B antibiotic enables functional testing of the transformants. This is important because we need to identify and select the cells that have taken up the vector. Their presence in the vectors can be incorporated into segments of DNA that cannot be detected by functional assay, and transformants containing non-selectable DNA may then be isolated by selection with hygromycin B antibiotic. These non-selectable DNA segments include, but are not limited to: human insulin A chain, human insulin B chain, human proinsulin, human pre-pro-insulin, human growth hormone, porcine growth hormone , avian growth hormone, human interferon, and non-human interferon coding segments.

More specifically, a non-selectable DNA-carrying segment of a gene is inserted into a plasmid, such as pIT144 or pIT208. The non-selectable DNA may be inserted into an EcoRI site of pIT144 following partial SalI digestion of pIT144, or inserted into the SmaI site of pIT208. Among the cells transformed with the ligation mixture, the given recombinant is identified by colony hybridization using a radioactive probe. After demonstrating the presence of non-selectable DNA, the vectors are amplified in E. coli cells and then introduced into yeast cells in plasmid pIT208. Yeast transformants are readily isolated by selection with hygromycin B. Thus, selectivity for antibiotic resistance allows for the efficient isolation of a very rare cell carrying a non-selectable DNA.

Functional assays based on hygromycin B resistance may also be performed to identify DNA segments that act as a gene expression regulator. Examples of such segments include promoters, attenuators, repressors, inducers, ribosome binding sites, and others. These are used to regulate economically important genes. In addition, the method of the invention can be used to identify origin of replication. This is done by cloning DNA fragments into the vectors containing the aph (4) gene of the invention and then transforming the host cells with hygromycin B selection. Cells resistant to hygromycin B antibiotic are selected and transformed with E. coli cells; this allows us to isolate a replicon of any microorganism of interest to us.

The resistance vectors of the invention may also be used to demonstrate that linked DNA fragments are stably retained in E. coli, yeast, or other transformants.

Preferably, the genes or DNA fragments linked to the aph (4) gene of the present invention are maintained by digesting the transformants in the presence of hygromycin B, the concentration of hygromycin B being selected to be toxic to untransformed cells. Thus, transformants that have lost the vector and the covalently linked DNA are thereby unable to grow and are eliminated from the culture. Particularly important in large scale fermentation, where the product must be expressed with maximum efficiency.

The DNA, cloning vectors and transformants of the present invention are particularly useful for the cloning of genes that directly or directly define specific functional polypeptides or fusion gene products, such as human insulin A chain, human insulin B chain, human proinsulin, , human growth hormone, non-human growth hormone, human and non-human interferon and others; enzymes involved in the metabolic metabolism of processes or compounds of practical importance; regulatory elements that enhance gene expression or vector replication; any physiologically active enzyme which has research or practical value. For example, enzyme function-determining DNA sequences catalyze the synthesis of cephalosporin antibiotics, the synthesis of actaplanin, penicillin, penicillin derivatives, or tylosin antibiotics. One of ordinary skill in the art will appreciate that the method of the present invention is widely applicable and is not limited to cloning the genes described above.

The invention is further illustrated by the following examples. Where appropriate, the detailed procedure, and in some cases a general description thereof, will be described.

Example 1

A) Construction of the starting plasmid pKC222

Isolation of plasmid pKC203 and production of E. coli KI2 BE827! PKC203

E. coli JR225 (ATCC 31912) was grown in TY medium (1% tryptone, 0.5% yeast extract, 0.5% sodium chloride, pH 7.4) in the presence of 100 pg / ml of antibiotic hygromycin B, a well-known microbiological using methods. After 18 hours of culture, 0.5 ml of the culture was transferred to a 1.5 ml Eppendorf tube and centrifuged for 15 seconds. Unless otherwise specified, all steps are performed at room temperature. Carefully remove the supernatant with a thin-ended pipette and suspend the pellet in 100 μΐ of freshly prepared lysozyme solution. Its composition is as follows:

pg / ml of lysozyme, mmol of glucose, mmol of CDTA (cyclohexane diamine tetraacetic acid), mmol of tris hydrochloride, pH 8.0.

incubate at 0 ° C for 1 minute and then add 200 μΐ of alkaline sodium

EN 200366 Β decyl sulfate solution (0.2 N sodium hydroxide, 1% sodium dodecyl sulfate) was added. The centrifuge tubes are gently stirred and kept at 0 ° C for 15 minutes. 150 ml of a 3 molar sodium acetate solution are prepared by adding 3 mol of sodium acetate to the minimum amount of water and the pH of the solution is 4,8 with glacial acetic acid. and then adjust the volume to 1.0 L. The solution is stirred for a few minutes by gentle agitation of the centrifuge tube, whereupon a precipitate of DNA is formed.

The solution is kept at 0 ° C for 1 minute, then centrifuged for 5 minutes, after which time an almost completely supernatant is obtained. 0.4 ml of the supernatant was transferred to another centrifuge tube, previously filled with 1 ml of cold ethanol. After 30 minutes at -20 ° C, the precipitate was collected by centrifugation. The centrifugation was carried out for 2 minutes. The supernatant was removed. The resulting precipitate was dissolved in 100 μΐ 0.1 M sodium acetate / 0.05 M Tris hydrochloride (pH 8.0) and re-precipitated after addition of 2 volumes of cold ethanol. The suspension is kept at 20 ° C for 10 minutes and then the E. coli JR225 plasmid DNA precipitate is isolated by centrifugation.

The pellet of E. coli JR225 DNA was dissolved in 40 μΐ of water or dilute buffer and transformed with E. coli K12 BE827 cells, essentially as described by Wensink (1974, Cell 3: 315). E. coli strain K12 BE827 is found in ATCC 31911 from the American Type Culture Collection, Rockville, Maryland, USA.

The resulting transformants were selected on TY agar. The agar has the following composition:

% tripton,

0.5% yeast extract,

0.5% sodium chloride,

1.5% agar, pH 7.4,

200 pg / ml hygromycin B antibiotic.

According to the gel electrophoretic assay (Rao and Rogers, Gene, 3, 247 (1978)), some transformants contain large and small (15 kB) plasmids and are resistant to ampicillin and hygromycin B antibiotics. Some transformants contain only the smaller 15 kB plasmid and are resistant to hygromycin B and G418 antibiotics, but are sensitive to ampicillin.

Transformants of the latter type are plated on TY agar containing 1 mg / ml hygromycin B and cultured using standard microbiological techniques. The resulting cells are used to isolate the hybromycin B and G418 antibiotic resistance plasmids described above, which are then designated pKC203. The presence of genes conferring resistance to the hygromycin B and G418 antibiotics present in plasmid pKC203 was subsequently confirmed by transformation and selection analysis,

B) Construction of plasmid pKC222 and isolation of E. coli KI2 JA221'pKC222

1) Isolation of the 2.75 kB SalIBGlll fragment of plasmid pKC203 was treated with restriction enzymes SalI and Bgin according to the manufacturer's instructions [restriction enzymes, T4 DNA ligand and polynucleotide The Klenow fragment was obtained from New England Biolabs, 32 Tozer Road, Beverly, MA 01915, USA]. For the use of restriction enzymes, see also Maniatis et al. (Molecular Cloning, Cold Spring Harbor Laboratory, Cold Spring Harbor, New York, 1982). As described in Maniatis et al. (1982), supra, a 2.75 kb fragment containing genes and regulatory elements for expression of hygromycin B and G418 antibiotics is isolated.

