12. G E N E T I C C Y T O L O G Y
417
film is developed, the labeled and the exposed areas are one and the
same. This autoradiographic technique is, therefore, extremely useful
for cytologists.
When utilizing isotopes to study nucleic acid synthesis by means of
autoradiography, two major factors, the type of compound and the
type of isotope, must be considered. Since D N A and R N A share three
common bases—adenine, guanine and cytosine—these compounds are
therefore not suitable to use, for they will be incorporated by both systems, unless enzymatic digestion is applied. D N A and R N A differ in
one base: a pyrimidine. Thymine is present in D N A and uracil is
present in R N A . Although in living systems the normal pathway of
manufacturing thymidylic acid is not a direct synthesis from thymine,
it is possible to supply the tissues or cells with thymine or its deoxyriboside, thymidine ( T D R ) , as precursors of thymidylic acid and thus DNA.
Similarly, the cells utilize uracil or its riboside, uridine ( U R ) , to form
RNA. Incorporation of thymidine can be more efficient if the cell
system is a thyrnine-requiring mutant; or one can artificially interfere
with thymine metabolism by adding an inhibitor such as 5-fluorodeoxyuridine or aminopterin. Generally, however, this is not necessary.
Among the convincing evidence that thymine is present only in
D N A is the work by Reichard and Estborn (1951) who supplied N
1 5
-
labeled thymidine to a synthetic medium to grow Escherichia coli. It
was found that thymidine was incorporated exclusively, efficiently and
stably into the new D N A of the bacteria. Friedkin, Tilson and Roberts
(1956) found that thymine-2-C
14 was utilized by chick embryos for
DNA synthesis. Other investigators using C
1 4
- or P
3 2
- label on a variety
of cells supported the notion that D N A synthesis is performed just
prior to mitosis (Howard and Pelc, 1953; Lajtha, 1954; Lajtha, Oliver
and Ellis, 1954; Hornsey and Howard, 1956).
Thus if a cell undergoing D N A synthesis utilizes isotope-labeled
thymidine, the nucleus, not the cytoplasm, should bear the label.
D N A has been shown to be relatively stable in the nucleus; therefore
once the cell is labeled, the label should remain in the nucleus for long
periods of time until many division cycles later, when the label is
highly diluted by new D N A in the filial cell generations. It appears,
therefore, safe to use radioactive thymidine as a precursor to study
D N A synthesis of living tissues or cells.
The choice of isotope for autoradiograph has been tritium ( H
3 ) .
Isotopes such as C
1 4 and S
3 5
, though emitting weak /?-rays with relatively short pathways, still expose large film areas overlaying the cells
or tissues so that exact locations are obscured. They also may exert
radiation damage upon the cells. Fitzgerald, Eidinoff, Knoll and
Simmel (1951) suggested that tritium, with its very short ^-emission,
417
film is developed, the labeled and the exposed areas are one and the
same. This autoradiographic technique is, therefore, extremely useful
for cytologists.
When utilizing isotopes to study nucleic acid synthesis by means of
autoradiography, two major factors, the type of compound and the
type of isotope, must be considered. Since D N A and R N A share three
common bases—adenine, guanine and cytosine—these compounds are
therefore not suitable to use, for they will be incorporated by both systems, unless enzymatic digestion is applied. D N A and R N A differ in
one base: a pyrimidine. Thymine is present in D N A and uracil is
present in R N A . Although in living systems the normal pathway of
manufacturing thymidylic acid is not a direct synthesis from thymine,
it is possible to supply the tissues or cells with thymine or its deoxyriboside, thymidine ( T D R ) , as precursors of thymidylic acid and thus DNA.
Similarly, the cells utilize uracil or its riboside, uridine ( U R ) , to form
RNA. Incorporation of thymidine can be more efficient if the cell
system is a thyrnine-requiring mutant; or one can artificially interfere
with thymine metabolism by adding an inhibitor such as 5-fluorodeoxyuridine or aminopterin. Generally, however, this is not necessary.
Among the convincing evidence that thymine is present only in
D N A is the work by Reichard and Estborn (1951) who supplied N
1 5
-
labeled thymidine to a synthetic medium to grow Escherichia coli. It
was found that thymidine was incorporated exclusively, efficiently and
stably into the new D N A of the bacteria. Friedkin, Tilson and Roberts
(1956) found that thymine-2-C
14 was utilized by chick embryos for
DNA synthesis. Other investigators using C
1 4
- or P
3 2
- label on a variety
of cells supported the notion that D N A synthesis is performed just
prior to mitosis (Howard and Pelc, 1953; Lajtha, 1954; Lajtha, Oliver
and Ellis, 1954; Hornsey and Howard, 1956).
Thus if a cell undergoing D N A synthesis utilizes isotope-labeled
thymidine, the nucleus, not the cytoplasm, should bear the label.
D N A has been shown to be relatively stable in the nucleus; therefore
once the cell is labeled, the label should remain in the nucleus for long
periods of time until many division cycles later, when the label is
highly diluted by new D N A in the filial cell generations. It appears,
therefore, safe to use radioactive thymidine as a precursor to study
D N A synthesis of living tissues or cells.
The choice of isotope for autoradiograph has been tritium ( H
3 ) .
Isotopes such as C
1 4 and S
3 5
, though emitting weak /?-rays with relatively short pathways, still expose large film areas overlaying the cells
or tissues so that exact locations are obscured. They also may exert
radiation damage upon the cells. Fitzgerald, Eidinoff, Knoll and
Simmel (1951) suggested that tritium, with its very short ^-emission,