CS240392B1 - A method of preparing a highly polymeric 14C-labeled uniformly labeled chromosomal dsoxyrlbonucleic acid - Google Patents

Abstract

A method for preparing highly polymeric deoxyribonucleic acid uniformly labeled with the radioisotope 14C, based on the cultivation of cyanobacteria of the genus Synechococcus in an atmosphere of 14COs and subsequent isolation of deoxyribonucleic acid from radioactive biomass. The essence of the invention lies in the fact that the source of deoxyribonucleic acid is biomass obtained by a controlled cultivation process at a specific growth rate μ' of 0.20 to 0.25 li"* 1, with a total utilization of 14COj from the environment of more than 75% and with a minimum DNA content of 1% in dry matter, whereby the isolation yields deoxyribonucleic acid with a molecular weight of 10s to 107 atomic mass units.

Description

(54) A method for the preparation of a highly polymeric ¹⁴ C chromosamous dsoxyrbonucleic acid uniformly radiolabelled

2

A process for the preparation of a highly polymeric ¹⁴ C radio-labeled deoxyribonucleic acid, based on the cultivation of Synechococcus cyanobacteria in an atmosphere of ¹⁴ COs and subsequent isolation of deoxyribonucleic acid from radioactive biomass. The invention is based on the fact that the source of deoxyribonucleic acid is biomass obtained by a controlled cultivation process at a specific growth rate μ 'of 0.20 to 0.25 li ^{* 1} , with a total utilization of ¹⁴ COj from an environment of more than 75% and a minimum DNA content of 1. isolation to obtain deoxyribonucleic acid having a molecular weight of 10 ^s to 10 ⁷ atomic mass units.

The invention relates to a method for preparing high 'polymeric chromosomal deoxyribonucleic acid uniformly labeled with radioisotope ¹⁴ C, based on the cultivation of cyanobacteria of the genus Synechococcus, preferably Synechococcus elongatus (NAG./NAG. Var. Thermalis Kovrov strain 1972/8), under which ¹⁴ and subsequent isolating deoxyribonucleic acid from radioactive biomass.

Cyanobacteria are among prokaryotic microorganisms with photoautotrophic nutrition and are able to use CO2 (or carbonates) as the sole carbon source. In addition, they are highly resistant to radioactive radiation [Carr, N. G., Whitton,

BA, The Biology of Blue-Green Algae (1973), Kraus, Μ. P., Radiation Botany, 9, 481 (1969)]. Thus, their main advantage in terms of their use in the production of ¹⁴ C-labeled organic substances is their ability to grow in a ¹⁴ CO2 environment for several generations at high growth rates and to produce biomass with a high radioisotope content.

As mentioned above, cyanobacteria of the genus Synechococcus belong to prokaryotes, so they resemble in many bacteria: cell wall construction, high specific growth rate, high nucleic acid content in dry matter. However, the chemical composition of biomass strongly depends on the culture conditions and growth rate of the culture [Stanier, R.Y., Van Niel, C. B., Arch. Microbiol., 42, 17 (1962)]. A specific feature of cyanobacteria is the photoautotrophic diet (photosynthesis) and the resistance of some species to extreme cultivation conditions - high temperature, with a cultivation optimum of up to 343 K [Castenholtz, R. W., Bacteriol. Rev., 33, 476 (1969)], over a wide range of light intensities [Beljanin, V. N., Trenkensu, A. P., Arch. Hydrobiol. Suppl. 51, Alological Studies 18, 46 (1977)], and radioactive radiation [Kraus, Μ. P., Radiaton. Botany, 9, 481 (1969); Sparow, A.H., Radiation Research, 32, 915 (1967)].

