AU593296B2 – A method for the expression of genes in yeast, and DNA fragments as well as plasmids comprising said DNA fragments to be used when carrying out the method
– Google Patents
AU593296B2 – A method for the expression of genes in yeast, and DNA fragments as well as plasmids comprising said DNA fragments to be used when carrying out the method
– Google Patents
A method for the expression of genes in yeast, and DNA fragments as well as plasmids comprising said DNA fragments to be used when carrying out the method
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Publication number
AU593296B2
AU593296B2
AU64338/86A
AU6433886A
AU593296B2
AU 593296 B2
AU593296 B2
AU 593296B2
AU 64338/86 A
AU64338/86 A
AU 64338/86A
AU 6433886 A
AU6433886 A
AU 6433886A
AU 593296 B2
AU593296 B2
AU 593296B2
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AU
Australia
Prior art keywords
sequence
gene
heme
dna
derived
Prior art date
1985-10-24
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AU64338/86A
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AU6433886A
(en
Inventor
Erik Ostergard Jensen
Kjeld Adrian Marcker
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Danske Sukkerfabrikker AS
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Danske Sukkerfabrikker AS
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1985-10-24
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1986-10-23
Publication date
1990-02-08
1986-10-23
Application filed by Danske Sukkerfabrikker AS
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Danske Sukkerfabrikker AS
1987-05-07
Publication of AU6433886A
publication
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patent/AU6433886A/en
1990-02-08
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1990-02-08
Publication of AU593296B2
publication
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patent/AU593296B2/en
2006-10-23
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Ceased
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Classifications
C—CHEMISTRY; METALLURGY
C07—ORGANIC CHEMISTRY
C07K—PEPTIDES
C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
C07K14/415—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from plants
C—CHEMISTRY; METALLURGY
C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
C12N15/09—Recombinant DNA-technology
C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
C12N15/67—General methods for enhancing the expression
C—CHEMISTRY; METALLURGY
C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
C12N15/09—Recombinant DNA-technology
C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
C12N15/80—Vectors or expression systems specially adapted for eukaryotic hosts for fungi
C12N15/81—Vectors or expression systems specially adapted for eukaryotic hosts for fungi for yeasts
Abstract
A method of expressing genes in yeast by introducing into a yeast cell a recombinant DNA segment containing both the gene to be expressed and a 5 min flanking region comprising a promoter sequence, and culturing of the transformed yeast cells in a growth medium, using as the recombinant DNA segment a segment, of which the 5 min flanking region comprises a first DNA fragment containing a promoter sequence in combination with a second DNA fragment containing a leader sequence regulated on the posttranscriptional level by heme, heme analogs, or heme precursors. Furthermore DNA-fragments and plasmids to be used when carrying out the method. When carrying out the method according to the invention an increased expression of genes is obtained by a reduced genetic load of the host cell and a more optimal utilization of the protein synthesis apparatus and the energy metabolism.
Description
r 1> Form COMMONIWEALTH OF AUSTRALIA PAT0’S ACT 1952-69 COMPLETE SPECIFICATION
(ORIGINAL)
Class I t. Class Application Numbep,: q 9″S Lodged: 59296 Completi,’ Specification Lodged; Mer;, Priority: ‘Related Art: Accepted:- -u~tz~w~n* u ai~d under Siction 49, and Ix cor”. tOr pLating.
I
J’amieof Applicant: AKTIESELSKABET DE DANSKE SUKKERFABRIIKER C Address of Applicant: 5, Langebrogade, DK-KL411 Copenhagrin Denmark Actual Inventor-, Address for Service: IJELD ADRIAN MAROKER and ERIK OSTERGARD JENSEN EDWD. WATERS SONS, 50 QUEEN STREET, MELBOURNE, AUSTRALIA, 3000.
Complete Specification for the Invention entitled: A METHOD FOR THE EXPRESSION OF GENES IN YEAST, AND DNA FRAGMENTS AS WELL AS PLASMTDS COMPRISING SAID DNA FRAGMENTS TO BE USED WHEN CARRYING OUT THE~ METHOD The following statement is a full description. of this invention, including the best method of performing It kcnown to; IvU 1 A method for the expression of genes in yeast, and DNA fragments as well as plasmids comprising said DNA fragments to be used when carrying out the method.
The invention relates to a novel method for the expression of genes in yeast, and DNA fragments as well as plasmids comprising said DNA fragments to be used when carrying out the method.
In the description i.a. the following terms are S* 10 used: r’ SPromoter region: A DNA fragment containing a pro- S” moter and target sequences for RNA polymerase as Swell as possible activation regions comprising target sequences for transcriptional effector substances.
Effector substance: Substances exerting or mediating a regulatory function. Thus the effector substances also include substances influencing the concentration of substances exerting or mediating a regulatory function.
S i c Leader sequence: Generally is meant a DNA sequence Sbeing transcribed into a mRNA, but not further translated into protein. The leader sequence comprises thus the DNA fragment from the transcription start to the ATG translation start codon.
Leader sequence: In relation to t present invention is- meant a short DNA frAgment typically having 40-70 bp and comprising target sequences 11 i’6?
L
2 for a post transcriptional regulation exerted or mediated by intracellular home.
Furthermore the following terms generally known to persons skilled in the art of molecular biol.ogy are used.
CAP addition site: The site where 7-methyl-GTP is added on the corresponding mRNA.
DNA sequence or DNA segment: A linear array of nucleotides interconnected through phosphodiester 10bonds between the 3′ and 5′ carbon atoms of adjacent pentoses.
Expression: The process undergone by a struct ial r gene to produce a polypeptide. It is a combination of transcription and translation as we.l as possible posttranslational modifications, Flanking regions: DNA sequences surrounding coding regions. 5′ flanking regions contain a promoter.
3′ flanking regions may contain transcriptional terminator signals etc.
Gene: A DNA sequence composed of three or four parts the coding sequence for the gene product, the sequences in the promoter region which control whether or not the gene will be expressed, those sequences in the 3′ end conditioning the transcriptional termination and optional polyadenylation, as well as intervening sequences, if any.
K i r: intervening sequences are transcribed into pre-mRNA and are eliminated by modification of precursorRNA Sinto mRNA.
Cloning: The process of obtaining a population of organisms or DNA sequences deriving from one such organism or sequence by asexual reproduction, or more particular: process of isolating a particular organism or part ther and the propagation of this subfrac- Stion as a homogeneous population.
