IT8922065A1 - CLONING OF THE NMT1 ADJUSTABLE GENE OF SCHIZOSACCHAROMYCES POMBE AND USE OF THE CONTROL SEQUENCES OF THE TRANSCRIPTION REGULATION OF THIS GENE FOR THE ADJUSTABLE EXPRESSION OF HETEROLOGICAL GENES - Google Patents

Description

DESCRIPTION

The present invention relates, in general, to a chromosomal DNA fragment of Schizosaccharomyces pombe (S.pombe) which comprises the structural gene that encodes a new protein (nmtl) whose transcription? completely regulated by the presence or absence of thiamine in the culture medium and the sequences of the regulatory elements necessary for the control of the thiamine-mediated transcription of said gene, vectors containing it and microorganisms transformed with said vectors.

In particular, the present invention relates to the upstream (promoter) and downstream (terminator) regions of said gene comprising, respectively, the sequences of the regulatory elements necessary for the control of thiamine-mediated transcription and those of transcription termination and their use for the construction of recombinant DNA molecules for the adjustable expression of heterologous proteins of interest in host organisms.

Recent developments in genetic engineering have made it possible to prepare a protein of interest in high yields by culturing host organisms transformed with an expression hybrid vector containing the heterologous gene encoding said protein, where such gene? placed under the control of host-recognized regulatory sequences.

It is known that obtaining a heterologous product in high yields strictly depends on the choice of both the host organism and the regulatory sequences (promoter and terminator) placed upstream and downstream of the structural gene which codes for said product.

Strong, capable promoters are generally employed to give rise to messenger RNA with high frequency, and host microorganisms chosen from the group of bacteria, such as Escherichia coli and Bacillus, or yeasts such as Saccharomyces cerevisiae or mammalian cells.

Preferably the expression of eukaryotic genes? obtained using yeasts as host organisms as they have numerous advantages compared to bacteria (prokaryotes) and mammalian cells.

In fact, yeasts are eukaryotic microorganisms which, unlike cells, can advantageously be grown on a large scale in fermenters and also possess the eukaryotic mechanism necessary for the functional expression of cloned eukaryotic genes.

From the numerous studies conducted on genes that encode yeast proteins and human proteins, it appears likely that many of the sequences involved in the transcriptional control mechanism have been highly conserved through the evolution of eukaryotes.

A typical eukaryotic gene? constituted by a 5 'terminal region (promoter) that is not transcribed, a coding region that can? be interrupted by introns and a non-transcribed 3 'terminal (terminator) region. The transcript, what? catalyzed by RNA polymerase li, it begins in the 5 'non-translated region and this first transcript includes coding and non-coding regions. Termination of transcription occurs in the 3'terminal region. Non-coding regions (introns) are removed from the primary transcript by the splicing process which eventually produces a translatable mRNA. The analysis of the primary structures of many eukaryotic genes revealed the presence of specific signals necessary for the transition from the primary to the functional transcript. Promoter analysis showed the presence of the TATA sequence at 25-32 bp from the transcription start site and of the CAAT sequence at approximately 80 bp upstream of the same site. Upstream sequences of TATA capable of affecting transcription efficiency have been highlighted in several cases; often these sequences, also called enahancer cio? enhancers, are repeated sequences.

The sequence downstream, corresponding to that? at the end? 3 'of the messenger extends beyond? of the translation termination site. Before the 3 'termination point, the AATAAA sequence is generally found which is, presumably, the recognition site of the enzyme that adds poly-A to messenger RNA.

In general, the organization of yeast promoters? similar to that of eukaryotes pi? complexes above. A TATA sequence? generally present upstream of the transcription initiation site, but at a distance from this variable from 40 to 120 base pairs in S .cerevisiae. Upstream of this sequence there are regulatory elements, analogous to eukaryotic enhancers, which have been shown to be specific sequences for the binding of positive and negative regulatory factors (see in particular Guarente, L. et al., (1987), Ann.Rev. Genet., 21, 425-452) and a homologous sequence? was found in region 3 '.

It is still known that the high level of transcription of a gene can, on the one hand, give rise to a product which, precisely why? expressed in large quantities, can? be toxic to the cell, and on the other hand it can? interfere with the mechanisms of DNA replication and lead to instability? plasmid.

Hence the need to have promoters whose activities? can be modulated by means of an induction / repression system connected to them. In this way the expression of a gene placed under the control of this promoter can? be obtained by means of an external modulator, chemical or physical, which allows the choice of the exact moment in which the expression must begin.

Numerous strong promoters isolated from yeast genes have been described in the technical and patent literature which can be controlled efficiently and conveniently by varying the culture conditions.

What? for example, the promoters of the inducible genes GALI, PH05 and ADH1 isolated from S. cerevisiae, a yeast that reproduces by budding (budding), have been used for the construction of vectors useful for the inducible expression of genes encoding heterologous proteins of interest, in particular of eukaryotic proteins.

Schizosaccharomyces pombe yeast? a versatile organism and extensively studied in the laboratory that like S.cerevisiae yeast with, which? only remotely related, it occurs in the form of a unicellular ascomycete. S.pombe reproduces by cell division and shows a similarity with the transcriptional system of eukaryotes pi? evolved even more? pronounced than that of S.cerevisiae

Indeed, the TATA sequence? placed in a more? uniform at 25-30 base pairs upstream of the transcription initiation site, exactly as in higher eukaryotic cells (Benoist, C. et al., (1980), Nucleic Acids Res., 8, 127-142).

Furthermore, the genes containing introns are more? frequent and the consensus sequence essential for CT-AC cutting, located near the 3 'terminal of the intron,? less stringent than that TACTAAC found in S. cerevisiae and d? similar to the consensus sequence found in eukaryotes pi? evolved (Mount, S.M., (1982), Nucleic Acids Res., 10, 459-472). For all these reasons S. pombe constitutes a very attractive alternative to budding yeasts as a host organism for the expression of heterologous genes and the production of proteins of interest.

However, the use of S pombe? limited by the fact that efficient transcription regulation systems in said yeast are not known and therefore not? It is possible to control the expression of the heterologous genes of interest.

Currently, only phol and pho4 encoding two acid phosphatases have been isolated as inducible genes of S.pombe (Maundrell, K. et al., (1985), Nucleic Acids Res., 13, 3711-3722; Scweingruber, A.M. et al. , (1986), J. Biol. Chem., 261, 15877-15882). However these genes are expressed in low yields, aren't they? in fact, an abundant transcript was determined, n? they are completely repressed in the state of non-induction.

Therefore, the control sequences of the transcription regulation of said genes do not seem suitable for the construction of vectors with inducible expression of heterologous proteins.

The present patent application reports for the first time the isolation and characterization in nucleotide and amino acid terms of a fully adjustable S. pombe gene that encodes a new protein, hereinafter referred to as nmtl, and the identification of the control sequences of the regulation of the transcription of said gene.

It represents one of the genes most efficiently expressed by S.pombe under induction conditions and? completely repressed in culture medium containing thiamine in a concentration equal to or greater than 0.5? iM.

Therefore, an object of the present invention is a cloned and sequenced fragment of S. pombe chromosomal DNA comprising the structural gene nmtl which encodes a new protein whose transcription? completely regulated by the presence or absence of thiamine in the culture medium and the sequences of the regulatory elements necessary for the control of thiamine-mediated transcription of said gene.

An object of the present invention is also an adjustable expression cloning vector containing said DNA fragment.

Another object of the present invention is a host organism selected from bacteria, yeasts or mammalian cells transformed with said vector.

