GB2153363A - Method of preparation of cloning vector - Google Patents

Abstract

A method of producing a cloning vector including the steps of, obtaining a nucleic acid molecule with a repeated sequence, and, cutting the nucleic acid molecule to obtain a length of nucleic acid posessing at least part of the said repeated sequence.

Description

SPECIFICATION Method of preparation of cloning vector, cloning vector and method of use of same The present invention relates to the preparation of a novel cloning vector, the cloning vector itself and a method of use of the cloning vector.

Streptomyces species produce many extracellular enzymes. Some of these are produced commercially e.g. pronase from S. griseus, and many others have considerable potential e.g.

xylanase. Streptomyces appear to export proteins very efficiently and the recent advances which have taken place in the molecular biology of the genus make them good candidates for the production of commercially important foreign proteins.

Up to now there are few reports of the cloning of extracellular enzymes within Streptomyces. The tyrosinase gene from S.antibioti- cus, normally an extracellular enzyme, has been cloned into S.lividens but most or all of the enzyme remained intracellular. The endoglycosidase "H" gene was cloned from S.

plicatus into E.coli and some extracellular enzyme was produced. However, there are no reports of the successful cloning of a Streptomyces extracellular enzyme gene to achieve high levels of secreted protein enzyme.

According to a first aspect of the present invention there is provided a method of producing a cloning vector including the steps of; obtaining a nucleic acid molecule with a repeated sequence, and, cutting the nucleic acid molecule to obtain a length of nucleic acid possessing at least part of the said repeated sequence.

Preferably the nucleic acid is deoxyribonucleic acid and the repeated sequence is obtained from a member of the genus Streptomyces. More preferably the sequence is obtained from a member of the strain Streptomyces lividans 66 strain TK64.

In an embodiment of the present invention the sequence is that obtained as tandem repeats in the chromosomal sequence of an Arg strain derived from a spontaneous chloramphenicol-sensitive mutant of the strain Streptomyces lividans 66 strain TK64.

According to a second aspect of the present invention there is provided a cloning vector including at least part of a repeated sequence of nucleic acid.

Preferably the particular nucleic acid is deoxyribonucleic acid, and more preferably the repeated sequence is one which occurs in a member of the genus Streptomyces In an embodiment of the present invention the cloning vector further comprises at least part of the nucleic acid sequence of the plasmid plJ702.

According to a third aspect of the present invention there is provided a method of cloning which comprises the steps of; obtaining a gene to be cloned; linking the said gene to be cloned to a nucleic acid sequence capable of existing as a multi-copy sequence within the chromosome of an organism into which the gene is to be cloned; and introducing the combination of the said gene and the said sequence into the said organism.

Preferably the combination of the said gene and the said sequence is introduced into the organism by a plasimid vector.

According to a fourth aspect of the present invention there is provided a nucleic acid sequence capable of integrating into the chromosome of an organism and multipling therein to establish the said sequence as a multi-copy sequence within the said chromosome.

Conveniently the sequence contains a marker capable of indicating the presence of the sequence within the chromosome. This marker may for example confer thiostreptone resistance upon the organism.

More preferably this sequence is that present as tandem repeats in the chromosomal sequence of an Arg strain derived from a spontaneous chloramphenicol sensitive mutant of S.lividans.

According to a fifth aspect of the present invention there is provided a method of gene amplification including the steps of; linking a gene to be amplified to a sequence capable of existing as a multi-copy sequence within the chromosome of an organism; introducing the said sequence into the chromosome of the organism; and, allowing the said sequence to multiply within the chromosome of the organism; whereby the combination of the said sequence and the gene to be amplified becomes amplified such that it exists as a multi-copy combination within the chromosome.

The present invention will be further described, by way of example, with reference to the accompanying figures, wherein; Figure 1 is a restriction map of an sequence present as tandem repeats in the chromosome of an Arg strain derived from a spontaneous chloramphenicol sensitive mutant of S.lividans.

Figure 2 is a circular restriction map of the plasmid pMT605.

Figure 3 is a representation of the isolation of an Arg - strain derived from spontaneous chloroamphenicol sensitive mutants of S.lividans.

Figure 4 is a representation of the method of Example 2a).

Figure 5 is a representation of the method of Example 2b).

Figure 6 is a representation of the method of Example 2c).

Figure 7 shows the agarose gel electrophoresis patterns of BamHI digests of the derivatives of strain 405.

Figure 8 shows the results of digesting total DNA containing the amplified fragment with different restriction enzymes.

EXAMPLE 1 ISOLATION OF THE CLONING VECTOR.

We looked for a suitable system to demonstrate high level extracellular expression of clone genes in streptomyces.