Coding sequence: DNA sequence determining the amino acid sequence of a polypeptide.
(mRNA): RNA molecule produced by transcription of a gene and possible modification of precursorRNA. The mRNA molecule mediates the genetic metsage determining the amino acid sequence of a polypeptide by part of the mRNA molecule being into said peptide.
Nucleotide: A monomeric unit of DNA or RNA con- S, sinting of a sugar moiety (pentose), a phosphate, and a nitrogeneous heterocyclic base. The base is linked to the sugar moiety via a glucosidic bond 25(1′ carbon of the pentose), and this combination of base and su4.r is a nucleoside. The base charac- I terises the nucleotide. The four DNA bases are adenine guanine cytosine and thymine The four RNA bases are A, G, C, and uracil 0 1 1 4 Plasmid: A nonchromosomal double-stranded DNA sequence comprising an intact replicon such that the plasmid is replicated in a host cell. When the plasmid is placed within a unicellular organism, characteristics of that organism are changed or transformed as a result of the DNA of the plasmid. For instance a plasmid carrying the gene for tetracycline resistance (TcR) transforms a cell previously sensitive to tetracycline into one which resistant to it. A cell transformed by a plasmid is called a transformant.
a Polypeptide: A linear array of amino acids interconnected by means of peptide bonds between the a c-amino and carboxy groups of adjacent amino acids.
S
c 15Recombination; The creation of a new DNA molecule by combining DNA fragments cf different origin.
Replication: A process reproducing DNA molecules.
Replicon: A self-replicating genetic element possessing an origin for the initiation of DNA reand genes specifying the functions necessary for controlling the replication.
F Restriction fragment: A DNA fragment resulting 1^ ~from double-stranded cleavage by an enzyme recognizing a specific target DNA sequence.
polymerase: Enzyme exerting the transcription of DNA into RNA.
Transformation,: The process whereby a cell is incor- 1 1 ^fr porating a plasmid.
Translation: The process of producing a polypeptide from mRNA or: the process whereby the genetic information present an mRNA molecule directs the order of specific amino acids during the synthesis of a polypeptide.
Transcription: The method of synthesizing a complementary RNA sequence froin a DNA sequence.
B* Vector: A plasmid, phage DNA or other DNA sequences of replication in a host cell and having ,one or a small number of endonuclease recognition a:o sites at which DNA sequences may be cleaved in a determinable manner without attendant loss of an essential biological function of the DNA, e.g.
production of coat proteins or loss of promoter or binding sites, and which contain a marker suitable for use in the identification of transformed colls in the form of for instance tetracycline resistance or ampicillin resistance, A 2Qvector is often called a cloning vehicle.
The production of a biologically active product by means of recombinant DNA technology is a complex Smatter which involves many process steps from the initiation of the transcription to the final aof the biologically active molecule.
Many of these process steps do not appear in procaryotic organisms the reason why eucaryotic production organisms must be used in many cases.
lt 6 Yeast is an eukaryotic organism, the synthesis apparatus of which comprises many of the processes and regulating mechanisms characteristic of higher organisms. In addition yeast cells have a short bgeneration time and a thousand-year old experience basis exists for the use of yeast as a culture organism.
Completely decisive factors for a biological synthesis of a desired gene product are a possibility lOand improvement of transcriptional initiation as a well as transcriptional and posttranscriptional 0 00 regulation of the gene expression.
ae These functions are mainly carried out by 5′ flankiing regions. A wide range of 5′ flanking regions tt 15of prokaryotic and eukaryotic genes ha_ been sequenced, and inter alia based thereon comprehensive knowledge has been provided of the regulation of gene expression and of the subregions and sequences being of importance for the regulation of v F the gene. Great differences exist in the regulatory mechanism in procaryotic and eucaryotic organisms, but within these two groups there are many common features, The regulation of the gene expression may take on the transcriptional level and then it is a preferably exerted by regulation of the initiation frequency of transcription, The latter is well known and described inter alia by Benjamin Lewin, Gene Expression, John Wiley Sbns, Vol, I, 1974, II, Second Edition 19801 vol. II, 1977. Alternatively the regulation may be exerted at the *t *i 4 as #4 44 4 ,*4 4444 4 t(r 4F 7 posttranscriptional level e.g. the regulation of the frequency of the translation initiation at the rate of translation and of the termination of translation.
Leghemoglobins are monomeric hemoproteins exclusively synthesized in the root nodules which develop through the symbiotic association of Rhizobia with leguminous plants. A logical candidate for an effector substance activating the leghemoglobin genes is heme produced, in Rhizobia and constituting the prostetic group of the leghemoglobins, The synthesis of several hemoproteins in the yeas’ Saccharomyces cerevisiae is also regulated by the level of intracellular heme which also forms the prostetic group 15 of these proteins. Thus the transcription of the isocytochrome c gene is heme depend–at while in the case of catalase T 1 the heme control is exerted both at the transcriptional and the posttranscriptional level.
In accordance with the present invention is presented the sequence of 5′ flanking regions of the four soybean leghemoglobin genes Lba, Lbcl, Lbc2, and Lbc3, The sequences are presented in the enclosed sequence scheme, Scheme 1, where the se- 25 quences are aligned in such a manner that the homology appears clearly.
In the sequence scheme indicates that no base is present in the position in question. The names of the genes and the base position counted upstream from the ATG start codon are indicated to the right of the sequence scheme, Furthermore the important 4 i 4 4 4 04 *0 060 t 4 sequences have been underlined, As it appears from the sequence scheme a distinct degree of homology exists 1Lxtween the four 5′ flanking regions, and in the position 23-24 bp upstream from, the CAP addition site they all contain a TATATAAA sequence corresponding to the “ITATA”‘ box which in eucaryotlic cells usually ar’i located a corresponding number of bp upstream from the CAP addition site, Furthermore a GGAAG sequence is present 64-72 bp upstream from the CAP addition site,, said sequence corresponding to the I”CCAAT”I box usually located, 70-90 bp upstream from the CAP addition site. From the CAP additign site to the translation start codon, ATO, leader sequences of 5-&$bp are present and shjow a distinct degree of homology of approx. 75-80%, In accordance with the present invention it. has furthermore been proved, exemplified by Lbc3f that the 5′ flanking regions of the soybean leghemoglobin genes are functionally ac.tive in yeast, The latter has been proved by ftsioning the IL,._qO~iL chloroamphenicol acetyl tr–nsferase (CAT) gene with the 5′ and V’ flaning regions of the soybtan Lbc3 gene in ouch a manner that the expression of, the CAT gene is controlled by the Lb promoter. The Ev.4ioning fragment was inserted in the yeast plasmid vector, YEP 24# comprising the yeast URA-3 gene as a selectable marker, The yeast strain Si cerevisiae TH’I wh~ich is URA-3 and unable to synthetize hetne due to a mutation in the 5..amino levultnic acid syntV etnse gene, $-ALA, was subsequenttly ttatisformed with the resulting constltuction, The transformed ‘Sir4
V.
