DK171215B1 - Plasmids which have a conditionally uncontrolled replication threshold, and a process for preparing a gene product by culturing a microorganism which harbours such a plasmid - Google Patents

Description

DK 171215 B1

The present invention relates to novel plasmids useful as cloning and production vectors in the field of recombinant DNA technology, the replication behavior of which is uncontrolled under conditions favorable therefor, and to a method of producing gene products by growing microorganisms containing such plasmids.

δ I Mol. Gen. Genet. 184. 1981, 56-61 disclose that the replication of plasmid RI is controlled by the genes copA. copB and repA. In contrast, it is not disclosed that by inserting a regulatable promoter transcribing into the replication region, a significant increase in the plasmid copy number can be achieved.

Plasmids with conditional uncontrolled replication behavior (so-called runaway plasmids) are known from & the specification of European Patent Application No. 78101877.5. The replication of these plasmids is temperature dependent, the plasmids having a controlled, constant, low copy number when host bacteria carrying the plasmids are grown at one temperature, e.g. 30 ° C, and an uncontrolled copy number when host bacteria are grown at a different temperature, & a temperature above ca. 36 ° C.

The plasmids described in the above patent application were isolated by chemical mutagenization of plasmid RI, which is a wild-type plasmid known to autonomously replicate in Escherichia coli. The mutagenization was performed in two steps, the first step comprising the isolation of a plasmid-bearing bacterial clone in which the plasmid copy number was about 1-2 at 30 ° C and 4-5 times higher at 40 ° C, and the second step included the isolation of a bacterial clone in which plasmid 20 had an uncontrolled copy number at the higher temperature. Thus, the plasmids exhibiting runaway behavior were double mutants of the parent plasmid.

The above-mentioned patent application also disclosed mini-plasmids which are useful as cloning vectors because they can be relatively easily transformed into host bacteria due to their small size. These mini-plasmids have retained the genes responsible for the temperature-dependent runaway replication behavior.

As cloning and production vectors, the plasmids described in the aforementioned patent application can be used to recover gene products from inserted fragments of foreign DNA.

The temperature-dependent replication of the pias agents can be utilized to produce amplified amounts of gene products from DNA fragments of Feukaiot origin. Such amplification of gene product has not been possible with the conventional plasmid DNA amplification techniques which act by adding chloramphenicol to the culture medium, with the presence of chloramphenicol causing protein synthesis to stop.

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The plasmids described in the aforementioned patent application can advantageously be used as cloning and production vectors in cases where an inserted foreign gene encodes a product which is harmful to the bacterium to which the plasmids are transformed, since the plasmids at low temperatures have a low copy number. and as a result, only slight re-expression. This means that during the propagation step of the culture, which is carried out at low temperatures, the bacteria are not likely to be damaged.

The present invention relates to novel plasmids carrying at least one regulatable promoter inserted in such a way that transcription thereof regulates plasmid replication. The invention relates to plasmids in which the regulatable promoter causes substantially increased or uncontrolled replication when host microorganisms containing the plasmids are cultured under conditions which ensure increased transcription from the promoter. The plasmids are useful as cloning and production vectors.

It is known to insert a regulatable promoter to control the expression of a structural gene inserted into the plasmid that encodes a desired gene product. However, it is believed to be novel to insert a regulatable promoter into plasmids, by means of which it is possible to regulate the number of plasmid copies formed.

In a first aspect, the invention relates to a plasmid useful as a cloning vector and having a size in the range of ca. 2.5 - 15.0 x 10 6 daltons, which plasmid a) an inserted regulatable promoter in a position and in a direction that allows transcription from the promoter into the replication region; b) replication region comprising a gene encoding a gene product which is positively necessary for replication, with increased transcription from the regulator promoter leading to an increased level of the gene product which is positively needed for replication, the increased level resulting in a plasmid replication rate which is higher than the rate of growth of the host cell, which leads to an increased plasmid capital, which will usually be increased by a factor of 25-1000 after 25 to 4-5 cell divisions. This specification and claim designates the term "adjustable promoter" a promoter whose activity with respect to to the frequency at which the transcription is initiated can be regulated by adapting the conditions under which host microorganisms containing the promoter-bearing plasmids are grown. Such an adjustable promoter may be, for example, a foreign promoter, i.e. a promoter not naturally associated with the plasmid into which it is inserted. The promoter may itself be adjustable due to a particular DNA structure within the promoter region. An example of such a promoter is the promoter found in the known runaway plasmids (see description below). Alternatively, the promoter may be controllable by a regulatory factor which can either exert positive control, i.e. the plasmid does not achieve uncontrolled replication unless the promoter is positively induced, for example by adding an inducing agent to the medium in which the host cells are grown or the regulatory factor can exert negative control. The latter type of controlling factor is also known as a repressor, the activity of which can also be regulated by an adaptation of host cell growth conditions. The repressor gene may be located on the plasmid itself with the promoter, on a separate plasmid in the same host microorganism or on the chromosome of the host microorganism. The repressor gene encodes a product that inhibits transcription from the inserted promoter so that replication is kept at a low, constant level. When for some reason the repressor is inactivated, no such control exists and transcription increases.

Unless otherwise indicated, the term "promoter" as used in the present specification and claims shall usually include both a promoter whose activity itself responds to changes in host cell growth conditions and a promoter controlled by a regulatory factor. The term "a plasmid into which a regulatable promoter is inserted" is also intended to include plasmids of the invention in which the promoter and the promoter-regulated replicon have been inserted on the same DNA fragment where the promoter and replicon ultimately originated from various sources; this means that, at some stage during the construction of the desired end plasmid, the promoter has been inserted into the plasmid from which the replicon originates. The term "substantially increased or uncontrolled plasmid copy" refers to the fact that when the conditions for culturing the host microorganisms are changed to ensure increased transcription from the promoter, so that the plasmid loses its replication control, the plasmid copy number increases exponentially with a generation time of approx. 40-50%, or less, of the generation time of the microbial population. This increase is continued until the host cell ceases to grow, usually after 4-5 cell divisions or less, at which stage the content of plasmid DNA in the cell is approx. 40-70% of the total amount of DNA, ie the plasmid copy number is increased by a factor of approx. 25-1000 or even more. In other words, the plasmid copy number does not reach a stable level. This type of uncontrolled replication behavior is also referred to as "runaway" behavior.

The knowledge of the Rl-type plasmid replication control principle recently obtained by the present inventors is utilized to construct plasmids according to the invention (a further explanation of this principle is given in the section "DETAILED DESCRIPTION OF THE INVENTION"). When a regulatable promoter is inserted into the plasmid in accordance with the principle of the invention, increased transcription from the promoter leading to a substantially increased or uncontrolled plasmid copy may be caused, for example, by derepression of the promoter. This means that a repressor gene inserted with the promoter that encodes a gene product that controls the promoter under certain conditions is inactivated under other conditions so that transcription from the promoter is no longer inhibited, which ultimately results in a uncontrolled plasmid copy.

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This represents a significant improvement over the known runaway plasmids which, under varying conditions, may exhibit differences in their replication properties. There is no evidence that this is the case with the plasmids of the invention, since the foreign promoter inserted will usually be a promoter that is so strong (i.e., the frequency of transcription initiation is high) that such changing conditions are without effect.

One of the conditions of culture which allows the activity of the regulator to be regulated is the temperature at which the host microorganisms are grown. The activity of the promoter may itself be temperature dependent, i.e. react to temperature changes during cultivation, or the regulating factor may be temperature sensitive, & a temperature sensitive 10 repressor.

The regulatable promoter may be a promoter from the bacteriophage λ, which may be inserted as part of a DNA fragment which additionally carries the gene for a temperature-sensitive λ d repressor that controls transcription from the promoter. The λ promoter can be either λΡΒ or XPL, especially λΡΒ. However, other promoter systems inserted into the plasmids in accordance with the principle of the invention and under the influence of external factors other than temperature may also be used, such as promoters which can be induced by chemicals or regulated by metabolites such as lac, faith and deo promoters.

A preferred embodiment of the plasmid according to the invention is an embodiment in which the promoter control system causes a temperature-dependent replication behavior of the plasmid. In particular, the temperature at which the plasmid has a substantially increased or uncontrolled plasmid copy capital is a temperature higher than the temperature at which the copy number is low. Plasmids of the invention which carry an adjustable promoter, whose transcription pattern is temperature dependent, and due to the presence of a temperature sensitive repressor, have a constant, low copy number at one temperature such as a temperature of approx.

25 at 30 ° C because the promoter at this temperature is suppressed and the transcription thereof is kept at a low level and a substantially increased or uncontrolled copy number at a higher temperature such as a temperature in the range of about 36-42 ° C, as the regulatory system is inactivated in this temperature range, causing derepression of the promoter and consequently increased transcription from the promoter. It is preferred that runaway replication occurs at temperatures above 30 ° C. 39 ° C.

The following description refers only to plasmids whose replication is controlled by a control system which is temperature sensitive, especially by a temperature sensitive repressor, although it is clear that other types of conditions under which plasmid replication can be regulated can also be utilized in an analogous manner as described above.

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One type of plasmid of the invention is a plasmid into which a regulatable promoter has been inserted in place of a portion of a natural replication regulatory gene in the plasmid. In this specification and claims, such a plasmid is referred to as a type A plasmid. When the plasmid is a derivative of the plasmid RI, the regulatory gene deleted from the plasmid is the copB gene encoding one of the 5 plasmid's natural replication inhibitors. Deletion of the conB gene results in a small, stable increase in the plasmid copy number. Thus, the plasmid has a copy number of ca. 20-40, preferably 20-30 and especially 20-25 copies, per. cell at approx. 30 ° C. When the temperature is raised to approx. However, the repressor function of the adjustable promoter is inactivated and the replication becomes uncontrolled, resulting in a plasmid copy in the region of at least about 500-1000 copies per cell, which depends on the size of any foreign DNA fragment inserted into the plasmid (the larger the DNA fragment, the lower the number of plasmid copies formed), the effect of gene products of any such foreign DNA and / or the microbial strain to which the plasmid is transformed. In some cases, however, the plasmid can produce up to several thousand copies per cell at the higher temperature. Whatever the case, the content of plasmid DNA at the high temperature relative to the total DNA content is approx. 40-75%, usually 50-60%.

Another type of plasmid according to the invention is a plasmid with a copy number of approx. 3-5 copies per cell at one temperature, such as a temperature of ca. 30 ° C, and an uncontrolled copy number in the range of at least 500-1000 copies per cell, depending on the above factors, and in some cases up to several thousand copies per cell. cell at a higher temperature such as a temperature of ca. 42 ° C. Such a plasmid can be obtained by inserting the regulatable promoter above the plasmid's natural replication control gene (s). In this specification and claims, such a plasmid is referred to as a type B plasmid. As for plasmid R1 derivatives, the regulatable promoter has been inserted above both the conB and copA gene, so that both of these natural replication inhibitors have been conserved, causing the plasmids to exhibit the above copal pattern. However, if the plasmid is a plasmid lacking the par region (the presence of the plasmid of the region responsible for distribution has the effect that the plasmid is inherited stably; this effect can be attributed to one or more genes in the nar region), the plasmid is lost at a frequency of approx. 1% per generation at the low temperature due to the low copy capital at the low temperature. Therefore, it may be advantageous to insert the pair region into such a plasmid to ensure that the runaway replication of the plasmid occurs later in all cells of the population.

