Study of transcription in measles virus-infected Vero cells using cDNA probes prepared from poly(A)RNA from uninfected and infected cells.
From af primary plasmid cDNA library prepared from measles virus-infected Vero cell poly(A)RNA, 435 clones selected at random were used to examine the sensitivity and specificity of cDNA probes derived from total poly(A)RNA from uninfected and infected Vero cells. The correlation between the abundance level of a particular species in the cDNA probe and the hybridization signal strength generated by the corresponding cDNA clone on a filter was reliably determined only when at least three independently prepared filters were examined. Variation in the amount of target plasmid was the most important cause of spurious signals. Variation in cDNA insert length did not disturb the signal strength within certain limits. cDNA species with abundance levels down to 0.08-0.01% were able to produce a hybridization signal above background. Unspecific cross-hybridization was shown to define the sensitivity limit of mixed cDNA probes. Despite the many false signals present at different stages, cDNA probes provided valuable information: the cDNA probes were used to monitor relative RNA expression levels and to clone five different measles virus transcripts and 2 host cell transcripts more abundantly expressed in infected cells. The abundance levels of the measles virus nucleocapsid, phosphoprotein, matrix, fusion protein and haemagglutinin genes were 1.5%, 1.5%, 1%, 0.75% and 0.5%, respectively, of the total cDNA library.
Kalland
K H
KH
Department of Microbiology and Immunology, Gade Institute, University of Bergen, Norway.
Kalvenes
M B
MB
Oyan
A M
AM
Haukenes
G
G
D000818:Animals; D004247:DNA / Q000032:analysis*; D015342:DNA Probes; D008459:Measles virus / Q000235:genetics*; D009693:Nucleic Acid Hybridization; D010957:Plasmids; D011061:Poly A / Q000235:genetics*; D012333:RNA, Messenger / Q000032:analysis / Q000235:genetics*; D012367:RNA, Viral / Q000235:genetics; D014158:Transcription, Genetic; D014709:Vero Cells
D015342:DNA Probes; D012333:RNA, Messenger; D012367:RNA, Viral; D011061:Poly A; D004247:DNA