A,1 r -il r- 9 yeast cells showed a CAT activity under all growth conditions tested. The conclusion can therefore be made that the 5′ flanking regions of soybean hemglobin genes are functional in yeast.
In accordance with the present invention it has furthermore been proved that the 5′ flanking regions act; as target for a regulation exerted or mediated by intracellular heme. The CAT activity is thus 20-40 fold higher in the yeast S. cerevisiae TM1 grown in the presence of 6-ALA than the CAT activity in yeast grown without this heme precurser S* in the growth medium. Similar high CAT activities were present in the yeast S. cerevisiae TM1 grown in the presence of heme, protoporphyrin IX, or the )e 15 heme analog deuteroporphyrin IX, The effect of heme on the CAT activity is specific since the amount of the URA-3 geie product remains constant under all the conditions tested. Furthermore the transcriptional level does not apparently change because of the presence of hers as the CAT-mRNA level remains constant independent of changes in tl, intracellular heme concentration. It can therefore be concluded that the regulatory mechanism exerted or mediated by heme occurs on the posttranscriptional level, The observed increase of CAT activity is depenaent on protein synthesis, The halft l tf oi *cl AT enzyme is furthermore independenh o th e.ce i of heme, and in vitr- CAT aciv- y d” heme. Therefore hene mos 1 gene expression on the ransal ion of the 5′ flanking region of tLc3 rit
SI:
of the neomycine phospho transferase (neo) gene, is controlled by heme in a completely similar manner as the CAT gene fused with a 5′ flanking region of the Lbc3 gene. The effect of heme is thus not mndiated by heme interacting with the coding sequence, but rather by heme interacting with the 5′ or 3′ flanking Lbc3 sequences present in the CATmRNA, The expression of a gene only containing the Lbc3 flanking region and the neo gene is controlled in a similar manner by heme. The effect of intracellular heme on the gone expression can thus be mediated by an interaction with the leader sequence, S, The short leader sequences do not contain translation start codons. The regulatory mechanism exer- 15 ted or mediated by intracellular heme is therefore not related to regulatory mechanisms involving false start codons, cf, the disclosure of Hunt, T, Nature, Vol. 316, 580-581, (1985). The regulatory mechanism described in relation to The present invention is exerted or mediated by heme interacting with a leader sequence and is there2ore a novel regulatory mechanism.
The presence of plasmids in a cell present in a natural environment provides the cell with a property which is only -d’4antageous for the cell under certain circumstances. Plasmid encoded properties may for instance be resistance to an antibiotic 2 present in the surrounding environment, The presence of a plasmid and synthesis of the plasmid encoded gene products do, however, also load the energy metabolism of the cell and the protein synthesixs apparatus, and a cell containing a plasmid is there- I /i; Uf’ 4/ i Ii I 11 fore ousted and lost in an environment not needing the plasmid encoded properties, The above mentioned instability is additionally increased by using plasmids as vectors for synthesis of a desired gene product not usually produced by the cell in question. The latter implies that cells synthesizing such products must be bjected to a selection pressure in order to ensure that the desired gene product can still be synthesized. The previous method of achieving a high expression of a certain gene product is that the expression is S, controlled by a strong promoter causing a high Sconcentration of the mRNA being translated into the gene product in question, t 15 A high concentration of gene product can, however, t also be obtained by a more efficient translation of the mRNA in question. The latter implies that the gene product, synthesis is controlled both at the transcriptional and on the translation level, which means that the genetic load on a cell synthesizing a certain gene product can be distributed on two activities instead of one as usually.
The two activities can therefore be manipulated in such a manner that the same result concerning concentration of gene product can be obtained though the promoter is not as strong as the promoters usually employed, Such a distribution of the two activities implies that the cell is not as genetically loaded as when the gene product synthesis is only controlled by one strong promoter. As a result the selection pressure on the cell in ques- 4
IS*
‘S 1
L
r V! I 12 tion can be reduced.
It is furthermore of importance to obtain the utilization most rational for the cell of the energy metabolism and of the protein synthesis apparatus in the phase where the synthesis of the desired gene product occurs. Such a rational utilization of the energy metabolism and of the protein synthesis apparatus is improved preferably by optimating the late steps of the synthesis of a gene product rather than optimating the early steps.
An important feature of a gene expression system is therefore that the expression of the desired product can be increased from an initially low i expression to an overproduction by a manipulation 15 of the external environment c- the cell as dis- St V closed by the present invention. Furthermore an inducible optimating of the posttranscriptional synthesis steps which has been disclosed by the present invention is more advantageous than an induced optimation of the transcription.
1 Previous methods for the expression of genes in yeast employ a range of promoters and expression vectors, cf. for instance EP 120 551 A2, in which the use of GAPDH- or PyKyeast promoters is disclosed, as well as of expression vectors comprising Sthese promoters.
GB 2,137,2.7 A discloses furthermore the use of the promoter GAL1 in several expression vectors.
When using these previously known promoters, the i 1 1 13 expression can only be increased by increasing the transcription-initiation frequency. The of these promoters involves consequently a high genetic load on the cell, an irrational utilization of the energy metabolism and the synthesis apparatus, as well as a resulting instability necessitating a high selection pressure on the host organism when using these promoters.
The object of the present invention refore to disclose a method of using promoters active in yeast, as well as leader sequences subjecting a novel manner the expression of the following gene to a regulation at the posttranscriptional level.
