|||||||||||||| O US005436141A United States Patent (19) 11 Patent Number: 5,436,141 Miyata et al. (45) Date of Patent: Jul. 25, 1995 54 METHOD FOR SYNTHESIZING STABLE 56) References Cited SINGLE-STRANDED CONAN EUKARYOTES BY MEANS OF A U.S. PATENT DOCUMENTS BACTERAL RETRON AND PRODUCTS 5,079,151 1/1992 Lampson et al. ................ 435/91.51 75 Inventors: Shohei Miyata, Misato, Japan; FOREIGN PATENT DOCUMENTS Atsushi Ohshima, Highland Park, 0132309 1/1985 European Pat. Off. ... C12N 15/00 N.J.; Sumiko Inouye; Masayori Inouye, both of Bridgewater, N.J. OTHER PUBLICATIONS Y o Dhundale et al., J. Bact, vol. 170, 1988, pp. 5620-5624. 73) Assignee: University of Medicine and Dentistry Lampson et al., Science, vol. 243, 1989, pp. 1033-1038. of New Jersey, Newark, N.J. Sambrook et al., Molecular Cloning: A Laboratory Man ual, Cold Spring Harbor Laboratory Press, 1989, pp. 21 Appl. No.: 753,110 16.15-16.16. 22 Filed: Aug. 30, 1991 Primary Examiner-Richard A. Schwartz Assistant Examiner-James Ketter Related U.S. Application Data Attorney, Agent, or Firm-Weiser & Associates (63) Continuation-in-part of Ser. No. 315,427, Feb. 24, 1989, 57 ABSTRACT Pat. No. 5,979,151, and a continuation in part of Ser. A method for producing in vivo stable single-stranded No. 315,316, Feb. 24, 1989, Pat. No. 5,320,958, and a DNAs in eucaryotic cells. The DNAs are multicopy Eup ii.g 1. single-stranded DNA (msDNA) structures constituted 517,946, May 2, 1990, and a continuation-in-part of Ser. by a RNA and a DNA portion. The group of genes No. 518,749, May 2, 1990. (retrons) producing said coupled RNA and DNA por tions of the msDNAs and the gene encoding reverse 51) Int. Cl.......................... C12N 1/19; C12N 5/10; transcriptase (RT). The transformed eucaryotes harbor C12N 15/81; C12P 19/34 ing these retrons. The new msDNAs which are en 52 U.S. C. ................................ 435/91.1; 435/240.2; coded by the new retrons. The msDNAs can be used as 435/254.2; 435/254.21; 435/320.1; 536/25.2 vectors for antisense DNA and for amplification of 58) Field of Search ................... 435/91.1, 69.1, 320.1, inserted genes. 435/252.33, 194, 254.21, 256, 240.2, 240.21, 240.4, 254.2; 536/27, 25.2 45 Claims, 17 Drawing Sheets o2 b2 b 1 o 1 CHROMOSOMEE-7a nSr X- RT msd ( 1) 2 RNA POLYMERASE 5 OH (RIFAMPICIN-SENSITIVE) 3' -(6)—- AUG - 74 (2) SELF ANNEAL ING y 5 RT PRODUCTION - Neo-cloo2 b2 O 1 b 1 (3) REVERSE TRANSCRIPTION RNA PROCESSING (4) TERMINATION 3. p RNA e *...........' DNA U.S. Patent July 25, 1995 Sheet 4 of 17 5,436,141 FIG. 2C 25 CTTCTCACCTGGG-1 NGGAGAGTGTCCTGC-3 S& RNA •„ººso Sa163 ||||Q o—oo3,\ Q Qò U.S. Patent July 25, 1995 Sheet 8 of 17 5,436,141 / CN C C C A 2G U.S. Patent July 25, 1995 Sheet 9 of 17 5,436,141 A AA-40C-G 5 T-A C A-T A A-T G T C AntiSense DNA for CDC 28 T-A1 A (50 base) C-G U 3O OTo G G C-Go50 AOU 70 TAG N G CTCGAG. A A G TG / C C-G A C-G 19 2OOG-C 60 25 TCCTTCGCACAGCACACCT-1 NGATTCCTCCTGCC-3 3'-AGGACGG - A U U AOSO A — -C-C-U-U-1 UOG UOG 2OOG OU UOG U-AO 4O G-C G-C U-A U-A G-C UOG (G-C JOOA YA1 FIG. 2H U.S. Patent July 25, 1995 Sheet 10 of 17 5,436,141 8 RT RNosed - Ho- - Co 25O - 26O 12O - 15O Eukaryotic RT nsLONA-Mx 162 ms/DNA-Mx65 nSDNA-Ec67 nnsLONA-EcS6 msDNA-Ec75 U.S. Patent July 25, 1995 Sheet 11 of 17 5,436,141 ORI INSERT GENE M INSERT REP3 al NIGENE blo U.S. Patent July 25, 1995 Sheet 12 of 17 5,436,141 EcoRI ECO RI Pst I Pst I (d) -Q S (d) -Q Pvu SSC -Y O s Pst 1 (c) - S Pst 1 (c) Pst I (b) Pst I (b) U Pst I (o) S Hind III V V V Hird II S V Q \ g V Bol I V N \ 2 \ Pvu I S Pst I (o) O N J. Eco RI . O Q) FIG. 6 U.S. Patent July 25, 1995 Sheet 13 of 17 5,436,141 JSLIJ•— 8.^dH-D&H. Q>1!–—--— psu—>lè? --•?–?????????????????????????????????????????•!--JSll/ H91779S67H8 †ZZZZZZZZZZZZZZZZZZZZZZZZZZZZZZZZZZZE psu-> lè?psu-> Hj 87##9,Twº ZZZZZZZZZZZZZZZZZZZZZZZZZZZZZZZZZZZZZZZZZZZZZZZZZZZZZZZZZZZZZZZZZZZZZZZZZ ØO1779 <No.w •—JS11/ ·„Ol770 padgl–10d U.S. Patent July 25, 1995 Sheet 14 of 17 5,436,141 123456 sums FIG. BA U.S. Patent July 25, 1995 Sheet 15 of 17 5,436,141 nSDNA-Ec67 AMV-RT-4dNTP (band a) RNose A (band b) FIG. BB U.S. Patent July 25, 1995 Sheet 16 of 17 5,436,141 O o O y CO 3. CN sm O) Q5 S8 N G Y N CO C\ U.S. Patent July 25, 1995 Sheet 17 of 17 5,436,141 YEp521-M4 "CONSTRUCTION OF YEp521-M5" gol 10P CD2 YEp521-M4 RT "msd add Xhol site into msd region by PCR method gal foP Xhol E2 YEp521-M4 Xhol RT "msd digeted by Xhol / Anti Sense DNA for(5Obp) CDC28 (Xhol Fragment) | 10P Ligation go E>2 2 YEp521-M5 RT Emsd This plasmid (YEp521-M5) was transformed into Yeast (SP-1) sicGATGTAATTTCCTAATTCACCCCTCATGTTCCAAGGATAGTICTATTTGATC AcAfiaAAccAiiAATCGCGAGTACAAccriccia TCAAATAAACTAGACCT AntiSense DNA for CDC 28 FIG. 10 5,436,141 1. 