L-dopa decarboxylase (DDC) is another useful marker in SCLC tumors, especially in "classical 
SCLC" (82,83). The DDC assay will be performed using the method published previously (84). Briefly, 
the supernatant extract from the cell homogenate will be mixed with [carboxyl] 14 C labeled dopa together 
with pyridoxal phosphate and 0.1M Tris buffer pH 9.0 in an Eppendorf tube placed inside a glass 
scintillation vial containing hyamine hydroxide for trapping l4 C02. Perchloric acid (2N) is added to 
terminate the reaction and to convert H 14 C02 to free ,4 C0 2 . Enzyme activity will be determined by 
measurement of C0 2 release from 14 C labelled L-dopa. One unit of enzyme activity is defined as 1 nmol 
of 14 C0 2 /hr/mg of protein. i 
4.4 IL-2 cDNA CONSTRUCT AND EXPRESSION VECTOR: 
4.4.1 IL2 cDNA constructs 
Human 112 cDNA is modified by PCR in the following way. The 5’ untranslated sequence 1-89 is 
truncated. Position 90 is preceded by the Sal 1 recognition sequence for cloning. Position 90 to 572 
correspond to the natural 112 sequence followed by the Sal 1 recognition sequence. Compared to the 
natural 112 mRNA the construct lacks 89 nucleotides of the 5’ untranslated sequence (normal length 1 17) 
and contains three nucleotides of the 3’ untranslated sequence (normal length 278). 
The construct contains the complete unmodified 112 coding sequence. The 3’ sequence has been 
deleted and is replaced by the rabbit beta-globin 3’ untranslated sequence and poly A signal contained 
in the vector. The 3’tail of lymphokine messages is known to destabilize the mRNA. (Figure 5, see 
Appendix D). 
i 
The human 112 cDNA construct is cloned into the Sal 1 site of the pBMG-neo plasmid. 
4.4.2 The Plasmid Carrier. The complete 112 expression unit consists of the murine 
metallothioneine promotor, the 112 cDNA, the second rabbit /3-globin intron, and the rabbit /8-globin 
poly A signal. As shown above this expression unit is quite active (5720U/mlx5xl0 6 cells x24hrs) in 
human SCLC upon transfection. 
The plasmid carrier is pBMG-Neo. It contains the neomycin resistance gene expressed under the 
thymidine kinase promotor (for eukaryotic cells) and the Neo R promotor (for prokaryotes). The 
thymidine kinase poly A signal is used as termination signal (Figure 6, see Appendix D). For more 
efficient eukaryotic expression the plasmid contains 2.7kb of the human j3-globin sequence. The 
eukaryotic replicating unit consist of a 69% fragment of the bovine papilloma virus 1 (BPV1). (Figure 
7 see Appendix D; Figure 8, see Appendix D). The LI and L2 sequences coding for the major 
structural proteins have been deleted. The 69% fragment contains the El to E8 early genes and | 
regulatory sequences including the origin of replication. For eukaryotic replication El and E2 are 
absolutely required and are responsible for the episomal replication as nuclear plasmid (85). The copy 
number is controlled by the cell type and replication appears to occur in synchrony with cell division. 
The E6 and E7 gene products of some human papilloma subtypes (HPV16, 18) have been shown 
to interact the Rb protein and p53, respectively (86). E5 of BPV1 activates EGF receptors in the 
absence extraneous growth factors (87). 
The final piece of pBMG-Neo is a fragment of pBR322 containing the origin of replication for E. 
coli and the ampicillin resistance gene. 
4.4.3 Literature: Evidence against replication competence of the 69% BPV 
Is the 69% bovine papilloma virus fragment replication defective for infectious virus particles? 
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Recombinant DNA Research, Volume 18 