S* Furthz objects of che invention are to provide com- 15 binations of the promoter and leader sequence, 4′ whereby these combinations have been obtained from 4 4 5′ flanking regions of plant leghemoglobin genes i and proved to be functional in yeast, as well as it is an object of the invention to provide plasmids comprising the above combination of promoter and leader sequences.
The method according to the present invention of expressing genes in yeast by introducing into a yeast cell a recombinant DNA segment containing both the gene to be expressed and a 5′ flanking r t region comprising a promoter sequence, and culturing of the transformed yeast cells in a growth medium i characterised by using as the recombinant DNA segment a segment, of which the 5′ flanking region comprises a first DNA fragment containing a promoter sequence in combination with a second DNA fragment aontaining a leader sequence regulated on the postg u itr t it ft tr ft transcriptional level by heme, heme analogs, and heme precursors. In this manner it is possible by inserting a gene downstream from the combination and in a suitable vector being able to replicate in yeast to obtain a synthesis of a biologically active product. This method allows an increase of the expression of a desired gene by a novel regulatory mechanism acting at the posttranscriptional level. As a particular result a reduced genetic strain of the host cell and an optimal utilization of the protein synthesis apparatus and the energy metabolism of the host cell is obtained and consequently an increased stability of the expression vector in the host cell.
15 A particular embodiment of the method according to the invention uses as a first DNA fragment an isolated of synthesized promoter sequence to be combined with a second isolated or synthesized leader sequence. In this manner it is possible to combine any leader sequence from yeast, plants or animals under natural conditions being subjected to a posttranscriptional regulation according to claim 1 with any suited yeast promoter, plant promoter or another promoter being functional in yeast, 25 According to a particular embodiment of the method according to the invention the intracellular concentration of heme is increased by adding to the growth medium such carbon sources, especially nonfermentable carbon sources, which cause increased intracellular concentrations of heme. J;n this manner an induction of the expression c f thi desired gene is obtained by adding a carbon souWA to the growth ii {f i r r
I
E
pc r c r r I
B
i lU,
J
L
medium. Examples of such carbon sources are glycerol and succinate which are particularly preferred because they are inexpensive and easily available carbon sources. Furthermore ethanol can be used.
Under certain conditions the yeast itself can produce this ethanol while growing. After termination of the growth the ethanol is utilized whereby the translation is increased.
According to a special embodiment the same effect can be obtained by the intracellular concentration of heme being increased by adding to the growth medium one or several substances selected from the group consisting of heme, heme analogs, and heme Sprecursors. An example of a heme analog is deuteroporphyrin IX, and an example of a heme precursor is 6-amino levulinic acid.
A special embodiment of the method according to the invention uses a DNA fragment comprising a promoter sequence and a leader sequence, said DNA fragment being identical with, derived from or comprising 5′ flanking regions of plant leghemoglobin genes, yeast genes or other genes subjected to an expression regulation under natural circumstances, said expression regulation being exerted or mediated by intracellular heme. In this manner a simple access to a combination of leader sequence r) and promoter sequence is obtained, said combination being proved according to the present invention to be functional in yeast. Examples of such DNA fragments are the four 5′ flanking regions of the soybean leghemoglobin genes, viz, i I Lba with the sequence:,
GAGATACATT
GATATATACC
TCTTTTATTT
ATTTTGAAAA
GGTTAAATCT
GTAGAGTCTA
AAAGTTGGTT
TTTTTTTTGG
CTAACCATAT
1D) TAATTAAAAA
TCATCATGCT
TGGTTTTCTC
TTGTTGCATA
AAAGAAATAT
ATAATAAtCT
TTCTCGTATA
TTATAAAAAA
CATGCTCTTT
CATAGTGCCT
CATAAAATTT
TTTCTCGAGG
ATTAATAGTT
TAAATTTAGA
ATTATTTGAT
GATTGACACC
ACT’CTCCAAG
ACTTGCATCG
CTCTAGTGTC
CTGTTATTTT
GACTTTATTT
GACAATTTTC
CTATTCAATA
ACCTTAATAG
AAGAAAGGAA
ATGTTTATAT
ACAACACTTC
TAAATTTTTT
CTCCACAAGC
CCCTCTATAT
AACAATTAAT
TATTTATTAT
TTCAATCTTG
TTTTAAAAAA
TGTTTCCTTT
ATTTGGGCTC
TAGAGAATAG
ATGTTAAAAA
GAAAACTGAA
AATTATTTTT
AAAAGATCGT
CAAGAGAAAC
AAACAAATAT
AGAAATAACA
TTTATCTGGT
TAGATTTACT
AATAAAG TGA
TTCATCATTG
?ATTTAATTA
AGA’3TCTTGG
CTGTGATATT
AATAAATAAA
TTAATTTGAT
TGTTTCTTCT
ACATAAGCTT
TGGAG’rGAAG
GAAPAATTAAA
o @o 00 0 0 oO 00 0 0 0 *4 00 0 4 0 0 0 15 Lbcl with the sequence:
TTCTCTTAAT
AATCTCTAGT
CTTTAATATT
TTTCCAAACC
2 0ACTTCAGAAA
TAATAAACTT
TATTGGGTGA
TTATTAGTAA
GAAAGGGAGC
TAATTATGTT
AATACTTAAA
TAGGATTTTG
CACAAGCCAA
ATATAAACAT
TAG AAAATAA
ACPJ\TGGAGT
GTCTATTTAC
ATTATATC.CT
TGTAGATTTA
AGTAATTACA
TAAAATCAAA
AATCTCATAG
AGTCTGCATC
GAATGTTAAA
TACATGAAAA
ATATTTATTT’
AAAAGATCAT
GAGAAACTTA
GTATTGGATG
CAAAAAAAAG
TTTTGTTGAA
CCGGTGAGAA
CAACCCCACA
TTTATTTATT
TAAAGATAGT
CTTTTTTATA
TGAAGCCATT
AAATTTAACT
AAGTGTGATA
CATACAAAAA
GCTTAATTGA
TGGCTCTTCG
AGTTGTAAAC
TGAAGTTATT
TAAAAAAGTA
CATACATACA,
GCCTTCTCGT
AAAAAGAAITA
TATTTATTTT
GAACATCATT
TTTTTTGTTA
AAATAATTTG
TAACAATAGA
TTAT4ATTTTA
AATACTTTTA
TTAACTGAAA
TCATGCCGAT’
TTTCTCACTC
GCATAACTTG
GAAAAGAAAT
TTTAAAAAAA
GTTTTACACA
CTGTTATATC
TACAAAGGAG
TTATTTATTA
CCCTTTTCAT
GGCTCAAGTT
GAGAGTTTTC
TTTCGATTAA
AATTCAGAAT
ATTATTTGAT
TGACACCCTC
CAAGCCTTCT
CATTGAAcAA
ATG,
Lbc2 with the, sequence:, 17.
