Method for synthesizing stable single-stranded cDNA in eukaryotes by means of bacterial retron, products and uses 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 1 wherein the retron for msDNA contains the msr-msd region and the gene encoding for RT.

Claim 7

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

Claim 8

The method of claim 3 wherein in the retron, a non-coding DNA fragment of the 5′ end upstream of msr which contains several ATG codons, has been deleted but for the initiation codon AUG of the RT gene.

Claim 9

The method of claim 8 wherein in the retron, the RT gene is upstream of the msr - msd region.

Claim 10

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

Claim 11

The method of claim 10 wherein the promoter is a foreign strong promoter.

Claim 12

The method of claim 11 wherein the strong promoter is GAL10 .

Claim 13

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

Claim 14

The method of claim 9 wherein a foreign DNA fragment is contained in the msd region of the retron which codes the DNA of the ms DNA.

Claim 15

The method of claim 1 wherein the retron is in a vector selected from the group consisting of YEp521-M1, YEp521-M3, YEp521-M4 and YEp521-M5.

Claim 16

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 17

The method of claim 16 wherein the stable msDNA which is produced is selected from the group consisting of msDNA-Ec67, msDNA-Mx162, msDNA-Mx65, msDNA-Ec107, msDNA-Mx86 and Sa163.

Claim 18

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

Claim 19

The method of claim 18 wherein the stable msDNA which is produced is msDNA-Ec67.

Claim 20

A DNA expression vector capable of replication in an eucaryotic host which comprises a retron which is capable of 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 21

The DNA expression vector of claim 20 wherein in the retron, the RT gene is downstream of the msr-msd region.

Claim 22

The vector of claim 21 wherein in the retron a non-coding fragment of the 5′ end upstream of msr which contains several ATG codons has been deleted but for the initiation codon AUG of the RT gene.

Claim 23

The vector of claim 22 wherein in the retron, the RT gene is upstream of the msr - msd region.

Claim 24

The vector of claim 20 which includes a promoter for the msr - msd region and the RT gene, which promoter is located upstream of msr .

Claim 25

The vector of claim 24 wherein the promoter is a foreign strong promoter.

Claim 26

The vector of claim 25 wherein the promoter is GAL10 .

Claim 27

The vector of claim 23 in which a foreign DNA fragment is contained in the msd region of the retron which encodes the DNA of the msDNA.

Claim 28

The vector of claim 27 wherein the fragment is 50-bp long.

Claim 29

The vector of claim 20 which is YEp521-M1, YEp521-M3, YEp521-M4 or YEp521-M5.

Claim 30

A eucaryotic host cell transfected with a DNA expression vector capable of replication, which vector contains a retron capable of msDNA synthesis.

Claim 31

The eucaryotic host cell of claim 30 which is selected from the group consisting of yeast, plant cells and mammalian cells.

Claim 32

The eucaryotic host cell of claim 31 which is yeast.

Claim 33

The eucaryotic host cell of claim 32 wherein the yeast is of the genus Saccharomyces .

Claim 34

The eucaryotic host cell of claim 30 wherein the retron contains the msr-msd region and the gene encoding for RT.

Claim 35

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

Claim 36

The eucaryotic host cell of claim 41 wherein in the retron a non-coding fragment of the 5′ end upstream of msr which contains several ATG codons, has been deleted but for the initiation codon AUG of the RT gene closest to the 5′ end.

Claim 37

The eucaryotic host cell of claim 36 wherein in the retron, the RT gene is upstream of the msr - msd region.

Claim 38

The eucaryotic host cell of claim 31 wherein the expression vector contains the promoter for the msr - msd region and RT gene, which promoter is located upstream of msr .

Claim 39

The eucaryotic host cell of claim 38 wherein the promoter is a foreign strong promoter.

Claim 40

The eucaryotic host cell of claim 39 wherein the strong promoter is GAL10 .

Claim 41

The eucaryotic host cell of claim 37 which contains a retron having an msd region which contains a foreign DNA fragment.

Claim 42

The eucaryotic host cell of claim 41 wherein the DNA sequence is 50-bp long.

Claim 43

A new msDNA molecule constituted of a single-stranded RNA and a single-stranded DNA portion which 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 ms DNA is encoded by a primary RNA transcript, pre-msDNA which contains an open-reading frame (ORF) upstream of the msr locus, the ORF encoding a polypeptide which has sequence similarity with retroviral RTs and a highly conserved sequence common to all known RTs and which msDNA is encoded by an msr-msd region, the msDNA containing a foreign DNA fragment in its 5′end strand of the DNA in the region which was encoded by the msd region of the retron which encoded the DNA of the msDNA.

Claim 44

The new msDNA of claim 43 wherein the msDNA is encoded by either plasmid YEp521-M4 or plasmid YEp521-M5.

Claim 45

The new msDNA of claim 43 which contains in its DNA portion which terminates by the 5′end a foreign DNA fragment which is capable of generating an RNA transcript which is complementary to a specific mRNA of a target gene or fragment thereof.

Claim 46

The new msDNA of claim 45 wherein the complementary sequence is (SEQ ID N°: 1) :

Claim 47

The new msDNA of claim 46 wherein the generated RNA is complementary to cdc28.