2 METHOD FOR SYNTHESZING STABLE SUMMARY OF THE INVENTION SINGLE-STRANDED CIDNA NEUKARYOTES BY In accordance with the invention, a fundamental MEANS OFA BACTERIAL RETRON AND finding has been made. It has been discovered that sin PRODUCTS gle-stranded DNAs which are stable can be produced in vivo in eucaryotic cells. RELATED PATENT APPLICATIONS Briefly described, the invention provides a method (or process) for producing in vivo stable, single This is a continuation-in-part of patent applications stranded DNAs in eucaryotic cells like yeasts or plant Ser. No. 315,427 filed Feb. 24, 1989, now U.S. Pat. No. cells or mammalian cells. The method of the invention 5,079,151; 315,316 filed Feb. 24, 1989, now U.S. Pat. produces a single-stranded cDNA by means of a retro No. 5,320,958; 315,432 filed Feb. 24, 1989, now aban element called a retron. The single-stranded DNA is doned; 517,946 filed May 2, 1990 and 518,749 filed May produced as an integral part of a branched RNA-linked 2, 1990. multicopy single-stranded DNA (msDNA) structure. FIELD OF THE INVENTION 15 These structures are stable, i.e., detectible after produc tion and isolation in spite of the fact that they are consti The invention concerns the field of recombinant tuted of RNA and DNA, both single-stranded. The DNA. More particularly, the invention relates to an in method of the invention also provides such msDNAs vivo method of synthesis of stable single-stranded which contain foreign DNA and RNA fragments in the cDNA in eucaryotic cells by means of a bacterial retro 20 DNA and RNA portions, respectively, of the RNA element called a retron. The invention also relates to DNA structure. Though different from the known bac new eucaryotic vectors carrying the necessary elements terial msDNAs, these molecules are designated as to produce the single-stranded DNA-RNA hybrid msDNAs or "modified’ msDNAs, because they have structures. Moreover, the invention relates to trans the characteristics and unique features of msDNAs as fected eucaryotes, e.g., yeast, plant cells and mamma 25 described herein. lian cells. Uses are described for the new products. The invention also provides retrons. Retrons are BACKGROUND genetic elements which contain the coding region msr for the msRNA and msd for the msdDNA of the Gram-negative bacteria such as Myxococcus xanthus, msDNA molecule, respectively, and the gene for re Stigmatella aurantiaca and Escherichia coli have been 30 verse transcriptase (RT). The retrons which are new in found to contain a retroelement called a retron. In accordance with the invention, have sequences which TIBS, 16, 18–21 (1991a), the authors report on a pecu are different from known bacterial retrons in that the liar type of satellite DNA, named multicopy single non-coding region has been shortened, specifically the stranded DNA (msDNA). These molecules are charac region between the transcriptional initiation site of the terized by a structure which comprises a single-stranded 35 selected promoter and the initiation codon of the RT DNA branching out of an internal guanosine residue of gene. a single-stranded RNA molecule by a unique 2',5'-phos The invention also provides retrons which are new phodiester linkage. These molecules are thus single by virtue of the fact that, unlike known bacterial re stranded DNA-RNA hybrids. Reverse transcriptase is trons, the RT gene is positioned upstream of themsr required for the synthesis of these msDNAs. In Ann. msd region, in reverse relationship of that in bacterial Rev. Microbiol, 45, 163-186 (1991b), the authors present retrons. These new retrons produce greater yields of a comprehensive review on msDNAs. Also see msDNAs. msDNA in Bacteria, Lampson et al., Progress in Nucleic The invention further provides new types of Acid Research and Molecular Biology, 60, 1-24. msDNAs which are new by virtue of having been pro The production of single-stranded cDNA by reverse 45 duced by the novel retrons.