CS238268B1 - A method of cloning bovine prochymosin genes - Google Patents

Abstract

The subject of the invention is a method of cloning genes for bovine prochymosin, obtained by reverse transcription of polyA+ -mRNA - fraction with sedimentation coefficient 12 to 17S from the mucosa of the fourth stomach of two to three-week-old calves. Using terminal -fcransferase, single-stranded homopolymer ends with an average length of 55 to 200 nucleotides are attached to double-stranded complementary DNA molecules, which are connected to a linear plasmid vector, which after the transferase reaction also contains homopolymer single-stranded, but complementary ends with an average length of 10 to 20 nucleotides, and transferred to recipient cells intended for cloning, in particular E. coli, Bacillus subtilis or Streptococcus cells, after which the selection of recombinant clones is carried out.

Description

The invention relates to the cloning of prochymosin genes of bovine origin phsynonymous with prenenine.

Cloning of e-caryotic nucleotide sequences as carriers of biological information, e.g. in E, coli, is a method used increasingly / R. Wu / ed./: Meth. Enzymol. £ 1979 (1979), which applies to various gene technology techniques and solutions.

A precondition for the production of specific vectors ensuring the expression of selected animal proteins is the molecular cloning of a specific coding sequence. The procedure described and published by Nishimori et al. / J. Biochem. 22 (901-904, 1981), using a homopolymer technique (tailing), exhibited low efficacy (relative to the number of recombinants depending on the DNA used) and furthermore only resulted in clones having a maximum of 80% of the length of the entire coding sequence. . By using a special selection procedure for long double-stranded cDNA molecules from a heterogeneous population, a more efficient procedure for cloning the complete coding sequence of the bovine prochymosin gene can be devised (U.S. Patent No. 143,794 (NDR)).

In a number of cases, eukaryotic nucleotide sequences have been manipulated under the control of prokaryotic expression control elements, thereby producing efficient microbial synthesis of eukaryotic proteins:

Human insulin, A and B chains / Goeddel et al., Proc. natn.

Acad. Sci. USA, Jš, 106,

1979 /

238 268 | 3-endorphin

Human growth hormone

Human Elucocyte Interferon

Human Fibroblast Interferon (Shine et al., Nature 25 _of 456, 1980) (Goeddel et al. Nature _{of the Z} 2§J 544 1979 / / Goeddel et al. _from Nature 2SZz 411 _of 1980 / / Goeddel et al. _of Nucl. Acids Res. 3 _of 4057, 1980].

The method for cloning functional gene for prochymosin bovine origin _from the assistance terminal transferase connects to double-stranded molecules complementary DNA single stranded Homo to polymer ends an average length of 35-200 nucleotides, and combine these with linear plasmid vector which contains the following trans Luciferase reaction also homopolymer _of single stranded complementary ends but having an average length of 10 to 20 nucleotides, and transferred to recipient cells intended for cloning, in particular E. coli, Bacillus subtilis or Streptococcus, followed by re selection, by recombinant clones.

According to the invention, the cloning procedure for the complete functional prochymosin gene of bovine origin is based on the material obtained by the transcription of the polyA ⁺ -mRNA (fraction 12'17S information mRNA fraction) from the mucosa of the fourth stomach of two to three weeks of calves. The method consists in attaching single-stranded ends with an average length of 35 to 200 nucleotides to the double stranded complementary DNA molecules of the DNA population thus obtained, which is heterogeneous in length and sequence, using terminal transferase. It is then coupled to a linear plasmid vector which, after the transferase reaction, has homopolymeric, complementary single stranded ends with an average length of only 10 to 20 nucleotides, and transferred to recipient cells to be cloned, preferably E. coli, Bacillus subtilis or streptococci. Recombinant clones are then selected.

The selection is either

in situ hybridization according to Grunstein and Hogness / Proc. natn. Acad. Sci. USA 22 (3961, 1975) by means of a fractionated polyA * -RNA from a calf stomach that contains a highly enriched translationally active chymosin mRNA, or

238 268

- in situ hybridization or SNA-DNA hybridization with chemically synthesized oligonucleotides corresponding to the bovine chymosin coding portion, or

RNA-DNA hybridization techniques / Alwine et al., Proc. natn. Acad. Sci., USA I (5350, 1977).

These techniques are suitable for selecting individual clones or clone families in a single screening procedure. In combination with specific translational assays / e.g. hybrid stopped translation, Paterson et al., Proc. natn. Acad. Sci. USA Z4, 4370-4374 (1977) allow specific detection of chymosin clones.

The procedure for cloning single-stranded end-point populations of different lengths applied to two stranded complementary DNAs / cDNAs, respectively. vector DNA leads to the enrichment of long inserts, by recombinant DNA re clones as compared to cloning based on the stocks of similar length at the single-stranded termini of double stranded cDNA _y conn. Vector DNA / GDR patent publication no. 143,794 /. Recombinant plasmids consist of pBR322-PstI-dG _n ; The double stranded chymosin cDNA / partial or complete specific nucleotide sequence < dC > pBR 322 is a PstI vector molecule. is a restriction site in this molecule to which a single-stranded sequence of deoxyguanol nucleotides is attached, and dC denotes a similar sequence of deoxycytidol nucleotides.

Yet, when cloning very long mRNA molecules, e.g. in the case of chymosin = RNA 12%; 17S; when compared to a globin or proinsulin new mRNA = RNA 9S / it is possible that the specific encoding is topotogically distributed on more than one recombinant plasmid. By means of common restriction sites within the inserts, it is possible to construct in a simple manner the corresponding recombinant plasmid which carries the complete coding sequence for prochymosin (pre-prochymosin).

