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United States Patent (19) 11 Patent Number: 5,405,775 Inouye Et Al

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United States Patent (19) 11 Patent Number: 5,405,775 Inouye Et Al USOO5405775A United States Patent (19) 11 Patent Number: 5,405,775 Inouye et al. 45 Date of Patent: Apr. 11, 1995 54 RETRONS CODING FORHYBRID ATCC Catalog (1985), pp. 66-79. DNA/RNAMOLECULES Yee et al. Multicopy Single-Stranded DNA. Isolated from a Gram-Negative Bacterium ... etc. Cell 38, pp. 75 Inventors: Masayori Inouye; Sumiko Inouye, 203-209 (1984). both of Bridgewater, N.J. Dhundale et al. Distribution of Multicopy Single-S- 73) Assignee: The University of Medicine and tranded DNA amoung Myxobacterial and Related Spe Dentistry of New Jersey, Piscataway, cies J. Bacteriol. 164, pp. 914-917 (1985). N.J. Furuichi T. et al. Biosynthesis and Structure of Stable 21 Appl. No.: 518,749 Branched RNA Covalently . etc. Cell 48, pp.55-62 (1987). 22 Filed: May 2, 1990 Dhundale et al. Structure of msDNA from M. xanthus ... etc. Cell 51, pp. 1105-1112 (1987). Related U.S. Application Data Dhundale et al. Mutations that Affect Production of 63 Continuation-in-part of Ser. No. 315,432, Feb. 24, 1989, Branched RNA-Linked msDNA.M. xanthus ... etc. J. abandoned, Ser. No. 315,427, Feb. 24, 1989, Pat. No. Bacteriol., pp. 5620–5624 (1988). 5,079,151, Ser. No. 315,316, Feb. 24, 1989, Pat. No. Lin and Maas, Reverse Transcriptase-Dependent Syn 5,320,958, and Ser. No. 517,946, May 2, 1990. thesis of a Covalently Linked . etc. Cell 56, pp. 51. Int. Cl. ....................... C12N 1/21; C12N 15/70, 891-904 (Mar. 1989). C12N 15/52 Primary Examiner-Richard A. Schwartz 52 U.S.C. ............................ 435/252.33; 435/320.1; Assistant Examiner-James Ketter 536/23.2; 536/25.2 Attorney, Agent, or Firm-Weiser & Associates 58) Field of Search ....................... 536/27, 33.1, 35.2, 536/23.2, 25.2; 435/91, 172.3, 172.1, 252.3, 57 ABSTRACT 252.33, 320.1 A multicopy single-stranded DNA (msDNA) synthe sizing system in E. coli is disclosed. The use of the 56) References Cited msDNA system to synthesize cDNA in vivo is dis PUBLICATIONS closed. Construction of synthetic msDNA is also dis Masui et al., “Multipurpose Expression Cloning Vehi closed. Also processes for gene amplification and for cles in Escherichia coli', in Experimental Manipulation of producing a stable RNA are disclosed. Gene Expression, Academic Press, New York, 1983, Inouye, ed., pp. 15-32. 29 Claims, 17 Drawing Sheets U.S. Patent Apr. 11, 1995 Sheet 1 of 17 5,405,775 cy c) N EcoRI Sn N Pst 1(a) S Pvu II v ( \ Q V U V S Bol I \ S \ ( V Vs Hindo I I I \ V V Q NL Hind I I I s Pst I (a) Pst I (b) Hind III Pst I (b) S Pst I (c) s Pst I (d) ----- :EcoRI K-12.4% EcoRI FIG. 1 U.S. Patent Apr. 11, 1995 Sheet 2 of 17 5,405,775 5 U Ao 50 A FIG. 2A U.S. Patent Apr. 11, 1995 Sheet 3 of 17 5,405,775 FIG. 2B • U.S. Patent Apr. 11, 1995 Sheet 5 of 17 5,405,775 pINIII(lppp.)Ec67-RT 1O. 7kb Xbo I BAP + Synthetic msDNA (196bp) 1 ppp-NS loc PO -- RTV pINIII(lppp.ms10ORT 1.O. 9kb FIG. 4 U.S. Patent Apr. 11, 1995 Sheet 7 of 17 5,405,775 Dro I # Synthetic msl)NA (196bp) Xbo I Xbo I Soc II SocII Km R IacP ox Xibal, Sac II pUCKms 1 OO anem-ame + ms C1, C2 FRAGMENTS pUCKns 101 FIG. 6 U.S. Patent Apr. 11, 1995 Sheet 9 of 17 5,405,775 I -Sloc Z' pUC19-Ec67-20 FIG. B U.S. Patent Apr. 11, 1995 Sheet 10 of 17 5,405,775 1-yC Idc Z' pUC19-Ec67-20 TRANSCRIPTION OF Icic Z 5' 80 BASES 20 BASES 3' mRNA cDNA synthesis in vivo