An ideal condidate was the agarase gene of S.coelicolor A3(2). This agarase hydrolyses glycosidic bonds linking the alternating units of (1 -3)fl-D-galadopyranosic and (1-4) 3,6 anhydro L-galactoside which constitute the agarase polymer. Detection of extracellular agarase activity is relatively simple because agarase producing colonies sink into the surface of an agar plate following digestion, by the agarase, of the agar beneath the colony.

Flooding such plates with Gram's iodine solution reveals zones of clearing surrounding the colonies. In addition, there is a quantitative assay which determines agarase activity by measuring the release of reducing sugar units by the enzyme when it cleaves agarase.

S.coelicolor A3(2) which is genetically the best characterized streptomycete produces an extracellular agarase, whereas S.lividans 66, a good host for cloning experiments has little or no detectable agarase activity. The two species are closely related so that expression and export of a cloned extracellular enzyme seemed unlikely to present problems.

Although the example given herein is largely restricted to the abovementioned strains it is believed that the performance of the present invention may be extended to other strains of Streptomyces.

There are many unstable genes in Streptomyces species. Unstable properties include antibiotic resistance (Freeman and Hopwood, 1978; Federenko and Danilenko, 1980; Matsubara-Nakano et al, 1980), pigment synthesis (Gregory and Huang, 1964; Scrempf, 1983), sporulation and arginine biosynthesis (Redshaw et al, 1979). Unstable genes often mutate at frequencies of about 1 % of spores tested. In some cases the mutations revert (Freeman et al, 1977), whereas, in other cases they do not revert and are sometimes due to delection (Schrempf, 1983). As well as deletions, DNA amplifications seem to occur often in Streptomyces.Amplification sometimes accompanies mutation of unstable genes (Ono et al, 1982; Schrempf, 1983) and sometimes accompanies genetic manipulation such as protoplast fusion (Robinson et al, 1 981) or selection for increased antibiotic production (Orlova and Danilenko, 1983).

Freeman et al, (1977) found unstable chloramphenicol resistance in S.coelicolorA3(2) and in S.lividans 66. The biochemical basis of chloroamphenicol resistance in S.coelicolor A3(2) is unknown; it does not seem to be due to chemical modification of the antibiotic, (Shaw and Hopwood, 1976). Sermonti et al, (1978) found that some chloramphenicol-sensitive mutants in S.coelicolorA3(2) had an arginine gene instability.

We decided to investigate S.lividans 66 because it is the Streptomyces strain best suited for in vitro genetic manipulation (Hopwood, Bibb et al, 1983) and also has conjugation systems (Hopwood, Kieset et al, 1983).

a) ISOLATION OF MUTANTS We have shown that spontaneous Cmls mutants of the S.lividans 66 strain TK64 are very unstable, producing Amy- Arg- mutants at high frequency, this procedure is described in greater detail below. Sporulation was restored by adding arginine to minimal medium, which suggests that the non-sporulating phenotype may be a direct consequence of arginine auxotropy. Vargha etna!, (1983) found an Argmutant of S. fra diae that did not sporulate unless -high levels of the argini;ne precursor citrulline were present in the medium. The lack of sporulation on Complete Medium and R2 medium ight be due to inhibition or arginine uptake by other amino acids as seen in S.hydrogenans and as reported by Gross and Burkhardt, 1973, but we do not wish to limit ourselves to this hypothesis.Growth tests with arginine precursors suggest that the Arg mutation is in the gene for argininosuccinate synthetase like mutations found in several other Streptomyces species (Redshaw et al, 1979).

For surface cultures R2YE (Thompson et awl., 1980), CM (Hopwood, 1967) or M40 mini mal medium (Polsinelli and Beretta, 1966) was used. Glucose was substituted by 0.2% Glyceroi in M40. For auxotrophic strains single amino acids were added to a final concentration of 60 g/ml or as casamino acids to 0.2g/l. To determine the step in arginine biosynthesis that was blocked in Arg- strains, colonies were grown on M40 containing either ornithine, citrulline or argininnosuccinate at a concentration of 60 g/ml. Chloramphenical was used in M40 medium at 8 g/ml/ Liquid cultures were made in L-broth (Lennox, 1955) on rotary shakers. All incubations were performed at 30"C.

We used S.lividans 66 strain TK64 that has been cured of both known indigenous plasmids (Hopwood, Kieset et al, J.General Microbiology 1 29, 2257, 1983). We selected three chloramphenicoi resistant TK64 clones and isolated the spontaneous mutants in parallel in the three independent lines. Fig. 3 summarises the different derivatives isolated in each line.

Between 100 and 1 50 spores were plated on M40 supplemented with casamino acids.

After sporulation the colonies were replica plated onto M40 with and without chloroamphenical. Those colones were only able to grow on plates without chloroamphenicol were again tested. If they remained Cmls, they were used for further experiments.