The type B plasmid has the additional advantage of having a large copy spread. Because the copy number at the low temperature is very low, it is possible to insert foreign genes into the plasmids whose products are partially toxic (slowing the growth rate) or even lethal to the 35 microorganisms to which the plasmid has been transformed. This is sometimes the case with products from eukaryotic genes, which are usually the products that have the greatest interest, for example, in the pharmaceutical industry. Because of the low replication frequency at the low temperature, foreign genes are either not expressed at all or only in quantities so small that the cells are not damaged therefrom and can be grown at the low temperature under normal conditions. This greatly facilitates or facilitates the process of producing certain polypeptides which have not hitherto been possible or have been associated with great difficulties.

A third type of plasmid according to the invention is a plasmid with a copy number in the range of approx. 0.5-1 copy per cell (the number 0.5 means that the replication rate is less than 1 per cell cycle) at one temperature such as a temperature of approx. 30 ° C and an uncontrolled copy capital in the range of at least about 500-1000 copies per cell depending on the above factors and in some cases up to several 10 thousand copies per cell. cell at a higher temperature such as a temperature of ca. 42 ° C. Such a plasmid may be a plasmid carrying the regulatable promoter in the replication region from which a portion of a natural replication regulatory gene is deleted, which gene has then been inserted outside the replication region, i.e. in a place where it is not naturally located. In this specification and claims, this plasmid is referred to as a type C plasmid. As for plasmid 15 R1 derivatives from which the copB gene has been deleted, the copB gene is not reinserted at its original site but at the EeoRI site outside the region of replication. Even then, the copB-in-hibitor protein influences the replication of the plasmid, but in such a way that the plasmid exhibits the above copal pattern. However, if the plasmid is a plasmid lacking the pair region, the plasmid is lost at a frequency of approx. 5% per generation at the low temperature due to the extremely low copy capital at the low temperature. As a result, it is even more advantageous for this type of plasmid than for type B plasmiderae to insert the pair region into the plasmid to ensure that the plasmid runaway replication will occur later in all cells of the population. The replication behavior at the low and high temperatures, respectively, and thus the advantages of using this plasmid are similar to the replication behavior of the type B plasmids. In another aspect, the invention relates to a plasmid in which, in addition to the first regulatable 2 promoter which regulates transcription from the replication region, a second promoter is inserted in a manner which allows the insertion of a structural gene whose expression is controlled by the other 4 promoter. This second promoter may be controllable, viz. it may be possible to regulate its function by adapting the conditions under which the host cell is grown 6 in a manner similar to that of regulating the first regulatable promoter. Currently, 7 different promoters are believed to be useful for this purpose such as an Igc promoter, trp promoter. deo-8 promoter, recA promoter. XPL promoter, etc. The XPL promoter may prove to be particular to 9 as another promoter, which controls the expression of the structural gene, as it is also regulated by the temperature-sensitive cl repressor, so the amplification conditions are the same, so that the culture of the host cells is simplified and product amplification takes place simultaneously with the amplification of plasmid DNA. It should be noted that the second promoter may also be identical to the first adjustable promoter.

The second promoter can be inserted into type A, type B and type C plasmids. Such a plasmid has a constant, low copy number and very little or no gene expression at ca. 30 ° C and a substantially increased or uncontrolled plasmid copy capital and gene expression at a temperature in the range of ca. 36-42 ° C. The plasmid preferably has an uncontrolled plasmid copy and a very high gene expression at temperatures above 39 ° C.

The plasmids of the invention can be used as cloning and production vectors, i.e. plasmids which can be used in the field of recombinant DNA technology for the purpose of producing a wide variety of products for technical or medical purposes, in particular polypeptides and proteins or fragments thereof, enzymes and non-proteinous products of reactions of enzymes with a compound in the nutrient medium , low molecular weight products such as hormones, and nucleic acids; products of eukaryotic genes, especially mammalian genes, are particularly interesting. To be useful as a cloning and production vector, it is advantageous, although not essential, that the plasmid has at least one restriction endonuclease that has a unique site that can be cleaved by this endonuclease. The site should be a site that, when inserting a fragment of foreign DNA, allows the resulting recombinant plasmid to replicate autonomously, and which allows the regulator promoter inserted into the plasmid to regulate transcription of the replication region, which, when transcribed from this promoter is increased, leading to a significantly increased or uncontrolled plasmid copy capital. Thus, restriction sites located within the replication region are not allowed as cloning sites, as this would cause the plasmid to lose its replication properties.

The plasmid of the invention is constructed by a method comprising inserting a regulatable promoter into a plasmid in such a way that transcription from that promoter regulates replication and identification of the thus treated plasmid by screening for those plasmids which have a significantly increased or uncontrolled plasmid copy capital when transcription from the promoter is increased. When the regulatable promoter is XPR), it can be inserted by mixing plasmid and phage DNA, cleaving with an appropriate endonuclease, heat inactivating and ligating the mixture and transforming the ligation mixture into a microorganism.

The construction of such plasmids can be done by inserting the promoter at randomly different sites on the plasmid. When screening for plasmids with uncontrolled replication behavior under conditions that ensure increased transcription from the regulatable promoter, the correct insertion is identified. A simple screening for such plasmids includes analysis of the growth characteristics of the plasmid-bearing cells under the various conditions. The amount of plasmid DK 171215 B1 8 DNA in the host cells can be measured in various ways, especially by agarose gel electrophoresis in a manner known per se. This screening method is performed easily and quickly.

In a specific embodiment of the invention which is based on the construction of type A plasmids, the construction method comprises deletion of a portion of a natural regulatory gene and its promoter, in the case of the copB gene from plasmid RI by cleavage with the restriction endonuclease BglII. and inserting a DNA fragment such as a BglII fragment comprising the regulatable promoter at this site.

In another embodiment of the invention embodying type B plasmids, the construction method comprises inserting the adjustable promoter into the plasmid above the natural replication control system, in the case of plasmid R1 derivatives at the BglII site above the copB gene at by partial restriction with BglII, ligation and transformation to the microorganism concerned such as E. coli. Alternatively, the plasmid can be constructed using the type A plasmid with the partially deleted natural replication regulatory gene as starting material and reinserting the natural regulatory gene at its original site, in the case of R1 type plasmids by reinserting the copB gene into the BglII site.

A third embodiment of the invention, which is based on the construction of type C plasmids, comprises inserting - using the type A plasmid as the starting material - a natural regulatory gene into a region of the plasmid where it is not located in the wild-type stem plasmid. case of R1 type plasmids by inserting the copB gene as an EcoRI fragment at the 20 EcoRI site outside the replication region of the plasmid by restriction with EcoRI. ligation and transformation.

A fourth embodiment of the invention comprises inserting a second promoter in addition to inserting the adjustable promoter at an appropriate restriction site, which allows insertion of a structural gene whose expression can be regulated by the second promoter. As mentioned above, this site must not be located within the region of replication of the plasmid.

The plasmids of the invention are mini-plasmids, i.e. they are substantially smaller than their wild-type stem plasmids. They have a size in the area of approx. 2.5-15.0x10® daltons, often 2.5-10.0x10® daltons, usually 4.0-12.5x10® daltons, especially 4.0-5.0x106 daltons.

The uncontrolled replication behavior of the known runaway plasmids apparently depends on the particular bacterial strain to which they are transformed, the culture medium in which the bacteria are grown, and the inserted DNA. This dependence is probably due to the fact that the runaway replication behavior of the known plasmids is partly caused by substitution of a single 9 nucleotide in the copB promoter. which causes a slight increase in the transcription frequency which may be affected by environmental changes in the bacterial species and strain used and / or the culture medium, as well as foreign DNA inserted into the plasmids. The plasmids of the invention do not appear to be correspondingly limited, since the strong foreign promoter, when activated / depressed, increases the transcription frequency to such an extent that such changes are immaterial. Accordingly, these plasmids can be transformed into a wide variety of strains and are also more reliable to handle than the known runaway plasmids.

The present invention also includes plasmids derived from wild-type plasmids other than RI, and / or found in plasmid-carrying microorganisms other than E. coli. It is also possible to imagine the possibility of constructing a plasmid of the type described above that can be held in microorganisms which do not in nature contain plasmids. Several different microbial plasmid replication systems have been shown to be similar in many ways. For example, all known plasmids require transcription for replication to take place. By utilizing the principle of the invention, it is possible to transmit the uncontrolled replication phenotype to other plasmids, so that plasmids already used as cloning vectors and known to function well in a desired microorganism can achieve runaway behavior. Thus, by inserting a regulatable promoter, e.g., λΡχ, into a suitable site in such plasmids in such a way that transcription from that promoter regulates replication, uncontrolled replication will occur under conditions that ensure increased transcription from the promoter.

DETAILED DESCRIPTION OF THE INVENTION

Rl-type plasmids replication control system

The basic replicon

Until recently, the replication control system of R1-1 type plasmids was unknown. It has now been found that the genetic information needed for plasmid R 1 replication and replication control is carried in a 2500 base pair region consisting of three Pst I fragments located within the resistance transfer factor (Molin et al., J. Bact. 138. 1979, pp. 70-79).

This region is defined as the basal replicon and contains four important elements, three genes (copA. CopB and repA) and origin of replication (Molin et al., Microbiology. 1981, pp. 408-411). A physical, genetic and functional map of the basal replicon is shown in FIG. 1. The gene repA encodes a function that is positively needed for replication. The gene product (according to the DNA sequence) is a protein of 278 amino acids and a molecular weight of approx. 33,000 daltons (Rosen et al., Mol. Gen. Genet. 179. 1980, pp. 527-537). This protein has not been uniquely identified, but the ability to form protein from the repA gene has been clearly demonstrated using translational gene fusions with the lacZ gene (Light & Molin, Mol. Gen. Gen. 184. 1981, pp. 56-61). The biochemical function of the RepA protein has not yet been fully investigated, but the RepA protein is thought to be a replication initiation factor.

The gene cqpA encodes a small (80-90 nucleotide long) unstable RNA molecule with a high degree of secondary structure (i.e., it forms hole targets) (Stougaard et al., Proc. Natl. Acad. Sci. USA 78. 1981 , pp. 6008-6012). The CopA RNA is a replication inhibitor; mutations that affect the nucleotide sequence of the CopA RNA may lead to an increased copy number (cop mutants). DNA sequence analysis of copA mutants shows that all mutations are strikingly assembled in a loop region within 10 5 base pairs. These mutations result in a drastic reduction or complete loss of inhibitor activity against the wild-type plasmid. Therefore, it can be concluded that the specificity of the inhibitor is in the loop. It is also striking that in all the copA mutants whose nucleotide sequence is known, a cytosine in the CopA RNA has been replaced with a uracil (or in one case with an adenine). This strongly suggests that the CopA RNA acts by nucleic acid / nucleic acid interaction.

This conclusion is further supported by the fact that even those copA mutants that have lost all CopA activity against the wild-type plasmid retain some CopA activity against themselves. Furthermore, the wild-type CopA RNA most often has decreased inhibitory activity against copA mutant plasmids. As a result, a copA mutant is altered not only in the inhibitor but also in the target, copT: a single base substitution thus leads to what is phenotypically a double mutant.