J 4 7z~ 17 TCGAGTTTTT ACTGAACATA CATTTATTAA AAAAAACTCT CTAGTGTCCA TTTATTCGGC GAGAAGCCTT CTCGTGCTTT ACACACTTTA ATATTATTAT ATCCCCACCC CCACCAAAAA AAAAAAAACT GTTATATCTT TCCAGTACAT TTATTTCTTA TTTTTACAAA GGAAACTTCA CGAAAGTAAT TACAAAAAAG ATAGTGAACA TCATTTTTTT AGTTAAGATG AATTTTAAAA TCACACTTTT TTATATTTTT TTGTTACCCT TTTCATTATT GGGTGAAATC TCA’YAGTGAA ACTATTAAAT AGTTTGGGCT CAAGTTTTAT TAGTAAAGTC TGCATGAAAT TTAACTTAAT AATAGAGAGA GTTTTGGAAA GG.TAACGAAT GTTAGAAAGT GTGATATTAT TATAGTTTTA TTTAGATTAA TAATTATPGTT TACATGAAAA TTGACAATTT ATTTTTAAAA TTCAGAGTAA TACTTAAATT ACTTATTTAC TTTAAGATTT TGAAAAGATC ATTTGGCTCT TCATCATG CC GATTGACACC CTCCACAAGC CAAGAGAAAC TTAAGTTGTA. ATTTTTCTAA CTCCAAGCCT TCTATAThAA CACGTATTGG ATGTGAAGTT GTTGCATAAC TTGCATTGAA CAATAGAAAT AACAACAAAG AAAATAAGTG AAAAAAGAAA TATG, and Lbc3 with the sequerce: *0 0 4 04 0,0 4 0 4 44 04 0* 0 4
TATGAAGATT
GTACTATTTA
GTAGATTTAT
ATAAAAA TAG 20 AAATATAATT
TTGTTTAAAT
TATAAAAAAA
CATTATATTA
AATTTTAACT
25 GTGATATTAG
CTAAAAAAAT
TTATTTACTG
TTCACCATAC
GTTTTATTAG
TTGGATGTC-‘A
CAGAAAAGTA
AAAAAATACA
AGAAAAGAAA
TTCTTTTATT
TGAACATCGT
TTTTTGTCTA
TGGATAAGAT
ATTGTTTCCC
AAAAAATTAG
TAAAAATAGA
AAATTTGTCG
ATATATTAAA
AAAATGAGTT
CAATTGATCA
“TTATTCTGAT
AGTTGTTGCA
GAAA A GAAA T CTCATATATA TGCCATAAGA ACCAACAAAA AAAAAAACCT GCTACATAAT TTCCAATCTT TTT’ATAAAGG AGAGTTA.AAA AAATTACAAA CTAA.GCATTT TTATATAAGA TGAATTTTAA AATCGTATGT ATCTTGTCTT AGAGCCATTT CACIACTATAA AGTTCTTCCT CCGAGTTTGA TTTTGATTAT TGGATAAAAT CTCGTAGTGA GGCTCAATTT TTATTAGTAT AGTTTG CATA GAAAATCTGG AI AAGGGACT GTTAAAAAGT GATATATTAA TATTTTATTT TATATGGAAA ATTTTAAATT CAGAATAATA CTTAAATTAT GATTTAAGTT TTTGAAAAGA TGATTGTCTC CCCTCCTCCA ACAAGCCAAG AGAGIACATAA CACTCTTCAA GCCTTCTATA TAAATA-AGTA TAACTTGCAT TGAACAATTA ATAGAAATAA
ATG.
A further embodiment of the method according to the invention uses a DNA fragment identical with, derived from or -,omprising 5′ flanking regions of the YEP Lb CAT geno with the sequence: TATGAAGATL AAAAAATACA, CTCATATATA TGCCATAAGA ACCAACAAAA GTACTATTTA AGAAAAGAAA AAAAAAACCT GCTACATAAT TTCCAATCTT GTAGATTTAT TTCTTTTATT TTTATAAAGG AGAGTTAAAA AAATTACAAA ATAAAAATAG TGAACATCGT CTAAGCATTT TTATATAAGA
TGAATTTTAA
AAATATAATT TTTTTGTCTA AATCGTATGT ATCTTGTCTT
AGAGCCATTT
18 TTGTTTAAAT TGGATAAGAT CACACTATAA AGTTCTTCCT CCGAGTTTGA TATAAAAAAA ATTGTTTCCC TTTTGATTAT TGGATAAAAT CTCGTAGTGA CATTATATTA AAAAAATTAG GGCTCAATTT TTATTAGTAT AGTTTGCATA AATTTTAACT TAAAAATAGA GAAAATCTGG AAAAGGGACT GTTAAMAAGT GTGATATTAG AAATTTGTCG GATATATTAA TATTTTATTT TATATGGAAA CTAAAAAAAT ATATATTAAA ATTTTAAATT CAGAATAATA. CTTAAATTAT TTATTTACTG AAAATGAGTT GATTTAAGTT TTTGAAAAGA TGATTGTCTC TTCACCATAC CAATTGATCA CCCTCCTCCA ACAAGCCAAG AGAGACATAA GTTTTATTAG TTATTCTGAT CACTCTTCAA GCC.TTCTATA TAAATAAGTA TTGGATGTGA AGTTGTTGCA TAACTTGCAT TG \ACAATTA ATAGAAATAA CAGAAAAGTA GAATTCTAAA ATG Still a further embodiment of the method2 according to the invention discloses a method of preparing a polypeptide by introducing into a yeast cel.l a recombinant plasmid characterised by using, as re- 4* combinant plasmid a plasmid containing a promoter sequence and a leader sequence in a DNA fragment with a 5′ flanking reion of a plant leghemoglobin gene., Another embodiment of the method according to the invention discloses a method of using as recombinant plasmid a plasmid containing a pr~omoter sequence and a leader sequence in a DNA fragment with a flanking region as well as a 3′ flanking region 2225 a plant leghemoglobin gene.