We isolated spontaneous chloramphenicol sensitive mutants (Cmls) at an average frequency of 0.8% of spores (5/620, 1/173 and 2/175 colonies tested for the strains 402, 403 and 405 respectively). One of the Cmls mutants (strain 407) no longer produced aerial mycelium (Amy- phenotype) and did not sporulate on minimal medium supplemented with casamino acids. The other strains were Amy+ but gave rise spontaneously to Amy- derivatives at high frequencies: five strains (406, 408, 411, 412, 413) were very unstable, giving an average frequency of Amy- mutants of about 25% of spores (average of ten measurements) whereas strain 408 only yileded 1.3% Amy- mutants (average of three independent measurements).The Amycolonies also had a growth requirement for arginine (Arg- phenotype): 35/40 Amycolonies tested were Arg- (the other 5 did not grow after transfer to fresh medium) whereas 80/80 Amy+ were Arg+.

All seven of the CmlS strains tested (406, 408, 409, 410, 411, 412, 413) gave chloramphenicol resistant revertants; the reversion frequencies, which we observed, varied between 10-8 and 10-5 per spore. The revertants were strongly pigmented and had lower levels of chloramphenicol resistance than the wild type (inhibition zones with a chioram- phenicol disc were 15mm (wild type), 43mm (Cmls) and 26mm (revertants)). These partial revertants are indicated by Cml' in Fig. 1, rather than as CmlR. The intermediate level of chloramphenicol resistance in Cml strains made it impossible to test for mutation to a Cmls phenotype.

The Cml- strains gave rise to spontaneous Amy- Arg mutants at high frequency (an average of 20% of spores in four independent measurements) like their Cmls parents. These Arg- strains seem to retain a chloramphenicol resistance level characteristic of the Cml* strains.

b) CHARACTERISATION OF Cm!s and Arg MUTANTS The three Cml phenotypic classes (CmlR, Cmls and Cml") were tested for their resistance to a range of antibiotics using antibiotic discs.

Of 1 7 antibiotics tested only two showed any difference between the strains: The Cmls and Cml strains were more resistant to fusidic acid than the CmlR wild type (the wild type had 9mm diameter inhibition zones compared to no inhibition zones around a 6mm disc in the Cmls and Cml mutants), whereas the Cmls was slightly more sensitive to kanamycin (32 diameter inhibition zone) than the CmlR (26mm diameter) or Cml* (28mm diameter) strains.

The Cmls Arg- and Cml* Arg- strains will grow on minimal medium supplemented with arginine or arginiosuccinate, but do not grow when supplemented with ornithine or citrulline. This suggests loss of the enzyme argininosuccinate synthetase but we do not wish to restrict ourselves to this hypothesis. The Amy- Arg- mutants sporulated (and also became phenotypically Amy+) on minimal medium supplemented with high enough concentrations of arginine (more than 0.1 mM; good sporulation with 0.25mM), but did not sporulate with argininosuccinate. They did not sporulate on Complete Medium or on R2 medium, even, in the latter case, if supplemented with up to 0.85mM arginine.

We did not obtain any Arg+ revertants from any of the three cells Arg - strains (415, 418, 420) or the thice Cml" Arg strains (422, 426, 429). This showed that the reversion frequency to Arg + was less than 5 X 108 per spore (poor sporulation on minimal medium prevented us testing a larger number of spores).

c) ANALYSIS OF DNA ALTERATIONS All the Arg- strains (7 tested) contained an amplified DNA-sequence described in more detail below, whereas none of the Arg + (8 tested) did. 250-500 copies per chromosome of the amplified sequences were present in tandemly repeated arrays. We do not know how long the arrays are (the maximum fragment size of 23kb seen in partial digests only corresponds to 4 copies of the amplified sequence). The arrays could be present at one or more sites in the chromosomeor they could be in large extrachromosomal DNA circles, too large to be isolated as CCC-DNA.

The high frequency of the amplification event and the high copy number suggest that amplification is due to over-replication rather than unequal crossing over during recombination. If amplification also involves transposition to new sites this might inactivate the arginine gene. Alternatively there may be deletions as in the tyrosinase gene of S.reticuli (Schrempf, 1983).

In order to characterise the specific DNA alterations we prepared total DNA from our strains and digested with various restriction enzymes.

Cells from strains 414, 415, 418, 420, 422, 426 and 429 were grown for five days in 25 ml 1-broth, harvested by centrifugation and washed with STE-buffer (10.3% sucrose, 25mM Tris, 25mM EDTA, pH 8). After resuspending in 5ml STE-buffer containing 1 Omg/ml lysozyme the cells were incubated for 60 mins at 37"C. Lysis was performed by adding 2ml 5% SDS and heating up to 65"C for 10 mins. After cooling to room temperature the solution was extracted with phenol/ chloroform (Hopwood et al, 1983) and the aqueous phase precipitated for 1 5 mins at room temperature by addition of 0.7ml 3M sodium acetate (unbuffered) and 7ml isopro panol.The DNA was re-dissolved in 3ml TE (1OmM Tris, 1mM EDTA, pH 8) and incubated with 20mg/ml RNase for 45 mins. at 37"C.-The solution was again extracted with phenol/chloroform and precipitated with isopropanol. Then the DNA was washed three times with cold 70% ethanol, dried and resuspended in 3ml TE buffer.

