Gene transfer to animal cells - Principles of Gene Manipulation - page 178

Salvage
pathway

De novo
pathway

Salvaged bases
and nucleosides

Nucleotides De novo source

APRT

Adenine

AMP

AK

Adenosine

( Aminopterin, methotrexate )
DHFR

Inosine

HPRT Glycine +

deoxyribose

Hypoxanthine IMP

IMPDH ( Mycophenolic acid )

XGPRT (bacteria l)

Xanthine XMP

HPRT

GMP

Guanine

CTP UTP

TK

TMP

Thymidine

DNFR
( Aminopterin,
methotrexate )

CAD

TK Aspartate +

deoxyribose

Deoxyuracil

dUMP UDP

(PALA)

Fig. 10.1 Simplified representation of the de novo and salvage nucleotide synthesis pathways. Top panel: purine synthesis.
De novo purine nucleotide synthesis (shown on the right) initially involves the formation of inosine monophosphate (IMP)
which is then converted into either adenosine monophosphate (AMP) or, via xanthine monophosphate (XMP), guanosine
monophosphate (GMP). The de novo synthesis of IMP requires the enzyme dihydrofolate reductase (DHFR), whose activity can
be blocked by aminopterin or methotrexate. In the presence of such inhibitors, cell survival depends on nucleotide salvage, as
shown on the left. Cells lacking one of the essential salvage enzymes, such as HPRT or APRT, therefore cannot survive in the
presence of aminopterin or methotrexate unless they are transformed with a functional copy of the corresponding gene. Thus,
the genes encoding salvage-pathway enzymes can be used as selectable markers. Note that the enzyme XGPTR, which converts
xanthine to XMP, is found only in bacterial cells and not in animals. Bottom panel: pyrimidine synthesis. De novo pyrimidine
nucleotide synthesis (shown on the right) initially involves the formation of uridine diphosphate (UDP). This step requires a
multifunctional enzyme, CAD, whose activity can be blocked by N -phosphonacetyl-l-aspartate (PALA). UDP is then converted
into either thymidine monophosphate (TMP) or cytidine triphosphate (CTP), the latter via uridine triphospate (UTP). De novo
TMP synthesis requires DHFR, so the reaction can be blocked in the same way as in de novo purine synthesis, making cell survival
dependent on the salvage enzyme thymidine kinase (TK). Thus, the Tk gene can be used as a selectable marker. There is no salvage
pathway for CTP.

can only be used with mutant cell lines in which the
corresponding host gene is non-functional. This
restricts the range of cells that can be transfected.
Endogenous markers have therefore been largely
superseded by so-called dominant selectable markers ,
which confer a phenotype that is entirely novel
to the cell and hence can be used in any cell type.
Such markers are usually drug-resistance genes of
bacterial origin, and transformed cells are selected on

a medium that contains the drug at an appropriate
concentration. For example, Escherichia coli trans-
posons Tn 5 and Tn 601 contain distinct genes encod-
ing neomycin phosphotransferase, whose expression
confers resistance to aminoglycoside antibiotics
(kanamycin, neomycin and G418). These are protein-
synthesis inhibitors, active against bacterial and
eukaryotic cells, and can therefore be used for selec-
tion in either bacteria or animals. By attaching the