2) Ligation and preparation of the final product pg pKC7 (ATCC 37084) was treated with restriction enzymes SalI and BglII. The plasmid can be constructed according to Rao and Rogers (1979, Gene 7, 79). To inactivate the enzymes, keep the mixture at 70 ° C for 5 minutes and then mix 1 µg of DNA with a 2.75 kB SalI / BglII fragment of plasmid pKC203. The fragments were ligated with T4 DNA according to the manufacturer's instructions (see Example 1, B-1). See also Maniatis et al., 1982, supra, for the use of the restriction enzymes given above. The resulting plasmid pKC222 was transformed into E. coli K12 JA221 cells (NRRL B-15211) according to Example 1 (A). The resulting tomformants were selected on TY agar with the following composition:

% tripton,

0.5% yeast extract,

0.5% sodium chloride,

1.5% agar, pg / ml ampicillin antibiotic.

The transformants were assayed for the presence of the plasmid produced.

3) Isolation of plasmid pKC222

Purified transformants were cultured on TY medium (1% tryptone, 0.5% yeast extract, 0.5% sodium chloride, pH 7.4, 50 pg / ml antibiotic ampicillin). The culture is carried out using known microbiological methods. After 18 hours of culture, 0.5 ml of the culture was pipetted into a 1.5 ml Eppendorf centrifuge tube and centrifuged for 15 seconds. Unless otherwise specified, all experimental steps are performed at room temperature. The resulting supernatant was carefully removed and the cell suspension suspended in 100 µl of freshly prepared lysozyme solution. It has the following composition:

pg / ml of lysozyme, mmol of glucose, mmol of cyclohexane diamine tetraacetic acid, and mmol of tris hydrochloride, pH 8.0.

After incubation at 0 ° C for 1 minute, 200 µl of alkaline sodium dodecyl sulfate solution is added as follows:

0.2 N sodium hydroxide, 1% sodium dodecyl sulfate.

Gently stir the centrifuge tubes and keep at 0 ° C for 15 minutes. Then 150 µl of 3 molar

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EN 200366 Β sodium acetate solution was prepared by dissolving 3 moles of sodium acetate in as little water as possible, adjusting the pH of the solution to 4.8 with glacial acetic acid, and then adjusting to 1 l. Final volume. Stir the solution gently for 60 minutes at 0 ° C and centrifuge the precipitated DNA for 5 minutes to obtain an almost pure supernatant. Transfer 0.4 ml of the supernatant into another centrifuge tube, add 1 ml of cold ethanol and keep the solution at -20 ° C for 30 minutes. The precipitate was collected by centrifugation for 2 minutes and the supernatant was aspirated off. The precipitate thus collected was dissolved in 100 μΐ of the following composition:

0.1 mol sodium acetate and

0.05 M Tris hydrochloride, pH 8.0.

The solution was reprecipitated by adding 2 volumes of cold ethanol. After 10 minutes at -20 DEG C., the precipitate was collected by centrifugation. According to agarose gel electrophoresis (Rao and Rogers, 1978, supra), the precipitate contains plasmid pKC222 DNA.

Example 2

Construction of plasmid ρΓΓ123 and E. coli K12 JA22111plT123

A) Isolation of the HphI-PstI fragment of pKC222 Plasmid pKC222 is digested in 100 µl of the following formulation, 20 New England Biolab. unit with restriction endonuclease Hphl:

mmol of potassium chloride, mmol of tris hydrochloride, pH 7.4, magnesium chloride, dithiothreitol;

the solution is called IX Hphl solution.

Digestion was checked by electrophoresis using a 2% reaction mixture on an agarose gel. An appropriate amount of 5M sodium chloride solution was added to adjust the sodium chloride concentration to 60 mM and then 20 units of PstI restriction endonuclease was added. Digestion completeness was again checked by agarose gel electrophoresis. Using the known methods (Schlief and Wensink, Practical Methods in Molecular Biology, Springer-Verlag, New York, 1981), an HphI-PstI fragment having 325 bp and a single-stranded end was isolated from an acrylamide gel.

The purified 325 bp fragment was treated with a large E. coli DNA polymerase I fragment (New England Biolabs.). 1.5 µl (1 µg) of the fragment, 0.5 µl of 10X buffer (0.5 molar Tris, pH 7.5, 0.1 molar magnesium chloride), 0.5 µl (200 µg) dCTP, dATP, dTTP and dGTP plus 1 µl of a large fragment of DNA (Klenow fragment) containing 1 unit enzyme and incubated for 15 minutes at 37 ° C. The polymerase enzyme is heat inactivated and then BamHI linkers are added according to Roberts and Lauer (1979, Methods in Enzymology, 68, 473). The BamHI linker-containing DNA is known to be digested with BamHI restriction enzyme in BamHI salt solution IX (0.15 moles sodium chloride, 6 mM tris hydrochloric acid, pH 7.9, 6 mM magnesium chloride). The solution was then raised to 100 mM Tris-HCl by addition of an appropriate amount of a 2M Tris-HCl solution (pH 7.4), followed by further digestion with Eco RI. The resulting cleaved DNA was purified by electrophoresis on a 7% acrylamide gel, and the 250 base pair fragment was isolated as described above.

Β) Construction of plasmid ρΓΓ122 and E. coli K2 JA22HpIT122 pg pBR322 was digested with BamHI and then restriction enzyme EcoRI according to Example 2 (A). The enzyme was inactivated by incubation at 70 ° C for 5 minutes, followed by 1 µl (1 µg) of pBR322 DNA with 1 µl (1 µg) of purified 250 bp fragment, 37 µl of water, 5 µl (10 mmol) of adenosine triphosphate. With 5 µl of ligation mixture (0.5 mole Tris-hydrogen chloride, pH 7.8, 0.1 mole dithiothreitol, 0.1 mole magnesium chloride) and 1 µl T4 DNA (about 100,000 New England Biolabs Unit). The reaction mixture was incubated for 2 hours at 15 ° C, then quenched for 5 minutes at 70 ° C, cooled in an ice bath and transformed with the resulting ligation mixture according to Wensink (1974), supra. Transformed E. coli K12 JA221 (NRRL B-15211) cells were isolated on TY agar containing 200 pg / ml ampicillin. The identity of the transformants to be isolated is assayed for the expected phenotype (AmpR, Tet ^s ) ^and the corresponding Eco RI-Bam HI fragment is examined. The resulting E. coli K12 JA221 / pIT122 transformants were cultured in known manner to isolate plasmid pIT122.

C) Linkage of the 1.45 kb Eco RI fragment of pKC222 to Eco RI digested pIT122 Plasmid pKC222 and pIT122 were independently cleaved with 40 units of Eco RI in different reaction mixtures. 200 µl reaction mixtures (IX EcoRI reaction mixture) have the following composition:

0.1 molar Tris hydrochloride, pH 7.5,

0.05 moles of sodium chloride,

0.005 mol of magnesium chloride.

The 1.45 kb EcoRI fragment is isolated from a gel containing 7% acrylamide in a known manner and ligated with Eco RI-cleaved pIT122 DNA.