To detect supercoiled plasmids the DNA was dissolved in 1 Oml TE buffer after the first isopropanol precipitation and 10g CsCI and 0.25ml ethidium bromide (1 Omg/ml) were added. Centrifugation was carried out in a Beckman Ti 50 rotor at 36,000 rpm, 1 7"C for 60 hours. DNA was visualized under UVlight.

Restriction Endonuclease Analysis and Gel Electrophoresis were performed as described previously (Schoffl et al., 1981).

The tracks run were as follows, and are shown in figure 7.

Track 1. DNA digested with Hindlll (marker) Track 2. 405 DNA digested with BamHI (Cml) Track 3. 412 DNA digested with BamHI (Cmls) Track 4. 420 DNA digested with BamHI (Cmls Arg-) Track 5. 421 DNA digested with BamHI (Cml*) Track 6. 422 DNA digested with BamHI (Cml Arg-) The agarose gel electrophoresis patterns of BamHI digests of the derivatives of strain 405, as shown in Fig. 7, revealed that, in the Cmls Arg - strain and the Cml- Arg - strain there was an intense 5.75kb band that was not present in the three Arg + parent strains: All the Arg - strains that we tested (414, 415, 418, 420, 422, 426, 429) had the same 5.75kb BamHI band, although in strain 426 the band was weaker than in the other strains.The strains tested included Cmls Arg - and Cml- Arg- derivatives of all three independent lines. We estimated by densitometer scanning that the new bands accounted for 14-29% of total DNA (four strains measured), which would give a copy number of 250-500 copies per chromosome (assuming a genome size of 104kb, Benigni et al, 1975).

None of the Arg + derivatives tested (402, 405,409,411,412,421, 425, 428) contained the amplified band.

Chromosomal DNA's containing amplified sequences were cleaved with Bglll and run on 0.7% agarose gels. The DNA-distribution was measured according to Ono et al (1980).

Standard curves were obtained using plJ702 DNA (Katz et al., submitted), cleaved by Bglll.

The results of digesting total DNA containing the amplified fragment with different restriction enzymes are shown in Fig. 8., and the tracks are identified as follows; Track 1. DNA digested with Hindlll (marker) Track 2. 420 DNA digested with Bglll and Pstl Track 3. 420 DNA digested with Pstl and BamHI Track 4. 420 DNA digested with Pstl Track 5. 420 DNA digested with Bglll Track 6. 420 DNA digested with Bglll and BamHI Track 7. 420 DNA digested with BamHI Track 8.DNA digested with Hindlll (marker) Bglll (track 5 and BamHI (track 7) both gave one 5.75kb band, whereas a Bglll + BamHI double digest (track 6) gave two bands of sizes 2.7kb and 3.05kb. This implies a circular restriction map for the amplified fragment. Pstl digestion (track4) gave two bands of sizes 1.57kb and 4.1 8kb and three bands appear in Pstl + Brill (track 2) and Pstl + BamHI (track 3) double digests, again giving a circular restriction map. These data and other digestions with Sacll and Smal (data not shown) allowed us to construct the restriction map in Fig. 1.

A circular restriction map can arise from tandemly repeated sequences as well as from circular DNA molecules. There were no extra bands on agarose gels of undigested total DNA that could correspond to circular plasmid DNA (data not shown) and no CCC-DNA band was seen in caesium chloride-ethidium bromide density gradients of total DNA (this showed there were less than 10 CCC-DNA molecules per chromosome). However, partial digests with Bglll of total DNA gave bands of sizes 11 .4kb, 1 8kb and 23kb as well as the unit length 5.75 kb band (data not shown).

This shows that the amplified sequence is present in tandem repeats.

We also ran caesium gradients in the presence of a dye that binds differentally to DNA of different GC-content.

The GC-content of chromosomal DNA containing amplified sequences was estimated by using CsCI bisbenzimide gradients (Hoechst dye No 33258 from Calbiochem-Boehring Corp., La Jolla, California, U.S.A.). 8.3ml DNA-solution (60 g DNA) were mixed with 10.29 CsCI and 1m1 bisbenzimide (2mg/ml).

The solution was centrifuged for 60 hours at 35,000 rpm at 17"C in a Beckman Ti 50 rotor. Marker DNA's (20 g each) of Staphylococcus aureus (33% GC), 'phage lambda cl 857(50% GC) and Streptomyces coelicolor (72% GC) were run separately and used for a standard curve. No satellite band was seen, which showed that the amplified fragment differs less than 5% in GC-content from total S.lividans DNA.