The 20 copB gene encodes a basal protein of 86 amino acids with a molecular weight of 11,000 daltons. This protein is formed in significant amounts. Deletion of the copB gene and its promoter results in an 8-fold increase in copy number; it is therefore concluded that the copB protein acts as a replication inhibitor, ie. as a negative-acting control element in the regulation of plasmid R1's replication (Molin et al., Mol. Gen. Genet. 181. 1981, pp. 123-130).

A double mutant (copAcopB) is unconditionally fatal to the host microorganism due to the so-called runaway replication. Thus, the two control gene products act synergistically.

Transcriptional activity in the basal replicon

There are three transcriptional initiation sites in the basal replicon, the copB promoter, the repA promoter (Light & Molin, Mol, Gen. Genet. 184. 1981, pp. 56-61) and the promoter for the copA gene 30 (Stougaard et al., op. cit.). The latter transcribes away from the replication initiation site, whereas the former two transcribe towards the replication initiation site. As a result, both strands are transcribed in the copA region. The copB transcript proceeds past the copB gene, and this transcript and the transcript starting at the repA promoter both transcribe the repA gene.

Deletions of the copB gene constructed by restriction with Bell! results in an 8-10 fold increase in copy number of plasmid RI. This is surprising as the copB gene promoter and the proximal portion 5 of the copB structural gene are deleted, resulting in 1) loss of copB activity and derepression of the repA promoter and 2) loss of the copB promoter contribution to overall repA expression . so that the depressed reoA promoter effectively takes over the total repA transcription from the copB promoter. The total effect should only be a 2-fold increase in the transcription frequency of the repA gene, as the repA promoter has a strength that is 2-fold increased over the copB-10 promoter.

This apparent paradox is explained by the activity of the copA gene. CopA RNA is terminated in a normal termination structure with a stem ending in a series of U's. CopA-RNA control of the formation of RepA protein depends on such correct termination. If transcription of the opposite strand is strong (convergent transcription), the termination efficiency is decreased.

Increased transcription from the renA promoter results in an extension of the CopA transcript to ca. 150-200 nucleotides. The extended CopA transcript does not function as a replication inhibitor, ie. it is essentially inactivated, leading to an increased amount of RepA protein and, finally, to an increased plasmid copy (Stougaard et al., EMBO Journal 1. 1982, pp. 323-328).

Replication control 20 The products of two genes, cqpA and copB. negatively controls the replication of the plasmid RI, viz. they act as replication inhibitors. By constructing and analyzing repA-lac fusions, it can be shown that the two cop gene products act by negatively controlling the expression of the repA gene (Light & Molin, Mol. Gen. Genet. 184. 1981, pp. 56-61 ). Therefore, plasmid R1 replication appears to be controlled by the regulation of the formation of a protein which is positively needed for presumably initiation of the plasmid replication.

Using lac gene fusions, it has also been shown that the copA and copB products have different targets. The target of the CopB protein is the renA promoter. where it works by inhibiting the transcriptional initiation. However, the CopB protein is unable to interfere with transcription initiated above the repA promoter. The CopA RNA does not affect transcription, but acts at a post-transcriptional level, i.e. by inhibiting translation of RepA mRNA.

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Runaway mutant replication

The replication system of the plasmids described in published European Patent Application No. 78101877.5 is substantially the same as that described above with respect to the wild-type R1 plasmids. Recent research by the present inventors has now partially determined the cause of the existence of temperature-dependent plasmid mutants with runaway replication behavior. As explained above, increased transcription toward the repA gene can lead to inactivation of the CopA RNA molecule. In the runaway mutant plasmids described in the above patent application, the copB promoter is mutated by substitution of a single nucleotide in a sequence, as shown: 10 a) Wild-type plasmid 5 '-... TTCTCAAGTCGCT ... -3' b) Runaway -mutant 5 '-...... TTCTCAAGTTGCT ...- 3'

The italicized letters show the nucleotide substitution. The region shown is the RNA polymerase binding site.

This mutation was found to cause increased transcription from the copB promoter. especially at higher 15 temperatures.

Replication of plasmids carrying a regulatable promoter

To construct the plasmids of the present invention, the recently acquired knowledge of the present inventors on the wild-type R1 plasmids replication system has been utilized. However, the replication of the plasmids of the present invention is not made dependent on mutations in the plasmid, as is the case with the runaway plasmids described in published European Patent Application No. 78101877.5.1, a foreign promoter has been inserted such that transcription of the replication region is at least partially controlled from this promoter. The promoter may be adjustable by adaptations in the conditions under which the host microorganisms are grown, and the regulation of the promoter activity may be carried out in various ways such as by changing the culture temperature or the composition of the growth medium.

When the regulatable promoter is a promoter selected from temperature dependent promoters, it is preferably a promoter derived from bacteriophage λ such as XPR or XPL, especially XPR. In phage λ, the PR promoter is controlled by the cl repressor, which in accordance with the principles of the invention is obtained from phag λ together with XPR and inserted into a plasmid in the form of a BglII fragment (cf. the plasmid map shown in Figs. 4-11). ). The wild type cl repressor is not in itself temperature sensitive, so to make the replication system adjustable by adjustments in the culture temperature, the specific cl repressor used (cf. the examples below) is temperature sensitive encoded by the mutant allele cl0t. "(Sussman & Jacob, Compt.

Fuck. Acad. Sci. 254. 1962, p. 1517). The repressor is active, ie. it represses the XPR promoter, 5 at low temperatures such as a temperature of approx. 30 ° C, whereas at higher temperatures such as temperatures above ca. 39 ° C is denatured, causing inactivation of repressor function and derepression of XPR. As the inserted promoter is very strong, this results in a drastically increased transcription towards repA. which in turn leads to uncontrolled plasmid replication.

Alternatively, the regulation of the promoter may be carried out by chemical means, i.e. by adjusting the composition of the medium in which the host microorganisms are grown, either by adding a chemical that induces the promoter, by causing inactivation of the repressor which controls promoter activity, and as a result, derepression of the promoter or by ensuring the formation of a promoter inducing metabolite leading to activation of the promoter. A specific example of a chemically inducible promoter is the E. coli deo promoter. which is subjected to negative control by two chromosomal repressor proteins, namely the products of the deoR and cvtR genes. The deo promoter can be depressed by adding cytidine to the growth medium, since cytidine acts upon affinity to the repressor and causes a conformational change thereof to cease to act as a repressor. In addition, positive control of the expression ice from the deo promoter can be exerted via the effect of catabolite repression. Activation of the crp (catabolite repressor protein) protein by cyclic AMP (cAMP) leads to stimulation of deo promoter activity; high cellular levels of cAMP are known to be associated with low levels of glucose or lack of glucose in the growth medium, which can be achieved by adding only a limited amount of glucose to the medium, & an amount of approx. 0.01% along with another carbon source such as glycerol or succinate. When the glucose is consumed by the cell, the resulting increase in the intracellular level of the cAMP activates the deo promoter. causing an increased frequency of replication of the plasmid carrying the promoter. Thus, if the deo promoter is inserted above the copB gene into a plasmid RI derivative, induction of the promoter leads to increased or completely uncontrolled replication. The uncontrolled replication behavior can be reversed by adding glucose to the growth medium, so that the plasmid copy number is slowly reduced to the pre-induction level. Another example of a chemically controllable promoter is the lac promoter, which is induced by the presence of lactose in the nutrient medium, resulting in increased or uncontrolled replication. Also in this case, the process can be reversed by dosing the amount of lactose added to the medium so that it is eventually consumed by the cells.

Insertion into the correct position and orientation of a DNA fragment carrying a regulatable promoter instead of part of a natural replication regulatory gene, fe instead of the BglII-B1 fragment which covers part of the conB unit on plasmid R 1 leads to a plasmid derivative whose replication frequency is at least partially controlled by the inserted promoter (a type Λ plasmid).

Insertion into the correct orientation of a DNA fragment carrying a regulatable promoter 5 immediately above the natural replication regulatory gene, which is the copB gene in the case of plasmid R1 derivatives, leads to a plasmid with slightly different replication properties (a type B plasmid).

As mentioned above, type B plasmids lacking the pair region are lost at a frequency of approx. 1% per generation. Without selection for the presence of the plasmids in a culture, the plasmid is eventually lost from cells grown at ca. 30 ° C. In order to stabilize the plasmid, it is possible to insert a DNA fragment carrying a pair of regions from, for example, a wild type R1 plasmid into a type B plasmid, resulting in complete stability (no loss) of the plasmid from cells grown under similar conditions . Plasmid stability can be determined by first culturing cells containing type B plasmids into which lac genes are inserted as well as the pair region and as control cells containing type B plasmids carrying the lac genes but not the pair genes. the region, selectively on plates containing an antibiotic (against which the plasmids mediate resistance) to ensure the presence of both types of plasmids in the parent colonies. Samples of both colonies are then ironed onto plates containing no antibiotic, allowing cells to grow without selection for a number of generations (cell divisions) such as & 25 generations. Finally, cells from both of the resulting colonies are plated out on McConkey lac tose indicator plates, where the cells that originally contained plasmids carrying the pair region form red colonies, showing that the plasmids have been preserved, whereas the control cells, which originally contained plasmids without para. the region forms both red and colorless colonies, which means that the plasmids are lost from some of the cells during the selection-free period. Thus, it has been shown that plasmids into which the pair region has been inserted have become completely stable (cf. Example 11). This stabilizing effect is particularly important if an inserted DNA fragment causes a decrease in the growth rate of the plasmid-bearing cells compared to plasmid-free cells.

Finally, insertion of a DNA fragment carrying the native replication regulatory gene, such as the copB gene on plasmid RI, into a site outside the replication region of a type A plasmid results in a plasmid having particular replication properties (a type C plasmid).

The desirability of plasmid stability described above with respect to type B plasmids is even more pronounced in the case of type C plasmids since, as mentioned above, these have an even lower copy number at the low temperature and therefore are lost with a DK 171215 B1 higher frequency than type B plasmids if they lack the pair region. The Type C plasmids can be similarly stabilized by inserting the pair region into the plasmids in the case of plasmids where it is not already present.

According to a particular aspect of the invention, in addition to the first adjustable promoter, the plasmid carries a second promoter located outside the replication region, which regulates the expression of an inserted foreign structural gene, which encodes, for example, a desired polypeptide. This additional promoter may be, for example, the cl promoter, which is located on the fragment which further carries the XPR promoter and the cl gene, which cl promoter is controlled by the cl gene product and transcribes in the opposite direction of λΡβ. Below the cl promoter, it is possible to insert a structural gene whose expression can be regulated by this promoter. For example, lac genes (known as lac operon) lacking the lac promoter have been inserted below the d gene, and the gene product amplification obtained suggests that the transcription of the lac genes is at least partially controlled by the cl promoter (see Example 8). Hereby, it was demonstrated that it is possible to use the plasmids of the invention as cloning vectors for the insertion of a foreign gene.

The 15 cl promoter has the advantage that it can also be regulated by the cl repressor so that amplification of the gene product occurs simultaneously with amplification of plasmid DNA.