The resulting linked DNA is designated pIT123 and transformed with E. coli K12 JA221 (NRRL B-15211). Ligation and transformation were performed as described in Example 2 (B). Ampicillin-resistant transformants were confirmed by restriction enzyme (EcoRI) cleavage and agarose gel electrophoretic analysis to confirm the proper orientation of the 1.45 kb EcoRI fragment. Except for the first 9 base pairs, plasmids containing the entire aph (4) gene are designated pIT123. E. coli K12 JA221 / pIT123 transformants were used to construct plasmid pIT123 and

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HU 200366 Β. A restriction map of plasmid pIT123 is presented in the accompanying Figure 1.

Example 3

Construction of Plasmid ρΓΓ144 and E. coli K12 RRlAMl5! PlTl44

A) Preparation and Isolation of the Bb HI-BglII Ib kb Plasmid p / T123

The digestion and isolation was carried out as in Example 2 (A) except that the restriction enzymes BamHI and BglII and the reaction mixture were used instead of the restriction enzymes Hphl and PstI and the reaction mixtures thereof.

B) Digestion of pUC7 with BamHI

Example 2 (A) except that 1 µg of plasmid pUC7 (available from Bethesda Research Laboratories, 8717 Grovemont Circle, PO Box 6009, Gaithersburg, MD 20877) was used in the presence of the BamHI linker. instead of ribonucleic acid.

C) Ligation and Transformation

One pg of the 1.3 kb BamHI-BglH fragment of plasmid ρΓΓ123 was ligated with 1 pg of BamHI-digested pUC7 DNA and transformed with E. coli K12 RR1AM15 cells. (The strain is found in the National Region Research Laboratory, Peoria, Illinois, Permanent Collection under NRRL B-15440). Both procedures, both ligation and transformation, were performed as described in Example 2 (B). The transformed cells contained 50 µg / ml ampicillin, 100 mmol isopropylthio-fLD-galactoside (IPTG) and 0.02% 5-bromo-4-chloro-3-indolyl - () - D-galactoside (X-gal). Plated on TY agar. The white colonies resistant to ampicillin were replicated on TY plates containing 50 pg / ml ampicillin, 200 pg / ml antibiotic hygromycin B and 100 mM IPTG. The hygromycin B antibiotic resistant cells are E. coli K12 RRlAM15 / pIT144 transformants. Their identification is confirmed by restriction enzyme treatment and agarose gel electrophoretic analysis after plasmid isolation. The resulting E. coli K12 RR1AM15 / pIT144 transformants were cultured for production and isolation of plasmid pIT144. Plasmid pIT144 is readily transformed into E. coli cells, such as E. coli K12, E. coli K12 RR1, E. coli K12 JA221, or E. coli K12 HB101. Transformations were performed as described in Example 2B. A restriction map of plasmid pIT144 is provided in Figure 2 of the accompanying drawings.

Example 4

Construction of plasmid plT208 and E. coli K12 JA221 pIT208

A) Construction of plasmid pIT207

1) Construction and isolation of a 750 bp BamHI-BglII fragment of plasmid plTH8

a] Isolation of plasmid pITH8

Plasmid pITl18 was isolated from E. coli strain K12 JA221 / pITl18 according to Example 1 (A). The above strain is found in the Northern Region in a permanent collection of Research Labo12 ratory, Peoria, Illinois, under the accession number NRRL B-15441.

b] Digestion

Digestion and isolation were carried out as in Example 2 (A) except that BamHI and BglH were used instead of Hph1 and PstI.

2) Digestion of pMC1587 with BamHI

The digestion was carried out as in Example 2 (A), except that 2 µg of plasmid pMC1587 was used in place of the BamHI linker-containing DNA. Plasmid pMCl587 can be readily isolated from E. coli K12 strain JA221 / pMC1587 by the procedure of Example 1 (A). The strain is a permanent strain collection of the Northern Region Research Laboratory, Peoria, Illinois under the accession number NRRL B-15442. Because pMC1587 has a single BamHI site, digestion can be easily followed by agarose gel electrophoresis. When a single band of about 16 kb is displayed, digestion is complete.

3) Ligation and transformation of E. coli K12 JA221

One pg of the 750 bp BamHIBglII fragment of plasmid pIT118 was ligated with 1 pg of BamHI-digested plasmid pMC1587 and the resulting ligation mixture to transform E. coli K12 JA221 (NRRL B-15211). Both steps a

Example 2 was performed as described for ligation and transformation. Transformants resistant to ampecillin are assayed in a known manner to determine the proper orientation of the 750 base pair fragment by restriction enzyme and agarose gel electrophoretic analysis. Plasmids with the BamHI / BglII linkage (formed by ligation of the 750 base pair fragment) are located near the leu 2 gene of plasmid pIT207. E. coli K12 JA221 / pIT207 transformants were cultured for production and isolation of plasmid pIT207.

B) Construction of plasmid plT208

1) Digestion of plasmid plT207 with BamHI

Digestion was performed as in Example 2 (A) except that 1 µg of plasmid pIT207 was used in place of the BamHI linker containing DNA.

2) Preparation of E. coli K12 JA221IpIT208 after ligation

2 pg of the 1.3 kb BamHI-BglII fragment of plasmid pIT123, prepared as described in Example 3A, was ligated with 2 pg of pIT207 after BamHI digestion. The ligation mixture is used to transform E. coli K12 JA221 (NRRL B-15211) cells. The ligation and transformation were carried out as in Example 2 (B). Ampicillin-resistant transformants are assayed for

Presence and orientation of a 1.3 kb fragment. The assay is performed by restriction enzyme and agarose gel electrophoretic analysis. In the 1.3 kb fragment, a plasmid containing the internal Eco RI site for the leu 2 gene

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HU 200366 Β are located in the plasmid pIT208. E. coli K12 JA221 / pIT208 transformants were cultured for production and isolation of plasmid pIT208.

Plasmid pIT208 contains:

1) the truncated YG101 gene linked in a translation reading phase to the DNA of the invention;

2) the yeast leu 2 gene, which allows plasmid selection by complementing the leu 2 auxotrophs;

3) a 2 μ sequence of the yeast, which allows independent replication in the yeast;

4) the origin of replication from plasmid pBR322, which allows autonomous replication in E. coli cells; finally

5) the B-lactamase gene from plasmid pBR322, which allows selection of the plasmid in E. coli cells.

A restriction map of plasmid ρΓΓ208 is provided

3.

Example J.

Preparation of Saccharomyces cerevisiae / pIT208

Saccharomyces cerevisiae cells were transformed with pIT208 according to Hinnen et al., Proc. Nat. Acad. Sci., USA, 75, 1929 (1978)]. Although different yeasts may be used, we use the strain Saccharomyces cerevisiae DBY746. The strain can be obtained from Yeast Genetics Stock Center, Department of Biophysics and Medical Physics, University of Califomia, Berkeley, Califomia 94720.