EXAMPLE 2 PREPARATION AND USE OF THE CLONING VECTOR a) CLONING OF THE AMPLIFIED SEQUENCE Total DNA from a derivative of TK64 bearing the amplified sequence was isolated and digested with Bglll.

The digest was separated as a low melting point agarose gel, the amplified sequence 5.75kb band cut out of the gel and the DNA purified.

The amplified sequence DNA was then ligated into the Bglll site of plJ702 DNA which had been cut with Bglll (all enzymes and instructions for use available from BCL-The Boehringer Corporation (London) Ltd, Bell Lane, Lewes, East Sussex BN7 11 G) and treated with alkaline phosphatase to prevent recircularisation (phosphatasing method as disclosed in U.S. Patent No. 4 264 731).

The purified DNA (amplified sequence) was ligated with the plJ702 DNA and transformed into TK64 protoplasts with a final growth in the presence of thiostreptone, to select for transformants. Approximately 4000 thiostreptone resistant transformants were obtained. Of these, more than 95% were meianin-negative, indicating a high frequency of amplified sequence DNA insertion into the Bglll site of plJ702 with consequent disruption of the tyrosinase gene which straddles this restriction site and whose product enzyme produces the melanin pigment.

Plasmids isolated from such clones would transform TK64 to ThioR and give the nonsporulating Arg- phenotype. The plasmids differed slightly from each other: one plasmid (pJOE 752) was consistent by restriction mapping with the product of cloning the Brill amplified fragment into plJ702, a second one (pJOE753) had only one Bglll site, but had lost little else (it may be that a deletion, or a bad religation had occurred) and a third plasmid (pJOE754) had lost about 2kb of the amplified sequence. It therefore appears that only one part of the sequence is necessary to introduce the Arg non-sporulating phenotype The thiostreptone-resistant clones were streaked out several times successively on R2 agar containing thiostreptone and non-sporulating colonies appeared.These non-sporulating colonies were Arg-. Total DNA was prepared and showed amounts of DNA in a CCCform, but large amounts of the plasmid sequence were seen on digestion with appropriate enzymes, suggesting incorporation and amplification of at least some of the plasmid sequence in the chromosome.

The Arg- clone did not grow very well.

However, the thiostreptone resistance was not lost during and after growth without selection.

This stability is unusual as derivatives of plJ702 such as plJ752, 753 and 754 are usually readily lost. We concluded that integration and amplification within the chromosome had stabilized the plasmids and therefore made them acceptable as cloning vectors.

b) ISOLATION OF THE AGARASE GENE BY DELETION AND CLONING.

Total DNA from S coelicolorA3 (2) bearing the agarase gene was isolated and digested with Bglll.

The digest was separated as a low melting point agarose gel, the agarase gene 5kb band cut out of the gel and the DNA purified.

The DNA was then ligated into the Bglll site of plJ702 DNA which had been cut with Bglll (all enzymes and instructions for use available from BCl-The Boehringer Corporation (London) Ltd, Bell Lane, Lewes, East Suggest BN7 1 G) and treated with alkaline phosphatase to prevent recircularisation (phosphatasing method as disclosed in U.S. Patent No. 4 264 731).

The purified DNA (agarase coding sequence) was ligated with the plJ702 DNA and transformed into TK64 protoplasts with a final growth in the presence of thiostreptone.

Approximately 4000 thiostreptone resistant transformants were obtained. Of these, more than 95% were melanin-negative, indicating a high frequency of agarase DNA insertion into the Bglll site of plJ702 with consequent disruption of the tyrosinase gene which straddles this restriction site and whose product enzyme produces the melanin pigment.

The transformant colonies were then replicated onto minimal medium without a carbon source to facilitate identification of agarose clones. An area of sinking could be seen on one of these plates. Restreaking of spores from this region yielded colonies which sank into the surface of agar. Flooding such plates with Gram's lodine solution revealed clearing zones considerably larger than those produced by S.coelicolor.

Transformation of S.lividans protoplasts with plasmid DNA isolated from these colonies, therefore showed that most (more than 95%) of thiostreptone-resistant transformants produced agarase.

The small Pstl 1.9kb fragment of pMT605 was deleted and the resulting plasmid (designated pMT606) was found to have retained agarase activity, which was to some extent enhanced in correlation with the increased plasmid stability.

Restriction analysis of plasmid pMT605 demonstrated a 7kb insert into the Bglll site of the plJ702 and allowed the construction of the restriction map shown in Fig. 2.

