JPS6235757B2 - - Google Patents

Description

[Detailed description of the invention]

The present invention relates to plasmids. More specifically, the present invention relates to a novel plasmid obtained from Cliffaromyces lactis. Plasmids, which are extrachromosomal genes, are widely used in recent genetic engineering as vectors for in vitro genetic recombination. In vitro genetic recombination techniques have a wide range of applications, including the breeding of useful microorganisms. Currently, prokaryotes such as Escherichia coli are mainly used as hosts in genetic engineering. For example, it may be advantageous to use eukaryotes such as yeast as hosts rather than E. coli. Furthermore, since yeast is harmless to the human body, it has the advantage of being a highly safe genetic manipulation system. The present inventors have previously investigated cell fusion, mitochondrial fusion, plasmid isolation, etc. using the protoplast polyethylene glycol method using Saccharomyces cerevisiae, with the aim of developing a gene manipulation system using yeast. We succeeded in isolating a new yeast plasmid from Ises lactis. That is, the gist of the present invention is to obtain a compound having a molecular weight of 5.3±0.1 obtained from Cliffaromyces lactis (ATCC8585) and having the following degrading properties with respect to restriction enzymes.
× ¹⁰⁶ Dalton linear plasmid (A) and molecular weight 8.3±0.1× ¹⁰⁶ Dalton linear plasmid (B)
The plasmid is selected from the group consisting of: (B) Linear plasmid (A) Digested into two fragments of 3.0×10 ⁶ Daltons and 2.3×10 ⁶ Daltons with EcoR, and 2.8× fragments with BamH.
broken down into two pieces of 10 ⁶ daltons and 2.6 x 10 ⁶ daltons, Hind 3.4 x 10 ⁶ daltons, 1.5
Decomposed into 3 pieces of ×10 ⁶ Daltons and 0.510 ⁶ Daltons, Pst 3.9 × 10 ⁶ Daltons, 1.0 ×
It is degraded into three fragments of 10 ⁶ Daltons and 0.5×10 ⁶ Daltons and is not degraded by Hpa. (B) Linear plasmid (B) Digested by BamH into two fragments of 4.8 × 10 ⁶ Daltons and 3.6 × 10 ⁶ Daltons, Hind, Pst
and Hpa, respectively. To explain the present invention in detail, Kluyveromyces lactis
ATCC8585, or IFO1267-) is used as a plasmid test strain. Plasmid separation is performed using the following methods (A) and (B). (A) Density gradient centrifugation-agarose gel electrophoresis. (B) Alcohol precipitation-agarose gel electrophoresis. (A) Outline of method: Cliffaromyces lactis cells are inoculated into a liquid complete medium from a preservative slant medium, cultured with shaking, and then harvested. PH including EDTA (ethylene diamine tetraacetic acid)
After washing thoroughly with 7.0 buffer, convert into protoplasts using a cell wall lytic enzyme (e.g. zymolyase or helyase), add SDS (sodium dodecyl sulfate) to lyse the cells, add 1M NaCl and leave overnight (4.
°C). After centrifugation, the supernatant was subjected to cesium chloride-ethidium bromide density gradient centrifugation, and the DNA
(deoxyribonucleic acid) is banded and separated.
The conditions for density gradient centrifugation were to add and dissolve ethidium bromide (2 to 4 mg/ml) and cesium chloride to the supernatant to give a final density of 1.57 to 1.58 g/ml;
Centrifuge at 38,000 rpm and 16°C for 48 to 72 hours.
Furthermore, circular DNA commonly found in bacteria
Plasmids can be separated from chromosomal DNA by cesium chloride-ethidium bromide density gradient centrifugation. However, since the plasmid of Cliffaromyces lactis, which is a linear DNA, cannot be separated from the chromosomal DNA by the above-mentioned centrifugation method, the plasmid and chromosomal DNA are separated using the following electrophoresis method. The DNA separated and concentrated by density gradient centrifugation is collected using an appropriate method (e.g., extracted from the side wall of the sedimentation tube with a syringe needle, or a small fraction of the sample is collected from the bottom of the sedimentation tube using a peristaltic pump, and the DNA is separated. ), remove ethidium dye with butanol, dialyze and purify, and prepare 0.7-1.5% agarose.
