DD153893A1 - METHOD OF NUCLEOTIDE SEQUENCING CLONING OF THE VIRUS OF ENZOOTIC BOVINE LEUCOSE (BLV) - Google Patents

Abstract

It is the object of the invention to further develop the previously known cloning techniques so that BLV nucleotide sequences of different length u. be cloned from different genome areas of heterogeneous or homogeneous population with good yield. According to the invention BLV-deoxyribonucleic acid (BLV-DNA) of different origin is assumed. The coupling takes place m. a vector DNA to the recombinant DNA according to a) the synthesis of homopolymeric Einstrangenden (terminal transferase reaction) b) the addition of short, doppelstraengigen sequences with certain restriction sites (adapter sequences) or c) the exploitation of natural restriction sites in the BLV DNA using restriction endonucleases , Subsequently, a transfer of the recombinant DNA is carried out in specific for the cloning host cells and the BLV clones are selected.

Description

1 8 3 A - * -

Dr. Liebscher (GDR)

Dr. Kettmann (BB) ·

Prof. Burny (BE)

Prof «, S. Rosenthal (GDR). ,

Method for the nucleotide sequence cloning of the enzootic bovine leucosis virus (BLV ) .

Field of application of the invention

The invention relates to a method for the cloning of JTucleotidsequenzen the enzootischen bovine leucosis (BLV) virus _β The method is in the microbiological-pharniakologi-Bchen industry usable, and the invention has significance for medicine, veterinary medicine and the "agriculture"

Ch ^ aj ^ k.teristjLk .der known technical solutions

The cloning of eukaryotic nucleotide sequences as biological information carriers in prokaryotic recipients such. B. Escherischia coli is an internationally increasingly used methodology since 1975 (Xit _e T ₉ Maniatis et al., Cell 8 (2), 163 (1976)), the various genetic engineering solutions (review in WA Scott and R. Werner " (Eds.), Molecular cloning of recombinant DITA, Ac ad. Press, ITew York 1977; Klingmüller, Genetics and Gene Therapy, Springer-Verlag Heidelberg 1976). These possibilities are dependent on the starting material (MS, single-stranded DNA, double-stranded DUS) , from the vector (plasmid, phage, virus DHS), from the site of integration and from the cloning target (eg ₀ expression of the foreign information in the recipient or only replication of the DIiS) selected.

The Riis sequences of bovine leukemia virus (A "Burn3r et al _e, BLV molecular biology and epidemiology, in: Viral oncology, ed" G, Small, Raven Press, Hew York, 1980) have not been kloniertc The virus genome has a length about

10 !!!} ,! inriii . ' ·:

22 1 834

9,000-10,000 nucleotides whose sequence is unknown { The length of the sequences is given in single-stranded nucleic acid segments or single-stranded nucleic acids in nucleotide numbers in double-stranded nucleic acid segments in base pairs (bp)). A restriction map of the BLV virus genome has not yet been published. In contrast, the cloning of Hukleotidseqüenzen some other RUS viruses such. B, Influenza (J _e S «Emtage et al., Nature 2§2 (.5743)» .171 (1979) and M ₀ J. Sleigh et al., Nucl. Acid. Res; Ulis 879 (1979) and vesicular stomatitis virus (J * K. Rose and L, J _Iverson? * Virol. £ 2J £ X succeeded _S 404 (1979), "Even RUS tumor virus sequences (Moloney sarcoma virus), after restriction-endonukleolytischem Aussclineiden the provirus DHS sequences from the Y / irts DUS cloned (G "I"'"Van de Woude et al., Proo * Nati; Acad _e Sei, USA J & (9) .4464

The cloning of nucleotide sequences of the RNA tumor viruses is complicated in contrast to the cloning of DITS virus B. hepatitis B virus, since the starting materials (viral RITS, integrated provirus DliS or non-integrated viral DITS) only in Extremely limited amounts are available. "Other difficulties arise because 100% purification of the starting material is practically impossible. In addition, nucleic acid breaks occur during the purification steps. For the cloning of nucleotide sequences derived by reverse transcription, a pre-selection of relatively long double-stranded cDITS molecules from a heterogeneous population is required. This preliminary fractionation by size using gel electrophoresis or density gradient (Villa-Komaroff et al, Prooν Natl Acad Sci USA 22 "y 727 -.... 3731 (1978)) is laborious and has losses' · material used result. In DD - patent

794- "was proposed as a principal solution to select differentiated tails of double-stranded cDUS- ¹ molecules, the longest molecules from a heterogeneous population. ·

Zie l the invention inventions

The object of the invention is to produce clones with BLV nucleotide sequences of different length and different genome regions.