To further localize the agarose gene on the cloned fragment, we attempted to subclone Xholl and Sau3A fragments from pMT605 into the Bglll site of plJ702. As a single Bgl II site was reconstituted at the border of the insert and vector DNA in pMT605, it was possible to linearise the plasmid with Bglll and then to partially digest with either Xholl or Sao3A. The resulting fragments were then ligated into the Bglll site of plJ702 and the mixture transformed into S.lividans. Several agarase producing transformants were obtained. Two of the smallest of these, pMT607 and pMT609 were picked out for further studies.Restriction analyses of these plasmids gave results that were incompatible with insertion of fragments into the Brill site of plJ702. It appeared that both were derivatives of pMT605 containing deletions removing the Bglll site. Presumably these deletions had arisen in vivo and were accidentally selected for by the sub-cloning procedure. Both plasmids retained the 1.9kb Pst I fragment.

Deletion of this fragment created pMT608 and pMT600 respectively.

c) COMBINING THE AMPLIFIED SEQUENCE AND THE AGARASE GENE IN THE SAME PLASMID.

The plasmid pJOE753 which had only one Bglll site was used as a vector to clone another gene. As an example we used the agarase gene of S.coelicolor. S.lividans 66 has not produced an agarase. The agarase gene of S.coelicolor A3(2) is expressed more strongly and produces large quantities of extracellular agarose when cloned in the plasmid vector plJ702 into TK64. The agarase is easily measured by standard methods because it produces reducing agarose from agarose.

The agarase can be cloned as a 4kb BamHI fragment (BamHI and Bglll produce the same sticky ends GATC).

We cloned the 4kb BamHI agarose fragment into the Bglll site of pJOE 753 and transformed into TK64 selecting for ThioR.

Some colonies produced agarose (and thus sank into the agar medium) and streaking out several times gave ThioR Aga+ Arg - colonies.

The agarase-production phenotype was stable when streaked out without Thiostreptone selection.

PMT605, the initial isolate, as was found to be lost from cells at high frequency, particularly when grown without thiostreptone selection, hindering initial purification of the plasmid. Experience with the later derivatives suggested that this segregation was less marked in those with increased agarase activity. To investigate this phenomenon, spore suspensions were prepared from cultures grown on thistreptone-containing agar. Suitable dilutions of the spores were plated onto LB without thiostreptone and the germinating colonies scored.

Culture supernatants of the parent strains S.coelicolorM130 and S.lividansTK64 and of the various plasmid-carrying strains were, assayed for agarase activity using reducing sugar assay methods, and for protein using standard techniques.

We measured the agarase production in shaken liquid cultures with and without Thiostreptone selection. Taking arbitrary units of enzyme activity, the best plasmid, PMT608, gave 58 unit/ml( + Thio) against 2.63 un it/ml( - Thio), whereas the derivative from step 9 gave 1 6.9 unit/ml( + Thio) against 11.37 unit/ml( - Thio) without any significant difference in the assay conditions used. It thus produced large quantities of agarase.

S.coelicolorA3(2) strain M130 the source of the agarase gene, produces at least 100-fold less and was stable without Thiostreptone selection.

Approximately 100 micrograms of total extracellular protein from each strain was subjected to electrophoresis on 10% SDS polyacrylamide gels. Bands of MW 28000 daltons could be seen in many of the tracks, the intensity of which correlated well with the agarase activity of each supernatant. In addition, preliminary purification data (results not shown) indicated co-purification of agarase activity with a 28kD SDS-PAGE band. Scanning of gels indicated that up to 50% of total extracellular protein contained within that single band.

A striking feature of this embodiment of the present invention is the over-production of extra-cellular agarase. Whereas S coelicolor A3(2) produces barely detectable levels of agarase, S. lividans harbouring the smallest derivative clone, PMT608, secreted large quantities of agarase; the specific activity in the supernatant was at least 500 fold greater than that found with S.coelicolor. This is probably due to a gene damage effect (p13702 is normally present at between 40 and 800 copies per chromosome). It is intersting that a much smaller increase in activity (36-fold) was reported with a cloned tyrosinase gene according to the methods of the prior art listed above. Further studies of induced and growth phase effects on production may allow even greater levels of production to be attained.

The 28kb band seen on SDS-PAGE of total extracellular protein is almost undoubtedly the agarase protein as its intensity correlated well with agarase activity for each culture supernatant and it is co-purified with agarase activity.

Significantly more bands are visible on the gels than one would expect from extracellular enzymes above. Many of these are likely to be intracellular proteins released due to cell lysis.

A 28kb protein would require a coding sequence of around 800 base pairs. Thus, the maximum length of the entire gene should be less than 1 kb. The gene has been localized to a 2kb segment such that another 1 kb of dispensable DNA remains.

The instability of many of the plasmids was particularly noticeable. Those plasmids with the greatest stability produced the most agarase suggesting that stability, and therefore presumably copy number, is a major factor in over production and also that the instability is not caused by the agarase gene. Plasmid size alone does not account for stability (PMT609 is extremely unstable), thus the phenomena must be due to the presence of deleterious DNA sequences within the cloned fragment.