Subject to gel electrophoresis analysis. Two species of Cliffaromyces with different molecular weights
lactis plasmids electrophores at a faster rate than chromosomal (nuclear and mitochondrial) DNA, and their respective migration positions can be easily recognized by emitting fluorescence (yellow-red) under UV irradiation. Outline of Method B: According to Method A, protoplasts are prepared from bacterial cells and lysed with SDS. Add NaCl to a final concentration of 1M to the lysate, leave it overnight at 4°C, centrifuge it, and add about twice the volume of cold alcohol to the supernatant to precipitate the nucleic acids. The precipitate was dissolved in Tris-HCl buffer (PH
7.5), treated with ribonuclease, and subjected to agarose gel electrophoresis to separate the plasmid from the chromosomal DNA using the difference in mobility. Regardless of whether method A or method B is used, as a result of agarose gel electrophoresis analysis, two types of plasmids with different molecular weights (electrophoretic migration distances) are identified as Cliffaromyces lactis (ATCC8585 or
IFO1267), but after fractional extraction from agarose gel and purification,
When analyzed using an electron microscope (Model JEOL-1OOB), uniform particles of 2.6 to 2.7 μm (hereinafter referred to as plasmid (A)) and 4.1 to 4.2 μm (hereinafter referred to as plasmid (B)) were found. Two types of linear DNA with similar sizes were observed. (Lambda) λDNA restriction enzyme (EcoR One) (EcoR, or (Hindsley Hind)
) The molecular weight of the plasmid was determined by comparing the mobility by agarose gel electrophoresis using a DNA fragment of known molecular weight obtained by digestion with plasmid (A) as an internal marker.
5.3±0.1× ¹⁰⁶ Daltons, number of bases 8.9Kb, plasmid (B) = 8.3±0.1× ¹⁰⁶ Daltons, number of bases 13.4Kb
The molecular sizes were consistent with those obtained by electron microscopy (the molecular weight of 1 μm DNA is approximately 2×10 ⁶ Daltons). In addition, the results of investigating the degradation characteristics of the above plasmid with various restriction enzymes (EcoR, Hind, BamH, Pst, and Hpa) are as follows. In other words, plasmid (A) (molecular weight 5.3 ± 0.1 × 10 ⁶ Daltons, number of bases 8.9 Kb) is
), BamH, EcoR,
In BamH, Hind Three (Hind
), Pust One (Pst), Hind, Pst 3
Etsuchipawan (HpaI), Hpa
is not decomposed. Also, plasmid (B) (molecular weight
8.3±0.1×10 ⁶ Daltons, base number 13.4 Kb) is degraded into two fragments by BamH, but not by Hind, Pst, or Hpa. Furthermore, the molecular weights of the fragments degraded by restriction enzymes are as follows. About Plasmid (A) Echo R One (EcoR): 2.3 x 10 ⁶ Daltons, 3.0 x 10 ⁶ Daltons Hind: 0.5 x 10 ⁶ Daltons, 1.5 x 10 ⁶ Daltons, 3.4 x 10 ⁶ Daltons Pust One (Pst): 0.5×10 ⁶ Daltons, 1.0
×10 ⁶ Daltons, 3.9 × 10 ⁶ Daltons BamH: 2.6 × 10 ⁶ Daltons, 2.8 × 10 ⁶ Daltons About plasmid (B) BamH: 3.6 × 10 ⁶ Daltons, 4.8 × 10 ⁶ Daltons or less , the present invention will be explained by way of examples. In addition, in the following examples, plasmid (A)
PGKl-1 plasmid, plasmid (B)
It is called pGKl-2 plasmid. Example 1 (1) Separation and purification (Method A) Kluyveromyces lactis ( Kluyveromyces lactis ATCC8585 or
IFO1267) was cultured in a complete medium, and 1 g of the obtained bacterial cells was converted into protoplasts according to the method described in Japan J. Genetics 53 , 41-49 (1968). That is, 1 clifaromyces lactis
After culturing in a medium (PH5.3) containing % yeast extract, 2% peptone, and 2% glucose, it was washed with water and cultured in 0.2% β-mercaptoethanol and 0.06 MEDTA solution for 30 min.