The vector plasmid pBR322 used is suitable for cloning foreign DNA, preferably in E. coli. However, the specific plasmids of the recombinant pBR322 - chymosin - double stranded cDNAs may be altered / e.g. by inserting the corresponding replication regulatory or expression regulatory sequences, so that recombinant plasmids can also be used for efficient cloning in Bacillus subtili or streptococcal strains.

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Recombinant clones obtained by this cloning procedure that contain specific coding for bovine chymosin can be used in the following regions:

- as a molecular probe and / for analytical, specific detection of chymosin nucleic acid as additionally labeled (nick-translation) DNA fragments / DNA = specific chymosin gene fragment of calf /.

There, specific sequences from genomic banks (cloned genomic DNA), double-stranded cDNA banks (cloned double-stranded cDNA mixture) can be selected (harvested). but also from tissue extracts (genomic DNA or RNA) or from a mixture of cDNA of bovine origin. The selected positive clones from the respective flasks can then be cultured in the usual manner.

b) for preparative enrichment / harvesting of the 7 nucleotide sequences that represent chymosin gene fragments, particularly from tissue extracts. Conventional affinity chromatography methods _from e.g. agarose type chromatography (Sepharose) with DNA bound (DNA = specific chymosin gene fragment after cloning of double-stranded cDNA) come into consideration here.

as specific primers for the specific synthesis of chymosin cDNA into polyA ⁺ -mRNA, eg, from the gastric mucosa at various physiological periods of animal growth. Partial sequences of already cloned specific chymosin DNA in single-stranded form are used as the origin of replication (primer, primer) for specific reverse transcription.

as a starting material in the expression vector, such that specific coding is brought under the control of an efficient prokaryotic expression regulatory element. These regulatory elements originate either from the early region of T7 or T3 phage, or from lactose, tryptophan operons, but also from lambda promoters or hybrid regulatory elements. In this way, efficient transcription and translation of a specific chymosin sequence can be achieved, which can be used in a technical fermentation process for the microbial synthesis of chymosin.

The procedure will be explained in more detail in the following examples, without limiting the invention.

- 5 Examples

Preparation of double stranded cDNA 238 268

Fractionated polyA ⁺ -mRNA / fraction 12-17s; S = Swedberg's unit of sedimentation (from the fourth gastric mucosa of two to three week old calves) is converted by reverse transcriptase from a single myeloblastosis virus (AMV) in complementary DNA (cDNA) in a volume of 1 ml reaction mixture (containing 34 µg RNA, 10 µg oligo) dT / _{12- 18} , 50 mM Tria-HCl pH 8/2/50 mM KCl, 7.5 mM MgACg, 2 mM dithiotreitol (DTT), 0.5 mM each of the deNNTPs (Gunin-derived deoxynucleoside triphosphates), cytosine (C) _from adenine (A) and thymine (17) (G, C, A, 1), 600 µCi 1 H-dCTP and 30 AMV reverse transcriptase units (Liebscher). Within 2 hours at 37 ° C, 0.91 µg of cDNA (ca. 2.8% transcript) was transcribed respectively. 850,000 cpm in cDNA.

This material is used immediately (without prior fractionation) to synthesize double stranded cDNA using a specific portion of the E. coli DNA polymerase I enzyme (composition of the Boehringer Klenow fragment) in a reaction mixture containing 1.5 µg / ml cDNA _from 30 mM Tris-HCl pH 7/5/4 mM MgACg, 0.5 mM β-mercaptoethanol _from 0 _of 5 mM each dNTP (G, C, A, Τ), 70 mM KCl and 62.5 units of enzyme / ml. After 4 hours incubation at 20 ° C, the mixture was freed from protein / salt, and double stranded cDNA by preparative affected nucleophilic sum S ^ _of that material removed from the single-stranded DNA segments. In a volume of 300 µl of the reaction mixture containing 0.15 µg / ml cDNA / radioactive label c DNA serves as a reference value // 70 mM sodium acetate pH 4.5, 250 mM NaCl _of 2 mM ZnSO 4 and 100 units / ml nuclease after 10 minutes of incubation at 60 ° C still% _of the labeled cDNA, this means that 60% of the cDNA, nuclease-resistant S ^, respectively. it is present in a double-stranded form. (In a parallel analysis, this value was 75% of chymosin cDNA, unaffected by polymerase, showed only 16% resistance to Sp nuclease.)

After nuclease treatment, double stranded cDNA was separated and fractionated in a preparative neutral linear sucrose linear density gradient / 5-20% linear gradient, 0.1 M NaCl, 10 mM Tris-HCl pH 7.5, 20 ° C, 25,000 rpm. Beckman Rotor SW41, 6 pm /. Fractions 10-14 (long molecules) and 15-20 (middle molecules) were collected separately and further used. Globin wild-type 9S cDNA was found in fraction 23 (peak fraction) of a total of 30 fractions. 238 268

The average length of double stranded cDNA molecule corresponding to 600 bp / 10 is calculated from the data. «<. 14th fraction.

The invention can be used in the food industry, eg in cheese production, in animal feed production and in veterinary medicine (gene transfer to organism).

Claims (1)

SUBJECT OF THE INVENTION 238 268 Method for cloning bovine prochymosin genes obtained by reverse transcription of polyA ⁺ -mRNA - a fraction with a sedimentation coefficient of 12 to 17S from the fourth gastric mucosa of two to three weeks of calves, characterized in that by terminal transferase 35 to 200 nucleotides in length and linked to a linear plasmid vector which also contains, after the transferase reaction, homopolymeric single-stranded but complementary ends with an average length of 10 to 20 nucleotides and transferred to recipient cells intended for cloning, particularly E. coli cells _from Bacillus subtilis or Streptococcus, followed by selection of recombinant clones.