FR2469454A2 - Hybrid plasmid(s) of yeast - contg. DNA from 2 mu plasmid of yeast, URA3 plus gene and bacterial plasmid pBR 322 - Google Patents

Abstract

Hybrid plasmid within the scope of the main claim of the parent patent, 78.32100, has all or part of the DNA of the 2 mu plasmid of yeast, DNA segment contg. URA3 plus gene and all or part of the DNA of the bacterial plasmid pBR 322. The DNA segment contg. the URA3 plus gene is pref. inserted into the DNA of plasmid pBR 322 at the Hind III site. The 2 mu DNA fragment is pref. the 2 mu D fragment and is inserted at the Eco R1 site of the DNA of plasmid pBR 322. Yeasts contg. the hybrid plasmids are also claimed. These plasmids allow the cloning of long DNA fragments while they themselves are shorter and hence are less fragile and easier to handle and extract.

Description

 The present addition relates to new hybrid plasmids according to the main patent useful for modifying the properties of strains of microorganisms and in particular of yeasts as well as the microorganisms comprising these hybrid plasmids.

 The present addition relates to a hybrid plasmid composed of at least all or part of the DNA of yeast plasmid 2, a DNA segment containing the yeast URA3 gene and a DNA of the bacterial plasmid pBR 322.

 It may be useful to recall that the yeast plasmid 2 is known and a very complete study can be found in "Viruses and Plasmid in Fungi" editor Paul A. Lemke, this work can also be used as a reference for the definition of certain terms whose complete definition would not be given in the present description.

The URA3 + gene is the gene encoding 1 'orotidine-5' -phosphate-decarboxylase, in its absence the yeast can only develop on a medium containing uracil. The presence or absence of this gene makes it possible to " to screen "yeasts using a medium with and without uracil.

Plasmid pBR 322 is available at Bethesda
Research Laboratory Inc., Rockville, Maryland.

 In one embodiment of the plasmids according to the present invention, the DNA segment containing the yeast URA3 + gene is inserted into the DNA of the bacterial plasmid pBR 322, preferably at the Hind III site.

 The Hind III restriction sites correspond to the places of the DNA molecule which are cut by a particular enzyme, the endonucl se Hind III (which will be called hereinafter by abbreviation Hind III).

 There is only one Hind III restriction site on plasmid pBR 322.

The plasmids according to the present invention can comprise the entire DNA of plasmid 2 but, preferably, contain only part of the DNA of plasmid 2, the part called 2 D (the other part being called 2 D A), relative to the Eco R1 restriction sites.

The Eco R1 restriction sites correspond to the places in the DNA molecule which are cut by a particular enzyme, the Eco R1 endonuclease.

 In a particularly advantageous embodiment of the plasmids of the present invention, the total DNA or the DNA part of the plasmid 2 is inserted at the Eco RI site of the DNA of the plasmid pBR 322.

In another particularly advantageous embodiment of the present invention, the hybrid plasmid, in particular at the level of the DNA of the bacterial plasmid, comprises the insertion of an exogenous DNA originating from a prokaryotic organism or especially from an organism eukaryote such as yeast.

In the context of the present addition, it is also possible to provide hybrid plasmids in which the plasmid 2p includes an insertion of a
Exogenous DNA from a prokaryotic or especially eukaryotic organism.

 The vector plasmids according to the present invention can be prepared by known techniques which are already described in the main patent.

Hybrid plasmids can be stored as is or in yeast or bacteria where they will multiply and can be extracted on demand.

 These plasmids can be integrated into a yeast by the method described in the main patent.

 The following example is intended to illustrate a process for the preparation of hybrid plasmids and microorganisms according to the present invention without, however, that it can be considered as limiting the scope of said invention.