Another object of the present invention is the promoter and terminator regions of the nmt1 structural gene containing, respectively, the sequences of the control elements of the regulation of the thiamine-mediated transcription and the termination sequences of the transcription.

It is also an object of the present invention to use said regions for the fully adjustable expression of heterologous structural genes which encode a polypeptide of interest in host organisms.

A further object of the present invention is a recombinant DNA molecule for the adjustable expression of heterologous polypeptides in host organisms, where said molecule contains the promoter region or the terminator region of the nmt1 structural gene or both, the heterologous structural gene that encodes the polypeptide of interest whose transcription? regulated by the regulatory elements contained in the aforesaid regions and at least one marking gene useful for the selection of the organisms transformed with said recombinant DNA molecule.

A further object of the present invention is a host organism transformed with the aforementioned recombinant DNA molecule where said organism? selected from bacteria, yeast or mammalian cells.

A further object of the present invention is a process for the fully adjustable production of heterologous polypeptides which comprises the cultivation of a host organism transformed with a recombinant DNA molecule as defined above in a suitable culture medium in the presence or not of thiamine.

Another object of the present invention is the protein encoded by the nmtl gene characterized by an assumed molecular weight of 39 KD and by the amino acid sequence deduced from the nucleotide sequence shown in Figure 3A.

Further objects of the present invention will become evident from the following description, text and examples.

In order to better illustrate the present invention, a brief description of the figures is given below.

(A) Northern blotting analysis shows the effect of thiamine concentration on nmtl expression.

The analysis? was carried out on mRNA extracted from S.pombe cells 972 h cultured in minimal medium alone (1) or added with different concentrations of 0.01 thiamine; 0.02; 0.05; 0.1; 0.2; 0.5; 1.0; and 2.0 juM (2 to 9).

(B) Northern blotting analysis of mRNA extracted from the same cultures reported above for the determination of the cytochrome C transcript. The results show that equal quantities? of RNA are loaded in each line and that the nmtl mRNA, when fully expressed,? about 50-100 times pi? abundant of cytochrome C mRNA. These data indicate that nmtl? one of the major cellular transcripts.

Figure 2:

(A) Northern blotting analysis shows the kinetics of nmtl transcription repression by thiamine.

The analysis? was conducted on mRNA extracted from S.pombe cells 972 h? grown first in minimal thiamine-free soil (To) and then in the same medium supplemented with thiamine (2jilfl). The samples taken from the culture (To) and at intervals of 1, 2, 3, 5 hours from the same culture added with thiamine show the repressive effect of thiamine which? complete after about 3 hours.

(B) Northern blotting analysis shows the kinetics of nmtl transcription induction by thiamine.

The analysis? was carried out on the mRNA extracted from 5 pombe 972 h- cells cultured first in minimal medium added with 2 µM of thiamine up to the logarithmic phase (To) and then in minimal medium free of thiamine. The samples taken from the To culture and after 4, 8, 10, 12, 14 and 16 hours from the thiamine-free one show that the first appearance of nmtl mRNA occurs after 10 hours, while after 16 hours the appearance of all 1 'is observed. mRNA.

Figure 3:

(A) The nucleotide sequence of the chromosomal DNA fragment of S. pombe containing the adjustable gene nmtl is reported. The structural gene (1 to 1038) that encodes the mature nmtl protein? also characterized by the amino acid sequence in which the amino acids are indicated with the single letter code where:

The horizontal arrow indicates the transcription start site which? placed 27 bp downstream of the TATAAA sequence (boxed). The two underlined sequences are homologous to those found in the PH03 gene of S.cerevisiae whose expression? regulated by thiamine. The vertical arrow downstream of the nmtl structural gene (142 bp from the stop codon) indicates the transcription termination site.

(B) The restriction map of the 4.1 kb fragment containing the nmtl gene is reported; the boxed area represents the region of the sequenced 4.1 kb fragment (Figure 3A); the dotted area represents the region used for the construction of plasmids with adjustable expression; the shaded area represents the nmtl coding region.

Northern blotting analysis shows the expression and repression of nmtl in a plasmid in multiple copies.

Lines a and b = cells containing the plasmid DB262 as is (control); lines c and d = cells transformed with the pNMT41 plasmid containing the 4.1 kb HindlII fragment comprising the nmt1 gene; lines e and f = cells transformed with the plasmid pNMT ^ 41 containing the HindlII fragment of 4.1 kb devoid of the 185 bp BamHI-BamHI sequence. Cells were grown in minimal medium in the absence (lines a, c, e) or in the presence (lines b, d, f) of thiamine.

Figure 5:

(A) Southern blotting analysis of the diploid strain containing one copy of the intact nmtl gene and one copy of the nmtl gene destroyed by insertion of the ura4 gene (nmt :: ura4) and of the four spores from this dipolid.

Column D shows the DNA obtained from the diploid digested with HindlII. The two fragments of 2.4 kb and 3.4 kb that hybridized correspond to the wild type and the destroyed genes, respectively. The diploid was sporulated and the DNA isolated from each of the four spores. The wild type gene (spores 1 and 3) and the destroyed one (spores 2 and 4) segregate with the Ura (-) and Ura + (+) phenotype, respectively.

(B) Growth curves showing the dependence of the strain containing the nmtl :: ura4 gene on thiamine.

Each of the 4 spores (A) was grown in minimal medium (supplemented with adenine, leucine and uracil) both in the presence (filled symbols) and in the absence (empty symbols) of thiamine. Cells were counted at regular intervals using a hemacytometer. The graph above shows nmtl + cells derived from spore 1 (K) and spore 3 (A *) - The graph below shows nmtl :: ura4 cells derived from spores 2 (? 0) and 4 (1D).

Figure 6:

The construction of the adjustable expression vectors in S.pombe pMNS36L and pMBS36L is reported.

(A) Schematic representation of the 4.1 kb HindlII fragment comprising the nmtl gene where: the solid area represents the nmtl coding region; the upstream (promoter) and downstream (terminator) dotted areas represent the sequences used for the construction of the vectors with adjustable expression; the sequences around the Ndel site (-768) and the ATG translation start codon are shown; the arrow defines the extension of the nmtl transcript.

(B) Shows the construction of the pMNS36L plasmid. The promoter of the nmtl gene? modified to the ATG to contain the Ndel restriction site (5'CATATG 3 ') and the Sali site (5'GTCGAC 3'). The edited sequence is shown. The Ndel site in position -768? was destroyed by deletion of the sequence 5'TATGTC 3 '. The transcription termination region of the nmtl gene that goes from the TAA stop codon to the? C-terminal residue at position 2030 (Fig. 3 A)? was copied by PCR and the restriction sites BamHI, Smal and SstI were added as indicated. The pMNS36L vector contains the S.cerevisiae LEU2 gene. as a selection marker, and the arsi sequence of S.pombe as an origin of cloned plasmid replication, respectively, in the HindiII and EcoRI sites of the bacterial vector pUC119.

(C) Shows the construction of the pMBS36L plasmid. This vector? identical to the. vector pMNS36L except that the sequence around the ATG? been modified to contain the blunt end Ball restriction site (5'TGGCCA 3 ') instead of the Ndel site. The Ndel endogenous restriction site at position -768 does not? been changed.

The pMINS6L and pMIBS6L integrating vectors were obtained by deletion of si from the pMNS36L and pMBS36L plasmids, respectively.

Figure 7:

The construction of the hybrid plasmids pMCMA (replicating plasmid) and pMCM (integrating plasmid) containing the CAT heterologous gene is reported. Figure 8:

Shows the analysis for the determination of the activity CAT on extracts prepared from S. pombe cells transformed with the plasmid pMCMA (lines a and b) or pMCM (lines c and d). The transformants were grown in the absence (lines a and c) and in the presence (lines b and d) of thiamine.