By controlling a cyanobacterial culture regime that is able to grow and reproduce in a ¹⁴ CO ² atmosphere for several generations, biomass (and hence deoxyribonucleic acid) with varying degrees of radioisotope incorporation can be obtained. On the one hand, very low specific radioactivity (corresponding to tracer), but on the other hand, highly radioactive biomass, in which more than 95% of the carbon replaced by the radioisotope can be assumed. The specific radioactivity of the resulting product can be controlled by the fact that CO 2 and ¹⁴ CO 2 are used as a source of carbon. However, the radioisotope content of biomass can also be varied by cultivating the culture exclusively in a ¹⁴ CO2 environment, but allowing it to grow only to a certain density (only one or two generations). For example, the relationship between the degree of incorporation of ¹⁴ C into deoxyribonucleic acid and the lethality (or deformation of the morphological structure) of the cells could be monitored as described.

The question of the ability of organisms to regenerate and their ability to withstand environmental conditions is currently of major concern. The possibility of studying DNA isolated from these particular microorganisms is therefore particularly attractive.

In the context of the development of molecular biology and molecular genetics, preparations of nucleic acids with preserved biological activity, macromolecules and intact chemical as well as physical structures are increasingly in demand (Watsen, J. D., Molecular Biology of Gene,. Academy Prague 1982). For this reason, the demands on separation and isolation methods in biochemistry also increased strongly. It has been shown that a suitable procedure for DNA isolation can be designed only on the basis of a good knowledge of the source of nucleic acids (microorganism) used - knowledge of the structure and chemical composition of cells, physiology. To design the isolation procedure on a preparative scale, it is also necessary to guarantee a standard biological material with a constant nucleic acid content. The chemical composition of cyanobacterial biomass and its growth rate depend not only on the specific characteristics of the selected strain, but also on the composition of the nutrient solution, the culture temperature and also the intensity and spectral composition of light [Carr, NG, Whitten, BA] 1973), Beljamin, VN, Trenchen, AP, Arch. Hydrobiol./Suppl. 51, Algological Studies 18, 46 (1977)]. Therefore, standard biological material can only be obtained by appropriate selection and strict adherence to culture conditions.

The basic principles of isolation of highly polymeric DNA from microorganisms were formulated in 1961 by Marmur and in 1964 by Kirby J Marmur, J., J. Mol. Biol., 3, 203 (1961); Kirby, K. S., Biochem. J., 93, 5c (1964)]. The study of the properties of deoxyribonucleic acid from cyanobacteria and hence its isolation was later addressed by a number of authors: Edelman, M, Swinto, D., Schiff, J.A., Eppstein, Η. T., Zeldin, B., Bacteriol. Rev. 31, 315 (1967), Graig,

I.W., Leach, C.K., Carr, N.G., Arch. Microbiol., 85, 218 (1969), Stanier, R.Y., Kunisava, R., Mandel, M., Cohen-Bazire, G., Bacteriol. Rev., 35, 171 (1971). However, these were mainly experiments on an analytical scale on a non-radioactive material and on their basis only individual operations of the isolation process can be determined in general.

Background information on cyanobacterial DNA preparation can be summarized as follows:

A. the procedure can be divided into operations:

1. preparation of a suitable DNA source

2. chemical lysis or mechanical cell disintegration

3. inactivation of deoxyribonucleases

4. extraction of nucleic acids

5. deproteinace

6. separation of DNA and RNA

7. DNA purification and specifications.

Β. Different methods and reagents have been proposed by the various authors for each operation without specifying specific conditions, but they cannot be used in general. It is always necessary to choose a method only according to the selected type of microorganism.

C. If a detailed procedure is given, it applies only to a specific species and strain of micro-organisms.

D. Isolation of cyanobacteria DNA has been described only generally from non-radioactive material and only in some selected strains.

The preparation of ¹⁴ C-radiolabelled high-polymer DNA from cyanobacteria of the genus Synechococcus has some specific peculiarities that have not been addressed. The design of the technological process must be taken into account.