Explanation of the essence of the invention

It is an object of the present invention to further develop the hitherto known cloning techniques in such a way that BLV-hucotide sequences of different lengths and from different genome regions can be cloned from heterogeneous or homogeneous population with good yield. , The procedure according to the invention for the cloning of nucleotide sequences of enzootic bovine leucosis is characterized in that a BLV deoxyribonucleic acid (BLV-DIS) for the coupling with a vector DITS, if necessary after splitting of coupling sites or structure present in the BLV-DIS molecule. prepared new coupling sites on the BLV-DHS molecule, with a. Vector DNA is coupled to recombinant DNA, the recombinant DNA is transferred to host cells, cloned there and the BLV clones selected.

The invention is based on BLV deoxyribonucleic acid (BLV-DITS) of different origin;

, a) from a homogeneous or heterogeneous population of complementary double-stranded BLV-DE ^- S obtained by reverse transcription of BLV-ribonucleic acid (BLV-RNA)

or b) BLV DNA in replicative form present in a host cell, unintegrated in the host cell genome (replicative form means both circular and linear forms of BLV DNA)

or c) virus-transformed BLV DNA, which is excised after integration from a host genome.

The DNA thus obtained varies in length within the range of 50-10,000 base pairs.

': - 4 - 22 18 3 4

This DHS is going through

04) the synthesis of homopolymeric single-stranded sequences (terminal transferase reaction), β) the addition of short double-stranded sequences

certain restriction sites (adapter sequences) by exploiting natural restriction sites in the BLV-DHS using restriction endonucleases

prepared for coupling with a vector to recombinant DHS. The in vitro recombinant DHS is then transferred to host cells intended for cloning, preferably E. coli cells, after which the BLV clones are selected.

The reverse transcription of BLV-DHS. and the double strand cDNA synthesis are performed according to known techniques. A heterogeneous population of double-stranded c'DHS molecules is obtained which is either used directly for the next steps (preparation of the coupling) or prefractionated first according to molecular weights by means of density gradients or by gel electrophoresis. However, this requires a sufficient amount of double-stranded cDHS molecules. Starting from a heterogeneous population of DoppelstrangcDHS molecules, the process according to the invention is carried out so that after the transferase reaction averaging relatively long homopolymeric ingrane ends having average lengths of 50 to 220 nucleotides are formed. (The value of 50 for the average length of the homopolymeric inguinal endings means, on the one hand, very short ends (10-20 nucleotides) on relatively long duplex cDHS molecules, on the other hand very long ends (100-200 nucleotides) a relatively short duplex cDHS The value of 220 means corresponding limit values of 10-20 or 300 400 nucleotides.The cause of this length distribution is the dependence of the activity of the transferase on the length of the double-stranded cDHS molecules Molecules, the activity is higher and leads

- 5 - 2 2 1 8 3 A

therefore relatively long intrinsic to these molecules and vice versa). Subsequently, the thus-extended duplex cDEfS is end-capped with a linear plasmid vector having short homopolymeric complementary single-stranded nucleotides with an average length of only 8-12 nucleotides after the transferase reaction, and Escherischia coli cells treated with CaCl ₂ Bacteria transferred. For security cells of E coli ₀ p £ 1,776 are particularly suitable.

When comparing the present invention cloning with average, relatively long duplex double-stranded eDNA molecules (average length about 200 nucleotides) with the cloning of double-stranded cDnS molecules, which are only averaged with 15-25 nucleotides, the higher efficiency of the new procedure

 From the average relatively long (200 nucleotides) tailed heterogeneous population, long double-stranded cDUS sections (more than 6OO base pairs) are obtained approximately four times more frequently.

When starting from a homogeneous population of double-stranded cDnS molecules, it is sufficient according to the invention if enzymatic attachment of short homopolymeric single-stranded endings with average lengths of only 8-10 nucleotides is contemplated. The v / eitere process management then takes place as described above.

