predicted, is of equal size. Fig. 2A also shows that DE-NA/S infected cells 
contain 5-10 fold less of the spliced RNA form as compared to cells harboring 
the DE-NA/X vector. Consistent with this observation is that DE-NA/X infected 
cells contain measurable levels of human ADA activity, about 10% of the 
endogenous mouse ADA activity, whereas no human ADA activity can be detected in 
cells infected with DE-NA/S virus (Fig.3B). Other DE vectors containing the 
human beta interferon or the mouse DHFR cDNAs in the envelope position have also 
been shown to be dependent on the presence of additional intron sequences for 
the efficient expression of the spliced gene product. (unpublished results). 
These results are consistent with our previous findings that sequences within 
the M-MuLV intron are essential for the efficient formation of the spliced RNA 
form (5) and, together with similar studies of others (7,13,16), further 
emphasize the importance of internal sequences in the biogenesis of M-MuLV RNA 
species. 
The N2 vector system . Fig. IB shows the structure of two M-MuLV derived 
vectors in which the selectable Neo gene is directly flanked by two LTRS. In 
N4, the viral sequences present downstream from the 5' LTR encode the packaging 
signal (11) but exclude the first functional viral initiation codon so that the 
selectable gene is expressed from an LTR-initiated unspliced RNA species. N2, 
on the other hand, differs from N4 , by containing sequences downstream from the 
LTR which extend into the viral gag gene coding sequences. Therefore, the Neo 
gene is preceded by a functional initiation codon as well as by 418 bp of coding 
sequences (determined by direct sequencing) containing a total of nine AUG 
codons. In addition, the Neo and gag initiation codons are out of frame. 
The titer of infectious Neo containing virus generated from N2 ranges from 
1-5X10^ Neo cfu/ml, which is 10-40 fold higher than the titer of virus generated 
from N4 . The difference in titer of infectious Neo virus generated by N2 and N4 
Recombinant DNA Research, Volume 12 [253] 