msDNA 3' cDNA 5' st an ame a so - - w mRNA Primer for PCR (P1, P2) d KC Pf P2 ApCR H P X U.S. Patent Apr. 11, 1995 Sheet 11 of 17 5,405,775 U.S. Patent Apr. 11, 1995 Sheet 12 of 17 5,405,775 U.S. Patent Apr. 11, 1995 Sheet 13 of 17 5,405,775 U.S. Patent Apr. 11, 1995 Sheet 14 of 17 5,405,775 U.S. Patent Apr. 11, 1995 Sheet 15 of 17 5,405,775 IGG CA TO AGA TAC GGA TTT ICA CTTCCT. IGA (AG 16 AIGACTAIG CIG CAT GAAAIC 60 GA IA ICATT GAG (AT CT CIT ICC ICA GAT (GCA GAA CIG GC GC TTT IGC ICA 120 IGT CAT GA IGT GA IGAAAA CA CIG (ATAAA G. GGC AGG CCI GGC GGG GATACGAGC 180 GCG (GC IAT CAC CAAAA IAG CAAAA IAC IIC IGGAAAACA GAA AGT ICA ACT GATAIC 240 ce RNA O2 TIC AIA AACAC CAT GIA CAC III GT GT ICT CAIC CACA GIG GIA AIG 300 AAG AT TIG 76 CIA (ATCO ICT AAA (AA (AACA CTTAGC CTT GT CAC CGG AAT IAC GAGGGGAAIC GCC. ICC CIA AAA ICC IIG ATT (AGAGC IATACG GCA GT GIG CIG IGC 360 (GT CCT (CT TAG CAS GATTTTAGGAA. IAA GIC 106 AIA 16 CCT CA (AC GAC ACG -- ol GAA GA CIG CCT GCA IGC GTT ICT (CI TGG CCT IIT TIC CIC IG GAT GAA GAA GAA AIG 420 CIT CICAC GGA CGT ACG CAA AGA CGA ACC CGAAAAAAG GAG ACC CIA CIT CTI CTT IAC K Ko DNA ACA AAA ACA ICT AAA CTTGAC GA CTTAGG GCI GCIACT ICA CGI GAA GAC TIG GCTAAA 480 T K T S K L D A L R A A T S R E D L. A. K. AIT IIA G4T AIT AAG IIG GIA IIT IIA ACT AAC GT CIA TAT AGAATCGGC ICG GAT AAT 540 I L. D 1 K L V F L J W W. L. Y. R. I. G. S D W CAA IAC ACT CAA TTT ACA AIA (GAAGAAA GAAAA GGG GIA AG ACTATT ICT GCA CT 600 0 Y T 0 F T 1 P K K G K G W R T | S A P abO ACA GAC CGG, TIGAAG GACAIC CAA CGA AGA AIA IGT GAC IIA CTI ICT GAT ICT AGA GAT 660 T D R L. K. D 1 0 R R N C D L L S D C R D CAGAIC TTT GCTATA AGAAAAITAGTAACAAC IAT ICC TTT GCI ITT GAG AGG GAAAA 720 E / F A 1 R K 1 S M N Y S F. G. F. E. R. G. K. at 100 ICA AIA AIC CIA AAT GCT IATAAG CAT AGAGCCAAA (AAAAAIA TIA AAIAIA GT CIT 780 S 1 I L N A y R H R G K Q 1 I L N J D H AG CAT IIT III GAAGC IITAT IIT G CoA GT AG. G. IAT III CTT ICC AAI (AG 840 K D F F. E. S. F. N. F. G R W R G Y F L S N 0 GATTTTTIA TIA AAT (CT GIG GIG GA ACG ACA CTTGAAAA GCT CA IGC IATAATGA 900 D F L L N P W W A T T L A K A A. C. Y. M. G. af30 FIG. 13A U.S. Patent Apr. 11, 1995 Sheet 16 of 17 5,405,775 ACC CIC CCC CAA CGA AGT CCA IGI ICT CCTATTAIC ICA AAT CIA ATT IGC AATATTAIG 960 T L P O G. S P C S P 1 1 S M L I C W 1 K GAI AIGAGA TIA GCTAAG CIG GCI AAAAAA IAI GA IGT ACT IATAGC AGA IAT GCT GAT 1020 D N R L. A K L A K K Y C C T Y S R Y AD a 200 GAI AIA ACA AIT ICT ACA AATAAA AAT ACA ITT CCG IIA GAA AIG GCIACT GIG CAA CCT 1030 D I T 1 S T N K N T F P L E N A T W G P GAA GGG GTI GTT TIG GAAAA GTI IIG GIA AAA GAA AIA GAA AAC ICT GGA TIC GAA AIA 1140 E. G. W. W. L. G K W L W K E.E / E. W. S G F E I AAI CAT ICA AAG ACTAG CTIAC IATAAG ACA IA AG (AA GAA GTAACGGGA CTTACA 1200 N D S K J R L J Y K I S R 0 E W T G L T a250 GTTAAC AGA AJC GTTAAT ATI GAT AGA TGT IAT IATAAAAAA ACT (GG GCG TIG GCA W M R / W N I D R C Y Y K K I R A L. A CA A 1 Y R T G E Y K W P D F M G W L V S GT CIG GATAAA CTTGA6 GG AIG TTT GT TITATIGAT (AA GTT (ATAAG TTT AACAAT 1380 G. L. D. K L E G N F G F | D 0 W D K F W W AIA AAGAAAAAA CIG AACAAG CAA CCT GAT AGA TAT CIA IIG ACT AAT GOG ACT TIG CAT 1440 I K K K L. M K C P D R Y W L T W A T L H GGT TITAAA TIA AAG TIGAATGG (G GAA AAA GA IATACTAAA TTIATI IAC IATAAA 1500 G F K L. K. L. M. A. R. E. K. A. Y S K F | Y Y K 50 TTI ITT CAT GOC AAC ACC IGT (CTACGAIA AIT ACA GAA. GGG AAG ACT GAT CGG AIA IAT 1560 F F H G W T C P T I I I E G R T D R Y TIGAAG GCT GCT TIG CAT ICT TIG GAG ACA ICA IAT (CTGA6.
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