EXAMPLE
Analogously to Examples 1 and 2 of the main patent
- a plasmid pBR 322 is prepared with the fragment carrying the URA3 + gene inserted at the Hind III site
- partial digestion of the plasmid obtained with the enzyme Eco R1 is carried out
- partial digestion of plasmid 2 of the Sacharomyces cerevisiae strain is carried out
FL 100 ATCC 28383 by the enzyme Eco R1
- The two digestion products are mixed after inhibition of the Eco R1 enzyme and the fragments of the mixture are ligated with the ligase overnight at 100C;
- The plasmids obtained are extracted and a strain of yeast S. cerevisiae ura3 receptor, that is to say that cannot grow without uracil, is transformed and the transformants capable of growing on medium without uracil are selected. Plasmids are extracted from these URA3 transformants which, as has been described, can be used to transform a strain
Escherichia coli URA3, which by selection of the + URA3 strains obtained constitutes a reservoir of plasmids, the structure of which is given in Figures 1 and 2 attached hereto.

FIG. 1 represents the plasmid pFL 1 which comprises the DNA of the plasmid pBR 322 (represented in dotted lines) with insertion of the DNA of the + gene
URA3 (shown in double line) at the site level
Hind III of pBR 322 DNA and insertion at the Eco R1 site of pBR 322 of a fragment of DNA from plasmid 2, the 2 D fragment (fragment defined with respect to Eco R1 sites).

I1 was isolated 3 other plasmids, pFL 2, + pFL 3 and pFL 4. For pFL 2, the DNA of the URA3 gene has the opposite orientation, that is to say that the Pst 1 site is at 0, 8 kb from the Eco R1 site taken as origin.

The plasmids pFL 3 and pFL 4 have the 2 D fragment in the opposite orientation to that observed for pFL 1 and pFL 2 respectively. The total length of this plasmid is 7.652 kb.

Plasmid pBR 322 carries a gene for
(Amp resistance to ampicillin (Ampr), gene which codes for a penicillinase. In a yeast transformed by a plasmid of this type (like pFL 1 or pFL 2) this bacterial gene is expressed and one identifies an excretion of penicillinase by transformed yeasts, which shows the advantage of the plasmids described in the present addition for the excretion of proteins (enzymes in particular), (CHEVALLIER MR and AIGLE M.,
Qualitative detection of penicillinase produced by yeast strains carrying chimeric yeast-coli plasmids.

FEBS Letters, November 1979).

Another advantage of these plasmids is that they demonstrate that the entirety of plasmid 2 is not essential for its replication and for its maintenance in yeast. The advantage of this construction is that it makes it possible to consider cloning large foreign DNA fragments since the receptor plasmid is smaller. Indeed, too large plasmids are more fragile, more difficult to handle and to extract bacteria or yeasts.

 Similarly, the plasmid pMA 1 is obtained, represented in FIG. 2.

 This plasmid has the same structure as the previous ones but has the entire DNA of plasmid 2 (2 A + 2 D) instead of the 2 D fragment.

Claims (9)

1) Hybrid plasmid according to claim 1 of the main patent comprising at least all or part of the DNA of the yeast plasmid 2p, a DNA segment containing the URA3 gene and all or part of the DNA of the bacterial plasmid pBR 322.  2) Plasmid according to claim 1, characterized in that the DNA segment containing the yeast URA3 + gene is inserted into the DNA of the bacterial plasmid pBR 322.  3) Plasmid according to claim 2, characterized in that the DNA segment containing the yeast URA3 gene is inserted into the DNA of the bacterial plasmid pBR 322 at the Hind III site. 4) Plasmid according to one of claims 1 to 3, characterized in that it comprises the 2 D DNA fragment of the plasmid 2p. 5) Plasmid according to one of claims 1 to 3, characterized in that it comprises all of the DNA of the plasmid 2p.  6) Plasmid according to one of claims 1 to 5, characterized in that the total DNA ou.la portion of DNA from plasmid 2 is inserted at the site Eco R1 of the DNA of the plasmid pBR 322. 7) Plasmid according to one of claims 1 to 6, characterized in that it comprises the insertion of DNA from a prokaryotic or eukaryotic organism in addition to the DNA segment containing the yeast URA3 gene. 8) A microorganism comprising a hybrid plasmid according to one of claims 1 to 7. 9) A yeast according to claim 8.