Yeast cells are grown in 100 ml culture at 30 ° C in YPD medium containing 1% Bacto yeast extract, 2% Bacto-peptone and 2% D-glucose. Growth is carried out until A100: 1. The cells are centrifuged under sterile conditions and washed twice with 15 ml of 1.2 M sorbitol solution and resuspended in 15 ml of sorbitol solution. To the suspension was added 100 μΐ of a 2.5 mg / ml solution of zimolase (60,000 units in 5 mM potassium phosphate, pH 7.6, supplemented with 1.2 M sorbitol). Zymolysis was obtained from Miles Laboratory, P.O. Box 2000, Elkhart, IN 46515. The extent of protoplast formation is monitored by adding 180 μ 180 of 10% sodium dodecyl sulfate by adding the above solution to 20 mL of protoplast solution and examining it under phase contrast microscope. When 90% of the cells turn black, the cells are harvested by gentle centrifugation and washed twice with 15 ml of 1.2 M sorbitol, 10 ml of 1.2 M sorbitol-5X YPD and suspended for 40 minutes at room temperature. incubate. Cells are harvested by gentle centrifugation and resuspended in 600 μΐ of 0.5X YPD solution, 1.2 M sorbitol solution, 10 mM calcium chloride and 10 mM tris hydrochloride, pH 7.5.0.2 ml aliquots of the suspension and containing 20 μΐ of DNA,

Add to a 1.2 M solution of sorbitol. The transformation mixture is incubated for 10 minutes at room temperature and then 1 ml of a solution of 20% polyethylene glycol 4000, 10 mmol calcium chloride, 10 mmol tris hydrochloride, pH 7.5 is added. The mixture was incubated for 60 minutes at room temperature and then divided into four portions. Inoculated portions are inoculated onto inclined agar with 3% agar, 0.67% Difco yeast nitrogen base, no amino acids,

1.2 mol sorbitol, 2% glucose, 2% YPD and other known media components. In addition, histidine, leucine and tryptophan are added to select for leucine prototrophs. The cells were gently mixed and immediately poured into an empty sterile petri dish and, after solidification of the agar, incubated at 30 ° C with appropriate humidity. After 3 days, leucine prototrophs were isolated and plated on YPD solid medium containing 500 pg / ml of hygromycin B antibiotic. The resulting hygromycin B antibiotic-resistant yeast cells are Saccharomyces cerevisiae / pIT208 transformants. The identification of the transformants is further confirmed by restriction enzyme and agarose gel electrophoretic analysis.

(Polyethylene glycol 4,000 used for transformation was obtained from Baker Biochemicals, 222 Red School Lane, Phillipsburg, NJ 08865).

Example 6

Construction of plasmid pKC307 and E. coli K12 RR1 & M151pKC307

A) Construction of plasmid pIT104 and E. coli K12 RR11p1104

The following mixture was incubated at 37 ° C for 15 minutes; 1.5 μΐ (1 pg) plasmid DNA cleaved with pKC222 SacI, 0.5 μΐ, 10X buffer (0.5 M Tris, pH 7.5, 0.1 M MCl), 0.5 - Solution containing 0.5 μΐ (200-200 mmol) dCTP, dATP, dTTP and dGTP and 1 μΐ, 1 unit DNA fragment (Klenow) fragment. The polymerase was heat inactivated and BamHI linkers were added according to Roberts and Lauer (1979, supra), BamHI linkers [d (CCGGATCCGG)] were obtained from Collaborative Research, 128 Spring Street, Lexington, Massachusetts 02173.

The resulting BamHI-linked DNA was digested with BamHI and then ligated as described in Example 2 (B). To reduce the number of starting plasmids, the enzyme was digested with Sac I and then transformed with the resulting pIT104 DNA according to Wensink (Wensink, 1974, supra). Transformed E. coli K12 RR1 (NRRL B-15210) cells were plated on LB plates [Rosenberg and Court, Ann. Port. Genet., 13, 319 (1979). The medium also contains 50 pg ampicillin per ml. The resulting ampicillin-resistant E. coli K12 RR1 / pIT104 cells were isolated in a known manner and cultured for production and isolation of plasmid pIT104. The structure of plasmid ρΓΓ104 was confirmed by transformation, selection, restriction enzyme treatment, and sequence analysis.

B) Digestion of plT104 with Hphl

The digestion was performed as in Example 2 (A) except that plasmid pIT104 was used instead of pKC222 and no PstI digestion was performed. The Hphl cleaved form of the resulting plasmid pIT104 was used without further purification.

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C) Digestion of plasmid pUC8 with HincII

The digestion was carried out according to Example 2 (A), except that plasmid pUC8 (available from Bethesda Research Laboratories, 8717 Grovemont Circle) was used in place of the plasmid pKC222 and the restriction enzymes Hph1 and PstI and their reaction mixtures. , PO Box 6009, Gaithersburg, MD 20877] as well as the restriction enzyme HincII and the following restriction enzyme reaction mixture: 50 mM sodium chloride, 10 mM tris hydrochloride, pH 7.5, 10 mM magnesium chloride, 1 mM dithiouritol. The resulting Hindi digested plasmid pUC8 was used without further purification.

D) Filling of the 3'-mononuclear region with T4 DNA polymerase

The remaining 3'-section after Hphl digestion was filled with blunt end bond. Proceed by incubating 1 µg of Hphl-digested plasmid pITlÓ4 in 10 μ eleg of the following composition at 37 ° C for 5 minutes: 33 mM Tris hydrochloride, pH 7.8, 67 mM potassium acetate, 10 mM magnesium acetate , 0.5 mM dithiothreitol, 0.1 mg / ml bovine serum albumin, 150-150 pmoles dCTP, dATP, dGTP and dTTP, and finally 3 units T4 DNA polymerase. To quench the reaction, incubate at 65 ° C and add 1 µl of 50 mM ethylenediaminetetraacetic acid.

E) Ligation and production of E. coli K12 RR1AM155ppC307

Ligation of the blunt-ended plasmid pIT104 and the digested plasmid pUC8 were performed by the ligation procedure described by Maniatis et al. (Maniatis et al. (1982), supra). The resulting plasmid was designated pKC307 and transformed with E. coli K12 RR1AM15 cells. Example B (b). Selection was carried out as in Example 3 (C). The resulting E. coli K12 RR1AM15 / pKC3O7 transformants were cultured in known manner for production and isolation of plasmid pKC307. For example, the following strains can be transformed with plasmid pKC3O7 as described in Example 2 (B): E. coli K12, E. coli K12 RR1, E. coli K12 JA221 or E. coli K12 HB101.

A restriction map of plasmid pKC307 is provided in Figure 4 attached.

A functional derivative of plasmid pKC307 is also prepared by cleaving a 1.7 kb Hindin fragment from the plasmid. The resulting plasmid was designated pKC308 and transformed with E. coli K12 JA221 cells. This further illustrates the plasmids of the invention.

Example 7

Construction of plasmid ρΓΤ125 and strain JA22llplTl25 of E. coli K12

1) Construction of plasmid ρΙΑ7Α4Δ1

A) Construction of plasmid pBRHtrp

Plasmid pGM1 carries the E. coli tryptophan operon containing the deletion of APL 1413 [Miozzari et al., J. Bacteriology. 1457-1466 (1978)], 14 and thus defines a protein which carries as a fusion protein the first 6 amino acids of the trp leader region and the last third of the trp E polypeptide (hereafter referred to as LE ') and the total trp D. polypeptide. All of these are under the control of the trp promoter operator system. E. coli K12 W3110tna2trpA102 / pGM1 was deposited with the American Type Culture Collection under accession number ATCC 31622. Plasmid pGM1 isolated from the strain is known to be used in the following procedure.

Plasmid pg was cleaved with restriction enzyme PvuII, which cleaved the plasmid at 5 sites. The gene fragments are then linked with Eco RI linkers (the linker contains a self-complementing oligonucleotide having the sequence: pCATGAATTCATG. For subsequent cloning, the plasmid will contain Eco RI cleavage sites. - treated with ribonucleic acid ligase in the presence of 20 picomoles of 5'-phosphorylated synthetic pCATGAATTCATG oligonucleotide and 20 μΐ T4 DNA containing 20 mM Tris pH 7.6, 0.5 mM adenosine -triphosphate, 10 mmol magnesium chloride, 5 mmol dithiothreitol The reaction was carried out overnight at 4 ° C. The mixture was kept at 70 ° C for 10 minutes to complete ligation. Fragments with EcoRI ends were separated by electrophoresis on a 5% polyacrylamide gel Juk.