The second promoter may also be an λ promoter such as XPL, or it may be a recA promoter. When the second promoter is XPL, it can be inserted into the plasmid as a phage λ BamHI-BglII fragment, thereby forming an appropriate cloning site (BamHD for insertion of DNA fragments generated by several restriction enzymes such as BamHI). Sau3A and Bell XPL can also be inserted on a suitable DNA fragment from a plasmid carrying the promoter. XPL is advantageous as a second promoter because it is also controlled by the temperature sensitive c1 repressor so that the amplification conditions are the same.

On the contrary, if the promoter is recA, it can be inserted into the plasmid of the invention as a BamHI fragment from, for example, pBEU14 (Uhlin and Clark, J. Bact. 148. 1981, pp. 386-390). This promoter functions in various ways depending on the microbial strain to which the recA promoter-carrying plasmid has been transformed. In a recA strain, the recA is controlled by the lexA repressor, which is located on the chromosome of the microorganism. For example, by a temperature induction of uncontrolled replication, the plasmid's copy number and thus the number of recA promoters are gradually increased. As the lexA repressor is only formed in small amounts and functions by binding tightly to the recA promoter region, the amount of lexA repressor present will gradually be titrated as the copy number increases. Thus, a gradual derepression of recA takes place and as the plasmid copy number becomes higher, the transcription from recA increases accordingly.

The plasmids of the invention as cloning and production vectors 16 In their capacity as cloning and production vectors, the plasmids according to the invention have a number of characteristic properties: 1) The plasmids are small with a molecular weight of approx. 4.0-12.5x10® daltons. The small size facilitates handling and improves transformation efficiency.

2) The plasmids have a number of unique sites for restriction endonucleases. The term "unique" means that for a specific restriction endonuclease, the plasmid has one and only one site which can be cleaved by the enzyme.

As already mentioned, restriction sites located within the replication region are not allowed as cloning sites. Thus, in the plasmids described in the Examples below, the restriction sites SalI and PstI are not suitable as cloning sites as they are located in the replicon of the plasmid, whereas restriction sites EcoRI and BamHI are not located in this critical region and thus provide useful sites for introduction. of foreign DNA. If an appropriate restriction site is not found on the plasmid itself, it can be constructed by inserting a small DNA fragment (known as a linker) containing such a site into the plasmid as described in Example 4.

However, to ensure the expression of the inserted structural gene, it is also necessary to have an appropriate translation start signal (ribosome binding site) located above the structural gene itself but below the other promoter. One way to ensure the presence of the ribosome binding site is to insert different synthetic DNA fragments (e.g., 20 as linkers), each of which carries the translation start signal readily for a different structural gene. Alternatively, a DNA fragment carrying a gene already known to be expressed in microorganisms can be inserted into the runaway cloning vectors in toto. thereby ensuring the presence of the correct translation start signal.

3) While, as mentioned above, it is advantageous to have as small a cloning vector as possible, it is convenient for many purposes that the cloning vector carry genes for expression of a so-called marker useful for identification and / or cell selection. , which carries the plasmid. The most useful marker is antibiotic resistance, & ampicillin resistance, as after treatment for transformation of a recombinant plasmid into a microbial host, it allows an easy counter-selection of microorganisms that have not received the recombinant plasmid. Another marker found in all the plasmids described in the examples below is λ immunity encoded by the cl gene. A further useful property of plasmids of the present invention may be the presence of a gene that mediates a selectable phenotype in which a unique restriction site is located. Insertion of a DNA fragment at this site causes insertion inactivation of the gene. An example of this is described in Example 9 with respect to the gene mediating the Lac + phenotype, and Example 12 with respect to the gene encoding chloramphenicol resistance. When it is desired to introduce a marker, & antibiotic resistance, into a plasmid of the invention to be used as a cloning vector, this can be done by transposition or by inserting a foreign DNA fragment in a manner known per se. An example of such a transposition is shown in Example 4.

Cultivation

The present invention also relates to a method for producing a gene product of 10 plasmid DNA, which comprises culturing microorganisms carrying a plasmid as described above in which an adjustable promoter has been inserted in such a way that transcription regulates replication. , in particular in such a way that increased transcription from this promoter leads to a substantially increased or uncontrolled plasmid copy, the method comprising at least one culture period under conditions which ensure increased transcription from the adjustable promoter leading to an increased level of the gene product that is positively needed for replication, which increased level results in a plasmid replication rate which is higher than the host cell growth rate, leading to an increased plasmid capital, which will usually be increased by a factor of 25 to 5 after cell division. 1000 or uncontrolled pla forging copy capital, and extracting a gene product of the plasmid from the 20 microbial culture. Cultivation itself is conveniently carried out using conventional techniques, including conventional nutritional media, which are known to be optimal for the particular microbial species. The recovery of the gene product is also carried out according to well-known methods which are adapted to the identity and properties of the particular manufactured gene product, the characteristics of the host microorganism, etc. The present invention further relates to a method for culturing the host microorganism to which the plasmid of the invention has been transformed under conditions. which results in a constant, low plasmid capital in the grafting and propagation steps until the desired number of cells has been obtained, after which the host microorganism is grown under conditions leading to a substantially increased or uncontrolled plasmid capital.

Thus, culture can initially be carried out under conditions where any transcription from the promoter does not significantly affect the copy number of the plasmid, after which a substance which induces promoter activity is added to the culture medium in an amount which leads to significantly increased or uncontrolled replication. If desired, these culture conditions can be reversed after a period of time sufficient to achieve a substantial amplification of the plasmid but short enough to avoid any significant damage to the microbial culture, removing the substance from the culture medium, leading to a slow reduction of the culture medium. the plasmid copy number until eventually 18 DK 171215 B1 Decreases the pre-induction level In this context, the term "multiple" need not mean a complete absence of the substance in question from the culture medium, but only a reduction in the concentration of the substance to an amount where it no longer exists. has any effect.

In an alternative method using a promoter that is inhibited rather than induced by a particular substance, the microbial culture can first be grown in the presence of the substance that inhibits the activity of the particular promoter used and then the concentration of the substance is decreased , for example, by diluting the culture medium or by gradually consuming it by the cells, to an extent which causes activation of the promoter, thereby obtaining a substantially increased or uncontrolled plasmid copy capital. If desired, the culture conditions can be reversed after a period of time sufficient to achieve substantial amplification of the plasmid, but sufficiently short to avoid any significant damage to the microbial culture by adding the substance which inhibits promoter activity to an amount which leads to a slow decrease in plasmid copolymer until it reaches the pre-induction level.

The conditions under which the microorganism is grown may also include at least one culture period at or near a temperature at which the plasmid copy number is substantially increased or uncontrolled.

If the adjustable promoter is temperature dependent or regulated by a temperature sensitive factor, it is preferred that the propagation of the microorganism transformed with a plasmid of the invention to a production size culture be carried out at or near the temperature at which the plasmid has a constant, low copy capital to avoid any inhibition of microbial growth due to increased plasmid and gene product concentration. Thereafter, the temperature can be changed to a temperature at which the plasmid has a substantially increased or uncontrolled copy number. After an appropriate production period, often until the growth of the microorganism has been inhibited due to the formation of plasmid DNA and / or gene products from the plasmid, the recovery of the gene product is made. Depending on the particular conditions, it may be desirable to carry out the production culture continuously at a temperature approaching only the temperature at which the plasmid copy number is substantially increased or uncontrolled, so that the survival and continued growth of the host microorganism are possible simultaneously with the formation of increased amounts of a gene product. of the plasmid (which can then be continuously or intermittently recovered from the culture in a manner known per se).

Alternatively, the microorganism can be propagated to a culture of production size at the temperature at which the plasmid copy number is constant, whereby the temperature is changed to a temperature at or near the temperature at which the plasmid copy number is substantially increased or uncontrolled, and this temperature is maintained for a period which is sufficient to achieve substantial amplification of the plasmid, but sufficiently short to avoid any substantial damage to the microbial culture, after which the temperature is again changed to a temperature which ensures a low, constant plasmid capital, and production culture is continued at this temperature. causing a slow decline in the plasmid copy number until it reaches the level of initiation. This culture method is particularly important when the foreign gene inserted into the plasmid originates from an organism that is not viable at a temperature above ca. 30 °, so that gene products from such a gene can be denatured at higher temperatures. Furthermore, the yield of gene product is usually higher when this culture method is used (cf. Example 16; compare Uhlin et al., Gene 6. 1979, pp. 91-106, Table II).

The temperature at which the plasmid copy number is substantially increased or uncontrolled may be higher than the temperature at which the plasmid has a constant low copy number. This higher temperature may be a temperature in the range of approx. 36-42 ° C.

DRAWING EXPLANATION

BRIEF DESCRIPTION OF THE DRAWINGS FIG. 1 is a schematic representation of the replication region of the wild-type plasmid R1; 2 is an explanation of the symbols used in the following figures; and FIG. 3-23 show restriction maps of the plasmids described in the Examples.

FIG. 1 schematically shows the basal replicon from plasmid RI, where the shaded areas denote the translated regions, the unfilled regions denote the transcripts, and the dashed lines denote possible transcripts. In the circle, the enlargement of the hairpin loops shows the high degree of secondary structure of the CopA RNA. Ori denotes the replication initiation site. The horizontally arranged arrows denote the direction of transcription.

FIG. 2 shows an explanation of the symbols used in the following figures, where A denotes DNA from R1-type plasmids; B represents DNA from bacteriophage λ (EDA4); C represents DNA from the Tn3 transposon (from EDATn3); D represents DNA from plasmid pBR322; E represents DNA from plasmid pMC871; F represents DNA from plasmid pVH1424; G represents DNA from plasmid pSKS104; H denotes structural genes and important sites; You denote a promoter with an indication of the direction of transcription; and J represents a measure of the size of the plasmids. The unfilled region represents DNA from plasmid pBEU14.

FIG. Figures 3-23 show linear restriction maps of the plasmids described in the Examples in which the genotypes of the R1-type plasmids shown are shown over the horizontal line. Thus, DK 171215 B1 20 denotes copB a gene encoding a polypeptide that represses transcription from the repA promoter between the copB and copA genes: copA denotes a gene encoding an RNA molecule that inhibits translation of RepA mRNA; repA represents a gene encoding a protein necessary for plasmid R1 replication; ori denotes the vegetative replication initiation site; 5 aphA represents a gene that encodes kanamycin resistance; bla denotes a gene that encodes ampicillin resistance; lacA. lacY and lacZ represent the inserted lac operon, of which lacA encodes transacetylase, lacY encodes permease, and lacZ encodes β-galactosidase; denotes a gene encoding a temperature-sensitive repressor of the XPR promoter; tnpR and tnpA denote genes encoding Tn3 transposition functions; pair represents the region responsible for plasmid distribution, recA represents a gene encoding a recombination protein; and PrecA denotes the recA promoter. Below the horizontal Urge, the sites of the restriction enzymes are shown where P represents PstI; B2 represents BglH: Sj represents Sal; E represents EcoRI: H3 represents HindHI; Bj represents Bam HI; S denotes Smal: B2Bj denotes a fusion of a BglII site and a BamHI site; and C represents Clal. In FIG. 11, the plasmid shown is drawn on the same scale 15 as the plasmids in the other figures, and the inserted insert denotes the inserted lac genes.

MATERIALS AND METHODS

The strain of Escherichia coli K-12 used was CSH50 (Δ pro-lac, rosL: cf. J. Miller, Exnerimants in Molecular Genetics. Cold Spring Harbor Laboratory, Cold Spring Harbor, New York, 1972). Several plasmids (Table 1) and bacteriophages (Table 2) were used.