This demonstration that such high levels of expression can be achieved with cloned streptomycete extracellular enzymes, has obvious commercial implications. In addition, further work may allow construction of "export" vectors so that the streptomycetes can become attractive alternative hosts to B.subtilis and E.coli for the export of foreign proteins.

We call this novel type of cloning vector carrying an amplifiable sequence an AMPLI CON. The simplest explanation of its nature could be that it is a part of the amplified sequence carrying a at least one cloning site.

We do not yet know if such a sequence without an extra plasmid replication origin would behave in this way and consequently we do not wish to be restricted to this explanation. A more generally useful vector would also contain a selective marker as Thio.

The procedure should be compatible with other plasmid replication origins such as SCP2 or SLP1 It is believed possible to exploit the amplifiable sequences in other species of Streptyomyces. The Arg - non-sporulating phenotype is useful (and may be fairly general as nonsporulating Arg mutants are common in many species), but easilly possible to detect amplification by other methods e.g.: (i) Another phenotypic effect, (ii) Selecting for stable transformants by continual streaking out, or (iii) Directly, using methods such as colony hybridisation.

The agarase gene itself has been shown to lie near an interesting region of the S.coelico lorchromosome. The NF high frequency donor strains of S.coelicolor have been shown to have arisen due to the integration of SCP1, a genetically identified but so far physically uncharacterised sex-factor of the species, into, or near the site of the agarase gene on the chromosome, inactivating the gene in the process. Use of PMT605 as a probe in hybridisation studies against DNA of various NF and SCPI-containing strains should provide some insight into many of the unusual features of SCPI and its ability for chromosomal integration.

Various modifications may be made within the scope of the present invention. For example, a Bglll fragment containing the amplified sequence has been cloned into the plasmid pBR325 in an E.coli host and Southern blot techniques have shown that TK64 has the amplified sequence present as a single tandem repeat. Furthermore a 5.7kb amplified sequence fragment has been ligated to a ThioR fragment and cloned into TK64, which demonstrates the possibility of making a vector which does not contain any plasmid replication sequences.

Finally, the original isolate of S. lividans 66 (John Innes strain number 1326) has been shown to give Chits mutants which yield highfrequency non-sporulating derivatives, indicating the wide applicability of the present invention.

REFERENCES Benigni R., Antonov R. P. and Carere A.

(1975) Estimate of the genome size by renaturation studes in Streptomyces. Appl. Microbiol. 30, 324-326.

Fedorenko V. A. and Danilenko V. N.

(1980) Instability of natural multiple drug resistance in actinomycetes (in Russian). Antibiotikii 25, 170-174.

Freeman R. F., Bibb. M. J. and Hopwood D. A. (1977) Chloramphenicol acetyltransferase-independent chloramephenicol resistance in Streptomyces coelicolor A3(2). J. Gen. Microbiol. 98, 453-465.

Freeman R. F. and Hopwood D. A. (1978) Unstable naturally occurring resistance to antibiotics in Streptomyces. J. Gen. Microbiol.

106, 377-381.

Gregory K. F. and Huang J. C. C. (1964) Tyrosinase Inheritance in Streptomyces scables 11 induction of tyrosinase deficiency by acridine dyes. J. Bacteriol. 87, 1287-1294.

Gross W. and Burkhardt K.-L. (1973) Multiple transport systems for basic amino acid transport in Streptomyces hydrogenans. Biochim. Biophys. Acta 298, 437-445.

Hopwood D. A. (1967) Genetic analysis and genome structure in Streptomyces coelicolor. Bacteriological Reviews. 31, 373-403.

Hopwood D. A., Bibb M. J., Bruton C. J., Chater K. F., Feitelson J. S. and Gil J. A.

(1983) Cloning Streptomyces genes for antibiotic production. Trends in Biotechnology 1, 42-48.

Hopwood D. A., Kieser T., Wright H. M.

and Bibb M. J. (1983) Plasmids, recombination and chromosome mapping in Streptomyces lividans 66. J. Gen.

Lennox E. S. (1955) Transduction of linked genetic characters of the host by bacteriophage P1. Virology 1, 190-206.

Matsubara-Nakano M., Kataoka Y. and Ogawara H. (1980) Unstable mutation of ss- lactamase production in Streptomyces lavendulae. Antimicrob. Agents Chemother. 17, 124-128.

Ono H., Hintermann G., Crameri R., Wallis G. and Hiitter R. (1982) Reiterated DNA sequences in a mutant strain of Streptomyces glaucescens and cloning of the sequence in Escherichia coli. Mol. Gen. Genet. 186, 106-110.

Orlova V. A. and Danilenko V. N. (1983) Multiplication of DNA fragment in Streptomyces antibioticus producing oleandomycin (in Russian). Antibiotikii 28, 163-167.

