TY - JOUR
T1 - Complementary DNA Cloning and analysis of carnation mottle virus RNA
AU - Carrington, J. C.
AU - Morris, T. J.
N1 - Funding Information:
The authors thank Bradley Hillman for helpful suggestions concerning this manuscript, and Dr. Kyle Willis for donating bacterial strains. We also thank Mary Ellen Morrin for typing this manuscript. This work was supported in part by a grant (83-CRCR-1-1227)f rom the U. S. Department of Agriculture.
PY - 1984/11
Y1 - 1984/11
N2 - A 3850-base pair (bp) cDNA fragment, representing most of the carnation mottle virus (CarMV) genome, was molecularly cloned in Escherichia coli using a modification of the plasmid-primer method of Okayama and Berg (H. Okayama and P. Berg (1982), Mol. Cell. Biol. 2, 161-170). Poly(dT)-tailed pUC8 was used to prime cDNA synthesis from polyadenylated CarMV RNA template. Oligo(dC) was added to the cDNA 3′ end, and second-strand synthesis was specifically primed using an oligo(dG)-tailed linker fragment. cDNA inserts of 3250 and 1950 by were identified from a clone bank as apparently overlapping and were fused at a common BglII restriction site to produce pCarMV-1C. Confirmation that this plasmid harbored CarMV cDNA sequences was achieved by Southern blot hybridization with a randomly primed cDNA probe. Nick-translated pCarMV-1C was used to probe virion and total single-stranded RNA extracts from infected Chenopodtium quinoa leaves by "Northern" blot hybridization. Besides the full-length genomic RNA, two additional species of approximately 1600 and 1750 bases were associated with CarMV. Based on a series of Northern blots with nick-translated subclones derived from pCarMV-1C as probes, these subgenomic RNAs were found to originate from the 3′ domain of the virus genome.
AB - A 3850-base pair (bp) cDNA fragment, representing most of the carnation mottle virus (CarMV) genome, was molecularly cloned in Escherichia coli using a modification of the plasmid-primer method of Okayama and Berg (H. Okayama and P. Berg (1982), Mol. Cell. Biol. 2, 161-170). Poly(dT)-tailed pUC8 was used to prime cDNA synthesis from polyadenylated CarMV RNA template. Oligo(dC) was added to the cDNA 3′ end, and second-strand synthesis was specifically primed using an oligo(dG)-tailed linker fragment. cDNA inserts of 3250 and 1950 by were identified from a clone bank as apparently overlapping and were fused at a common BglII restriction site to produce pCarMV-1C. Confirmation that this plasmid harbored CarMV cDNA sequences was achieved by Southern blot hybridization with a randomly primed cDNA probe. Nick-translated pCarMV-1C was used to probe virion and total single-stranded RNA extracts from infected Chenopodtium quinoa leaves by "Northern" blot hybridization. Besides the full-length genomic RNA, two additional species of approximately 1600 and 1750 bases were associated with CarMV. Based on a series of Northern blots with nick-translated subclones derived from pCarMV-1C as probes, these subgenomic RNAs were found to originate from the 3′ domain of the virus genome.
UR - http://www.scopus.com/inward/record.url?scp=0021683778&partnerID=8YFLogxK
U2 - 10.1016/0042-6822(84)90326-X
DO - 10.1016/0042-6822(84)90326-X
M3 - Article
AN - SCOPUS:0021683778
SN - 0042-6822
VL - 139
SP - 22
EP - 31
JO - Virology
JF - Virology
IS - 1
ER -