The experimental techniques used were standard techniques used in the field of microbial genetics (J. Miller, Experiments in Molecular Genetics. Cold Spring Harbor, New York, 1972) and genetic engineering (Davis, Botstein and Roth, A Manual for Genetic Engineering: Advanced Bacterial Genetics. Cold Spring Harbor, New York, 1980).

All cells were grown in LB medium (Bertani, J, Bact. 62. 1951, p. 293) or in A + B minimal medium (Clark and Maaløe, J. Mol, Biol. 23. 1967, p. 99) and the plates used were LA plates containing LB medium and 1.5% agar. Preparation and analysis of plasmid DNA was performed using "dye buoyant" density gradient centrifugation according to that of Stougaard & Molin, Anal. Biochem, 118. 1981, p.191 described. Labeling of DNA and preparation of lysates to determine plasmid content was performed according to Molin et al., J. Bact. 138. 1979, p. 70. The relative content of plasmid DNA was measured as the amounts of labeled thymidine present in the plasmid band relative to the amounts of labeled thymidine present in the chromosome band in the gradients.

DK 171215 Pg 21

The restriction endonucleases used in the Examples were used according to the manufacturer's instructions. Partial restriction was performed with a 10-fold dilution of the enzymes.

In addition, the following screening methods were used: 1. Screening for λ immunity (immX +) 5 In all of the examples described, the clgg ^ mutant allele is used. Plasmids carrying this gene render the K_coli host cell resistant to infection with lytic λ phages at 30 ° C. For TU selection of resistant cells, more than 108 Ab2 phage particles are added to the plates before the bacteria are ejected. Surviving colonies are further tested for their plasmid content.

2. Screening for codB + 10 Genetic fusions between the repA promoter and the lac operon from which the lac promoter has been deleted are constructed and inserted into a pl5 plasmid (pGA46). A resulting hybrid plasmid, pJL217, is responsible for a Lac + phenotype when carried by a Δ lac E. coli host strain.

The Lac + phenotype is easily detected on McConkey lactose indicator plates (red colonies). The TST site of a copB + gene in cells carrying pJL217 results in a Lac 'phenotype which appears as white colonies on the indicator plates. Thus, copB + hybrids are readily recovered as Lac "colonies when plasmid DNA is transformed into E, coli δ lac cells containing the plasmid pJL217.

TABLE 1

Plasmids used 20

Plasmid Source pKN1562 S. Molin et al., J. Bact. 138. 1979, pp. 70-79.

pBR322 F. Bolivar et al., Gene 2. 1977, p. 95.

25 pJL217 Constructed by inserting a Sau3A fragment from pKN1562 carrying the repA promoter into the Bol site of pJL207, a hybrid plasmid carrying lac genes without the lac promoter.

pOUl6 Light & Molin, Mol. Gen. Genet. 184, 1981, 30 p.57.

22 DK 171215 B1 pGA46 An & Friesen, J. Bact. 140, 1979, pp. 400-407.

pMC903 Casadaban et al., J. Bact. 143. 1980, p.971.

pMC871 Casadaban et al., od. cit .. pp. 971.

pVH1424 Constructed by P. Valentin-Hansen by insertion of a Sau3A fragment carrying the deo promoter from plasmid pVH17 (Valentin-Hansen et al., EMBO J "1982, p. 317) at the BamHI site of plasmid pMC1403 (Casadaban et al., op. cit. p. 971).

10 pSKS104 Constructed by M. Casadaban by insertion of a PvuII fragment containing the lac promoter and translation initiation region of pMl3mp7 (Messing et al., Nucleic Acids Res. 9).

1981, p. 309) in the Sma I site of pMC1403, followed by homologous recombination between the lacZ segments.

pKN184 Nordstrim et al., Plasmid 4, 1980, p. 322.

pJL3 Molin et al., Mol. Gen. Genet. 181, 1981, pp. 123-130.

20 pVH1451 Valentin-Hansen et al., Op. Cit.

pBEU14 Uhlin & Clark, J, Bact. 148. 1981, pp. 386-390.

pMC1403 Casadaban et al., J. Bact. 143. 1980, p.971.

TABLE 2 25 Bacteriophages used

Phag Source EDA4 Dempsey & Willetts, J. Bact. 126. 1976, p. 166.

30 EDXTn3 Nordstrom, Molin, Aagaard-Hansen, Plasmid 4.

1980, pp. 215-227.

23 DK 171215 B1

Stamplasmider

Plasmid pKN1562 is a derivative of plasmid Ri, constructed essentially as described in Molin et al., "Clustering of Genes Involved in Replication, Copy Number Control, Incompatibility, and Stable Maintenance of the Resistance Plasmid Rldrd-19".

J. Bact. 138, 1979, pp. 70-79, with the exception of a few modifications added by later research. pKN1562 has a molecular weight of 6.9x106 daltons and the following genotype: copA-k copB +. repA +. aphA-t-, Δ pair (cf. Fig. 3). It communicates resistance to kana-mycin. The plasmid has the wild-type plasmid replication properties, a copy number of 3–5 per. fast-growing cell and is lost at a frequency of 1% per cell. generation due to 10 deficiency of the pair gene (the gene for distribution).

By restriction of pKN1562 with BglII to delete part of the copB gene and ligation of the staple ends, another plasmid, pJL20, is constructed. The molecular weight of this plasmid is c 6.7x10 daltons and its genotype is as follows: copA +. Δ copB. reoA +. aphA +. Δ par.

The plasmid has a copy number of 25-40 per. fast-growing cell and the plasmid is not lost.

This plasmid also mediates resistance to kanamycin.

Plasmidbetegnelse

It should be noted that the plasmids designated pJEL ... in the examples and in the drawing in the base application have been renamed pOU ... in the present application.

EXAMPLE 1 Construction and characterization of nOU51 DNA from plasmid pJL20 and phage EDX4 was prepared according to standard methods. Plasmid and phage DNA were mixed at a final concentration of 20 pg / ml of each and at a final volume of 100 μΐ, digested with BglII for 30 min, heat inactivated at 70 ° C for 10 min, and ligated with T4 DNA ligase overnight at 15 ° C. Aliquots of the ligation mixture were transformed into E. coli strain CSH50. Transformants were selected on plates containing 50 µg / ml kanamycin, upon which a suspension of ca. 109 Xb2 particles had been ironed out. The plates were incubated for 20 hours at 30 ° C.

Surviving colonies were again isolated and tested for resistance to Ab2 by chewing iron at 30 ° C, growth at 42 ° C by streaking on kanamycin plates at 42 ° C and 30 for plasmid molecular weight by producing plasmid DNA and assay on agarose gels.

24 DK 171215 B1

Hereby a plasmid, pOU51, which mediates resistance to kanamycin and λ infection was found, makes the host bacterium temperature sensitive to growth and has an insertion of phage DNA into pJL20 corresponding to approx. I, 6x106 daltons. The total molecular weight of the plasmid was 8.4x106 daltons. These properties are all carried by the plasmid, as new E. coli recipient strains all have the phenotypic properties of the plasmid when transformed with pOU51 DNA.

DNA was prepared from pOUS1 and the plasmid mapped with restriction enzymes by purification of the plasmid DNA, cleavage with restriction enzyme (s) and analysis of the resulting fragments by agarose gel electrophoresis (cf. Fig. 4). Hereby, the 10 inserted 1,6x106 daltons long fragment was identified as a BglII fragment from the phage λ carrying the cl repressor gene (responsible for λ immunity at 30 ° C) and the λΡ promoter, and the orientation of the inserted fragment was determined. being as shown in FIG. 4. It is also apparent from the restriction map that pOU51 has a unique site for the restriction endonuclease EcoRI. which allows the plasmid to be used as a cloning vector.

The effect of the plasmid on cell growth was investigated by growing a culture in medium at 30 ° C. At a time when the growth was exponential, the temperature was changed to 42 ° C and the cell growth monitored (determined spectrophotometrically). The culture ceased to grow within 1.5-2 hours of growth at 42 ° C.

The content of plasmid DNA was measured as described under MATERIALS AND METO-20 DER from 10 ml cultures grown in A + B minimal medium enriched with 0.2% glucose and 1% casamino acids. One culture of 10 ml was labeled with 50 µCi of 3 H-thymidine at 30 ° C, the other receiving the isotope immediately after a temperature change to 42 ° C until the cells ceased to grow. The relative content of plasmid DNA was 2% at 30 ° C, corresponding to 25-40 plasmid copies per cell. At 42 ° C the relative content was more than 25 50% corresponding to over 1000 plasmid copies per cell.

The properties of this plasmid are shown in Table 5.

The strain E. coli CSH50 / pQU51 is deposited in the Deutsche Sammlung von Microorganism, Grisebachstrasse 8, D-3400 Gottingen, which is hereinafter abbreviated to DSM, under deposit number 2467. Landfill of this strain and other deposits of microorganisms mentioned in this description has been made in accordance with the Budapest Treaty.

EXAMPLE 2

Construction and characterization of dOU53 25 DK 171215 B1

Using the plasmid described in Example 1 as the starting material and by partial restriction of pOU51 with HindIII, the title plasmid was constructed from which the gene for encoding kanamycin resistance had been deleted but which had retained the gene for encoding λ immunity. The plasmid was transformed into E. coli strain CSH50. pOU53 had a molecular weight of 3.2x106 daltons and the following genotype: cqdA +. Δ codB. repA +. Δ pair, immX +, Δ aphA.

The plasmid was mapped with restriction enzymes as described in Example 1 (cf. Fig. 5).

10 It is apparent from the restriction map that pOU53 has a unique site for the restriction endonuclease EcoRI, which allows the plasmid to be used as a cloning vector.

To detect the presence of the desired plasmid in the host cells, cell cultures were grown on plates containing Xb2 phage particles, as described above, to test for resistance to λ infection. The cells were further tested for sensitivity to 15 kanamycin by replicating plaque colonies on plates containing 50 µg / ml kanamycin. To test for temperature-sensitive growth, cells were plated on plates at 42 ° C and grown as described in Example 1.

Since the content of plasmid DNA was measured in the manner described in Example 1, the cells were found to contain 20-40 plasmid copies at 30 ° C and more than 1000 plasmid copies 20 at 42 ° C. There was no loss of the plasmid.

The properties of this plasmid are shown in Table 5.

The strain E. coli CSH50 / pOU53 is deposited in the DSM under the deposit number 2468. EXAMPLE 3

Construction and characterization of pOU56 25 In vivo insertion of the Tn3 transposon from an X :: Tn3 phage containing the genes for transposition and ampicillin resistance (β-lactamase) (Nordstrom, Molin and Aagaard-Hansen, Plasmid 4. 1980, p. 215-227), in pOU53, the title plasmid was constructed. Plasmid DNA was isolated from a strain carrying the λ Tn3 phage in the chromosomes and plasmid pOU53. The plasmid DNA was transformed into E. coli strain CSH50 by selecting 26 amp 171215 B1 for ampicillin resistance and λ immunity. pOU56 had a molecular weight of 6.5x106 daltons and the following genotype: copA +. Δ copB. repA +. Δ aphA. Immunol. bla + (:: Tn3).

