producer cells correlates with a similar difference in the amount of Neo 
containing virus released into the medium (Fig.4), suggesting that the 
difference in the titer of infectious virus is real and is not due to nore 
efficient Neo expression in N2 infected cells. The difference between N2 and 
N4 does not reflect the size difference between the two vectors because 
additional DNA sequences downstream from the Neo gene present in LNL-2 (12), or 
DNA sequences inserted in both N4 and N2 (unpublished results and see below), do 
not abolish the difference in the titer of the corresponding viruses. Cells 
infected with N2 and N4 virus express an RNA transcript which corresponds in 
size to the predicted LTR driven transcript . N2 , but not N4 infected cells 
express a second, shorter transcript. Because this shorter RNA transcript 
hybridizes both to a Neo probe (panel C) and to a probe corresponding to the 5' 
end of the RNA transcript (panel B), it is probably a spliced RNA form. Further 
evidence to support this hypothesis is shown in Fig. 5, which is an SI nuclease 
protection assay designed to map the 3' splice site of this aberrantly spliced 
RNA form. Using a 5' end labelled probe extending from within the Neo insert 
into the upstream viral sequences, it is shown that N2 infected cells, but not 
N4 infected cells, contain a second SI protected band which is smaller them the 
probe used. Using an appropriate marker, shown in Fig. 5, the 3' end of the 
shorter protected band was mapped to the viral sequences, about 100 nucleotides 
5' to the Neo insert. These data suggest that the mRNA for the Neo gene is a 
spliced RNA form generated by the removal of about 500 bp intron resulting from 
the interaction of the naturally occurring viral 5' splice site present 68 
nucleotides downstream from the LTR, and a cryptic 3' splice site present in the 
viral gag coding sequences about 100 nucleotides upstream from the Neo gene (see 
figure IB). As a result of this splicing event, the gag coding sequences 
preceeding the Neo gene are removed. 
1254] 
Recombinant DNA Research, Volume 12 