℃ for 30 minutes. Then after washing with 0.8M sorbitol solution, 0.8M
Sorbitol, 0.01 MEDTA, 0.1M citrate -
Phosphate buffer solution (PH6.1) was used to obtain a cell concentration of 2×10 ⁹ /ml, and Zymolyase 60000 (Cell wall lytic enzyme manufactured by Kirin Brewery Co., Ltd., Archive of Biochemistry and Biophysics (Arch.
Biochem . The obtained protoplasts were collected by centrifugation at 1000 g for 10 min.
Suspended in 0.8M sorbitol and centrifuged again. 2.5 ml of 0.8 M sorbitol 0.1 MEDTA solution was added to the obtained protoplasts, and 5 mg/ml pronase E (TES buffer solution TrisHCl 30 mM,
0.35 ml of pretreated 1.5 hours at 37°C in 50mM NaCl and 5mM EDTA PH8.0 was added, and the mixture was allowed to react at 37°C for 30 minutes. Next, add 0.35ml of 10% SDS aqueous solution to lyse the bacteria.
0.7 ml of 5M sodium chloride was added. After standing at 4℃ overnight, centrifuge at 17,000 rpm for 60 minutes and supernatant (approximately 6 ml).
20 μg/ml ribonuclease was added to the mixture and treated at 37° C. for 30 minutes. Approximately 5.7 g of cesium chloride and 3 to 4 mg of ethidium bromide (dissolved in dimethyl sulfoxide) were added to the treatment solution to bring the final density to 1.57.
Adjusted to ~1.58g/ml, 38000rpm, 16
Centrifuged at 48°C for 70 hours. The generated DNA band was detected by irradiation with 350 nm UV light, and a small amount of the sample was taken out from the bottom of the sedimentation tube using a perister pump, and the DNA fraction was collected. Once the DNA sections were collected, they were treated with butanol to remove ethidium bromide, and then treated with 0.1x SSC buffer (SSC: 0.15M, NaCl, 0.015M sodium citrate) containing 1mM MEDTA.
After dialysis, it was subjected to agarose gel electrophoresis.
The pGKl-1 and pGKl-2 plasmids were separated and detected with 350 nm UV light. Cut out the DNA segments of pGKl-1 and pGKl-2 plasmids from agarose gel (several dozen bottles) with a sterilized razor blade, and use approximately 3 times the amount of buffer solution (10mM Tris HCl-PH8.0, 0.3M, NaCl,
0.01 MEDTA), put it in a syringe and injected it. The injection solution was left in a cold place (4°C) overnight with stirring, and then frozen at -20°C for 3 hours. After thawing at room temperature,
Centrifuge at 17,000 rpm for 30 minutes, filter the supernatant through a millibore filter, remove remaining ethidium bromide with butanol treatment, and reduce to 1mM.
Dialyzed with 0.1×SSC containing EDTA. Dialyzed and purified pGKl-1 plasmid,
The pGKl-2 plasmid was subjected to restriction enzyme digestion for molecular weight determination and electron microscopy analysis. (Method B) A final concentration of 1M NaCl was added to the protoplast lysate obtained in the same manner as in Method A, left overnight, and centrifuged at 17,000 rpm for 60 minutes. 2 to supernatant
Double the volume of cold ethyl alcohol was added, left at -20°C for 30 minutes, and centrifuged at 15,000 rpm for 15 minutes. 0.4% precipitation
It was slowly dissolved in 2 ml of TES containing sarcosyl. The mixture was centrifuged at 7000 rpm for 5 minutes and the supernatant was collected. Dissolve the precipitate in 2 ml of TES solution containing 0.4% sarkosyl,
The mixture was centrifuged again at 7000 rpm for 5 minutes, and the supernatant was combined with the previously collected supernatant, twice the volume of cold alcohol was added, and the mixture was left at -20°C for 30 minutes. Next, it was centrifuged at 15,000 rpm for 15 minutes, and the resulting precipitate was mixed with 0.01M Tris-HCl (PH
7.6) Dissolved in 300 to 500 μl of buffer solution and centrifuged at 7000 rpm for 5 minutes. The supernatant was collected and treated with ribonuclease (20 μg/ml) at 37°C for 60 minutes, and then subjected to agarose gel electrophoresis.
