Autonomous replication sequences and episomal DNA molecules
The present invention provides autonomous replication sequences (ARSs) isolated from Nannochloropsis that support the replication of episomal DNA molecules (EDMs) in eukaryotic cells. The ARSs and EDMs provided herein can be used for expressing genes in organisms including algae and heterokonts.
1. An isolated or recombinant nucleic acid molecule comprising an autonomous replication sequence (ARS) juxtaposed with at least one heterologous nucleic acid sequence, wherein the ARS has at least 95% identity to a nucleic acid selected from the group consisting of SEQ ID NO:5; SEQ ID NO:6; SEQ ID NO:7; SEQ ID NO:11; and SEQ ID NO:12, further wherein the ARS comprises a nucleic acid sequence having at least 98% identity to SEQ ID NO:14.
2. An isolated or recombinant nucleic acid molecule according to claim 1 , wherein the nucleic acid molecule does not comprise a 35S ribosomal RNA gene unit.
3. An episomal DNA molecule (EDM) comprising an ARS that comprises a sequence having at least 95% identity to a nucleotide sequence selected from the group consisting of: SEQ ID NO:1; SEQ ID NO:5; SEQ ID NO:6; SEQ ID NO:7; SEQ ID NO:8; SEQ ID NO:9; SEQ ID NO:10; SEQ ID NO:11; and SEQ ID NO:12.
4. An EDM according to claim 3 , wherein the ARS comprises a sequence having at least 98% identity to a nucleotide sequence selected from the group consisting of: SEQ ID NO:1, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:11, and SEQ ID NO:12.
5. An EDM according to claim 4 , wherein the ARS comprises the polynucleotide sequence selected from the group consisting of: SEQ ID NO:1, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9; SEQ ID NO:10; SEQ ID NO:11; and SEQ ID NO: 12.
6. An EDM according to claim 3 , further comprising a nucleic acid sequence encoding a polypeptide.
7. An EDM according to claim 6 , wherein the polypeptide is a reporter protein or selectable marker protein.
8. An EDM according to claim 7 , wherein the reporter protein is a fluorescent protein or a signal producing enzyme.
9. An EDM according to claim 7 , wherein the selectable marker protein is a protein that confers resistance to a drug, antibiotic, toxin, or herbicide; a protein that allows autotrophic growth on particular media; or a protein that prohibits growth on particular media.
10. An EDM according to claim 6 , wherein polypeptide is a metabolic enzyme, structural protein, kinase, phosphatase, nucleotide cyclase, phosphodiesterase, nucleotide diesterase, transcriptional regulator, transcriptional activator, transporter, secretory protein, ion channel, receptor, photosynthetic protein, chaperonin, ribosomal protein, or nuclear scaffold protein.
11. An EDM according to claim 6 , wherein the polypeptide is a meganuclease, a zinc finger nuclease, a TALEN protein, a cas protein, a recombinase, a topoisomerase, or a transposase.
12. An EDM according to claim 3 , further comprising a nucleic acid sequence encoding a functional RNA.
13. An EDM according to claim 12 , wherein the functional RNA is selected from the group consisting of an interfering RNA or a precursor thereof, an antisense RNA, a ribozyme, a micro RNA, and a guide RNA of a CRISPR system.
14. An EDM according to claim 3 , wherein the ARS is functional in an algal or heterokont cell.
15. An EDM according to claim 3 , wherein the EDM is circular.
16. An EDM according to claim 3 , comprising sequences for mediating homologous or site specific recombination, wherein the sequences for homologous recombination flank a gene, a gene disruption sequence, or a functional RNA.
17. A method for propagating a gene of interest through at least 8 generations of cell division, the method comprising incorporating the gene of interest into the episomal DNA molecule of claim 3 , transforming the episomal DNA molecule into a cell, and transforming the EDM into a host cell, and propagating the host cell for at least 8 generations.
18. A method according to claim 17 , wherein the transformed cell is an algal, heterokont, plant, animal, fungal, or yeast cell.
19. A method according to claim 17 , wherein the transformed cell is a Nannochloropsis cell.
20. A method for transiently expressing a gene or sequence of interest in a microbial cell, comprising transforming the EDM of claim 3 into a microbial cell and culturing the microbial cell under conditions in which the gene or sequence is expressed.
21. The method of claim 20 , wherein the episomal DNA molecule comprises a selectable marker, and further wherein the microbial cell is cultured in the presence of a selective agent for one or more generations.
22. The method of claim 21 , wherein the microbial cell is cultured in the presence of the selective agent for one or more generations and subsequently cultured in the absence of the selective agent for and additional one or more generations.
23. A method of transforming a microorganism, comprising: contacting at least one cell of the microorganism with an EDM according to claim 3 , and selecting transformed cells on a selective medium.
24. An isolated or recombinant nucleic acid molecule according to claim 1 , wherein the ARS has at least 98% identity to a nucleic acid sequence selected from the group consisting of SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:11, and SEQ ID NO:12.
25. An isolated or recombinant nucleic acid molecule according to claim 24 , wherein the ARS is selected from the group consisting of SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:11, and SEQ ID NO:12.
26. An isolated or recombinant nucleic acid molecule according to claim 1 , wherein the ARS has at least 95% identity to SEQ ID NO:6.
27. An isolated or recombinant nucleic acid molecule according to claim 26 , wherein the ARS has at least 97% identity to SEQ ID NO:6.
28. An isolated or recombinant nucleic acid molecule according to claim 27 , wherein the ARS comprises SEQ ID NO:6.