Figure 9:

(A) - Northern blotting analysis of mRNA extracted from S.pombe cells transformed with the plasmids pMS21L (a) and pMNS2lL (b) containing the human lipocortin 2 gene. Identical results are obtained with both vectors. Lipocortin 2? efficiently expressed in the absence (-) of thiamine and? completely repressed in the presence of thiamine (+).

(B) - Northern blotting analysis of mRNA extracted from S. pombe cells transformed with the pMBS36L plasmid comprising the HPV-16 E6 gene (a) or the HPV-16 E7 gene (b) cloned between the Ball and Sali sites. In both cases, high levels of expression are obtained in the absence of thiamine (-) and complete repression in the presence of thiamine (+).

In accordance with the present invention in order to detect the presence of genes of S.pombe which were completely adjustable, yes? proceeded with the construction of a representative library of the genome of said yeast and its screening using specific probes.

In particular, the probes were prepared by culturing a wild type strain of 5 pombe in minimal medium with or without thiamine (2 jUM) and, after having separated the RNA from each culture operating as reported by Maundrell, K. et al. , (1985), Gene, 39, 223-230, yes? proceeded to purification of mRNAs.

Since? many of the mRNAs present in the cytoplasm of eukaryotic cells contain a poly (A) sequence at the 3 'terminus, they can be recovered by passing over oligo (dT) cellulose, poly (U) sepharose or the like as described by Maniatis et al. (1982), "Molecular cloning: a laboratory manual", Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York.

The mRNAs like this? obtained were then used for the synthesis of single-stranded complementary DNA (cDNA) which? it was then marked with 32

JZP.

The probes cos? obtained were then used to identify within a library of S. pombe those clones containing the fragment of chromosomal DNA that hybridized with them in a different way.

In accordance with this, according to the present invention a representative library of the genome of S.pombe can be be obtained by complete or partial digestion of the chromosomal DNA of said yeast with suitable restriction enzymes.

For this purpose, different strains of S.pombe can be used.

In particular ? S. pombe strain (leul-32, weel-50, h minus) available from AFRC, Inst of Food Research was used. After culture in Yeast Extract medium added with glucose and adenine, the chromosomal DNA extracted from the lysed cells of said yeast? been partially digested with the HindiII restriction enzyme. The chromosomal DNA fragments derived from partial digestion with this enzyme were cloned in a cloning vector selected from those of the prior art containing a marker useful for the selection of transformants.

Preferably? The bifunctional plasmid vector was used in E.coli and in S.pombe DB262 (Wright, A. et al., (1986), Plasmid, 15.

156-158) and the transformants were selected on a selective medium by operating according to conventional techniques (see in this regard Maniatis et al., 1982).

In order to identify the fragments of chromosomal DNA of S.pombe that hybridized differently with the cDNA probes prepared from each culture, all the clones of the library were transferred on nitrocellulose filters and the DNA extracted from said clones? was hybridized by operating according to the technique reported by Maniatis et al. (1982). The clones that gave a different hybridization with the above probes were isolated and further characterized.

Was it so? identified a clone that hybridized strongly with the thiamine minus cDNA probe (obtained from a culture of S.pombe in a thiamine-free medium) and little with the thiamine pi cDNA probe? (obtained from cultivation of S.pombe in a medium added with thiamine). This clone contained a HindiII chromosomal DNA insert of about 2.4 kb which, upon subsequent analysis of the mRNA by Northern blotting, revealed the presence of a single transcript of about 1.3 kb which suggested the presence in said fragment of a sequence (structural gene) encoding a protein whose transcription was regulated by the presence or absence of thiamine.

In fact, Northern blotting analysis of RNA extracted from S.pombe cells grown in minimal medium in the absence or presence of different concentrations of thiamine had shown that thiamine was a potent inhibitor of the transcription of said gene (Figure 1A).

By increasing the concentration of thiamine in the minimum culture medium up to values of 0.05 µM, no effect on gene expression was observed.

An increase in the concentration of said vitamin above the values reported above resulted instead in a reduction of the transcript, while a complete inactivation of the gene expression was obtained for thiamine concentration values equal to or greater than 0.5 j.iM ( Figure 1A).

For the latter concentration, a 20-fold greater blotting exposure revealed no trace of mRNA hybridization. For this reason the gene was designated nmtl (no messenger in thiamine).

The same RNA samples were analyzed to determine the cytochrome C transcript which? known to be expressed efficiently. The results reported in Figure 1B showed that equal quantities? of RNA were loaded in each line and that the transcript of nmtl, when fully expressed, was 50-100 times more? abundant of cytochrome C transcript.

These data confirmed that nmtl represents one of the major cellular transcripts.

In order to study the kinetics of thiamine-induced transcription repression, a culture of S.pombe grown in minimal medium up to the logarithmic phase was supplemented with 2 µM of thiamine (To). Aliquots of this culture were taken at regular intervals (about 1 hour) and the mRNAs prepared from each culture were analyzed by the Northern blotting technique. The results obtained revealed that the nmtl mRNA disappeared completely after about 3 hours (Figure 2A), indicating that this is the case. a rapid transcriptional response and a high yield of 11? mRNA turnover.

In the inverse experiment, in which S.pombe cells grown in minimal medium containing thiamine (To) were removed from said medium and inoculated in minimal thiamine-free medium, the induction of nmtl transcription, determined by Northern blotting on samples taken at regular intervals from said culture, he showed that the first appearance of the nmtl mRNA occurred after about 10 hours while the maximum level of nmtl mRNA was observed after 16 hours.

The request for induction times pi? long was due, presumably, to the high intracellular concentration of thiamine, the disappearance of which required several generations to exhaust these internal reserves.

These experiments revealed that:

- under conditions of induction nmtl was one of the most? abundant mRNA present in the cell;

- its expression could be controlled by simple modification of the culture medium;

- in the state of non-induction the expression was completely repressed.

According to the present invention, the 2.4 kb HindiII fragment? It was then fully sequenced in both directions using a series of deletion mutants prepared by digesting the fragment with Exonuclease III and SI nuclease as reported by Henikoff, S., (1984), Gene, 28, 351-359.

Operating as described above? been found that said fragment? 2,478 kb long and within it? A single open reading landslide (open reading phase) of 1038 base pairs has been identified which potentially encodes a protein with an assumed molecular weight of about 39 kD and whose amino acid sequence, deduced from the nucleotide sequence, does not show any analogy with other proteins Note.

In accordance with the present invention, in order to identify the upstream and downstream regions of said gene, yes? mapped the terminals of the mRNA encoded by the clone containing the plasmid comprising the Hindi II fragment of about 2.4 kb.

Conventional techniques generally used in the field of recombinant DNA can be used for this purpose.

In practice the terminal 5 of the mRNA of nmtl? was identified using the "primer extension" method.

The method uses a synthetic oligonucleotide 5'CCTCAATCCCTTCACGCTCA 3 'complementary to the sequence of nmtl mRNA from 90 to 109 labeled at 5' 32

with P with polynucleotide kinase and "annealed" and extended with reverse transcriptase as described by Grimm et al. (Mol.Gen. Genet., 215, 81-86, (1988)) The 3'terminal region of the transcript? was identified by SI nuclease mapping using a probe prepared from an exonuclease-derived mutant containing a 3 'deletion extending into the clone from position 1310. The DNA of the single-stranded M13 phage containing this deletion was annealed with the universal primer 17mer, extended with the enzyme "Klenow" and digested with Ncol. The 371 nucleotide fragment complementary to the 3 'terminal of the nmtl mRNA? it was then purified and used for SI mapping.