The plasmid was mapped with restriction enzymes as described in Example 1 (cf. Fig. 6). The restriction map shows where the Tn3 transposon has been inserted into pOU56, and further shows that the plasmid has a unique site for each of the restriction endonucleases EcoRI and BamHI. While the plasmid is useful as a starting material for producing the plasmid pOU57 (cf. Example 4 below), it is not preferred as a cloning vector, as antihiotic resistance can be transmitted to other bacteria due to the presence of the transposition gene.

10 To detect the presence of the desired plasmid in the host cell, cell cultures were grown on plates containing Ab2 phage particles as described above to test for resistance to λ infection. The cells were further tested for ampicillin resistance on plates containing 50 µg / ml ampicillin. The temperature-sensitive growth test was performed in the manner described above.

15 pOU56 was found to have the same replication properties as pOU53.

The properties of this plasmid are shown in Table 5.

The strain E. coli CSH50 / pOU56 is deposited in the DSM under the deposit number 2469. EXAMPLE 4

Construction and characterization of pOU57 (a type A plasmid) 20 To eliminate the possibility of transposition of Tn3 from plasmid pOU56 as it carries the entire transposon, the plasmid was cleaved at the BamHI and EcoRI sites to delete the transposition gene, whereas the gene , which encodes β-lactamase, was preserved.

To join the staple ends, a small BamHI-EcoRI fragment from plasmid pBR322 was inserted, thus forming the plasmid pOU57. The plasmid was transformed into E. coli 25 strain CSH50. pOU57 had a molecular weight of 4.2x106 daltons and the following genotype: copA +. Δ copB. repA +, Δ par. Δ aphA. imm λ +, bla + (ΔΤη3).

The plasmid was mapped with restriction enzymes as described in Example 1 (cf. Fig. 7).

The restriction map shows that pOU57 has unique sites for each of the restriction endonucleases BamHI and EcoRI. making it useful as a cloning vector.

27 DK 171215 B1

To detect the presence of the desired plasmid in the host cell, cell cultures were grown on plates containing Xb2 phage particles as described above to test for resistance to phage infection. The cells were further tested for ampicillin resistance in the manner described above. The molecular weight of the plasmid was determined by agarose · 5 gel electrophoresis. The temperature-sensitive growth test was performed in the manner described above.

pOU57 was found to have the same replication properties as pOU53.

The properties of this plasmid are shown in Table 5.

The strain E. coli CSH50 / pOU57 is deposited in the DSM under the deposit number 2470.

EXAMPLE 5

Construction and characterization of pOU71 (a type B plasmid)

Using the plasmid described in Example 4 as the starting material and by inserting a BglII fragment from pKN1562 which included a portion of the copB gene in the BglII site of pOU57, the title plasmid was constructed, which contained both the cI-APR-15 repressor promoter system as the wild-type gene for the copB inhibitor from R1-type plasmids. The plasmid was transformed into E. coli strain CSH50. The molecular weight of pOU71 was 4.3x106 daltons and the genotype was as follows: copA-k copB-k repA +. Δ par. Δ aphA. imm λ +, bla +.

The plasmid was mapped with restriction enzymes as described in Example 1 (cf. Fig. 8).

20 The restriction map shows that pOU71 has the same unique restriction sites as pOU57.

To detect the presence of the desired plasmid in the host cell, cell cultures were tested for resistance to λ infection and ampicillin resistance in the manner described above. The plasmids were then screened for the presence of the copB gene by the method described in the section "Screening for copB +" (page 27 of this application). The temperature-sensitive growth test was performed in the manner described above. Finally, the number of plasmid copies at 30 ° C was determined by radiolabelling and gradient centrifugation.

28 DK 171215 B1

Since the content of plasmid DNA was measured in the manner described in Example 1, the cells were found to contain 3-5 plasmid copies at 30 ° C and over 1000 plasmid copies at 42 ° C.

At 30 ° C, the plasmid was lost at a frequency of 1% per day. generation.

The properties of the plasmid are shown in Table 5.

The strain E. coli CSH50 / pOU71 is deposited in the DSM under the deposit number 2471. EXAMPLE 6

Construction and characterization of pOU73 (a type C plasmid)

Using the plasmid described in Example 4 as the starting material and by inserting an EcoRI fragment from the pOU16 carrying copB gene into the EcoRI site of pOU57, the title plasmid was constructed which had the gene for the copB inhibitor as well as the cI-XPR repressor. sor / promoter system. The plasmid was transformed into E. coli strain CSH50. pOU73 had a molecular weight of 4.4x106 daltons and the following genotype: cop A +. CopB +. repA +.

Δ nar. Δ aohA. imm λ +, bla +.

The plasmid was mapped with restriction enzymes as described in Example 1 (cf. Fig. 9).

15 It appears from the restriction map that the copB gene has been inserted into an area of the plasmid where it does not naturally belong. Furthermore, it appears that the plasmid has a unique site for the restriction endonuclease BamHI. so that it can be used as a cloning vector.

The same screening methods as for pOU71 were used.

Since the content of plasmid DNA was measured in the manner described in Example 1, the cells were found to contain 0.5-1 plasmid copy at 30 ° C and over 1000 plasmid copies at 42 ° C.

At 30 ° C, the plasmid was lost at a frequency greater than 5% per day. generation.

The properties of this plasmid are shown in Table 5.

The strain E. coli CSH50 / pOU73 is deposited in the DSM under the deposit number 2472.

EXAMPLE 7

Construction and characterization of dOU75 (a type B plasmid) 29 DK 171215 B1

Using the plasmid described in Example 5 as the starting material and by partial restriction of pOU71 with HindIII, the title plasmid was constructed from which the Eco RI site 5 had been deleted. The plasmid was transformed into E. coli strain CSH50. pOU75 had a molecular weight of 4.2x106 daltons and the following genotype: copA +. copB +, repA +. Δ par.

Δ aphA. imm λ +, bla +.

The plasmid was mapped with restriction enzymes as described in Example 1 (cf. Fig. 10).

From the restriction map, it appears that the deletion of the HindIII fragment causes removal of the EcoRI site at pOU71.

To detect the presence of the desired plasmid in the host cell, cell cultures were tested for resistance to λ infection and ampicillin resistance as well as temperature-sensitive growth in the manner described above. The plasmid was also tested for loss of the EcoRI site by restriction with the enzyme.

15 pOU75 was found to have the same replication properties as pOU71.

The properties of this plasmid are shown in Table 5.

The strain E. coli CSH50 / pOU75 is deposited in the DSM under the deposit number 2473.

EXAMPLE 8 Gene Product Amplification The plasmid pMC903 (Casadaban et al., J. Bact. 143. 1980, p. 971) was digested with the restriction enzymes BamHI and Belli to generate a BamHI-BglII fragment carrying the lac genes. Plasmid pOU71 was digested with BamHI. and the above-obtained Bam HJ-Bgin fragment was inserted at this site as shown in FIG. 11 followed by ligation with T4 DNA ligase to generate the plasmid pOU106, which was transformed into 25 E. coli strain CSH50. The cells were plated on McConkey lactose indicator plates (cf.

J. Miller, Experiments in Molecular Genetics. Cold Spring Harbor, 1972), containing 50 µg / ml ampicillin, and incubated at 30 ° C to select for red colonies (Lac +).

The temperature was then changed to 42 ° C and the amplification of the gene product (β-galactosidase) was measured as shown in Table 3. The measurement was performed according to that of J. Miller, od. rit., described method.

TABLE 3

Anplificatian of gene product

Plasmid of the Invention Known Plasmid (pKN410)

Minutes Accumulated Minutes Accumulated after shift relative specific after shift relative specific to 42 ° C activity1 of to 42eC activity of gene product2 ger product3 0 10 0 10 10 21 25 34 55 62 60 20 83 107 90 47 1 Enzyme activity per total amount of protein, i.e. the increase of β-galactosidase per cell.

2 β-galactosidase mediated by plasmid pOU106.

Β-lactamase mediated by plasmid pKN410 (Uhlin et al., Gene 6, 1979, Table Π, p. 100).

The table shows that the level of gene product amplification is comparable to that achieved with other runaway vectors (Fe Uhlin et al., Gene 6. 1979, pp. 91-106).

The promoter from which the β-galactosidase gene is expressed is the cl promoter and / or tetracycline promoter located on the DNA fragment from pBR322 which, although relatively weak, cannot be excluded as an influencing factor and which transcribes in the same direction as the cl promoter.

pOU106 was mapped with restriction enzymes as described in Example 1 (cf. Fig. 11). It is apparent from the restriction map that the plasmid has a unique site for the restriction endonuclease BamHI immediately below the inserted lac genes, making the plasmid useful as a cloning vector.

The properties of this plasmid are shown in Table 5.

The strain E, coli CSH50 / pOU106 is deposited in the DSM under the deposit number 2474.

EXAMPLE 9

Construction and characterization of pOU79 (a TV B plasmid) 31 DK 171215 B1

Plasmid pOU71 was completely digested with BamHI and partially with PstI and ligated with a DNA fragment (carrying the lac operon) from plasmid pMC871 (Casadaban et al., J. Bact. 143, 1980, p. 971), which had been completely cleaved with BamHI and PstI.

The ligation mixture was transformed into E. coli strain CSH50, selected for resistance to ampicillin on McConkey lactose indicator plates containing 50 µg / ml ampicillin and incubated at 30 ° C. After 20 hours of incubation at 30 ° C, the plates were incubated for a short period (1-2 hours) at 42 ° C, and colonies switching from a Lac '(white) to a Lac + (red) phenotype were further analyzed. . The title plasmid was identified from such a colony. pOU79 had a molecular weight of 8.8x106 daltons and the following genotype: copA +. CopB +. repA. Δ pair, imm λ +, bla +. lac +.

The plasmid was mapped with restriction enzymes as described in Example 1 (cf. Fig. 12). It is evident from the restriction map that the plasmid has a unique site for the restriction endonuclease BamHI immediately above the lac genes. Insertion of promoter-carrying DNA fragments in the correct orientation at this site results in transcription of the lac genes, which results in a Lac-t phenotype of cells carrying such hybrid plasmids at 30 ° C. Lac T cells are readily identified on McConkey lactose indicator plates.

The plasmid also has a unique site for the restriction endonuclease EcoRI. which is located at one end of the lacZ gene. Insertion of EcoRI fragments at this site eliminates the activity of the lacZ gene product, β-galactosidase, resulting in a Lac 'phenotype which is readily observed as the formation of colorless colonies on McConkey lactose indicator plates after a temperature change from 30 ° C to 42 ° C. (see above description). Thus, the plasmid is useful as a cloning vector.

pOU79 was found to have the same replication properties as pOU71.

The properties of this plasmid are shown in Table 5.

The strain E. coli CSH50 / pOU79 is deposited in the DSM under the deposit number 2481.

EXAMPLE 10

Construction and characterization of dOU82 (a type B plasmid) 32 DK 171215 B1

Plasmid pOU79 was completely cleaved with BamHI and partially with SalI, and a BamHI-SalI fragment from the pSKS104 lac operon from which the lac promoter had been deleted and the first four codons 5 of the lacZ gene were inserted to construct pOU80 (not shown).

pOU80 is Lac 'and has a BamHI site and an EcoRI site immediately above the Jac genes. Furthermore, it lacks the EcoRI site. which are usually found in the lacZ gene, since the lac genes in pSKS104 are from pMC1403 (Casadaban et al., J. Bact. 143, 1980, p. 971).

