10. D N A A N D R N A S Y N T H E S I S
331
Until recently there was no reliable method for the determination
of the protein content of a single cell. Bloch and Godman (1955)
attempted to detect a correlation between D N A (Feulgen stain) and
histone content (fast-green) in rat fibroblasts in culture, but the specificity of the fast-green dye-binding method is not entirely clear (see
Walker and Richards, 1959). Again Harris (1960) incubated rat
fibroblast cultures in tritiated adenosine, valine and methionine precursors. He found that, considering the ratio of nuclear to cytoplasmic
label, there was no simple relationship between the rate of incorporation
of nucleoside into R N A and the rate of amino acid uptake into protein.
A serious disadvantage of methods that use labelled amino acids in an
attempt to observe protein synthesis in mammalian cells is that,
because of turnover, the relation between amino-acid uptake and net
protein synthesis is obscure. This fact was demonstrated by Eagle and
his collaborators (Eagle, 1959; Eagle, Piez, and Fleischman, 1957;
Eagle, Piez, Fleischman and Oyama, 1959), who grew cell cultures in
medium deficient in one or more amino acids essential to growth (see
Chapter 8). Although under these conditions there was no net increase
in mass or of protein in the cultures, the cells continued to incorporate
labelled amino acids into their proteins at a high level for periods up to
72 h. In addition, growing cultures were found to incorporate aminoacids at an appreciably faster rate than could be accounted for by the
amount of growth, and to possess a turnover rate similar to the nongrowing cells. In this respect, the situation appears to contrast with that
in bacteria or yeasts, where turnover in growing cells is much less than
in non-growing cells (see Mandelstam, 1958; Halvorson, 1958). All the
mammalian cell types tested by Eagle et al. behaved similarly (HeLa,
L-strain, KB-strain, a human conjunctiva line, and fresh monkey
kidney).
However, in 1952 the simultaneous development by Barer (1952)
and by Davies and Wilkins (1952) of the interference microscope for the
measurement of optical path-difference between a cell and its surroundings enabled the dry mass of an individual cell or nucleus to be determined in a relatively straightforward manner. In rapidly growing mammalian cells in culture, the nuclear protein constitutes about four-fifths
of the nuclear dry mass and so dry-mass measurements can be taken as
roughly equivalent to the protein content.
A great advantage of this cytological technique is that successive
measurements of different quantities can often be made on the same
population of fixed cells, in this way obviating variations that may
exist between different populations. For example, after dry-mass
measurements have been made on, say, a population of fixed cell nuclei,
the 2,600 A absorption can then be measured to give the total nucleic
331
Until recently there was no reliable method for the determination
of the protein content of a single cell. Bloch and Godman (1955)
attempted to detect a correlation between D N A (Feulgen stain) and
histone content (fast-green) in rat fibroblasts in culture, but the specificity of the fast-green dye-binding method is not entirely clear (see
Walker and Richards, 1959). Again Harris (1960) incubated rat
fibroblast cultures in tritiated adenosine, valine and methionine precursors. He found that, considering the ratio of nuclear to cytoplasmic
label, there was no simple relationship between the rate of incorporation
of nucleoside into R N A and the rate of amino acid uptake into protein.
A serious disadvantage of methods that use labelled amino acids in an
attempt to observe protein synthesis in mammalian cells is that,
because of turnover, the relation between amino-acid uptake and net
protein synthesis is obscure. This fact was demonstrated by Eagle and
his collaborators (Eagle, 1959; Eagle, Piez, and Fleischman, 1957;
Eagle, Piez, Fleischman and Oyama, 1959), who grew cell cultures in
medium deficient in one or more amino acids essential to growth (see
Chapter 8). Although under these conditions there was no net increase
in mass or of protein in the cultures, the cells continued to incorporate
labelled amino acids into their proteins at a high level for periods up to
72 h. In addition, growing cultures were found to incorporate aminoacids at an appreciably faster rate than could be accounted for by the
amount of growth, and to possess a turnover rate similar to the nongrowing cells. In this respect, the situation appears to contrast with that
in bacteria or yeasts, where turnover in growing cells is much less than
in non-growing cells (see Mandelstam, 1958; Halvorson, 1958). All the
mammalian cell types tested by Eagle et al. behaved similarly (HeLa,
L-strain, KB-strain, a human conjunctiva line, and fresh monkey
kidney).
However, in 1952 the simultaneous development by Barer (1952)
and by Davies and Wilkins (1952) of the interference microscope for the
measurement of optical path-difference between a cell and its surroundings enabled the dry mass of an individual cell or nucleus to be determined in a relatively straightforward manner. In rapidly growing mammalian cells in culture, the nuclear protein constitutes about four-fifths
of the nuclear dry mass and so dry-mass measurements can be taken as
roughly equivalent to the protein content.
A great advantage of this cytological technique is that successive
measurements of different quantities can often be made on the same
population of fixed cells, in this way obviating variations that may
exist between different populations. For example, after dry-mass
measurements have been made on, say, a population of fixed cell nuclei,
the 2,600 A absorption can then be measured to give the total nucleic