The three largest fragments were isolated from the gel by first staining with ethidium bromide and localizing the fragments with ultraviolet light. We cut out the gel from the right place. The gel pieces were placed in 300 µl of 0.1Χ TBE, then filled into a dialysis bag and subjected to electrophoresis at 100 V for 1 hour. The medium for electrophoresis is 0, ΙΧΤΒΕ-buffer. The TBE buffer has the following composition: 10.8 g of tris base, 5.5 g of boric acid, 0.09 g of disodium ethylenediaminetetraacetic acid in 11 water. The aqueous solution was collected from the dialysis bag with phenol and then extracted with chloroform and finally the sodium chloride concentration was adjusted to 0.2 mol. The DNA is then isolated by precipitation with ethanol and dissolved in water.

The EcoRI sticky end gene containing the trp promoter / operator system was identified by the procedure given below. The method involves inserting the fragments into a plasmid carrying a tetracycline susceptibility, which will be resistant to tetracycline after incorporation of the promoter / operator system. Subsequent isolations of the given DNA fragment are carried out following polyacrylamide gel electrophoresis as described above.

Β) Λ Construction of the pBRH trp plasmid, which controls the tetracycline resistance under the control of the trp promoter operator system and identification and amplification of the trp promoter operator system fragment isolated according to A) above.

For the construction of plasmid pBRH1 [see Rodriguez et al., Nucleic Acids Research,

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6, 3267-3287 (1979) and West et al., Gene, 7, 271-288 (1979)] expresses ampicillin resistance and contains a tetracycline resistance gene but is not linked to a promoter and thus does not express resistance Who. The plasmid-containing strain was deposited in the American Type Culture Collection Permanent Strain Collection under ATCC No. 37070. Thus, cells containing the plasmid are sensitive to tetracycline. If a promoter / operator system is inserted at the EcoRI site, the plasmid will determine tetracycline resistance.

Plasmid pBRH1 (ATCC 37070) was digested with EcoRI. The enzyme is removed by phenol extraction followed by chloroform extraction and the DNA is dissolved in water after precipitation with ethanol. The resulting DNA molecule was coupled in individually prepared reaction mixtures to the 3 DNA fragment produced in Example 7 (A) using T4 DNA ligase. The reaction mixture is used to transform competent E. coli KI2 294 strain (Backman et al., PNAS, USA, 73, 4174-4198 (1976), ATCC 31446). , 71, 3455-3459 (1974)). The bacteria were plated on LB (see above) medium containing 20 µg / ml ampicillin and 5 µg / ml tetracycline.

Several tetracycline-resistant colonies were selected and the plasmid DNA was isolated therefrom, designated pBRHtrp. The presence of the given fragment is confirmed by restriction enzyme analysis. Plasmid pBRHtrp defines a β-lactamase enzyme resulting in ampicillin resistance and further contains a DNA fragment carrying the tip promoter-operator system. The DNA fragment encodes the LE 'protein, consisting of the first 6 amino acids of the trp leader region and the last third of the trp E polypeptide, and a second protein (designated D'), which corresponds to the first half of the trp D polypeptide A fusion. The protein further comprises a third protein region encoded by the tetracycline resistance gene.

C) Construction of plasmid pSOM7A2

Plasmid pBRHtrp was digested with EcoRI and the resulting fragment isolated by polyacrylamide gel electrophoresis and electroelution was ligated into plasmid pSOM11 (Itakura et al., SCI. 198, 1056 (1977); British Patent No. 2,007,676A] to EcoRI-digested fragment. The ligation was performed with T4 DNA ligase and the resulting DNA was transformed into E. coli strain K12294 as described above. Transformant bacteria are selected on medium containing ampicillin and colonies isolated on ampicillin-resistant colonies (Gruenstein et al., PNAS. USA, 72, 3951-3965 (1975)) are tested. The fragment containing the trp promoter / operator system was isolated from the plasmid pBRHtrp and radiolabeled with P ³² . This is used as a probe for colony hybridization. Several colonies gave positive results during telephoto hybridization and these were selected. The plasmid DNA is isolated therefrom and the orientation of the inserted fragments is determined by analysis with the Bgin and BamHI restriction enzymes. Colonies were double digested and colonies containing the appropriate plasmid with the trp promoter / operator fragment were grown on LB medium containing 10 µg / ml ampicillin. The plasmid is designated pSOM7A2 and is used herein.

D) Construction of plasmid pTrp24

1) Construction of a gene fragment containing the coding regions for the distal regions of the LE 'polypeptide and BglII and EcoRI restriction sites at the 5' and 3 'coding ends, respectively.

Plasmid pSOM7A2 was digested with Hind III and repeated with lambda exonuclease (5'-3'-exonuclease). Digestion is performed under conditions that cleave at the BglII restriction site within the LE 'protein determining region. 20 gg of Hindin-digested pSOM7A2 DNA are dissolved in a buffer of 20 mM glycine buffer, pH 9.6, 1 mM magnesium chloride and 1 mM β-mecaptoethanol. Add 5 units of lambdaexonuclease and incubate for 60 minutes at room temperature. The reaction mixture was then extracted with phenol and then with chloroform and precipitated with ethanol.

An EcoRI site was inserted at the far end of the LE 'gene fragment. Crea et al., 1978, supra, synthesized the advanced phosphotriester method by priming pCCTGTGCATGAT ³² and hybridizing to the single stranded LE 'fragment obtained after lambda exonuclease treatment.

The hybridization was performed by dissolving 20 µg of the digested plasmid pSOM7Δ2 Hindin treated with lambda exonuclease in 20 µl of water and adding 6 µl, 80 picomoles of the 5'-phosphorylated oligonucleotide given above, to the 3 'end of the LE' protein coding sequence. hybridize and fill the remaining single strand on the LE fragment with Klenow polymerase I in the presence of dATP, TTP, dGTP and dCTP. Klenow Polymerase I is a fragment obtained after proteolytic cleavage of DNA. It has 5'-3'polymerizing activity and has 3'5'-exonucleotide activity but no 5'-3'-exonucleotide activity of the parent enzyme (Komberg, WH Freeman et al., San Francisco, Califomia, 1974). )].

The reaction mixture was heated to 50 ° C and then slowly cooled to 10 ° C after which 4 µl of Klenow enzyme was added. After incubation for 15 minutes at room temperature, the reaction was incubated for 30 minutes at 37 ° C, then quenched with 5 g of 0.25 M ethylenediaminetetraacetic acid. The reaction mixture is extracted with phenol and then with chloroform, and the DNA is precipitated with ethanol. The precipitated DNA is cleaved with BglII restriction endonuclease and the fragments are separated by polyacrylamide gel electrophoresis. The gel was autoradiographed with a P ³² -labeled fragment, which was recovered by electroelution. The product is 470 base pairs long. As in 15

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It has already been reported that this fragment of LE '(d) has a BglII end and also contacts a blunt end, which is the beginning of the primer.