Polsinelli M. and Beretta M. (1966) Genetic recombination in crosses between Streptomyces aureofaciens and Streptomyces rimosus. J. Bacteriol. 91, 63-68.

Redshaw P. A., McCann P. A., Pentella M.

A. and Pogell B. M. (1979) Simultaneous loss of multiple differentiated functions in aerial mycellium-negative isolates of Streptomyces.

J. Bacteriol. 137, 391-899.

Robinson M., Lewis E. and Napier E.

(1981) Occurence of reiterated DNA sequences in strains of Streptomyces produced by an interspecific protoplast fusion. Mol.

Gen. Gent. 182, 335-340.

Schöffl F., Arnold W., Pühler A., Altenbuchner J., Schmitt R. (1981). The tetracycline resistance transposon Tn 1721 and Tn 1771 have three 38-base-pair repeats and generate five-base-pair direct repeats. Mol. Gen.

Schrempf H. (1983) Deletion and amplification of DNA sequences in melanin-negative variants of Streptomyces reticuli. Mol. Gen.

Genet. 189, 501-505 Sermonti G., Petris A., Mecheli M. R. and Lanafaloni L. (1 978) Chloroamphenicol resistance in Stremptomyces coelicolor A3(2): Possible involvement of a transposable element.

Mol. Gen. Genet. 164, 99-103 Shaw W. V., and Hopwood D. A. (1976) Chloramphenicol acetylation in Streptomyces.

J. Gen. Microbiol. 94, 159-166.

Thompson C. J., Ward J. M. and Hopwood D. A. (1980) DNA cloning in Streptomyces: resistance genes from antibiotic producing species. Nature 286, 527-529.

Vargha G., Karsai T. and Szabo G. (1983) A conditional aerial mycellium-negative mutant of Streptomyces fradiae with deficient ornithine transcarbamoyl transferase activity.

J. Gen. Microbiol. 129, 539-542.

Claims (22)

1. A method of producing a cloning vector including the steps of; a) obtaining a nucleic acid molecule with a repeated sequence, and, b) cutting the nucleic acid molecule to obtain a length of nucleic acid possessing at least part of the said repeated sequence.

2. A cloning vector including at least part of a multi-copy sequence of nucleic acid.

3. A method of cloning which comprises the steps of: a) obtaining a gene to be cloned; b) linking the said gene to be cloned to a nucleic acid sequence capable of existing as a multi-copy sequence within the chromosome of an organism into which the gene is to be cloned; and c) introducing the combination of the said gene and the said sequence into the said organism.

4. A nucleic acid sequence capable of integrating into the chromosome of an organism and multipling therein to establish the said sequence as a multi-copy sequence within the said chromosome.

5. A method of gene amplification including the steps of; a) linking a gene to be amplified to a sequence capable of existing as a multi-copy sequence within the chromosomes of an organism; b) introducing the said sequence into the chromosome of the organism; and, c) allowing the said sequence to multiply within the chromosome of the organism; whereby the combination of the said sequence and the gene to be amplified becomes amplified such that it exists as a multi-copy combination within the chromosome.

6. The method of claims 1, 3 or 5 wherein the nucleic acid is deoxyribonucleic acid.

7. The vector of claim 2 wherein the nucleic caid is deoxyribonucleic acid.

8. The vector of claim 7, wherein the nucleic acid is obtained from a member of the genus Streptomyces.

9. The method of claim 6, wherein the nucleic acid is obtained from a member of the genus Streptomyces.

10. The vector of claim 8, wherein the sequence is obtained from a member of the species Streptomyces lividans.

11. The vector of claim 10, wherein the sequence is obtained from a member of the strain Streptomyces lividans 66 strain TK64.

1 2. The vector of claim 11, wherein the sequence is that obtained as tandem repeats in the chromosomal sequence of an Arg strain.

1 3. The vector of claim 12, wherein the Arg - strain is derived from a spontaneous chloroamphenicol-sensitive mutant of the strain Streptomyces lividans 66 strain TK64.

14. The vector of claim 2, 7,8, 10, 11, 1 2 of 13, further comprising at least part of the nucleic acid sequence of a plasmid.

1 5. The vector of claim 13, wherein the plasmid is plJ702.

1 6. The method of claim 2 or 5 wherein the combination of the said gene and the said sequence is introduced into the organism by a plasimid vector.

17. The vector of claim 2, 7, 8, 10, 12-15, further comprising a marker capable of indicating the presence of the vector within the chromosome.

1 8. The vector of claim 17, wherein the marker confers thiostreptone resistance upon the organism.

1 9. The vector of claim 2 wherein the host is a strain of the species Esherishia coli.

20. The method of claims 1, 3 or 5, wherein the host is a strain of the species Esherishia coli.

21. A method of gene amplification substantially as hereinbefore described with refer ence to the accompanying drawings.

22. A vector substantially as hereinbefore described with reference to the accompanying drawings.