The characterization of the resulting plasmid recombinants is carried out by determination of the molecular weight and by restriction analysis. 3 groups of recombinants are obtained according to the method of the invention

an EcoRI site,

a HindIII site or

- have neither an EcoRI nor a HindIII cleavage site.

BLV-DlS contains only a single EcoRI cleavage site that is 400 ~ 800 base pairs away from the 3 'end of the BLV genome. Accordingly, recombinin genes with an EcoRI cleavage site contain

- 6 ~ 22 183 4

Sections of the '3' end. Another unique site is the HindIII site in the middle of the genome. The plasmid recombinants obtained in this way contain BLV-DlTS fragments with a length of up to 1400 bp. They can be transferred into E. coli 1776 strains at any time without any special effort.

To obtain BLV clones with preferably very large fragments up to the complete genome, a variant of the method according to the invention is selected which is obtained from a BLV DNA in replicative form or from virus-transformed BLV DNA present in a host cell and not integrated in the host cell genome. DNA, which is cut out after integration from a host genome, runs out. .!. In the first case, the BLV DHS is the total genome in circular or linear form "To the BLV DHS if necessary after opening the circular shape adapter sequences added, d _e h, short double-stranded sequences 8 - 20 base pairs, the one Contain restriction site, which is also present in the proposed vector molecules. Preferred restriction sites are EcoRI, HindIII, Sail, PstI and BamHI sites. The coupling of 'BLV-DHS and vector occurs via the cohesive ends that result after restriction endonuclease treatment at the restriction sites. The addition of adapter sequences to prepare for coupling with plasmid DHS is possible even when there is a homogeneous population of double-stranded complementary BLV-DHS obtained by reverse transcription from BLV-RHS genomic and subsequent fractionation.

Likewise, the application of the homopolymer technique is suitable for the ionization of the unintegrated linear form. However, due to the overall length of the BLV-DHS molecule (10 kBp), single-strand bridges are to be expected. Therefore, although a population of molecules of homogeneous length is present, the terminal transferase reaction must be conducted in such a way that, based on terminal molecular ends alone, a

2 2 18 3 4

apparent length of 50 to 500 nucleotides is polymerized. However, due to the additional 3 'internal ends, there is a decrease in apparent length such that the approximate average length of the homopolymeric blocks relative to inner and terminal 3' ends is between 8-20 nucleotides. In the presence of. In a host cell present, in the host cell genome unintegrated BLV DUS -in replicative form or virus-transformed BLV DüTS, which is excised after integration of a host genome, according to the invention in the terminal regions of the BLV DlJS (they corresponding to the 3 ^f ~ or 5 'end of the single-stranded BLV-RIiS genome) existing restriction sites, preferably the EcoRI site, are exploited. The BLV DNA and a vector containing the same restriction site are cleaved with the appropriate restriction endonuclease and the coupling is carried out in a manner known per se. It may also be advantageous to have double cleavages at 2 unique restriction sites within the BLV molecule that are near the molecular ends, e.g. B ₉ Sail (5 'end) and EcoRI (3' ~ end), and to clone these fragments directly, for example, into the EcoRI-Sall region of pBR322 also non-integrated BLV-DlTS.

The latter starting materials are used to obtain BLV clones with preferably very large fragments up to the complete genome »

Thus, depending on the length composition of the starting material, BLV clones can be obtained up to the full genome length with the method according to the invention. Host organisms, in particular E. coli strains containing large fragments of the BLV genome, including the complete BLV genome, are used for the technologically simple production of the BLV genome. and large parts of d.avone These clones can be increasingly used and for diagnostic tasks (detection, integrated provirus DIP, detection of viral RIiS, detection of unintegrated viral DKS). Another one

_ ₈ _

option consists in the selection of individual DUS species of different lengths from a heterogeneous mixture. The clones can also be used for construction of expressing clones and recovery of specific proteins. It may be advantageous to use the cloned DNA fragments containing various regions of the BLV genome encoding, with the aid of overlapping sequences new (non-natural sequences) merge (e.g., B ₆ taking advantage of natural restriction sites).

The invention will be explained in more detail below by exemplary embodiments.