2) Construction of plasmid pTHct1 To construct plasmid pTHal I, the thymosin α 1 synthesized gene was inserted into pBR322. Following synthesis of thymosin, the protein 1 defining DNA, the oligonucleotide 16 (Ti-Tm) indicated by arrows in Figure 5 is ligated. At the N-terminus, an ATG codon defining methionine is inserted and b at the 5'-ends are made of single-strand cohesive sites for linkage with EcoRI and BamHI-cleaved plasmids. The BglII site in the gene clearly provides an opportunity for analysis of recombinant plasmids.

Ti-TiO oligodeoxyribonucleotides were prepared by the modified phosphotriester method using fully protected trideoxy ribonucleotide building blocks (Itakura et al., 1977) and Crea et al., 10 (1978), supra. The various oligodeoxyribonucleotides are shown in Table I below.

Table I

Synthetic oligonucleotides used to produce the thymosin gene

Compound Sequence Length Retention time by HPLC analysis (min) * You A-A-T-T-C-A-T-G-T-C 10 17.4 T2 T-G-A-T-G-C-T-G-C-T-G-T-T-G-A 15 24.3 T ₃ T-A-C-T-T-C-T-G-T-TOC of 12 20.3 T ₄ G-A-T-T-A-C-T-A-C-T-A-A-A 13 22.0 T ₅ G-C-A-G-C-A-T-C-A-G-A-C-A-T-G 15 24.8 T ₆ G-A-A-G-T-A-T-C-A-A-C-A 12 20.1 T ₇ A-G-T-A-A-T-C-T-C-A-G-A-A 13 22.6 T ₈ A-A-G-A-T-C-T-T-T-A-G-T 12 20.2 T ₉ G-A-T-C-T-T-A-A-G-G-A-G 12 20.4 Tio A-A-G-A-A-G-G-A-A-G-T-T 12 21.1 Needle G-T-C-G-A-A-G-A-G-G-C-T 12 20.5 T12 G-A-G-A-A-C-T-A-A-T-A-G 12 20.4 TL3 C-T-T-C-T-T-C-T-C-C-T-T 12 19.9 TL4 T-T-C-G-A-C-A-A-C-T-T-C 12 20.5 TL5 G-T-T-C-T-C-A-G-C-C-T-C 12 20.2 Tl6 G-A-T-C-C-T-A-T-T-A 10 17.2

* = At room temperature.

The attached Figure 6 summarizes the manner in which synthetic oligonucleotides were generated for the preparation of the Ti5 fragment. The various nucleotide fragments used to synthesize the Ti5 fragment are shown in figures. Abbreviations have the following meanings:

TPSTe = 2,4,6-triisopropyl-benzenesulfonyl-tetrazole;

BSA = bertzol sulfonic acid;

DMT = 4,4'-dimethoxytrityl;

CE = 2-cyanoethyl;

R = p-chlorophenyl;

Bz = benzoyl;

An - anisoyl; iBu = isobutyryl;

Py = pyridine;

AcOH = acetic acid, and Et 3 N = triethylamine.

mg (0.05 mmol) of fully protected 4 trideoxy ribonucleotides and 180 mg (0.1 mmol) of 2 fully protected trideoxy ribonucleotides were cleaved on 5'-hydroxyl groups with 2% chloroform: methanol (7: 3). made with benzenesulfonic acid.

The reaction was carried out at 0 ° C for 10 minutes. To quench the reaction, 2 mL of saturated aqueous ammonium bicarbonate was added, the mixture was extracted with 25 mL of chloroform and washed twice with 10 mL of water. The organic solvent phase was dried over magnesium sulfate, concentrated to 5 mL, and precipitated with petroleum ether (fraction 35-60 ° C). The colorless precipitate was collected by centrifugation and dried under reduced pressure to give 6 and 8. They are homogeneous by Merck 60 F254 silica gel chromatography (chloroform: methanol = 9: 1).

270 mg (0.15 mmol) 1 and 145 mg (0.075 mmol) of 3 trimers are converted to the phosphodiester (compounds 5 and 7) by stirring at room temperature for 25 minutes with a 1: 3: 1 mixture of triethylamine, pyridine and water Treat with 10 ml. The reagents were removed in a rotavapor and the distillation residue was dried (3 x 10 mL) with anhydrous pyridine. Trimer 8 and trimer 7 (0.05 mmol) were added to 2,4,6-triisopropylbenzenesulfonyl tetrazole (50 mg, 0.15 mmol) in anhydrous pyridine (3 mL) and allowed to stand at room temperature under reduced pressure for 2 hours. TLC showed 95% of the 8 trimers to be hexamer (assayed by

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HU 200366 Β Spray the gel with 10% aqueous sulfuric acid and heat to 60 ° C to detect the 4,4'-dimethoxytrityl group. Water (1 ml) was added to quench the reaction and the solvent was distilled off under reduced pressure. The pyridine is co-distilled with toluene, and the hexamer is cleaved at the 5 'end with 8 ml of benzenesulfonic acid as described for the preparation of Trimers 4 and 2. The product A10 was purified on a silica gel column (Merck 60 H, 3.5 x 5 cm). Elution was carried out stepwise with 98: 2 and 95: 5 chloroform: methanol. Fractions containing compound 10 were distilled to dryness.

Similarly, trimer 5 is coupled to compound 6 and the fully protected product is directly purified on silica gel. The latter compound is cleaved at the 3'-end as described for the preparation of fragment 9 in a mixture of triethylamine, pyridine and water.

Finally, hexamers 9 and 70 are coupled in 2 ml of anhydrous pyridine using 75 mg (0.225 mmol) of 2,4,6-triisopropylbenzenesulfonyl-tetrazole as a condensing agent. The reaction was carried out for 4 hours at room temperature. The reaction mixture was distilled off and the distillation residue was chromatographed on silica gel. The product (160 mg, 77 mg) is obtained by precipitation from its solution in petroleum ether. TLC showed the product to be homogeneous. Compound 77 (20 mg) was dissolved in pyridine (0.5 mL) and deprotected with concentrated ammonium hydroxide (7 mL). The reaction was carried out at 60 ° C for 8 hours. It was then treated with 80% acetic acid for 15 minutes at room temperature. The acetic acid was removed by distillation and the solid residue was dissolved in 4% aqueous ammonium hydroxide solution (4 mL) and extracted three times with 2 mL of ethyl ether. The aqueous phase was concentrated to 1-2 ml and a portion of the solution was fractionated by HPLC to purify Compound 72. The highest peak fractions (2.0 OD254 units) were collected and concentrated to a volume of about 5 mL. The final product 72 was desalted on a Biogel P-2 1.5 x 100 cm column eluting with 20% aqueous ethanol. After removal of the solvent, dissolve the substance in 200 μΐ of water to give the following absorbance: A ₂ 54:10.