The above-mentioned state is solved by a complex process of preparation of highly polymeric chromosomal deoxyribonucleic acid uniformly labeled with ¹⁴ C radioisotope based on cultivation of cyanobacteria of the genus Synechococcus in ¹⁴ CO ² atmosphere and subsequent isolation of deoxyribonucleic acid from radioactive biomass. cultivation process at a specific growth rate μ = 0.20 to 0.25 h ^-1 , with a total use of ¹⁴ CO2 from an environment of more than 75% and a minimum DNA content of 1% in dry matter, isolating to obtain deoxyribonucleic acid with a molecular weight of 10 ⁶ up to 10 ⁷ atomic mass units.

The advantages of the proposed procedure can be summarized as follows:

A suitable microorganism growing in a high radioactivity environment and capable of incorporating a ¹⁴ C radioisotope with high efficiency has been selected. The advantage of selected strain Synechococcus elongatus var. thermalis strain Kovrov is also a growth optimum at 56 ^° C. At this temperature the development of potentially infectious microorganisms is strongly suppressed; a higher cultivation temperature is also advantageous from an energy point of view, since intensive cooling with the thermophilic strain is not necessary when using an intense "warm" light source, where the suspension is overheated.

2. A suitable culture regime has been proposed, providing standard biological material as a source of DNA with a reproducible nucleic acid content.

3. A suitable lysis method and conditions have been proposed that lead to perfect DNA release from the cells.

4. By reducing sample handling (agitation, shaking) to the smallest extent that would result in mechanical degradation of the DNA, a preparation having a molecular weight of 10 ⁸ to 10 ⁷ atomic mass units was obtained.

Example

1. Cultivation of cyanobacteria

The following table shows the percentage of nucleic acids in cyanobacteria of Synechococcus elongatus (NAG./NAG. Var. Thermalis strain Kovrov 1972/8) as a function of specific growth rate. The culture was grown in a nutrient solution whose composition is as follows:

1 liter of nutrient solution contains: KNOj 1 G NaNCb 1 G K2HPO4 1 G NaHCCb 0.84 G MgSOi 0.25 G Fe ⁺³ (chelatonate) 18.4 mg Ca ⁺² (CaChj 15.6 mg

Microelements:

Cu ⁺² 0.635 mg

Mo ⁺⁶ 0.960 mg

Zn ⁺³ 0.650 mg

Co + ² 0.590 mg

Mn * ² 0.600 mg

EDTA (sodium salt) mg

Cyanobacteria of the genus Synechococcus elongatus (NAG./NAG. Var. Thermalis Kovrov 1972/8} are cultivated in ¹⁴ CD2 medium. The culture apparatus consists of a glass cylinder with a tempered jacket through which cooling water flows. The cylinder may contain about 700 ml of cyanobacteria suspension in nutrient solution. The thickness of the inner part of the cultivation roller is 45 mm The cultivation roller is illuminated by two Teslafot 500 W bulbs installed in a U-shaped illuminator with adjustable power input The cultivation roller is connected to another vessel in which a nutrient solution is prepared to dilute the cyanobacterial culture A ^1J CO2 (or CO :) generator is connected to the cultivation cylinder and the whole equipment is evacuated to prevent the escape of gases into the atmosphere.

Cultivation mode:

composition of the nutrient solution is shown in Table 2, cultivation temperature 56 ^C C external irradiance of 250 W culture. m ^-2 FAR, the gaseous mixture contains 2 to 3% by volume of ¹⁴ CO2, the average optical density of the culture p 0,5 cm ₌ . n CCJ ^ύ . 750 lira ^U ' ^0J ' mean specific growth rate of the culture μ = 0.209 h ^-1 , mean doubling time 3.3 h, DNA content in dry matter 1.11 ° / o, RNA 7.69 ° / o.