The title plasmid was constructed by replacing an EcoRI-Clal fragment in the lacZ gene (cf. map of 10 pOU79, Fig. 12) from pOU80 with an EcoRI-Clal fragment from pVH1424 which carried the deo promoter and the amino terminus of the lacZ gene. end. The replacement results in a reconstruction of the lacZ gene and, due to the presence of the deo promoter, the resulting hybrid plasmid pOU82 mediates a Lac + phenotype when transformed into E. coli strain CSH50. pOU82 had a molecular weight of 8, 1x10 6 daltons and the following genotype: codA +. cqpB +. repA +. Δ par> imm λ +, bla +. lac +.

The plasmid is eventually lost from the cells at 30 ° C due to the lack of the pair region which is readily observed on non-selective McConkey lactose indicator plates (cf. Example 11).

The plasmid was mapped with restriction enzymes as described in Example 1 (cf. Fig. 13). It is evident from the restriction map that the plasmid has a unique site for each of the restriction endonucleases EcoRI and BamHI. allowing the plasmid to be used as a cloning vector.

20 pOU82 was found to have the same replication properties as pOU71.

The properties of this plasmid are shown in Table 5.

The strain E. coli CSH50 / pOU82 is deposited in the DSM under the deposit number 2482.

EXAMPLE 11

Construction and characterization of pQU91 (a type B plasmid) 25 To construct a plasmid that is stable in distribution, pOU82 was digested with

EcoRI. and the EcoRI-A fragment, which comprises the wild-type pair region of a plasmid R1 derivative, pKN184 (Nordstrom et al., Plasmid 4. 1980, p. 322), was inserted at this site followed by ligation. The resulting plasmid (pOUdO, not shown) was transformed into E. coli strain CSH50 and the cells were selectively grown on plates containing 50 µg / ml ampicillin at 30 ° C. As control, cells containing pOU82 were cultured similarly to ensure the presence of the plasmids in both starting colonies. Then, samples of both colonies were plated on plates containing no antibiotic and the cells allowed to grow for 25 cell generations. For screening for the stable inheritance of the Lac + phenotype, cells from both of the resulting colonies were plated on McConkey lactose indicator plates at 30 ° C, where the cells to which pOU90 had been transformed formed red colonies, showing that this plasmid had been preserved, whereas the control cells to which pOU82 had been transformed formed both red and colorless colonies, meaning that pOU82 had been lost from some of the cells during the selection-free period.

The intermediate plasmid, pOU90, was then completely digested with BamHI and partially with Sau3A to reduce the size of the plasmid to form the title plasmid. After transformation into E. coli strain CSH50, the stability of the plasmid was determined in the manner described above. pOU91 had a molecular weight of 12.5x106 daltons and the following genotype: copA +. CopB +. repA +. couples +. imm 15 λ +, bla +. lac +.

The plasmid was mapped with restriction enzymes as described in Example 1 (cf. Fig. 14). From the restriction map, it appears that pOU91 has unique sites for each of the restriction endonucleases EcoRI and BamHI. making the plasmid useful as a cloning vector.

pOU91 was found to have the same replication properties as pOU71, but unlike pOU71 20, the plasmid is completely stable at 30 ° C.

The properties of this plasmid are shown in Table 5.

The strain E. coli CSH50 / pOU91 is deposited in the DSM under the deposit number 2483.

EXAMPLE 12

Construction and characterization of pOUIOI (a type B plasmid) 25 Using the plasmid described in Example 7 as starting material and inserting a

SauSA fragment carrying the gene encoding chloramphenicol acetyltransferase (cat +). in the BamHI site of pOU75, the title plasmid was constructed which conferred the host cell chloramphenicol resistance. The plasmid was transformed into E. coli strain CSH50. pOUIOI had a molecular weight of 4.7x106 daltons and the following genotype: copA-t-. copB-k repA-κ Δ par, Δ aphA, immX +, bla +. Cat +.

34 DK 171215 Bl

The plasmid was mapped with restriction enzymes as described in Example 1 (cf. Fig. 15). It appears from the restriction map that pOU101 has a unique site for the restriction endonuclease EcoRI. which allows the plasmid to be used as a cloning vector. Furthermore, insertion of EcoRI fragments inactivates the cat + gene, which allows screening of EcoRI hybrids.

5 To detect the presence of the desired plasmid in the host cell, cell cultures were tested for resistance to λ infection and ampicillin resistance in the manner described above. Furthermore, the cells were tested for resistance to chloramphenicol by growing cultures on plates containing 20 µg / ml chloramphenicol. The cells were also tested for temperature-sensitive growth in the manner described above. pOUlOl was found to have the same replication properties as pOU71.

The properties of this plasmid are shown in Table 5.

The strain E. coli CSH50 / pOUl01 is deposited in the DSM under the deposit number 2757.

EXAMPLE 13

Construction and characterization of pOUHO

The EcoRI-ClaI fragment containing part of the lacZ gene was deleted from plasmid pOU80 (described in Example 10; cf. map of pOU79, Figure 12) and replaced with an EcoRI-ClaI fragment ffa plasmid pVH1451 (Valentin-Hansen et al., EMBO Journal 1. 1982) which carried the deo promoter and amino terminus of the lacZ gene. The resulting plasmid was designated pOU83 (cf. Fig. 16).

Plasmid pOU83 was partially rescued with BamHI and BglII to delete the lac genes and the DNA fragment carrying the cl repressor gene and the XPR promoter. After ligation and transformation to CSH50 20, a plasmid with the desired properties was isolated, resulting in a close fusion of the deo promoter and copB gene. The plasmid, designated pOU110, had a molecular weight of 3.2x106 daltons and the following genotype: copA +. copB-k repA +. Δ pair, bla +.

The plasmid was mapped with restriction enzymes as described in Example 1 (cf. Fig. 17). From the restriction map it appears that the plasmid has a unique site for each of the restriction endonucleases 25 EcoRI and BamHI. which allows the plasmid to be used as a cloning vector.

As cultures of E. coli strain SO929 (cvtR. Lac. Deo; recA). to which pOU110 was transformed, was grown overnight in LB medium without glucose, uncontrolled plasmid replication occurred at a cell density greater than 2 x 10 8 cells / ml.

The strain E. coli CSH50 / pOU110 is deposited in the DSM under the deposit number 2758.

EXAMPLE 14

Construction and characterization of pQU130 35 DK 171215 B1

Plasmid pOU91 was digested with BamHI. and the BamHI site was deleted by the exonuclease Bal31 to generate the plasmid pOU92 (not shown). The plasmid was rescreened with EcoRI and Clal. and 5 a corresponding Eco RI-Cla I fragment from plasmid pMC1403 (Casadaban et al., J. Bact. 143. 1980, p. 971) was inserted to generate the plasmid pOU93 which carried the lac operon without the de At the BamHI site thus formed, a BglII fragment carrying a conB gene was inserted. to generate the plasmid pOU130 (cf. Fig. 18), followed by ligation and transformation to E. coli strain CSH50. The cells were plated on McConkey lactose indicator plates containing 50 µg / ml ampicillin and incubated at 30 ° C for selection for red colonies (Lac +). The temperature was then raised to 42 ° C and the amplification of β-galactosidase is shown in Table 4. After 45 minutes of incubation at 42 ° C, the temperature was lowered to below 38 ° C. The measured β-galactosidase amplification after the temperature change is shown in Table 4.

TABLE 4

Expression of β-galactosidase from pOU130 after amplification of plasmid DNA

Growth conditions dSpec. act. of Ampification β-galactosidase factor of 30 ° C; stable state 0.03 1 b 30 ° C 42 ° C 0.7 23 c 30 ° C 42 ° C 37 ° C 2.5 83 a Cells of CSH50 containing pOUl30 grown exponentially in LB medium at 15 30 ° C.

b After exponential growth at 30 ° C, the culture was grown at 42 ° C at which temperature growth continued for 2 hours.

c After exponential growth at 30 ° C, the culture was grown at 42 ° C for 45 minutes, then the temperature was lowered to 37 ° C for 90 minutes.

36 DK 171215 B1 d Cf. MATERIALS AND METHODS. Specific activities of β-galactosidase are expressed as described by Light and Molin, Mol, Gen. Genet .. 1981, pp. 56-61.

It can be seen from the table that the gene product amplification is substantially improved when switched back to a lower temperature, showing that this method is particularly useful for achieving a maximum expression of the gene products in question.

Since β-galactosidase is constitutively expressed from such a gene fusion (Light & Molin, J. Bact. 151. 1982, pp. 1129-1135), the levels of enzyme activity accurately reflect the gene product amplification ability of the plasmids of the invention.

The strain E. coli CSH50 / pOU130 is deposited in the DSM under the deposit number 2759.

EXAMPLE 15

Construction and characterization of pLC31 pOU75 was digested with BamH1. and a BamHI fragment from plasmid pBEU14 (Uhlin and Clark, J. Bact. 148. 1981, pp. 386-390) carrying the recA promoter and gene was inserted, followed by transformation into E. coli strain CSH50. The title plasmid had a molecular weight of 6.3x106 daltons and the following genotype: recA. bla +. codA +. cqpB +. repA +, immX +.

The plasmid was mapped with restriction enzymes as described in Example 1 (cf. Fig. 19).

From the restriction map, it appears that pLC31 contains a unique site for the restriction enzyme EcoRI below the recA promoter. which is suitable for insertion of foreign 20 genes.

pLC31 was found to have the same replication properties as pOU71.

When cells containing pLC31 were grown at 30 ° C, the promoter remained fully repressed, whereas after a temperature change to 42 ° C the lexA repressor was gradually titrated and transcription from recA was initiated, resulting in an amplified formation of the rgcA gene product. which could be detected by polyacrylamide gel electrophoresis.

The properties of this plasmid are shown in Table 5.

The strain E. coli CSH50 / pLC31 is deposited in the DSM under the deposit number 2718.

EXAMPLE 16

Construction and characterization of pLC32 DK 171215 B1 37

Plasmid pLC28 (Remaut et al., Gene 15. 1981, pp. 81-93) bearing the XPL promoter and Magenet and provided with a linker comprising the EcoRI restriction sites. Narrow.

5 BamHI. Sall. PstI and HindIII. was digested with Eco RI and inserted into the Eco RI site of plasmid pOU51 (cf. Example 1), followed by ligation and transformation to E, coli strain CSH50.

This plasmid, pLC32 '(cf. Fig. 20), having a molecular weight of 10.6x10 6 daltons, was partially digested with PstI followed by ligation and transformation to E. coli strain CSH50, selecting for plasmids which are kanamycin resistant and ampicillin-10 sensitive. The resulting plasmid was designated pLC32 and had a molecular weight of 7.7x106 daltons and the following genotype: cqpA +. copB-. repA +. aphA +. immX +.

The plasmid was mapped with restriction enzymes as described in Example 1 (cf. Fig. 21).

The restriction map shows that λΡ ^ ρΓΟίηοίΟΓβη is located immediately above the unique EcoRI site. making the plasmid convenient as a cloning vector.

15 pLC32 was found to have the same replication properties as pOU51.