The complete thymosin gene was prepared from the 16 synthetic oligonucleotides as described for the production of somatostatin (Itakura et al., 1977, supra) and growth hormone (Goeddel et al., Natur., 281, 544 (1979)). Quantitatively 10 pg T2-T15 oligonucleotides phosphorylated with ^(gamma-32 P) -ATP (New England Nuclear), T4 polynucleotide kinase in the presence of [Goeddel et al (1979), infra], where approximately 1 Ci / mmol specific activity product we get. Radiolabeled fragments were purified by electrophoresis on 20% polyacrylamide / 7 mol urea gel, and the sequence of the eluted fragments was confirmed by two-dimensional electrophoresis / homochromography (Jay et al., Nucleic Acids Sl., 7, 331 (1974)). Fragments were partially digested with snake venom before analysis. The Ti and Ti6 fragments are not phosphorylated as this minimizes unnecessary polymerization in subsequent ligation reactions. 2 to 2 pg of phosphorylated oligonucleotide in 4 groups containing 4-4 fragments are linked to T4 DNA according to known procedures (Goeddel et al. (1979), supra). The process is illustrated in Figure 7. The reaction products were purified by electrophoresis on a gel containing 15% polyacrylamide and 7 mol urea (Maxam and Gilbert, PNAS, USA, 77, 3455 (1977)). The isolated product 4 was coupled and the reaction mixture was separated by electrophoresis on a 10% polyacrylamide gel. The DNA (90-105 bp) corresponding to the thymosin gene is isolated by electroelution.

0.5 µg of plasmid pBR322 were treated with BamHI and EcoRI restriction endonucleases and the fragments were separated by polyacrylamide gel electrophoresis. A large fragment of the gel obtained by electroelution is ligated to synthetic DNA (Goeddel et al., 1979, supra). The mixture is used to transform E. coli strain K12294 (ATCC accession number 31446). 5% of the transformation mixture was plated on LB plates containing 20 pg / ml ampicillin. Ampicillin-resistant colonies are sensitive to tetracycline, indicating integration into the tetracycline resistance gene. Analysis of the plasmids obtained from the cells of the colonies indicated that the pTHal-labeled plasmids contain:

a BglII site not present in pBR322, i.e., incorporating the thymosin gene as shown in Figure 5; a 105 bp fragment obtained by BamHI and EcoRI cleavage.

The attached Figure 7 shows the construction of plasmid pTHal, the bold ends being the 5'-phosphate groups.

3) Reaction of treated pTHal with the fragment LE '(d)

Plasmid pTHal contains a gene for determining ampicillin resistance and a structural gene; the latter gene is cloned into an EcoRI site at the 5'-coding strand and a BamHI site at the 3'-end. The thymosin gene also contains a BglII site. To generate the plasmid harboring the LE '(d) fragment, pTHal DNA is digested with EcoRI and then reacted with Klenow polymerase I in the presence of TTP and dATP to populate the EcoRI end. Digestion with BglII yields a linear DNA fragment carrying the ampicillin resistance gene, which at its opposite ends has a sticky BglII fragment and a blunt end. The resulting DNA is encapsulated with a fragment of LE '(d) which has a BglII sticky end and a blunt end. Recirculation was performed with T4 ligase to give plasmid pTRp24. However, at the site of ligation of the blunt end, a region recognizable by the restriction enzyme EcoRI is restored.

E) Construction of plasmid pSOM7A2A4

Plasmid pTrp24 was digested with BglII and EcoRI, purified by polyacrylamide gel electrophoresis, and isolated by electroelution to obtain a fragment containing the codons for determining the LE '(d) polypeptide, BglII sticky end and EcoRI' sticky end. to the end 17

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HU 200366 Β close. The LE '(d) fragment can be cloned into the BglII of plasmid pSOM7A2 to obtain a LE' polypeptide-somatostatin fusion protein expressed under the control of the tryptophan promoter / operator system. First, plasmid pSOM7A2 is partially digested with EcoRI to cleave the EcoRI site further away from the tryptophan promoter / operator system, then the appropriate primary sequence is selected to maintain the codon reading pattern properly and to restore an EcoRI site.

Plasmid pSOM7A2 was dissolved in 200 µl, 20 mM Trist, pH 7.5, 5 mM magnesium chloride, 0.02% NP40 detergent and 100 mM sodium chloride, and 0.5 units of EcoRI was added. After incubation for 15 minutes at 37 'c, the mixture is extracted with phenol, followed by chloroform, precipitated with ethanol and digested with Bgin. The larger fragment was separated by polyacrylamide gel electrophoresis and isolated by electroelution. The fragment contains the so-called proximal end of the LE 'polypeptide. LE '(p) codons, i.e. the part to the right of BglII. This fragment was coupled to the previously generated fragment LE '(d) by T4 DNA to give plasmid pSOM7A2A4. Transformation of E. coli 294 cells with the plasmid yields fusion protein producing strains comprising the complete LE polypeptide and somatostatin, which is expressed under the control of the tryptophan promoter / operator system.

F) Preparation of a linear DNA gene containing a tetracycline resistance determining gene which has a PstI region at its 3 'end and a BglII region at its 5' end

Plasmid pBR322 was digested with Hindin and the Hindin ends digested with S1 nuclease. The S1 nuclease treatment was performed with 10 µg of HindIII. enzyme-cleaved pBR322 DNA is treated with 300 units of Si nuclease in 30 µl Si buffer for 30 minutes at 15 ° C. The Si buffer is composed of 0.3 molar sodium chloride, 1 mmol zinc chloride, 25 mmol sodium acetate, pH 4.5. To stop the reaction, 1 µl of 30X S1 nuclease stop solution was added. It is composed of 0.8 mol tris base, 50 mmol ethylene diamine tetraacetic acid. The mixture was extracted with phenol and then with chloroform, precipitated with ethanol and cleaved with EcoRI. The fragment is isolated by polyacrylamide gel electrophoresis and electroelution, which has an EcoRI sticky end and a blunt end, and its coding strand starts with a thymine nucleotide. The HindIIIII digested HindIII region of thymidine can be linked to the BglH region treated with Klenow polymerase I so that the BglII restriction site is restored after ligation.

Plasmid pSOM7A2 prepared in Example 7 IC was digested with BglII and the BglII sticky ends were double-stranded with Klenow polymerase I in the presence of 4 deoxynucleotide triphosphate. The resulting DNA fragment was cleaved with EcoRI and the small fragment was then isolated by polyacrylamide gel electrophoresis and electroelution. The resulting linear DNA fragment contains the tryptophan promoter / operator 18 system and the LE 'proximal sequence LE' (LE) (p) 'J. The product has an EcoRI end and a blunt end, which was created after filling the Bgin site. The BgUI site is reconstituted when the blunt end is linked to the blunt end of the S1-digested Hindin fragment prepared as above. The two fragments were ligated with T4 DNA to give the circled plasmid pHKY10. This plasmid was transformed into E. coli 294 cells. Tetracycline-resistant cells carry the recombinant plasmid pHKY10, the cells are selected, and the plasmid DNA is isolated. The product was digested with BglH and PstI, and the DNA was isolated by polyacrylamide gel electrophoresis and electroelution of the larger fragment to give the linear DNA having PstI and BglH. The DNA derived from plasmid pHKY10 contains the origin of replication and can thus be used to construct plasmid ρΙΑ7Δ4Δ1, in which both the fusion protein gene of trp LE 'polypeptide and the tetracycline resistance gene are under the control of the trp promoter / operator region.

G) Preparation of linear DNA containing the trp promoter / operator system

Plasmid ρ5ΟΜ7Δ2Δ4, prepared in Example 7 IC) was treated with EcoRI followed by PstI. The resulting fragment contains the trp promoter / operator region, which was isolated by electroelution following polyacrylamide gel electrophoresis. Partial Eco RI digestion is required to obtain the fragment which was cleaved near the 5 'end of the somatostatin gene but did not cleave the Eco RI site between the ampicillin resistance gene and the trp promoter / operator. The ampicillin resistance lost due to digestion with PstI can be restored by a ligation reaction with the linear derivative of plasmid pHKY10 prepared according to Example 7F.