The present Invention, deals furthermore with a DNA fragment to be used as a second DNA fragment when carrying out the method according to the invention, said fragment being characteri.9ed in that it is a short DNA fragment transcribed into a messenger RNA strand being A target for a regulation exerted or mediated by intracellu.1ar heme. Examples of such DNA fragments are DNA fragments identical with,
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19 derived from or comprising a leader sequence from plant loghemoglobin genes, yeast genes or other genes inI which said leader sequence under natural circumstances is a target for a regulation exerted or mediated by intracellular heme, Examples thereof are according to the invention DNA fragments which are identical with, derived from or which comprise a leader sequence from the soybean leghemoglobin genes, viz, Lba with the seqnience: AACTTGCATC GA1ACAATTAA TAGAAATAAC AGAAAATTAA AAAAGAAATA
TG,
Lbcl with the squence: AACTTGCATT GAACAATAGA AAATAACAAA AAAAAGTAAA AAAGTAGAAA AGAAATATGj .Lbc2 witb the sequence: AACTTGCATT GAACAATAGA AATAACAACA AAGAAAATAA GTGAAAAAAG AAATA 1G, and Lbc3 with tle sequence:4 2,)AACTTGCATT GAACMATTAA TAGAAATAAC AGAAAAGTAG AAAAGAAATA 9X Another example of such a DNA fragment is a fragment which is identical with, derived from or comprises the leader sequence from the YE? Lb CAT gene with the sequence GAACAATTAA TAGAAATAAC AGAAAAGTAG AATTCTAAAA
TG
The present invention deals furthermore with DNA fragments comprising the combination of a first DNA fragment and a second DNA fragment and to be use hen carrying out the method according to the inv(rlt ion.
These DNA fragments are characterised by comprising a promoter sequence and a leader sequence and by being identical with, derived from or comprising 5′ flanking, regions of plant leghemoglobin genes, Examples of such DNA fre ments according to the invention are DNA fragments comprising a promoter sequence and a leader sequence, and which are iden-.
tical with, derived from or comprise 5′ flanking regions of the soybean leghenioglobin genes, viz.
Lba with the sequence: GAGATACATT ATAATAA’TCT CTCTAGTGTC TATTTATTAT TTTATCTGGT GATATATACC TTCTCGTATA CTGTTATTTT TTCAATCTTG TAGATTTACT TCTTTTATTT TTATAAAAAA GACTTTATTT TTTTAAAAAA AATAAAGTGA ATTTTGAAAA CATGCTCTTT GACAATTTTC TGTTTCCTTT TTCATCATTG GGTTAAATCT CATAGTGCCT CTATTCAATA ATTTGGGCTC AATTTAATTA GTAGAGTCTA CATAAAATTT ACCTTAATAG TAGAGAATAG AGAGTCTTGG AAAGTTGGTT TTTCTCGAGG AAGAAAGGAA ATGTTAAAAA CTGTGATATT TTTTTTTTGG ATTAATAGTT ATGTTTATAT GAAAACTGAA AATAAATAAA CTAACCATAT TAAATTTAGA ACAACACTTC AATTATTTTT TTAATTTGAT 21
TAATTAAAAA
TCATCATGCT
TGGTTTTCTC
TTGTTGCATA
AAAGAAATAT
ATTATTTGAT TAAATTTTTT AAAAGATCGT TGTTTCTTCT GATTGACACC CTCCACAAGC -CAAGAGAAAC ACATAAGCTT ACTCTCCAAG CCCTCTATAT AAACAAATAT TUGGAGTGAAG ACTTGCATCG AACAATTAAT AGAAATAACA GAAAATTAAA Lbc2. with the sequenoet
TTCTCTTAAT
AATCTQTAGT
CTTTAATATT
TTTCCAAACC
ACTTCAGAAA
TAATAAACTT
TATTGGGTGA
TTATTAGTAA
13 GAAAGGGAGC
TAATTATGTT
AATACTTAAA
TAGGATTTTG
CACAAqgCAA 20J ATATAAACAT TAGAAkAATAA ACAATGGAGT TTTTGTTGAA CATACATACA TTTAAAAAAA GTCTATTTAC CCGGTGAGAA GCCTTCTCGT GTTTTACACA ATTATATCCT CAAQCCCAQA AAAAkAGAATA CTGTTATATC TGTAGATTTA TTTATTTATT TATTTATTTT TACAAAGGAQ AGTAATTACA TAAAGATAGT GAACATCATT TTATTTA\TTA TAAAATCAAA CTTTTTTATA TTTTTTGTTA CCCTTTTCA T AATCTCATAG TGAAGCCATT AAATAATTTG GQCTQAAGTT AGTCTGCATG AAATTTAACT TAACAATAGA GAGAGTTTTC GAATGTTAAA AAGTGTCATA TTATATTTTA TTTCGATTAA TACATGAAAA CATACAAAAA MATACTTTITA AATTCAGAAT ATATTTATTT GCTTAATTGA TTAACTGAAA AT32ATTTGAT AAAAGATCAT TGGCTCTTCG TCATGCCGAT TGACACCCTC GAGAAACTTA AGTTGTAAAC TTTCTCACTC CAAGCCTTCT GTATTGGATG TGAAGTTATP GCATAACTTG CATTGAACAA CAAAAAAAAG TAAAAAA(QTA GAAAAGAAAT ATGf I 7 4 Al $4 4 *4
A
A
It’ t Lbc~2 with the, sequenaot
TCGAGTTTTT
TT TAT T CGG C
ATCCCCACCC
TTATTTCTTA
ATAGTGAACA
TTATATTTTT
ACTATTA4AT
TTAACTTAAT,
GTGATATTAT
TTGACA.ATTT
TTTAAGATTT