Execution sbe i spi ele

1 ... Cloning of BLY-dscDUS

a) Preparation of double- stranded oDNS (ds-oDHS) · BLV-RHS, fractionated as a 7O-RITS complex in sucrose density gradient, then heat denatured and purified on oligo-dT-cellulose and containing about 5,000 to 10,000 nucleotides is long, is using AMV reverse transcriptase in complementary DlTS (cDUS) in a 40 axX. Approach of 87.5 / ug / ml BLV RNA, 20 / Ug / ml oligo CDT) ₁₂ _ _18, 50 mM

, Tris / HCl pH 8.2, 50 mM KCl, 5 mM Mg-acetate ₂ , 10 mM DTT,

1mM dGTP, 1mM dTTP, 1mM dATP, 0.5mM ³ H-dCT.P. (750 / UCi / ml), 40 μg / ml actinomycin D, 1 mg / ml Ifflase inhibitor (from G '. Vassart) and 1,500 units / ml AMV reverse transcriptase (from V /. Beard), in FIG Min. At 44 ⁰ O in 0.875 mg cDUS (5 _S 26 χ 10 ⁵ cpm) rewritten. In the alkaline linear sucrose gradient (5-20% sucrose, 0.33 M Haoh, 0.5 M HACL, 20 mM EDTA, 4 ⁰ C, "39000 U / min, Beckman SW41 rotor) gives a uniform, this material ' Spike representing an average length of the material of about 4,000 to 5,000 nucleotides (15x and 16x fraction from the bottom, total fractions; the 9S »standard globin cDHS is in the 19th and 20th fractions) Due to the uniformity of the spike, the uhfractionated cDltfS became more alkaline

2 2 1 8 3 A

Hydrolysis of RITS directly to double-stranded cDNA synthesis in a 125 / Ul assay with 2.24 / ug / ml cDITS, 30 mM Tris / HCl pH 7.5, 8 mM Mg-acetate g, 5 mM DTT, 70 mM KCl, 0.5 mM of each dNTP (G, C, a, T), and 120 units / ml Klenow Prague member of DITS-Polymerasel (Fa * Boehringer) was used and incubated for 9.5 hrs. at 20 ⁰ C.

This material is then incubated with S. nuclease to remove the remaining single-stranded areas in a 380 / μl batch with 0.5 μg / ml DNA (the reference is radioactive labeling of onDITS), 70 mM Ua-acetate pH 4.5, 250 mM UaCl, 2 mM ZnSO, and 1 unit / ml S ₁ -lituclease (from R. Kettmann) incubated for 10 min at 37 ° C. The resistance after double-strand synthesis was 57 %, whereas that for cDITS was controlled before this synthesis only 24j4 %. The resistance value results in an average length of about 2,000 base pairs for the DlTS double-stranded molecules, which serve as starting material for the synthesis of average relatively long homopolymeric clays to the ds-cDlTS.

b) Synthesis of hpmppol ymerer 3 'ends on dpjpj) of the elongated PITS 3 batches are 3.32 / ug / ml ds-cDUS each (this concentration corresponds to 5 mM 3' ends on the basis of an average length of the ds-cDIIS Molecules of 2,000 bp), 1 mM CoCl ₂ , 250 mM Hepes buffer pH 7.1, 170 units / ml of terminal transferase (from D ₀ -H. Lieböcher) and each with different nucleotide concentrations of 50, 143 or 500 / UM ³² P-dCTP (60 Ci / mmol) are incubated for 5 min at 37 ° C. This gives rise to homopolymeric prion rings having a length of 22 (carried out for comparison), 58 or 204 nucleotides per 3 '- = end.

For cloning, the vector used is the plasmid pBR32.2 (Bolivar et al., Gene, 2 (1977), S ₀ 95-113). After linearization of the vector with the help of the restriction endonuclease PstI, the plasmid only once in the ampicillin gene (this gene encodes a ß-lactamase, the