The properties of this plasmid are shown in Table 5.

The strain E. coli CSH50 / pLC32 is deposited in the DSM under the deposit number 2719.

38 DK 171215 B1 TABLE 5

Properties of MiniRl-XP ^ plasmids as cloning vectors

Relevant unique re-screening Capital Stable phenotype stricture (insertion per cell inheritance)

Plasnid site (s) inactivation) 30 ° C 42 ° C pOU51 Km, λ EcoRI No 25> 1000 Yes pOU53 λ 'EcoRI No 25> 1000 Yes pOU56 Αρ, λ EcoRI. BairHI None 25> 1000 Yes pCU57 Αρ, λ EcoRI. BamHI None 25> 1000 Yes pCU71 Αρ, λ EcoRI. BamHI None 3> 1000 No pCU73 Αρ, λ BairHI None 1> 1000 No pCU75 Αρ, λ BamHI None 3> 1000 No pOU106 Αρ, λ BairHI None 3> 1000 No pCU79 Ap, X, Lac {+) EcoRI. BairHI LaC (EcgRI) 3> 1000 No pCU82 Ap, X, Lac + EcoRI. BamHI None 3> 1000 No pCXJ91 Αρ, λ, Lac +, EcoRI. BairHI None 3> 1000 Yes

Par + pCUlOl Αρ, Οη, λ EcoRI Otf3 (EcoRI) 3> 1000 No pCU130 Ap, X, Lac + EcoRI None 3> 1000 Yes pLC31 Ap, RecA +, X EcoRI RecA '3> 1000 No pLC32 Km, λ EcoRI None 25> 1000 Yes pCUllO Ap 'EcoRI. BamHI None a) No a) Copy number is not temperature dependent.

BIBLIOGRAPHY

1. Published European Patent Application No. 78101877.5.

2. Molin et al., J, Bact. 138. 1979, pp. 70-79.

3. Molin et al., Microbiology. 1981, pp. 408-411.

4. Rosen et al., Mol. Gen. Genet. 179. 1980, pp. 527-537.

DK 171215 Bl 39 5. Light and Molin, Mol. Gen. Genet. 184, 1981, pp. 56-61.

6. Stougaard et al., Proc. Natl. Acad. Sci. USA 78. 1981, pp. 6008-6012.

7. Molin et al., Mol. Gen. Genet. 181. 1981, pp. 123-130.

8. Stougaard et al., EMBO Journal 1. 1982, pp. 323-328.

9. Sussman and Jacob, Compt. Fuck. Acad. Sci. 254. 1962, p. 1517.

10. Sninsky et al., Gene 16. 1981, p. 275.

11. Uhlin et al., Gene 6, 1979, pp. 91-106.

12. J. Miller: Experiments in Molecular Genetics, Cold Spring Harbor Laboratory, Cold Spring

Harbor, New York, 1972.

13. Davis, Botstein, and Roth: A Manual for Genetic Engineering Advanced Bacterial Genetics. Cold

Spring Harbor, New York, 1980.

14. Bertani, J. Bact. 62. 1954, p. 293.

15. Bolivar et al., Gene 2. 1977, p. 95.

16. An & Friesen, J. Bact. 140. 1979, pp. 400-407.

17. Casadaban et al., J. Bact. 143. 1980, p.971.

18. Dempsey & Willetts, J. Bact. 126. 1976, p. 166.

19. Nordstrom, Molin, Aagaard-Hansen, Plasmid 4. 1980, pp. 215-217.

20. Stougaard & Molin, Anal. Biochem. 118. 1981, p.191.

21. Nordstrom et al., Plasmid 4. 1980, p. 322.

22. Valentin-Hansen et al., EMBO Journal 1. 1982, p. 317.

23. Messing et al., Nucleic Acids Res. 9, 1981, p. 309.

24. Uhlin & Clark, J. Bact. 148. 1981, pp. 386-390.

Claims (29)

1. Plasmid useful as a cloning vector and having a size in the range of ca. 2.5 - 15.0 x 10 6 daltons, which plasmid has: a) an inserted regulatable promoter in a position and in a direction that allows transcription from the promoter into the replication region; b) replication region comprising a gene encoding a gene product which is positively necessary for replication, with increased transcription from the regulator promoter leading to an increased level of the gene product which is positively needed for replication, the increased level resulting in a plasmid replication rate , which is higher than the rate of growth of the host cell, leading to an increased plasmid capital, which will usually be increased by a factor of 25-1000 after 4-5 cell divisions. Plasmid according to claim 1, characterized in that the promoter is controllable by a regulatory factor. Plasmid according to claim 2, characterized in that the regulatory factor is a factor which exerts a positive control or a factor which exerts a negative control. Plasmid according to claim 1, characterized in that the increased transcription from the adjustable promoter leading to a substantially increased or uncontrolled plasmid copy capital is caused by derepression of the promoter. Plasmid according to any of the preceding claims, characterized in that the promoter is a chemically controllable promoter such as a lac promoter, faith promoter or deo promoter. Plasmid according to any one of claims 1-4, characterized in that the activity of the promoter is temperature dependent or controlled by a temperature sensitive regulating factor. Plasmid according to claim 6, characterized in that the promoter is an λ promoter and that the gene for a temperature sensitive λ cl repressor controlling transcription from the promoter is also present. Plasmid according to claim 7, characterized in that the λ promoter is either λΡκ or kTL, especially λΡκ. DK 171215 B1 Plasmid according to any one of the preceding claims, characterized in that transcription from the regulator promoter causes the plasmid to exhibit a constant, low copy number at low temperatures, a temperature of approx. 30 ° C, and a substantially increased or uncontrolled copy capital at higher temperatures, a temperature in the range of approx. 36-42 ° C. Plasmid according to any one of the preceding claims, characterized in that part of a natural replication regulatory gene has been deleted and replaced with the adjustable promoter. Plasmid according to claim 10, characterized in that when host microorganisms containing the plasmid are grown under conditions which ensure a constant, low plasmid copy, it has a copy number in the range 20-40, preferably 20-30 and especially 20-25, copies per . and that when the host microorganisms are cultured under other conditions which result in a substantially increased or uncontrolled plasmid copy, it has a copy capital in the region of at least ca. 500-1000 copies per cell. The plasmid according to claim 11, characterized in that it has a plasmid capital in the region of approx. 20-40, preferably 20-30 and especially 20-25, copies per. cell at one temperature such as a temperature of approx. 30 ° C, and an uncontrolled plasmid capital in the region of at least ca. 500-1000 copies per cell at a higher temperature such as a temperature of ca. 42 ° C. Plasmid according to any one of claims 1-9, characterized in that a regulatable promoter has been inserted above the natural replication control gene (s) on the plasmid. Plasmid according to claim 13, characterized in that, when host microorganisms containing the plasmid are grown under conditions which ensure a constant, low plasmid copy, it has a copy number in the range of approx. 3-5 copies per and that when host microorganisms are cultured under other conditions leading to a substantially increased or uncontrolled plasmid copy, it has a copy capital in the region of at least ca. 500-1000 copies per cell. Plasmid according to claim 14, characterized in that it has a plasmid capital in the region of approx. 3-5 copies per cell at one temperature such as a temperature of approx. 30 ° C and an uncontrolled plasmid capital in the region of at least ca. 500-1000 copies per cell at a higher temperature such as a temperature of ca. 42 ° C. DK 171215 Bl Plasmid according to any one of claims 1-9, characterized in that it carries an adjustable promoter in the replication region from which a portion of a natural replication regulatory gene has been deleted and into which the natural replication regulatory gene has been inserted. in an area where it is not naturally located. The plasmid according to claim 16, characterized in that when host microorganisms containing the plasmid are grown under conditions which ensure a constant, low plasmid copy, it has a copy number in the range of approx. 0.5-1 copy per and that when host microorganisms are cultured under other conditions leading to a substantially increased or uncontrolled plasmid copy, it has a copy capital in the region of at least ca. 500-1000 copies per cell. Plasmid according to claim 17, characterized in that it has a plasmid capital in the region of approx. 0.5-1 copy per cell at one temperature such as a temperature of approx. 30 ° C and an uncontrolled plasmid capital in the region of at least ca. 500-1000 copies per cell at a higher temperature such as a temperature of ca. 42 ° C. Plasmid according to any one of the preceding claims, characterized in that in addition to the first regulatable promoter regulating transcription from the replication region, a second promoter is inserted in a manner which allows insertion of a structural gene whose expression is controlled from the other promoter, which other promoter is controllable, and for example is the XPL promoter. Plasmid according to any one of the preceding claims, characterized in that it carries a gene which allows gene fusion by insertion of another gene, which allows detection of expression. Plasmid according to any one of the preceding claims, characterized in that it also carries one or more genes that are not naturally associated with the plasmid. A method of producing a gene product of plasmid DNA, characterized in that microorganisms carrying a plasmid according to any one of claims 1-21 are cultured, comprising at least one culture period under conditions which ensure increased transcription from the regulatable promoter, which results in an increased level of the gene product that is positively needed for replication, which results in a plasmid replication rate that is higher than the host cell growth rate, leading to an increased plasmid capital, which will usually be increased after 4-5 cell divisions. with a factor of 25-DK 171215 B1 1000 a substantially increased or uncontrolled plasmid copy capital, and a gene product of the plasmid is harvested from the microbial culture. A method according to claim 22, characterized in that the host microorganism to which a plasmid according to any one of claims 1-21 has been transformed is cultured under conditions which cause a constant, low plasmid capital during the grafting and propagation stages until the desired number of cells have been obtained and the host microorganism is then cultured under conditions leading to a substantially increased or uncontrolled plasmid copy number. Process according to claim 22 or 23, characterized in that the concentration of a substance which substantially inhibits promoter activity, after a period of cultivation in the presence of that substance, is reduced to an extent which causes activation of the promoter and a substantially increased or uncontrolled plasmid copy. Method according to claim 24, characterized in that, after the conditions which result in a substantially increased or uncontrolled copy number have been maintained for a period sufficient to obtain a substantial amplification of the plasmid, but sufficiently short for To avoid any significant damage to the microbial culture, the substance that inhibits promoter activity is added in an amount which leads to a slow decrease in plasmid copy capital to the pre-induction level. The method according to claim 22 or 23, characterized in that the conditions under which the microorganism is cultivated comprise at least one culture period at or near a temperature at which the plasmid copy number is substantially increased or uncontrolled. The method according to claim 26, characterized in that the culture at the temperature at which the plasmid copy number is substantially increased or uncontrolled is continued until the growth of the host microorganism is inhibited by the formation of plasmid DNA or gene products from the plasmids. A method according to claim 26, characterized in that, after propagating the microbial culture for production size, the temperature is changed to a temperature at or near the temperature at which the plasmid copy number is substantially increased or uncontrolled, and this temperature is maintained for a period which is sufficient to obtain a substantial amplification of the plasmid, but sufficiently short to avoid any significant damage to the microbial culture, after which the temperature is again changed to a temperature which ensures a low, constant plasmid copy, resulting in a slow decrease in the plasmid copy number until it reaches the beginning level. Method according to any one of claims 26-28, characterized in that the temperature at which the plasmid copy number is substantially increased or uncontrolled is higher than the temperature at which the plasmid has a constant, low copy number, a temperature of the area approx. 36-42 ° C.