The data obtained revealed that transcription begins at residue A in position -69 and ends at the 3 'terminus of 142 nucleotide mRNA after the translation stop codon.

The TATATAAA sequence? was found in the -34 to -27 position with respect to the transcription initiation site.

The predicted mRNA length based on terminal mapping? result therefore of 1252 nucleotides, very close to that? to the values obtained by Northern blotting (1.3 kb).

Based on these results, yes? Could I conclude that the transcription of the nmtl gene produces a simple "unspliced" mRNA and does not have a heterogeneity? determinable at both terminals In order to obtain a sequence pi? long upstream of the nmtl structural gene, the S .pombe library in HindiII? was re-analyzed using a probe consisting of the fragment of about 2.4 kb. Was it so? isolated a clone comprising a fragment of about 4.1 kb consisting of the original fragment of 2.4 kb and a fragment of 1.7 kb placed immediately upstream of this.

Southern blotting analysis of the chromosomal DNA of S.pombe indicated that these two fragments were present and contiguous in the yeast genome and that it was not an artifact due to the ligase reaction.

According to the present invention, most (about 1320 bp) of the 1.7 kb? it was then sequenced in both directions operating as reported above. The sequence and restriction map of this chromosomal DNA region are shown in Figure 3A and 3B.

Analysis of the promoter region of the nmtl gene for the localization of repetitive elements, which in genes of eukaryotic cells have often been identified as specific binding sequences of modulating factors acting in trans, revealed the presence of a sequence of 9 nucleotides 5 'AAATAATAC 3' in position -684 to -676 and in position -577 to -569 (Figure 3A underlined).

To verify whether the 4.1 kb HindlII fragment according to the present invention contained the control sequences for the transcription regulation of the nmt1 structural gene, the plasmid DB262 containing the whole fragment (pNMT41)? been used to transform S.pombe yeast.

The transformants, selected using conventional techniques, were then grown in the presence and absence of thiamine. Is the mRNA isolated from said yeasts? was then analyzed by Northern blotting (Figure 4). The results obtained showed that the cells cultured under induction conditions, free of thiamine, (line c) contained a quantity? of nmtl mRNA which was approximately double that determined for the control cells transformed with the plasmid DB262 as is (line a). The nmtl mRNA was not determinable n? in control cells n? in those transformed cultivated in conditions of repression (presence of thiamine).

In order to confirm the results obtained and verify that the increases in nmtl mRNA were due to the fragment, contained in the plasmid,? A new plasmid was prepared containing the 4.1 kb fragment devoid of the 185 bp BamHI - BamHI region contained in the nmtl structural gene (Figure 3A). Analysis of the mRNA showed, as expected, that the transcript of the deleted clone was 4.1 kb shorter than that of the normal fragment (Figure 4, lines e and f). By means of densiometric analyzes? It was possible to estimate that in the thiamine-free medium (line e) 45% of the transcripts were derived from the intact chromosomal gene, while 55% from the gene present in the plasmid. In the presence of thiamine, both the chromosomal and plasmid-derived transcripts were completely repressed (line b, d and f).

These results confirmed that the 4.1 kb chromosomal DNA fragment contained the structural gene nmtl and all the regulatory sequences required for the control of thiamine-mediated gene expression.

Furthermore, these results showed that the regulation of said cloned gene in the plasmids also took place in the presence of multiple copies of the promoter.

In fact, it was transcribed in high yields under induction conditions, while in the presence of thiamine it was thus repressed. such as the endogenous chromosomal gene.

The gene expression product of nmtl, whose animinoacid sequence does not show any analogy with that of known proteins, appears to be involved in the biosynthesis of thiamine. This hypothesis is supported by the fact that the nmtl :: ura4 strain, obtained by replacing the 820 bp Xhol-Ncol region of the nmtl structural gene with the ura4? an auxotrophic thiamine, that is? it requires the presence of thiamine in the culture medium to grow (Figure 5).

The promoter region of the nmtl gene, containing the elements that control thiamine-mediated regulation of transcription, thus such as the one downstream of the nmtl structural gene, containing the transcription termination sequences, appear particularly suitable for the construction of vectors for the inducible expression of heterologous genes in host organisms.

In accordance with the present invention, the promoter region can? be used as it is or modified in order to create restriction sites around the ATG useful for the insertion in the exact reading phase of heterologous structural genes without altering the functionality? of the regulation sequences contained therein.

Vectors suitable for the purposes of the present invention can be plasmids with a high number of replicating copies in an organism or with genomic integration selected from among those with expression in yeasts, bacteria or mammalian cells available commercially or at collection centers.

The structural genes can be selected from those encoding any polypeptide of interest such as hormones, proteins or enzymes useful in the pharmaceutical or food field.

According to an embodiment of the present invention, recombinant DNA molecules have been constructed comprising the suitably modified nmtl promoter region and / or the nmtl terminator region and the structural genes encoding CAT (Figure 8), human lipocortin 2 (Figure 9A) and the human papillomavirus transformation genes HPV-16 E6 and HPV-16 E7 (Figure 9B).

According to a further embodiment of the present invention, the promoter region of nmt1? was cloned into a genomically integrated plasmid in S-pombe by placing the heterologous structural gene immediately downstream of this. The transformation of yeast cells? was carried out by conventional techniques and the transformants were selected by operating as reported by Beach, D. and Nurse, P., (1981), Nature 290, 140-142.

Yeast cells transformed with the aforementioned recombinant plasmids were cultured in minimal medium in the presence and absence of thiamine. The results obtained showed, in both cases, an efficient regulation of the transcription of the heterologous genes located downstream of the nmtl promoter region.

The results obtained showed a complete regulation of the expression of the cloned heterologous gene and, moreover, the analysis of the 5 * terminal of the transcript revealed the exact beginning of the transcription of said genes.

The cloning vectors constructed in accordance with the present invention are shown in Table I.

According to the present invention, the chromosomal DNA fragment of 4.1 kb cloned in the plasmid DB262? was deposited as E. coli JA221 (pNMT41) at the American Type Culture Center as ATCC 68067.

The experimental examples which follow are illustrative and not limitative of the invention itself.

Example 1

Schizosaccharomyces pombe chromosomal DNA extraction.

S, pombe cells (leul-32, weel-50, h minus) were cultured in 200 ml of YEA culture medium (Yeast extract (DIFCO) 5 g / 1, glucose 30 g / 1, adenine 100 mg / 1 ), under mild agitation, at 30? C up to a density? of 8 x 10 6 cells per milliliter.

Then the cells were separated from the supernatant by centrifugation (10 minutes, 5000 revolutions per minute (rpm)) in an SS34 Sorvall RC-5B rotor at 4 ° C and resuspended in 10 ml of buffer (20 mM citrate phosphate, pH 5 , 6.40 mM EDTA, 1.2 M sorbitol, 0.2% 2-mercaptoethanol) containing 1 mg / ml of lysozyme. The shaken suspension? was incubated at 37 ° C for 30 minutes and then centrifuged at 5000 revolutions per minute (rpm) for 5 minutes.

The pellet cos? obtained ? was resuspended in 30 ml of TE buffer (10 mM Tris-HCl, pH 8, 0.1 mM EDTA) added with 1.5 ml of a 20% sodiumdodecyl sulfate (SDS) solution and maintained at 65 ° C for 10 minutes.