ίο -

Makes ampicillin inactive and thus causes the ampicillin resistance of the plasmid-carrying bacterium), dG is synthesized at its 3 'ends with terminal transferase. The mixture contains 5 mM 3 'ends of the plasmid, 0.15 M Hepes buffer pH 7.1, 1 mM GoGl ₂ and 102 enzyme units / ml. For the synthesis of the short-tailed vector (with dG _1Q ) only 20 / UM ³ H-dGTP (8 Ci / mmol) is used. The appropriate incubation time at 37 ^'0 O becomes analytically determined immediately beforehand and was 5 minutes

hybridization of endogenous and transfprmatilons Following synthesis of the homopolymeric single-nucleus fragments, 45 ng of vector DITS and 22 ng of BLV-ds-cDNA-dO (n = 22, 58, and 204 nucleotides, respectively ) are isolated in 3 separate batches (a 133 / oil) BaM Tris / HCl buffer (pH 7.5, 100 mM -IlClc, 0.2 mM EDTA) initially for 3 min. At 64 ⁰ C, then incubated for 2 hrs. At 43 ° C and finally in a period of 3 hr. cooled slowly to 25 ^° C. Under these conditions, the homopolymeric complementary Einrangenden the different DNA molecules (vector and ds-cDUS molecules in equimolar concentrations in the batch) to each other. This mixture is used directly for the transformation of CaClp-treated Έ.οοίί 1776 bacteria (using the method of Uorgard et al., Gene _5, 2 (1977), p. 279-292)

 d) Characterization of the clones

Table 1 shows the characteristic data of the obtained transformants (clones). From the table it can be seen that for the same ds-cDHS amounts (in each case 22 ng ds-cDNA) in the pail of ds-cDNA-dC _{20 /} , 4 times more frequent. strongly hybridizing clones (in situ hybridization according to Grünstein and Hogness, Proc., Uatl. Acad., USA 7j2 (1975), pp. 3961-3965) are found in comparison with the approach of ds-cDIS-dC22. Using the average of relatively long truancy to ds-CDUs long dscDlTS molecules are selected "preferred" (with shorter tails) by the vector pBR322-Pst-dG _10th

, - 11 - 22 1 8 3-4

Since the BLV-P cDNA with which the clones are backhybridized represents the entire BLV genome uniformly, the level of hybridization is a measure of the length of the integrated BLV DNA in the particular clone. Of the strongly hybridizing clones (13 clones), the relative

Mobility (P) of the recombinant DNA compared with the vector pBR322 (4361 bp) and the quotient 1 / F formed «, This quotient is similar for the strongly hybridizing clones to each other. After treatment with the restriction endonucleases EcoRI and HindIII, the recombinants-DUS is either linearized (1 cleavage site only in the vector molecule present) or cut into Spaltstükke (near the 3 ^f ~ end of the genomic BLV RLIs is sich'ein EcoRI cleavage site in the Center of the genome a HindIII site). The result is insertions of about 600 lb.sup.-4. The recombinant plasmids pLK29 (3820 + 1200 = 5020 bp, insert length about 660 bp) and pLK232 (4250 + 1320 = 5570 bp, insert length about 1210 bp) carry a .EcoRI- Cleavage site in the insert region and therefore represent the 3'-terminal region of the BLV RNA genome. The recombinant plasmid pLK42-453 carries a HindIII cleavage site in the

Insertion region (3800 + 1900 = 5700 bp, insert length about 1340 bp) and therefore represents the middle region of the genome. It also contains an internal Pstl location ",. Contains another plasmid pLKi66, a BamHI and BglI site within a 400 bp insert ~ _e Other plasmids containing fragments, which are either already represented by said Plasmide'in the kloniertenNukleotidsequenz with high probability or no internal restriction site, if examined, have.

22 1834

Table 1 characterize the molecular cloning of BLV-ds-CDHS in e _e coli

cloning pBR322-Pst ~ dG ₁₀ χ BLV-ds-cDHS-dC _n 1,065 1.075 204 , 9 dC _n (n) 22 (comparison) 58 1,075 137 IN THE. 1,078 1.052 Number of Tc ^r - 85 83rd 1,131 Transformants 8th 1,131 """""o ¹ ^ - - ~ Of which with - ^ P- BLV-cDITS strongly hybridizing clones'^; 2 3 - 5 1.07 ~ ~ ~ ~ T & fo ~ ~ ' - 2 J ------ "377 • pLK 422 425 1.07 Quotient of the rela- pJJK 1_ / Φ tive mobility (F) -i -i _octo-2-i of PLK the DHS - ^ ^jU strong hybridisieren- 235 1.075 £ M 29,138 42-453 1,131 the clones ' 166 456 1.15 465 488 493 522

Explanations:

'Strongly hybridizing clones blacken the

X-ray film already after 20 min. Exposure deep black. · · ·

'In comparison with the mobility of native · pBR322 in the agarose gel electrophoresis pLK Plasinid of BLV-Rekombinantenldons Hr. WH Tc ^r tetracycline-resistant (property of the plasmid).