H) Isolation of insulin A chain structural gene

The structural gene for the insulin A chain is the plasmid pIA1

EcoRI and BamHI (Goeddel et al., PNAS, USA, 76, 106 (1979)). The plasmid may also be prepared from E. coli strain K12 94 / pIAl (ATCC 31448). The fragment was isolated by polyacrylamide gel electrophoresis and electroelution, the product having Eco RI and Bam HI ends.

I) Ligation of the structural gene of the insulin A chain, the trp promoter operator region and the pHKY10 linear DNA fragment with PstI and BglII ends

The linear A fragment of the insulin A chain structural gene, the trp promoter / operator system produced in Example 7 (1G) and the linear pHKY10 DNA fragment obtained in Example 7 (IF) were ligated in the appropriate orientation. 8. Thus, plasmid ρΙΑ7Δ4Δ1 is obtained. This plasmid is easy to select due to the restoration of the sequence determining ampicillin and tetracycline resistance.

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2) LI inhibition of the 1.3 kb BamHI / BgllI plasmid ρΓΓ123 and the 45 kb BamHI / BgllI fragment of plasmid pIA7A4Al

A) Digestion of plasmid plA7D4D1 with BamHI / BglII and isolation of the 45 kb fragment

Digestion and isolation were performed as described in Example 2 (A) except that plasmid ρΙΑ7Δ4Δ1 and BglII and BamHI were used instead of pIT123 and Hph1 and PstI, respectively, and the corresponding reaction mixtures provided by the vendor are used.

B) Ligation and Transformation

Ligation and transformation were performed as in Example 3 (C) except that the 4.5 kb BamHI / BglII fragment of plasmid ρΙΑ7Δ4Δ1 and E. coli K12 were replaced by plasmid pUC7 and E. coli K12 RR1AM15 cells. Strain JA221 (NRRL B-15211) was used. The transformed cells are plated on TY solid medium containing 50 pg / ml ampicillin and then

Replicate with 200 µg / ml hygromycin B antibiotic medium. E. coli K12 JA221 / plT125 transformants resistant to the hygromycin B antibiotic were cultured in known manner for the construction and isolation of plasmid pIT125. Also, plasmid pIT125 can be used to transform E. coli strains such as E. coli K2, E. coli K2 RR1 or E. coli K12 HB101. The transformations are shown in Example 2

(B). A restriction map of plasmid ρΓΓ125 is given in the accompanying Figure 4. Additional illustrative plasmids and transformants that can be prepared as described above are described in a. and III. Table.

Π. Spreadsheet

Plasmids illustrating the invention

Example number Name Greatness about in Genetic mark E. coli yeast Preparation 8th pIT143 3.0 Ap MboII digestion of the 958 base pair Clal / HincII fragment of plasmid pIT141 followed by removal of the overhangs, ligation of the BamHI linker with the TGGATCCA sequence and insertion into the BamHI digested pUC8 9th pIT212 8.9 Ap URA3 Ligation of the 1.3 kb BamHI / BglII fragment of plasmid pIT103 with the BamHI digest of pRB5 (ATCC 37051) 10th pIT213 10.6 ApR Km ^R URA3 Ligation of the 1.7 kb PvuII fragment of pNG59 * into SmaI digested pIT212 11th pIT215 11.4 Ap URA3 The BamHI / BglH fragment of pIT1 18,750 base pair was coupled to the BamHI fragment of plasmid pIT213 in the orientation shown in Figure 8. 12th pIT217 11.7 ApR Km ^R URA3 Hm ^R Coupling of the 1 kb BamHI / BglII fragment of pIT120 to the BamHI linear fragment of plasmid pIT213 in the orientation shown in Figure 8 13th pIT219 10.8 Apr URA3 Coupling of the 230 bp BamHI fragment of plasmid pIT143 in the orientation shown in Figure 9 to the BamHI fragment of plasmid pIT213

* = E. coli K12 RR1 / pNG59 was cultured for the construction and isolation of plasmid pNG59. The strain is found in the Northern Region Research Laboratory, Peoria, Illinois, in a permanent strain collection under NRRL B-15604.

III. Spreadsheet

The transformants of the invention

E. coli / R

E. coli K12 / R

E. coli K12 JA221 / R

E. coli K12 HB101 / R

E. coli K12 RR1 / R

Saccharomyces cerevisiae / R ¹ , wherein R is pIT143, pIT212, ρΓΓ213, ρΓΤ215, pIT217 or pIT219, and wherein R ^{1 is a} plasmid pIT212, pIT2-3, pIT215, pIT217 or pIT219.

Claims (3)

Claims 1. A method for producing a plasmid comprising the last 338 amino acids of the hygromycin B phosphotransaminase enzyme, either alone or in a reading phase, together with a gene comprising a transcriptional and translational activation sequence. a) To construct plasmid pIT123, a 1.45 kb Eco RI restriction fragment of plasmid pKC222 was ligated with Eco RI digested 65 ρΓΓ122, and if desired -191 HU 200366 Β ai) To construct plasmid ρΠΓ125, plasmid pIT123 was digested with BamHI and BglH and the resulting 1.3 kb BamHI / BglH restriction fragment was ligated with the 4.5 kb BamHI / BglII restriction fragment of plasmid ρΙΑ7Δ4Δ1. ₂ ) Plasmid pIT123 for the construction of plasmid ρΓΓ144 A 1.3 kb BamHI / Bgin restriction fragment was ligated with the BamHI fragment of plasmid pUC7, or (a3) a 1.3 kb BamHI / Bgin restriction fragment of plasmid pIT123 was ligated with the BamHI fragment of plasmid pIT207, or a4) a ρ2 To construct the plasmid pIT123, a 1.3 kb BamHI / BglH restriction fragment was ligated to the BamHI site of pRB5, and optionally a4.i) The 1.7 kb PvuII restriction site of pN659 ^{x was} ligated into the SmaI site of pIT212 to construct pIT213. , a4.ii) ligation of the BamHI-cleaved plasmid pIT213 with the 750 bp BamHI / BglH restriction fragment of plasmid pIT215 to produce plasmid pIT215; Ligation with the restriction fragment of BamHI / BglII, or a4.i.3) deletion of plasmid ρΓΓ219. to construct the BamHI digested pIT213 plasmid pIT143 With a BamHI fragment of 230 base pairs, or b) ligating the Hphl-digested plasmid ρΓΓ104 with HincII-digested pUC8 to generate pKC307, and optionally bi) deleting the 1.7 kb HindIII fragment from pKC307 to generate pKC308, or c) ligating the 750 bp BamHI / Bgin fragment of plasmid pIT207 to the BamHI restriction site of plasmid pMC1587, or d) For the construction of plasmid pIT143, the 958 bp Clal / HincII fragment of plasmid pIT141 was digested with MboII, the single-stranded portions were cleaved by BamHI linkers, and the linker-containing fragment was ligated into the BamHI restriction site of pUC8. (Priority: July 20, 1984) The method of claim 1 for the construction of plasmids ρΓΤ125, pIT144, pIT207, pIT208, pKC307 and pKC308, characterized by starting from appropriate plasmids. (Priority: July 22, 1983) 3. The method of claim 1 for the construction of plasmids pIT143, pIT212, pIT213, pIT215, pIT217 and pIT219, wherein the plasmids are derived from appropriate plasmids.