CTCCACAAGC
3$ TCTATIATAAA
CAATAGAAAT
ACTGAACATA CATTTATTAA AAAAAACTCT CTAGTGTCCA GAGAAGCCTT CTCGTGCTTT ACACACTTTA ATATTATTAT CCACCAAMAA AAAAAAAACT GTTATATrQTT TOCAGTACAT TTTTTACAAA GGMACTTCA CGAAAGTMAT TACAANJ\AAG TCATTTTTTT AGTTAAGATG AATT1TTAAAA TCACACTTTT TTGTTACCCT TTTCATTATT GGGTGAAATC TCATAGTGMA AGTTT(GGCT CAAGTTTTAT TAGTAAAGTC TGCATGAAAT AATAGAGAGA GTTTTGGAAA GGTAACGAAW GTTAGAAAGT TATAGTTTTA TTTAGATTAA TAATTATGTT TACATGAAAA ATTTTTAAAA TTCAGAOTAA TACTTAAATT ACTTATTTAC TGAMAAGATC ATTTGGCTCT TCATCATGCC GATTGACACC CAAGAG~ kAC TTAAGTTGTA ATTTTTCTAA CTCCAAGCCT CACGTATTGG ATGTGAAGTT GTTGCATAAC TTGCATTGAA MACAACAAAG AAAATAAGTG AAAAAAGAAA TATG? 22 and Lbc3 with the sequence:
TATGAAGATT
GTACTATTTA
GTAGATTTAT
ATAAAAATAG
AAATATAATT-
TTGTTTAAAT
TATAAAAAAA
CATTATATTA
AATTTTAACX
GTGATATTAG
CTAAAAAAAT
TTATTTACTG
TTCACCATAC
GTTTTATTAG
.TGGATGTGA
CAGAAAAGTA
AAAAAATACA
AGAAAAGAAA
TTCTTTTATT
TGAACATCGT
TTTTTGTCTA
TGGATAAGAT
ATTGTTTCCC
AAAAAADT TAG
TAAAAATAGA
AA %iTTTGTCG
ATATATTAAA
AAkAATGAGTT
QAATTGATCA
‘TAT T C TGAT
AGTTGTTGCA
(.2AAAAGAAAT
CTCATATATA
AAAAAAACCT
TTTATAAAGG
CTAAGCATTT
AATCGTATGT
CACACTATAA
TTTTGATTAT
GGCTCAATTT
GAAAATCTGG
GATATATTAA,
ATTTTAAATT
GATTTAAGTT
CCCTCCTCCA
CACTCTTCAA
TAACTTGCAT
ATG.
TGCCATAAGA ACCAACAAAA GCTACATAAT TTCCAATCTT AGAGTTAAAA ‘”.AATTACAAA TT-ATATAAGA TGAATTTTAA ,ATCTTGTCTT AGAGCCATTT AGTTCTTCCT CCGAGTTTGA TGGATAAAAT CTCGTAGTGA TTATTAGTAT AGTTTGCATA AAAAGGGACT GTTAAAAAGT TATTTTATTT TATATGGAAA CAGAATAATA CTTAAATTAT TTTGAAAAGA TGATTGTCTC ACAAGCCAAG AGAGACATAA GCCTTCTATA’ TAAATAAGTA TGAACAATTA ATAGAALATAA, #4 4 4 41 4$ It 4$ 4 I #1 #4
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4111 I I Another example of such a DAfragment according to the invention is a DNA £ragnient which is id1en.
tical with, derived fromt or comprises 5′ flanking regions of the YFPP Lb CAT gene with the seqtteno~e
TATGAAGATT
GTACTATTTA
GTAGATTTAT
ATAAAAATAG
AAATATAATT
TTGTTTAAAT
TATAAAAAAA
T
TA
GTGATATTAG
CTAAAAAAAT
TTATTTACTG,
TTCACCATAC
GTTTTATTAG
TTGGATGTGA
CAGAAAAGTA
AAAAAATACA
AGAAAAGAAA
TTCTTTTATT
TGAACATCGT
TTTTTGTCTA
TGGATAAGAT
ATTGTTTCCC
AAAAAATTAG
TAAAAATAGA
AAATTTGTCG
ATATATTAAA
AAAATGAGTT
CAATTG AT CA
TTATTCTGAT
AGTTGTTGCA
GAATTCTAAA
CTCATATATA
AAAAAACCT
TTTATAAAGG
CTAAGQATTT
AATCG”&ATGT
CACACTATAA
TTTTGATTAT
GGCTCAATTT
GAAAATCTGG
GATATATTAA
ATTTTAAATT
GATTTAAGTT
CCCTCCTCCA
“‘ACTCTTCAA
IAACTTGCAT
ATG
UCCATAAGA ACCAACAAAA (“‘CTACATAAT, TTCCAATCTT AGAGTTAAAA AAATTACAAA TTATATAAGA TGAATTTTAA ATCTTGTCTT AGAGCCATTT AGTTCTTCCT CCGAGTTTGA TGGATAAAAT CTCGTAGTGA TTATTAGTAT AGTTTGCATA.
AAAAGGGACT GTTAAAAAGT TATTTTATTT TATATGGAAA CAGAATAATA CTTAAATTAT TTTGAAAAGA TGATTGTCTC ACAAGCCAAG AGAGACATAA GCCTTCTATA TAAATAAGTA TGAACAATA ATAGAAATAA In addition the invention relates to any plasmid to be used when carrying out the method according to the invention and characterised by comprising a tirst DNA fragment as previously defined, or a combination of a second DNA fragment and a second DNA fragment, also as previously defined. Suitable plasmids according to the invention are YEP Lb CAT and YEP 5 Lb Km. The plasmids according to the invention allow a high expression of of a desired gene product by inserting coding sequences 0 for these gene products.
EXAMPLE 1 Sequence determination of 5′ flanking regions of soybean leghemoglobin genes 5 From a soybean (glycine max. var. Evans) gene library t 15 the four soybean leghemoglobin genes Lba,Lbcl, Lbc2, S, and Lbc3 are provided as described by Jensen, E. 0. et al., Nature Vol. 291, No, 3817, 677-679 (1981). The flanking regions of the four soybean leghemoglobin genes are isolated, as described by Jensen, Ph D, Thesis, Institut for Molekylar Biologi, Arhus Universitet (1985), and the sequences of the four 5′ flanking regions are determined by the use of the dideoxy chaintermination method as described by Sanger, J. Mol.
Bio. 143, 161 (1980) and indicated in the sequence scheme.