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2. Cloning of unintegrated BLV-DHS

a) Preparation of the starting material

Hach cultivation of BLV-infected FLK cells in 12 scooter vessels was extra-2 mg DHS using HIRT technology! , hiert. The material contained only about 3 ng BLV-DIiS, analytically determined by specific RlTS-DUS hybridization. This corresponds to an enrichment ratio of about 1:10 or a ratio of on average less than 1 virus DUS molecule per cell genome. This determined value decides on the necessary number of clones to be selected, in order to discover with high probability BLV clones in a recombinant bank.

In order to improve the relatively poor enrichment ratio, the DHS was separated by preparative agarose gel electrophoresis (horizontal electrophoresis, 20χ 40 cm, 1 mg DHS per gel) and the DHS in the 10 kbp range (corresponding to 6χ10 D) Recovery of the DHS after agarose treatment of the gel band was about 60 % of the respective fraction The enrichment was about 35 fold (2 ng BLV-DH5 below 40, ng total DHS) compared to the original material, resulting in the finding that about 1 specific BLV clone should be detectable among 20,000 recombinant clones, which was sufficient to subsequently isolate 45,000 clones.

b) Synthesis of Homopolymeric 3'- Enes on Double-Stranded DHS The most effective coupling was carried out with BLV-DHS and plasmid molecules of different lengths (Pstl-linearis).

-sized plasmid pBR322) and then used for recombinant production. BLV-DHS-dCorQ or -Dog ₅ was hybridized with pBR322 Pst I dG ~ ^, -dG ₁₇ or -dG ^ and then transformed into E. coli} £ 1776th The reaction with terminal Tranafera.se was carried out in principle as shown in Example 1 with the in Table 2 ange-

give deviations. The different average lengths were generated essentially by varying the concentration of nucleotide in the reaction mixture.

Table 2. Synthesis of homopolymeric ends on BLV and pBR322 DHS

Concentration of the 3 'ends Nucleotide concentrates Enzymkonz Inkub. Time lengths (NM) (/AROUND) (U / ml) (Min) (B) A. BLV DNA 8.5 90 28 15 350 9 50 14 12 · 65 9 .25- 14 14 42 B. pBR322 ~ DNS 10 50 28 12 a) 11 b) 17 (pBR322 was through 2 consecutive reactions generated)

The concentration of the 3 'ends is solely on the terminal ends, i. H. related to the DNA molecule ends. The length (B) is given as the average base number, per molecule end. ,

The juxtaposition of the DNA molecules was carried out as already described in Example 1. As expected, the cloning efficiencies differed. ,

/ β., Overview of Cloning Efficiencies Combination of Transformation Efficiency DNA Molecules · cf, u ./ Ug plasmid

in %

pBR322 (alone, control) pBR322-PstI-dG ₁₁ (vector)

3 χ ιο

χ P-

BLV-dC £ ρ- χ 1 "-ανχ, *

BLV-dC _35O .x PhIG ₁₁ BLV-dC;, - χ P-dG,

Ί1

BLV-dC BLV-dC

42 x P-dG

χ P-dG

11 17

ί oo

10 13

, - ¹⁵ - 22 1 834

All approaches are compiled and edited under identical conditions.

From the data, it is concluded that the isolated DUS material must have single-strand breaks in a range of about 4 to 8 breaks per DNA molecule so that additional branching occurred after the terminal transferase reaction, which related the average length of the homopolymeric termini lower to terminal and internal 3 'ends of a BNS molecule. BLV-DNA-dCgj- contains accordingly about 4 to S single-strand breaks with homopolymeric ends in the length range of 11 to 6 bases

o) Selection and Characterization of the .BLV Clones All the recombinant clones produced were isolated and purified by the usual in situ hybridization method according to Grunstein and Hogness (see Example 1) for the purpose of selecting the BLV residues. analyzed. Among 45,000 clones, 2 BLV clones were detected and the. relevant recombinant from the depot P5-H4 and from the depot P47-F7 grown. After purification of the plasmids and after SOUTHERN transfer (J. Mol. Biol. £ 8, 503 (1975)), an insert length of about 800 bp was obtained for the specific BLV clone from depot P5-H4.