After adding 10 ml of 5 M potassium acetate to the solution, the resulting mixture? was kept on ice for 30 minutes.

11 so precipitated? obtained ? was separated by centrifugation and 30 ml of the supernatant were added to 30 ml of isopropanol and reacted at room temperature (20-25 ° C) for 5 minutes.

The resulting precipitate? was separated by centrifugation and resuspended in 6 ml of solution containing 100 mg / ml of proteinase K. The resulting mixture? was kept at 37 ° C for 2 hours and then extracted with phenol and phenol-chloroform. The nucleic acids, precipitated by adding 2 volumes of ethanol to the mixture, were separated and suspended in 500 µl of solution containing RNase A (50 µg / ml) and incubated at 37 ° C for 1 hour.

The solution cos? obtained? was extracted with phenol - chloroform and then added, drop by drop, with 2 volumes of ethanol. The precipitation? was carried out under gentle stirring, at room temperature. The precipitated DNA? was collected by precipitation and resuspended in lxTE buffer up to a concentration of 1 mg / ml.

Example 2

Construction of the S.pombe library in HindlII.

10 µg of chromosomal DNA obtained as reported in example 1 were diluted in 120 µl of 50 mM NaCl buffer, 10 mM MgC ^, 20 mM Tris-HCl, 1 mM of Dithiothreiotol (DTX) pH 7.5 and partially digested with 4 units of HindII restriction enzyme (BRL) at 37 ° C for 30 minutes.

In parallel, 3 jug of plasmid DB262 (Wright, et al., (1986), PLASMID, 15, 156-158) were digested in the same buffer reported above with 5 units? of HindII at 37 ° C for 3 hours.

Then 10µg of DNA fragments were ligated with 3µg of plasmid DNA in 300µl of ligase buffer (66mM Tris-HCl, pH 7.6, 1mM ATP, 10mM MgCl2, 10mM (DTT)) in the presence of 1,5 unit? of T4 DNA ligase (BRL) at 14? C overnight. Vector DB262 becomes tetracycline resistant when the HindiII site? destroyed and therefore allows the selection of positive transformants.

The ligase mixture? it was then used to transform E.coli JA221 cells (recAl, leuB6, trp delta E5, hsdR, hsdM, lacY, C600) made competent by treatment with 50 mM CaCl ^ as described by Mandel, M. and Higa, (1970), J.Mol.Biol., 53 ^ 154.

So the selection of transformants? was carried out on plates of LB agar medium (DIFCO) added with 5 pg / ml of tetracycline and incubated at 37 ° C for 18 hours.

Operating as reported above, 10 colonies resistant to tetracycline (positive colonies) containing a heterologous insert with an average length of about 4 Kb were selected.

Example 3

Purification of polyA mRNA and synthesis of cDNA probes.

The wild type S. pombe 972h strain? been cultivated, under agitation, in 200 ml of minimum soil, the composition of which? reported by Nurse, P. (1975), Nature 256,547 or in a minimum medium containing 2 mM of thiamine. The cultures were kept under gentle stirring at a temperature of 30 ° C, up to a density? of 8 x 10 6 cells / ml. Then the cells were recovered by centrifugation, washed in 0.9% NaCl and resuspended in 2 ml of 0 ° C buffer having the following composition:

0.32 M of sucrose;

20 mM Tris-HCl, pH 7.5;

10 mM EDTA;

0.5 mg / ml of heparin.

The solution ? was added with 3 ml of glass beads (Sigma, 500 microns in diameter), vortexed for 90 seconds and immediately diluted with 20 ml of the above buffer added with 1% SDS. The cells cos? lysates were extracted with phenol at 60 ° C for 3 minutes and then with phenol-chloroform at 22 ° C for 5 minutes.

Subsequently the RNA? been precipitated with 2 volumes of ethanol and the pellet? was washed with 70% ethanol, dried under vacuum and resuspended in sterile water at a concentration of 2 mg / ml. To select the polyA + fraction, the? RNA? was suspended in 1 mM EDTA and denatured at 65 ° C for 5 minutes, diluted with an equal volume of 2 x loading buffer (lx = 20 mM Tris-HCl, pH 7.5, 1 mM EDTA, 0.5 M NaCl, 0.1% SDS) and the resulting mixture? was loaded into a column packed with 0.05 g of oligo (dT) cellulose (Boehringer). The column ? was extensively washed with the buffer having the above composition. Polyadenylate mRNA? was eluted with a 10 mM Tris-HCl solution, pH 7.5, 1 mM EDTA, precipitated in 0.1 M NaCl, 66% ethanol and then resuspended in water at a concentration of 0.5 mg / ml.

2 jigs of polyA + mRNA were suspended in 10 JJ. 1 of a 100 mM KC1, 50 mM MgCl2, 10 mM Dithiothreitol (DTT) solution containing 0.5 mM of each of the deoxynucleotides (dGTP, dATP, dTTP) and 0.1 mCi of 32P-dCTP. The synthesis of cDNA probes? was conducted in the presence of 20 units? of AMV reverse transcriptase (Boehringer) at 42 ° C for 1 hour.

The reaction ? was blocked by adding EDTA to a final concentration of 40 mM. The RNA? was then hydrolyzed in 0.2 N NaOH at 65 ° C for 15 minutes and the resulting solution? was neutralized by adding an equal volume of 0.2 N HCl. The unincorporated nucleotides were removed by chromatography on Sephadex G-50 and the fractions containing high molecular weight cDNA obtained from the two cultures (plus or minus thiamine) were collected and used as probes.

Example 4

S. pombe library screening.

About 4x104 positive colonies obtained as reported in Example 2, were plated at a density? of 2500 colonies / plate on LB agar medium supplemented with tetracycline (5 pg / ml). Colonies that had reached a diameter of 0.5-1 mm were then transferred to nitrocellulose filters (Schleicher & Schuell, 0.45 µm) and lysed in situ. DNA? been adsorbed on the filter by subsequent exposure to:

- 10% SDS (5 minutes);

- 0.2 N NaOH, 1.5 M NaCl (5 minutes);

- 0.2 M Tris-HCl, pH 7.5, 2xSSC (lxSSC = 150 mM NaCl, 15 mM sodium citrate) (2x5 minutes).

The filters were dried first in air and then at 80 ° C for 2 hours. The prehybridization treatment? was conducted at 65 ° C for 5 hours in 100 ml of 6x SSC, 4 x Denhardt's (lx = 1% ficoll, 1% PVP, 1% BSA pentax fraction V), 0.5% SDS, 100 pg / ml of DNA of denatured calf thyme. Then the filters were transferred into 70 ml of the same buffer containing the cDNA / thiamine probe (10 cpm / ml) and the hybridization? was conducted at 65? C for 24 hours. Filters after washing in 2xSSC (2 washes for 5 minutes), 2xSSC, 1% SDS (2 washes at 65 ° C for 30 minutes) and 0, 1xSSC (1 wash at 50 ° C for 30 minutes) were exposed to autoradiography on a Kodak X-Omat AR plate. Subsequently the probe used? was removed by washing the filters in 1xSSC and 50% formamide at 70 ° C for 30 minutes. Then the filters were washed, dried and hybridized with the cDNA probe obtained from culture in minimal thiamine-free medium operating as described above.

Were they like that? isolated numerous colonies that hybridized differently with the two probes. One of these, which hybridized strongly with the minimal medium cDNA probe minus thiamine and weakly with the minimal medium cDNA probe more? thiamine,? was further characterized.

The recombinant plasmid, extracted from said colony and digested with HindlII, revealed the presence of a single 2.4 kb band.