24 EXAMPLE 2 Construction of YEP Lb CAT The construction has been carried out in a sequence of process sectiono as described below: the Lbc3 gene Tho Lbc3 gene was isolated on a 12kb EcoRI restriction fragment from a soybean DNA library, which has been described. by Wiborg et al. in Nuci. Acids Res. 10? 3487. A sect~Lon of the fragment is shown at the top of Scheme 2, This fragment wa-s digested by the enzymes stated so as subsequently to be ligated to pBR322 as indiepated at the Scheme, The resulting plasmid,- Lbc3HH and Lbc3HX were subse, quently digested by PvuII and religated, which resulted in two plasmids called pLpHH and pLpHXi Sub-cloning 51flankinpg sequences from the Lbc3 gene For this purpose pLpHH was used as shown in Scheme 3, This plasmid was opened by means of PvuII and treated. wi,:h exonuclease Bal3l, The reaction was I4k Ir 20 stopped at var~ious times and the shortened. plan-: mids iwere ligated into fragments from pBR322., These fragments had been treated in advance as shown in Saheme 3, in such a manner that in one end they had a DNA sequence TTC 25A4G After the lfgation a digestion wi#.h EcoRI took place, and the fragments containing 5 .ZLA nk ing a6 result the selection pressure on the cell. in- ques- Thes plsm.d weetasomdinoE oiKG i-s as folw 2k25 Forenthis prpoe, plgate wnto uedR dhige wsedip3R 32 2 byThe, phasmids were rasfrmedintoy fE col. and thcess pNAasievdsa hw in tetasoanSchwee. Tese bye seeed assow A n p tahi, pchem 5’Lb, contransformants contained a al-d flankind sequeceaterminaftinerbf the Lb ocdn, ATG sth codoaning suchoa,,anner that thqene seqrec -5 fanking A—AATGATCAAT Sub-hecontionn 3 fnin Treionr ofsstheLb3ge 4 Fo thisms purpoe 1pX as ued wiche waigested byavng XhoII t Th ns war partiallyr” faill ee o thand eces DA a remiiov ed, asm shln rcemed 4.fTh 26 Scheme 4. The ligated plasmids were transformed into E. coli K803. A plasmid in one of the transformants contained the correct fragments, and it was called pEJLb Construction of chimeric Lb/CAT gene The CAT gene of pBR322 was isolated on several smaller restriction fragments, as shown in Scheme The 5′ coding region was isolated on an Alul fragment whc-h was subsequently ligated into pBR322 and treated as stated in the Scheme. This was transformed into E. coli K803, and a selected trarisi formant contained a plasmid called Alull. The 3′ coding region was isolated on a TaqI fragment.
This fragment was treated with exonuclease Bal31, 15 whereafter EcoRI linkers were added. Then followed a digestion with EcoRI .nd a ligation into EcoRI digested pBR322. The latter was transformed into E.
coli K803 and the transformants were analy A plasmid, Taq 12, contained the 3′ coding region of the CAT gene plus 23 bp 3′ flanking sequences so as subsequently to terminated in the following sequence CCCCGAATTC. Subsequently the fol- S, lowing fragments were ligated together into EcoRI digested pEJLb5’- EcoRI/PvuII fragment from Alull, PvuII/DdeI fragment from pBR322 and DdeI/EcoRI fragment from Taq 12. The latter was transformed into E, coli K803. A selected transformant contained the correct plasmid called pEJLb CAT Cloning echmeri c LbLC&AT .Z t yeast piasmid /0 i 1 leader- sequ enc ru vated n tagment containing a leader sequence regulated on the pos t- 27 This chimeric gene was iso fragment from pEJLb C the yeast plasmid YEP24 cut i: i_ i jiI; !:1 ;-Cx
14. The method of claim 1 wherein the DNA fragment is identical with, derived from or comprises the leader sequence from the Lbc3 gene with the sequence: AACTTGCATT GAACPATTAA TAGAAATAAC AGAAA.AGTAG AAAAGAAATA TG The mrethod of claim I wherein the DNA fragment is identical with, derived from or comprises the leader sequence from the YEP Lb CAT gene with the sequence: AACTTGCATT GAACAATTAA TAGAAATAAC AGAANAGTAG AATTCTAAAA TG
16. A DNA fragment to be used when carrying out the method as claimed in claim 1, 2, 3, 4 or 5 characterised in that it comprises a promoter sequence anc a leader sequence an~d is identical with, derived from or comprises 5′ flanking regions of plant leghemoglobin genes.
17. A DNA fragment to be used when carrying out the methodi as claimed in claim 1, 2, 3, 4 or 5 chara’~terised in that it compri.ses a promoter sequence and a leader sequence and is identical with, derived from or comprises S’ flanking r egions of soy’)ean leghemoglobin genes.
18. A DNA fragment to be used when carrying out the vi ethod as claimed in claim 6, characterised in that it is identical with, derived from or comprises 5′ flanking regions of the Lba gene with the seque Ince: GAGATACATT ATAATAATCT CTCTAGTGTC TATTTATTAT TTTATCTGGT GATATATACC TTCTCGT1T CTGTTATTTT TTCAATCTTG TAGATTTACT TfCTTTTATTT TTATMAAAAA GACTTTATTT TTTTAAAAAA AATAAAGTGA ATTTTGAAAA CATG ICTCTTT GACAATTTTC TGTTTCCTTT TTCATCATTG GGTTAAATCP CIXTAGTGCCT CTATTCAATA ATTTGGGCTC AATTTAATTA GTAGAGTCTA CATAAAATTTI ACCTTAAIVAG TAGAGAPJAG t GAGTCTTGG AAAGTTGGTT TTTCTCGAGG AAGAAAGGA\FA ATGTTrAAAAA CTGTGATATT TTTTTTTTGG ATTAATAGTT ATGTTTATAT GAAAACTIGAA AATAAATAAA, CTAACCATTAT TAAATTTAGA ACAACACTTC AATTATTfTT TTAAT-TTGAT TAATTAAAAA ATTATTTGAT TAAM4′ ATTTT AM G’T ITTCTCT TCATCATGCT GATTGACACC CTCCAY ,AAGC CAAGA, AC. ACA*PTAAGCTT TdGTTTCTC ACTCTCCAAG CCCTCTATAT AA ACAAATLAT “CGGAGTGAAG 1TTGTT CATA ACTTGCATCG AACAATTAA’1V AGAPAATAACA CAAAATTAAA MAGAANTAT G. -39-
19. A DNA fragment to be used when carrying out the method as claimed in claim 7, characterised in that it is identical with, deriv el from or comprises 5′ flank