The localization of the cloned BLV DNA in the natural BLV genome is carried out by restriction analysis and comparison with the recombinants described in Example 1.

Claims (11)

22 183 4 - ^ - Srfindunff claim: 1, Method for the cloning of nucleotide sequences of the enzootic bovine leucosis virus (BIV), characterized in that a BLV deoxyribonucleic acid (BLV-DNA) for the. Coupling with a vector DNA, possibly after cleavage of coupling parts present in the BLV-DITS molecule or construction of new coupling parts on the BLV DNA molecule, prepared, coupled with a vector DNA to the recombinant DNA, the recombinant DNA in host cells transferred, there cloned and selected the BLV clones. 2 _e Method according to item 1, characterized in that the BLV DNA used is a homogeneous or heterogeneous population of double-stranded, complementary BLV DNA obtained by reverse transcription of BLV-ribonucleic acid (BLV-RNA) » 3. The method according to item 1, characterized in that used as BLV DNA present in a host cell \ unintegrated in the host cell genome BLV DNA in replicative porin. 4. The method according to item 1, characterized in that as a BLV DNA a virus-transformed BLV DNA verv / ends, which was cut out after integration of a host genome again. , 5. The method according to item 1-4 »characterized in that the preparation of the BLV DNA for coupling by synthesis of homopolymeric Einstrangenden (terminal transferase reaction) takes place. 6. The method according to item 1-4, characterized in that the preparation of the BLV DNA for coupling by attaching short double-stranded sequences with restriction sites (adapter sequences) and their cleavage takes place with Restrictions endonucleases. " 7 '. Method according to item 1-4, characterized in that the BLV DNA is prepared for coupling by cleavage • of restriction sites present in the BLV DNA molecule with resorption endonucleases. -? - 22 183 Λ 8. Method according to items 1, 2 and 5 »characterized in that long homopolymeric intrinsic lengths with an average length of 50 are assumed for a heterogeneous population of double-stranded, complementary BLV-DHS obtained by reverse transcription of BLV-MS genomic RNA "attached to 220 nucleotides enzymatically, then annealed with a linear plasmid vector having short homopolymeric complementary ingrown ends of only 8-12 nucleotides in length after the transferase reaction and transferred into CaClp-treated E.coli bacteria becomes. 9. The method according to item 1, 2 and 5, .Dadurch in that of a separated by molecular weight almost homogeneous population of double-stranded complementary BLV DlTS or of a non-integrated in a host cell in the host genome BLV DNA in replicative form or by virus-transformed BLV DNA, which is excised after integration from a host genome, is enzymatically attached to homopolymeric single-stranded rings of 8-20 nucleotides in length, followed by a linear plasmid vector which, after the transferase reaction, has short homopolymeric complementary ingrown sequences of average length has only 8-12 nucleotides, over the ends together stored and transferred to CaCl ₂ treated E. coli bacteria. 10.Verfahren according to item 1 to 4 and 6, characterized in that separated from a molecular weight nearly homogeneous population of double-stranded complementary BLV DNA or of a non-integrated in a host cell in the host genome BLV DNA in replicative form or of virus transformed -BLV DNA, which is excised after integration from a host genome, short double-stranded. Sequences of 8-20 bars senpaaren, which contain a restriction site, which is also present in the vectors for recombination vector molecules, preferably EcoRI, HindIII, BamHI, Sail or Pstl attached, and the coupling via the cohesive ends, which arise after treatment with restriction endonucleases at the restriction sites, he follows. 11 "method according to item 1, 3, 4 and 7>, characterized in that cut from a non-integrated in a host cell in the host cell genome BLV-DIS DIS in replicative form or virus-transformed BLV-DHS, after integration from a host genome again is assumed, in the end region of the BLV-DHS molecules existing Restriktiohsorte, preferably the EcoRI site in the 3 '~ region, are cleaved with restriction endonucleases, vector molecules with the same restriction sites also cleaved and with the BLV DUS in be coupled in a known manner. ,