Example 5

Sequencing of the 2.4 kb HindlII fragment. A) Subcloning of the 2.4 kb HindlII fragment in M13mpl9.

1 pg of plasmid M13mpl9 (BRL)? been digested in 20 µl of digestion mixture with 5 units? of HindII enzyme at 37 ° C for 1 hour.

The enzymatic reaction? was inactivated at 65 ° C for 10 minutes and the DNA precipitated with ethanol and separated by centrifugation.

0.4 pg of plasmid DNA cos? digested were ligated to 1 µg of the 2.4 kb HindlII fragment in 30 µl of ligase mixture in the presence of 0.2 units? of T4 DNA ligase, at 14? C overnight.

2.0 µl of the ligase mixture was used to transform 200 µl of competent E.coli JM101 cells.

The selection of the plates? It was then carried out on soft LB agar plates (3 ml) added with 400 µg / ml of X-GAL, 80 µg / ml of IPTG and 200 µg / ml of untreated E.coli JM101 cells. 6 positive clones containing the recombinant plasmids were isolated from 12 white plaques.

Two of said plasmids, indicated p5 and p7 containing the insert in the two orientations, were extracted and sequenced.

B) Sequencing of p5 and p7.

The p5 and p7 plasmids were completely sequenced in both directions by preparing a series of deletion mutants obtained after digestion with ExonucleasilII and SI nuclease operating as described by Henikoff, S. (1984), Gene, 28,351-359.

The presence of a single open reading phase of 1038 bp suggested the presence of a new gene, hereinafter referred to as nmtl, which potentially encoded a protein of 346 amino acids with a molecular weight of 39 kD.

Yup ? then proceeded to the localization of the 5 'and 3' terminals of the nmtl mRNA.

In practice, the 5 'region of the mRNA of nmtl? was determined by the "primer extension" strategy using the 5'CCTCAA-TCCCTTCACGCTCA 3 'oligonucleotide, complementary to the mRNA region between 90' and 109 (Figure 3A), terminally labeled as described by Grimm et al., (1988) , Mol. Gen. Gen., 215, 81-86.

The results obtained revealed that transcription started at residue A in position -69 and that the TATATAAA sequence extended from position -34 to -27 with respect to the transcription start site.

The 3 'terminal of the transcript? was determined by SI nuclease mapping (Berk A.P. and Sharp, P.A., (1977), Celi, 2, 721-737) using a 371 nucleotide probe corresponding to the downstream region of the Ncol (+935) site within the sequence coding nmtl. A single protected fragment of 244 nucleotides places the 3 'terminal of the messenger 142 nucleotides after the stop codon (Figure 3A).

The predicted length of nmtl mRNA, based on terminal mapping,? therefore of 1252 nucleotides very close to the value found by Northern blotting. From these data? Is it possible to conclude that the transcription of the nmtl gene produces a simple "unepliced" mRNA and has no heterogeneity? to the two terminals.

In order to obtain the entire sequence upstream of the nmtl gene, the genomic bank prepared as reported in example 2? was hybridized with the 2.4 kb fragment labeled with 32P operating as reported by Feinberg A.P. and Vogelstein, B., (1984) Anal.Biochem., 137, 266-267. From one of the positive clones? A plasmid was isolated containing a DNA fragment of 4.1 kb, hereinafter referred to as pNMT41, which, upon restriction analysis with HindlII, resulted to consist of the original 2.4 kb fragment and a 1.7 kb fragment. Southern blotting analysis of the chromosoral DNA of S.pombe, digested with the restriction enzymes BamHI and EcoRI, using as probes the two HindiII fragments of 2.4 and 1.7 kb separately, confirmed that in the yeast genome the fragment of 1.7 kb DNA was upstream and contiguous to the 2.4 kb fragment and was not the result of a ligation reaction during the cloning step.

Most of the fragment (1320 bp) of 1.7 kb? been sequenced in both directions and its sequence and restriction map are shown in Figure 3A and 3B.

Example 6

Determination of the control sequences of the nmtl gene regulation.

In order to verify if the 4.1 kb fragment contained the control elements of the transcription regulation of nmtl, the plasmid pNMT41? been used to transform Spaombe cells by operating according to the technique of Beach, D. and Nurse, P., (1981) Nature 290, 140-142. Transformants were selected on minimal medium by selecting for leucine.

Some of the transformed colonies were then grown in minimal soil in the absence and presence of 2 ^ ?? of thiamine at 30 ° C for 40 hours. The RNA prepared from each culture as reported in Example 3? was analyzed by Northern blotting using 32P-labeled nmtl probes. From the reported in Figure 4 it is observed that cells transformed with pNMT41 cultured under induction conditions (absence of thiamine) (line c) contain a quantity of double mRNA compared to cells transformed with the plasmid pDB262 as it is (control) (line a). Under conditions of repression (presence of thiamine) 11nmtl mRNA is not? determinable n? in control cells (line b) n? in those transformed with the pNMT41 plasmid (line d).

To confirm that the increases in mRNA were due to the presence of the plasmid containing the 4.1 kb fragment,? A plasmid was constructed containing the 4.1 kb fragment devoid of the 185 bp BamHI-BamHI region of the nmtl structural gene (????? 41). The analysis of nmtl mRNA isolated from S.pombe cells transformed with said plasmid showed the presence of a transcript pi? short (Figure 4: line e). Based on densiometric results,? It was possible to observe that in thiamine-free medium (line e) 45% of the nmtl gene product was derived from the transcription of the gene present in the chromosomal DNA of S.pombe, while 55% derived from the transcription of the nmtl gene present in the recombinant plasmid . In the presence of thiamine, both the transcript derived from the chromosomal gene and that derived from the plasmid gene were totally repressed (line f). On the basis of these data it was possible to conclude that the cloned nmtl gene and the adjacent regions include all the regulatory sequences of the thiamine-mediated gene transcription and, furthermore, show that this regulation also occurs in the presence of multiple copies of the promoter.

Example 7

Construction of a stump of S.pombe devoid of activity? nmtl {nmtl :: ura4) for the analysis of the function of the protein encoded by the nmtl gene. In order to obtain information about the function of the product encoded by the nmtl gene, it? been inactivated as reported by Grimm, C. et al., (1988), Mol. Gen. Genet., 215, 81-86.

The 816 bp Xhol-Ncol region contained in the nmtl structural gene? has been replaced with the ura4 gene (Bach, M.L., (1987), Curr.Genet., 12, 527-534).

The linear fragment obtained after digestion with HindlII? was isolated and used to transform the diploid strain (h / h, ura4-dl8 / ura4-dl8, leu-32 / leu-32, ade6-704 / ade6-704) to uracil prototrophy. The restriction analysis of 10 Ura + stable diploid transformants showed for two of these a pattern that confirmed that a homologous integration at the nmtl locus had occurred. molecules of a 2.4 kb band and a 3.4 kb band corresponding, respectively, to the wild type gene and to the destroyed one.

A sporulating diploid h + / h90? was selected by exposure to iodine vapors and the meiosis products were analyzed by tetrahedral dissection. In all cases the 4 spores were viable on YEA medium. DNA extracted from each spore was analyzed by Southern blotting and, as expected, showed two wild type and two destroyed nmtl alleles. The latter showed a Ura + phenotype (Figure 5A). The cultural characteristics of each of the 4 spores obtained from a single tetrad grown in minimal medium (supplemented with adenine, leucine and uracil) with 0 without thiamine are shown in Figure 5 B. All 4 spores grew in minimal medium containing thiamine, however in minimal medium alone, cells containing the deleted nmt.l gene (nmtl :: ura4) did not show substantial growth.