DNA was separated from chromosomal DNA and detected. Hereinafter, the fractional extraction of pGKl-1 plasmid and pGKl-2 plasmid is the same as that described in Method A. (2) Confirmation that it is a plasmid (A) Electron microscopy analysis shows that the length of the molecule is uniform.
A certain amount of linear DNA (2.6-2.7 μm for pGKl-1 plasmid and 4.1-4.2 μm for pGKl-2 plasmid) was observed. (B) Agarose gel electrophoresis analysis shows that the above linear
DNA of uniform molecular weight corresponding to DNA (pGKl-1
=5.3±0.1× ¹⁰⁶ Daltons, pGKl−2=8.3±
0.1 × 10 ⁶ Daltons) was detected. (C) pGKl-1 plasmid and
It was found that the pGKl-2 plasmid can be cleaved to a specific size. The total molecular weight of the cleaved DNA fragments matched the molecular weight size of each plasmid before cleavage. (3) Molecular weight measurement The molecular weight of pGKl plasmid DNA separated and purified by cesium chloride-ethidium bromide centrifugation was measured by agarose gel electrophoresis analysis. As a result, pGKl-1 plasmid = 5.3 ± 0.1 × 10 6 Daltons, pGKl-2 plasmid = 5.3 ± 0.1 × 10 ⁶ Daltons = 8.3±0.1×10 ⁶ Daltons. A DNA fragment with a known molecular weight obtained by digesting (lambda) λ DNA with (hindi) Hind was used as an internal marker for molecular weight measurement. Agarose gel electrophoresis using
The DNA detection conditions are as follows. Electrophoresis device: Mitsumi disk electrophoresis device (standard type)
It was used. Inject 0.7 or 1.5% agarose gel into a glass tube with an inner diameter of 0.5 cm and a length of 9 cm, place it between the upper and lower buffer baths of the electrophoresis device, and place the DNA sample on the top of the agarose gel. Electrophoresis: Buffer containing 0.5μg/ml ethidium bromide (0.04M Tris, 0.02M sodium acetate, 1mMEDTA, adjusted to pH 0.8 with acetic acid)
Run inside at 50V for 3 to 4 hours. After electrophoresis, remove the gel from the glass tube,
The position of DNA movement is detected by irradiation with 350nm UV light and photographed with Polaroid film 105 through an R1 filter. (4) Digestion with restriction enzymes The pGKl-1 plasmid and pGKl-2 plasmid isolated and purified by the method described in (1) were digested with restriction enzymes (EcoR, Hind, BamH, Pst, Hpa).
The mixture was reacted at 37° C. for 1 to 2 hours in the buffer shown in Table 1. The reaction was stopped by heating at 65° C. for 5 minutes, and the molecular weight of the DNA fragment was measured by agarose gel electrophoresis. As an internal marker, a DNA fragment obtained by digesting λDNA with EcoR was used. Table 2 shows the molecular weights of fragments obtained by digesting pGKl-1 and pGKl-2 plasmids with restriction enzymes.

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Claims (1)

[Scope of Claims] 1. A linear plasmid (A) with a molecular weight of 5.3±0.1×10 ⁶ Daltons and a molecular weight of 8.3±0.1× obtained from Cliffaromyces lactis (ATCC8585) and having the following degrading properties for restriction enzymes.
10 Plasmids selected from the group consisting of ⁶ Dalton linear plasmids (B). (B) Linear plasmid (A) Digested into two fragments of 3.0×10 ⁶ Daltons and 2.3×10 ⁶ Daltons with EcoRI, and 2.8× fragments with BamH.
broken down into two pieces of 10 ⁶ daltons and 2.6 x 10 ⁶ daltons, Hind 3.4 x 10 ⁶ daltons, 1.5
Decomposed into three pieces of × ¹⁰⁶ Daltons and 0.5 × ¹⁰⁶ Daltons, Pst 3.9 × ¹⁰⁶ Daltons, 1.0
It is degraded into three fragments of ×10 ⁶ Daltons and 0.5 × 10 ⁶ Daltons, and is not degraded by Hpa. (B) Linear plasmid (B) Digested by BamH into two fragments of 4.8 × 10 ⁶ Daltons and 3.6 × 10 ⁶ Daltons, Hind, Pst
and Hpa, respectively.