2. Isolation of deoxyribonucleic acid

The cyanobacteria culture is centrifuged immediately after harvesting and washed with solution. 0.1 M EDTA + 0.15 NaCl 10.2 M tris, pH = 8. The biomass obtained is mixed in 10x diluted 1: 1 preservative buffer and lysozyme at a concentration of 5 mg per 10 g of the mixture and 25% solution are added. sodium lauryl sulfate to a final concentration of 1.25%. The reaction mixture was allowed to incubate in a thermostat at 37 ° C for three hours. A solution of pronase enzyme (Streptomyces griseus) is then added in an amount of 0.5 mg per 10 g of the mixture. The mixture was left in the refrigerator for 20 hours. The ski is terminated when the viscosity of the liquid is greatly increased and only a few percent of the intact cells are detected microscopically. The viscous liquid was mixed with an equal volume of chloroform (containing isoamyl alcohol in a ratio of 24: 1) and gently mixed on a shaker in a flat container. After 30 minutes, the liquid was centrifuged at 5,000 x g for 5 minutes. Three layers are created. The upper aqueous phase is filtered off with suction and

E _o / OD

The non-radioactive culture is precipitated with cold ethanol (final concentration of 80 1% by volume) in NaCl at pH = 88. The fibrous nucleic acid precipitate is transferred to a solution of 0.15 M NaCl + 0.015 M sodium citrate, pH = 7 (hereinafter referred to as 1). The deproteinization (mixing with chloroform or phenol) is repeated until the middle layer containing the proteins disappears.

The separation of DNA and RNA is carried out by gel chromatography on a modified agarose column. 0.1 M NaCl + tris 0.02 M pH = 8 was used as the elution solution. Equilibration was carried out with 2.5 M NaCl -t-tris 0.02 M pH = 8.

The relative irradiance of the culture (E _o / OD) represents the ratio of the external irradiance of the culture (Ej, measured in Wm ^-2 photosynthetically effective radiation (FAR, 400-700 nm) to the mean optical density of the culture (extinction at 750 nm measured at 0.5 cm) Teslafot 500 W bulbs were used as the light source for the cultivation equipment.

TABLE

DNA% v p.

RNA% v s.

0.125 157 0.95 5.01 0.241 475 1.41 8.35 0.285 606 1.48 9.86 0.321 1013 1.76 10.94

¹⁴ CO ₂ culture

0.101 184 0.64 5.38 0,209. 455 1.11 7.69

(μ / h ^-1 ... mean specific growth rate of culture

E _o (Wm ² ) FAR ... external irradiance of the culture by photosynthetically effective radiation (400–700 nm)

OD ... optical density of the culture, measured as extinction at 750 nm in a 0.5 cm slurry layer

The table shows that the nucleic acid content of biomass increases with increasing growth rate. However, growth rates higher than 0.3 h ^-1 can only be achieved in a very diluted cyanobacteria culture on the cultivation equipment used, and the overall biomass production is then low. For this reason, it has been recommended for the production of cyanobacteria of Synechococcus as a source of DNA

Claims (1)

SUBJECT A method for the preparation of a highly polymeric chromosomal deoxyribonucleic acid uniformly labeled with ¹⁴ C radioisotope, based on the cultivation of Synechococcus cyanobacteria in a ¹⁴ CO ₂ atmosphere and subsequent isolation of deoxyribonucleic acid from radioactive biomass, characterized in that the deoxyribonucleic acid source is 20 to 0.25 h ^-1 (doubling time then corresponds to 2.5 to 3.0 h), which was achieved with a relative irradiation of the culture of 400-500 W. m ^-2 FAR and optical cultures 0.5-0.6. Under these conditions, biomass contained 1-1.5% DNA and 7.5-8.5% RNA in dry matter. INVENT the tivation process at a specific growth rate μ = 0.20 to 0.25 h ¹ , with a total use of ¹⁴ CO ₂ from an environment of more than 75 percent and a minimum DNA content of 1% in dry matter, isolating to obtain deoxyribonucleic acid with a molecular weight of 10 ⁶ to ΙΟ ² atomic mass units.