The data confirmed that the strain containing the nmtl :: ura4 fragment was auxotrophic thiamine.

Example 8

Effect of thiamine concentration on nmtl expression.

Spaombe cells 972 h ~ were cultured for 40 hours in minimal medium alone or added with different concentrations of thiamine (0.01; 0.02; 0.05; 0.1; 0.2; 0.5 ; 1.0 and 2.0 µl).

So 1 RNA? been extracted from the cells and after denaturation? was fractionated on 1.2% agarose gel in 25 mM phosphate buffer. After electrophoresis, the mRNA nmtl? was determined by Northern blotting using a P.

The results reported in Figure 1A show that the transcript of the nmtl gene was abundantly present in minimal thiamine-free medium, while it disappeared as the concentration of thiamine in the culture medium increased.

The same RNA samples were assayed in parallel for the determination of the cytochrome C transcript by Northern blotting using as probe the 0.54 kb EcoRI-SalI fragment containing the gene encoding cytochrome C isolated from the pPoCyc plasmid (0.54) (Russell, P. And Hall, B., (1982), Mol .Celi.Biol., 2,106-116).

The results reported in Figure 1B show that equal quantities? of mRNA were loaded into each line and that the nmtl mRNA, when fully expressed, was 50-100 times more? abundant than that of cytochrome C. Therefore these data indicated nmtl as one of the major cellular transcripts.

Example 9

Kinetics of repression and induction of nmtl transcription by thiamine.

A) To study the kinetics of repression, a 972 h-? was grown to the logarithmic phase in minimal thiamine-free medium. So, after taking an aliquot of said culture (T?) For mRNA analysis, the minimum medium? was added with 2 JJM of thiamine. Aliquots of said culture were taken at regular intervals for the preparation of the mRNA. The Northern blottingting results reported in Figure 2A reveal that the transcript disappears completely from the cells after approximately 3 hours.

B) To study the kinetics of induction, 972 h S.pombe cells were cultured in minimal medium supplemented with 2 µM of thiamine up to the logarithmic phase. So an aliquot of the crop? was taken for mRNA preparation (T?). The cells were then separated from the culture, washed with minimal medium and resuspended (106 cells / ml) in fresh minimal medium. Aliquots of this culture were withdrawn at 1 hour intervals and the mRNA? was assayed by Northern blottingting using the labeled nmtl probe.

The results reported in Figure 2B show the absence of the transcript at time T ?; the first appearance of the transcript after 10 hours and the appearance of the entire nmtl transcript after 16 hours.

In conclusion, these data indicate that the expression of nmtl pu? be controlled by simple modification of the culture medium and that in the state of non-induction the expression of this gene? completely repressed.

Example 10

Construction of vectors containing nmtl regulation sequences.

A) Construction of the multiple copy vectors PMNS36L and PMBS36L.

The 1.8 kb BclI-EcoRI fragment containing the nmtl promoter region and a part of the nmtl structural gene (Figure 3B)? been subcloned in the phage M13mpl9 (BRL) operating as described in example 5A and the region around the ATG? was modified by in vitro mutagenesis as follows:

1) the residue T in position 6 of the structural gene? was substituted with the nucleotide residue G in order to modify the endogenous restriction site Acci in the Sali site using the synthetic oligonucleotide 5 'CTTGTTAGTCGACATGATT 3? and operating as reported by Maundrell et al. (1988), EMBO J., 7, 2203-2209. In this way a promoter was obtained more? easily inserted; 2) the region immediately upstream of the ATG? modified to include the Ndel site (51CATATG 3 ').

For this reaction? The 5'AGTCGACATATGATTTAACA 3 'oligonucleotide was used.

In this construct the Ndel ATG encodes the methionine necessary for the initiation of translation and allows the precise fusion of heterologous nucleotide sequences containing a natural or constructed Ndel restriction site at the 5 'terminal.

The Ndel site? particularly suitable for constructs of this type since? it does not cause any modification of the N-terminal amino acid sequence of the expressed gene product.

Anyway in order to use this restriction site? It was necessary to modify the promoter in order to eliminate the endogenous Ndel site in position -768 upstream of the ATG. In practice such a change? was obtained by digesting the promoter with Ndel and then incubating with the enzyme "Klenow" (Boehringer). The activity? 5 '?) 3' exonuclease of this enzyme was used to eliminate the Ndel restriction site. The subsequent sequence analysis indeed showed that the original sequence of the nmtl 5'CCATATGTCTAA 3 'promoter had been modified to 5'CCATAA 3' (Figure 6 B) with consequent loss of the Ndel restriction site. This modification had no adverse effect on the sequences involved in the control of thiamine-mediated transcription (Figure 9A). The modified promoters as reported in 1 and 2, were then isolated as a 1.2 kb Pstl-Sall fragment and subcloned in the pIRT3 vector for expression in yeasts and E. coli, obtaining the pMS21L and pMNS21L plasmids respectively.

Said vectors contain the LEU2 marker of S. cerevisiae, useful for the selection of transformants, and the self of S. pombe which provides the origin of replication in eukaryotes necessary for the episomal maintenance of plasmids. LEU2 and arsi are cloned at the HindlII and EcoRI sites of the pUC119 polylinker, respectively.

The pIRT3 vector? was obtained from the pIRT2 vector (Hindley, J. et al., (1987), Mol.Cell.B-iol., 7., 504-511) by changing the orientation of the LEU label.

Finally, to provide the vector with a transcription termination signal, the 3 'region of nmt1 between the stop codon and the 990 bp site downstream of this? was amplified by PCR (polymerase chain reaction) and inserted in the BamHI and SstI sites of the pUC119 polylinker (Figure 6 B) obtaining pMNS36L.

The pMBS36L plasmid? identical to the pMNS36L plasmid except that the sequence around the ATG? been modified to contain the Ball site (5'TGGCCA 3?) instead of Ndel. Such a change? was obtained by in vitro mutagenesis using the 5'GTCGAC-ATGGCCAATTTAACAAAG 3 'oligonucleotide (Figure 6C). This construct allows by digestion with Ball to eliminate the ATG codon in the case in which the heterologous gene already contains? its translation start codon. B) Construction of pMINS6L pMIBS6L genomically integrated vectors.

Plasmids suitable for the integration of the heterologous DNA sequence into the chromosome of S.pombe were constructed by deletion of the E? ORI element arsi from pMNS36L and pMBS36L.

The plasmids obtained were indicated respectively pMINS6L and pMIBS6L.

The characteristics of the constructed vectors are shown in table I.

Table I.

Vectors Marker sites Origin of nmtl restrict. replication ends.

at the ATG

pMS21L Salts LEU2 burned NO

PMNS21L Ndel, Sal? LEU2 burned NO

pMNS36L Ndel, Sali LEU2 burned YES

pMBS36L Ball, Sali LEU2 burned YES

pMINS6L Ndel, Sali LEU2 YES

pMIBS6L Ball, Climb LEU2 YES

Example 11

Inducible expression of chloramphenicol acetyl transferase (CAT).

The 1,639 kb HindiII - BamHI DNA fragment containing the structural gene encoding CAT? was isolated from the plasmid pAlOCAT (DOnofrio et al., (1985), EMBO J. 4, 1981-1989) and subcloned in M13mpl8. So a Ndel site? was created at codon ATG by in vitro mutagenesis using the synthetic oligonucleotide 5'TAAGGAAGCTCATATGGAGAAAA 3 \ Subsequently the Ndel-XhoII fragment? was isolated from M13mpl8 and cloned in the Ndel and BamHI sites of the pMNS36L and pMINSSL plasmids to give the pMCMA and pMCM hybrid plasmids, respectively.

