Method for synthesizing stable, single-stranded cDNA in eukaryotes by transfection with a vector containing a bacterial retron and the vector used therefor

Claim 2

The method of claim 1 wherein the expression vector is a plasmid.

Claim 3

The method of claim 2 wherein the eucaryote host cell is yeast.

Claim 4

The method of claim 3 wherein the yeast is of the genus Saccharomyces.

Claim 5

The method of claim 1 wherein the eucaryote host cell is a plant or mammalian cell.

Claim 6

The method of claim 3 wherein in the retron, the RT gene is downstream of the msr - msd region.

Claim 7

The method of claim 3 wherein the retron contains a single AUG codon for the RT gene.

Claim 8

The method of claim 7 wherein in the retron, the RT gene is upstream of the msr-msd region.

Claim 9

The method of claim 3 wherein the expression vector contains a promoter for a retron consisting of msr - msd region and a RT gene, which promoter is located upstream of msr.

Claim 10

The method of claim 9 wherein the promoter is a foreign strong promoter.

Claim 11

The method of claim 10 wherein the strong promoter is GAL10.

Claim 12

The method of claim 2 wherein in the retron in the plasmid, the msd sequence contains a cloning site.

Claim 13

The method of claim 8 wherein a foreign DNA fragment is contained in the msd region of the retron which codes the DNA of the msDNA.

Claim 15

The method of claim 4 wherein the msDNA molecule comprises a branched single-stranded RNA which is covalently linked to a single-stranded DNA by a 2',5'-phosphodiester bond between the 2'-OH group and an internal rG residue and the 5'-phosphate of the DNA molecule, which RNA is non-covalently linked to the DNA by base-pairing between the complementary 3' ends of the RNA and DNA molecules, which RNA-DNA form stable stem-loop secondary structures, which msDNA is encoded by a primary RNA transcript, pre-msDNA which contains an open-reading frame (ORF) downstream of the msr locus, the ORF encoding a polypeptide which has sequence similarity with retroviral RTs and a highly conserved sequence common to all RTs.

Claim 16

The method of claim 15 wherein the stable msDNA which is produced is selected from the group consisting of msDNA-Ec67, mSDNA-h4x162, msDNA-Mx65, msDNA-Ec107, msDNA-Mx86 and msDNA-Sal63, as shown in Figure 2A.

Claim 17

The method of claim 4 wherein the ORF is located upstream of the msr locus.

Claim 18

The method of claim 17 wherein the stable msDNA which is produced is msDNA-Ec67, as shown in Figure 2A.

Claim 19

A DNA expression vector which replicates in an eucaryotic host which comprises a retron encoding a stable hybrid single-stranded RNA-DNA (msDNA) structure, which retron contains a gene encoding a reverse transcriptase (RT) and one coding region which contains two coding sequences, one " msr " and the other " msd " for encoding, respectively, the RNA and DNA portions of the msDNA.

Claim 20

The DNA expression vector of claim 19 wherein in the retron, the RT gene is downstream of the msr - msd region.

Claim 21

The vector of claim 20 wherein the retron contains a single AUG codon for the RT gene.

Claim 22

The vector of claim 21 wherein in the retron, the RT gene is upstream of the msr-msd region.

Claim 23

The vector of claim 19 which includes a promoter for the msr-msd region and the RT gene, which promoter is located upstream of msr .

Claim 24

The vector of claim 23 wherein the promoter is a foreign strong promoter.

Claim 25

The vector of claim 24 wherein the promoter is GAL10.

Claim 26

The vector of claim 22 in which a foreign DNA fragment is contained in the msd region of the retron which encodes the DNA of the msDNA.

Claim 27

The vector of claim 26 wherein the fragment is 50-bp long.

Claim 28

The vector of claim 20 which is YEp521-M1 (ATCC N° 74092), YEp521-M3, YEp521-M4 (ATCC N° 74093) or YEp521-M5 (ATCC N° 74094).

Claim 29

A eucaryotic host cell transfected with a DNA expression vector which replicates in the cell, which vector contains a retron encoding a msDNA, which retron contains msr and msd coding regions ( msr - msd ) for the msDNA and a gene encoding a reverse transcriptase (RT).

Claim 30

The eucaryotic host cell of claim 29 which is selected from the group consisting of yeast, plant cells and mammalian cells.

Claim 31

The eucaryotic host cell of claim 30 which is yeast.

Claim 32

The eucaryotic host cell of claim 31 wherein the yeast is of the genus Saccharomyces.

Claim 33

The eucaryotic host cell of claim 30, 31 or 32 wherein in the retron, the RT gene is downstream of the msr - msd region.

Claim 34

The eucaryotic host cell of claim 30 wherein the retron contains a single AUG codon for the RT gene.

Claim 35

The eucaryotic host cell of claim 34 wherein in the retron, the RT gene is upstream of the msr - msd region.

Claim 36

The eucaryotic host cell of claim 30 wherein the expression vector contains the promoter for the msr - msd region and RT gene, which promoter is located upstream of msr.

Claim 37

The eucaryotic host cell of claim 36 wherein the promoter is a foreign strong promoter.

Claim 38

The eucaryotic host cell of claim 37 wherein the strong promoter is GAL10.

Claim 39

The eucaryotic host cell of claim 35 which contains a retron having a msd region which contains a foreign DNA fragment.

Claim 40

The eucaryotic host cell of claim 39 wherein the DNA sequence is 50-bp long.