Cloning? was carried out in a mixture of ligases as described in Example 5A.

The S .pombe transformants containing the replicating plasmid pMCMA were cultured in minimal medium with or without thiamine, at 30 ° C for 40 hours.

Subsequently the cells were separated by centrifugation at 5000 rpm, at 22? C for 5 minutes. The pellet? was resuspended in 300 µl of 0.25 M Tris-HCl tamon, pH 7.5, 1 mM PMSF and after adding 500 µl of lysed glass beads by vortexing for 4 minutes. 400 µl of supernatant was transferred into Eppendorf tubes and centrifuged repeatedly (12,000 rpm for 2 and then 5 minutes) to remove cell residues. Finally the CAT essay? was conducted on 1 µl of the supernatant.

The results shown in Figure 8 show that in the absence of thiamine (line a), the CAT? actively produced by S. pombe, while in the presence of 2 ug / ml of thiamine its expression? drastically reduced (line b).

Again, estimates based on the dilution of the extracts and on the exposure time of the chromatogram to obtain signals of equal intensity? show about 200-300 times greater stimulation of expression for cells grown under induction conditions. Similar results were obtained using S.pombe cells transformed with the pMCM integrating plasmid (Figure 8 lines c and d).

The results indicate that these strains contain only one copy of the CAT gene stably integrated in the chromosomal DNA of the yeast.

For the integrating strain the difference in activity? CAT between induced and non-induced cells was approximately 500-600 times greater.

Based on what has been achieved? It is possible to conclude that the thiamine-mediated control system of nmtl gene expression can? be transferred to a different gene and that the regulation of that gene? effective both in high copy number plasmid vectors and when the construct, promoter region of nmtl and heterologous gene,? integrated into the yeast genome.

Example 12

Inducible expression of human lipocortin 2. A Sall-SstI DNA fragment containing the human lipocortin 2 gene? was cloned in the pMS21L and pMNS21L expression vectors by operating as described in Example 5A.

The recombinant plasmids were then employed to transform S.pombe. The transformants selected as reported above were grown in minimal medium in the presence and absence of thiamine. The results of Northern blotting mRNA analysis, shown in Figure 9A, show that high transcript levels of the lipocortin 2 gene are obtained under induction conditions (thiamine-free) (line -), while this transcript disappears completely under conditions of non-induction (presence of thiamine) (line).

The efficiency of thiamine-mediated human lipocortin 2 transcription regulation was identical for both pMS21L and pMNS21L vectors. This demonstrates that the introduction of the Ndel site at the 3 'end of the promoter and the elimination of the endogenous Ndel site in position -768 (pMNS21L), did not alter the activity. of the promoter himself.

Example 13

Inducible expression of the transforming HPV-16 E7 and HPV-16 E6 genes of the human papillomavirus.

The HPV-16 E7 and E6 genes were copied from the viral genome by PCR as described in Example 8A and the Ball and Sali sites were added respectively to the 5 * and 3 'terminals. These genes were then cloned into the pMBS36L plasmid previously digested with the same enzymes. The recombinant plasmids were introduced in S.pombe and the transformants cos? obtained were cultured for 40 hours in the presence (2 ^ JIM) or absence of thiamine. The mRNA? it was then prepared from the two cultures as reported in Example 3 and then analyzed by Norhtern blotting. The results confirmed that the gene products were expressed in high yields in the absence of thiamine and were completely repressed in the presence of thiamine (Figure 9B). Furthermore, in the mapping of the 5 * terminal of the mRNAs with SI it was shown that the transcript started correctly.

Claims (23)

CLAIMS 1. Schizosaccharomyces pombe chromosomal DNA fragment containing the nmtl structural gene encoding a new protein and the control sequences of thiamine-mediated transcription regulation of said gene where said DNA fragment? characterized by the nucleotide sequence shown in Figure 3A. 2. Promoter region of the nmtl gene or a fragment thereof containing the control sequences of thiamine-mediated transcription regulation characterized by the following nucleotide sequence: 3. Terminator region of the nmtl gene comprising the termination sequences of the transcript characterized by the following nucleotide sequence: 4. Cloning vector with adjustable expression in host organisms selected from the group of bacteria, yeast or higher mammalian cells comprising the DNA fragment according to claim 1. 5. Adjustable expression cloning vector according to claim 4, wherein the host organism? the yeast Schizosaccharomvces pombe. 6. Adjustable expression cloning vector according to claim 4, wherein the host organism? Escherichia coli. 7. Adjustable expression cloning vector according to claim 4, deposited at the American Type Culture Center as ATCC 68076. 8. Recombinant DNA molecule with adjustable expression in host organisms selected from bacteria, yeast or higher mammalian cells comprising the promoter region according to claim 2 and / or the terminator region according to claim 3, the heterologous structural gene encoding a polypeptide of interest where said gene? positioned in the exact reading phase under the control of said regions and at least one marking gene useful for the selection of the organisms transformed with said recombinant DNA molecule. 9. Adjustable expression recombinant DNA molecule according to claim 8, where the host organism? the yeast Schizosaccharomyces pombe. Adjustable expression recombinant DNA molecule according to claim 8, wherein the host organism? Escherichia coli. Recombinant DNA molecule according to claims 8 to 10, wherein the heterologous structural gene encodes an enzyme, hormone or protein of interest. 12. A recombinant DNA molecule according to claim 11, wherein the heterologous gene? chosen from the group of lipocortins, interleukins, interferon, human growth hormone or from the group of transformants of the human papillomavirus. 13. Microorganism selected from yeasts, bacteria or mammalian cells transformed with a vector according to claims 4 to 7 or with a recombinant DNA molecule according to claims 8 to 12. 14. Microorganism according to claim 13, wherein the yeast? Schizosaccharomyces pombe. 15. Microorganism according to claim 13, wherein the bacterium? Escherichia coli. Recombinant DNA molecule with adjustable expression according to claims 8 to 12, obtained by the union in the presence of the enzyme T4 DNA ligase of the promoter region according to claim 2 and / or the terminator region according to claim 3, with a vector of cloning chosen from the group of plasmids capable of replicating autonomously in the cells of host organisms or in the group of plasmids capable of integrating into the chromosomal DNA of said host organisms. Adjustable expression recombinant DNA molecule according to claim 16, wherein the host organisms are selected from the group of bacteria, yeast or higher mammalian cells. 18. A method for the adjustable production of the nmt1 protein in cine host organisms comprises cultivating in a suitable culture medium said organism transformed with a vector according to claims 4 to 7 in the presence or not of thiamine. 19. Method according to claim 18, wherein the production of nmtl? induced in absence of thiamine or in the presence of concentrations of this lower than or equal to 0.05 uM and? completely suppressed in the presence of thiamine concentrations above 0.05 yuM. A method for the adjustable production of a heterologous polypeptide in a host organism which comprises culturing in a suitable culture medium an organism transformed with a recombinant DNA molecule according to claims 8 to 12 in the presence or absence of thiamine. A method according to claim 20, wherein the production of the heterologous polypeptide? induced in the absence of thiamine or in concentrations of this lower than 0.05 yuM and? completely repressed in presence of thiamine concentrations higher than 0.05 µM. Method according to claims 20 and 21, wherein the heterologous polypeptide? human lipocortin 2, the transforming protein of the human papillomavirus, interleukins, interferon or human growth hormone. 23. New nmtl protein characterized by a molecular weight of about 39 Kilodalton and the following amino acid sequence: