T R A N S P L A N T A T I O N OF CELL N U C L E I
39
involved in new protein synthesis. As an example of this work, Greengard et al. (1963) have shown that new gene action is involved at least
in the case of the induced formation of tryptophan pyrrolase in rat liver
by cortisone. They found however, that the induction of this enzyme by
its substrate was not actinomycin-sensitive. It is too early to draw any
general conclusions regarding the molecular basis of induced enzyme
formation. However at least some cases of hormone-induced enzyme
synthesis demonstrate another example of reversible gene activity.
3. Histone Gene Suppression
The last kind of experiment to be discussed in this section concerns
the experimental suppression of gene-controlled RNA synthesis by
histone proteins. Huang and Bonner (1962) homogenized embryo shoots
from germinated pea seeds and separated the DNA as well as associated
protein from the homogenate by density-gradient and other fractionation procedures. Further fractionation yielded separate preparations of
DNA, histone protein, and RNA polymerase enzyme. The synthetic
activity of the DNA and histone, either singly or in combination, was
then determined by adding them to an incubation mixture which
included labelled nucleotides. The amount of RNA formed by incorporation of nucleotides for a given period of incubation was then
measured. It was found that the DNA from which the histone had been
removed stimulated much more nucleotide incorporation than did the
DNA which was still complexed with histone over the same incubation
period. Thus the removal of histone from DNA greatly increased its
synthetic activity. The histone could be recomplexed with the DNA
again by gradually lowering the ionic strength of a strong salt solution
to which the two components had been added. The recomplexed DNAhistone promoted much less RNA synthesis in the incubation mixture
than did the DNA without histone. This was not due to a denaturing of
the DNA by the histone as can happen under certain conditions since
the complexed nucleo-histone had a higher sedimentation constant and
melting point temperature than did the isolated DNA. Direct evidence
that the original state of gene repression is to some extent conserved in
the isolated nucleo-histone or chromatin comes from the remarkable
finding that isolated pea seed chromatin makes the characteristically
large amount of pea seed globulin—a protein which is made only in very
small amounts by other cell types and by chromatin extracted from
these cells (Bonner et al., 1963).
Some comparable work by Allfrey et al. (1963) has shown that the
amount of RNA synthesized by isolated thymus nuclei can be increased by
trypsin removal of histones from the nuclei and decreased again by adding histone. Furthermore the RNA made appeared to be messenger-RNA.
39
involved in new protein synthesis. As an example of this work, Greengard et al. (1963) have shown that new gene action is involved at least
in the case of the induced formation of tryptophan pyrrolase in rat liver
by cortisone. They found however, that the induction of this enzyme by
its substrate was not actinomycin-sensitive. It is too early to draw any
general conclusions regarding the molecular basis of induced enzyme
formation. However at least some cases of hormone-induced enzyme
synthesis demonstrate another example of reversible gene activity.
3. Histone Gene Suppression
The last kind of experiment to be discussed in this section concerns
the experimental suppression of gene-controlled RNA synthesis by
histone proteins. Huang and Bonner (1962) homogenized embryo shoots
from germinated pea seeds and separated the DNA as well as associated
protein from the homogenate by density-gradient and other fractionation procedures. Further fractionation yielded separate preparations of
DNA, histone protein, and RNA polymerase enzyme. The synthetic
activity of the DNA and histone, either singly or in combination, was
then determined by adding them to an incubation mixture which
included labelled nucleotides. The amount of RNA formed by incorporation of nucleotides for a given period of incubation was then
measured. It was found that the DNA from which the histone had been
removed stimulated much more nucleotide incorporation than did the
DNA which was still complexed with histone over the same incubation
period. Thus the removal of histone from DNA greatly increased its
synthetic activity. The histone could be recomplexed with the DNA
again by gradually lowering the ionic strength of a strong salt solution
to which the two components had been added. The recomplexed DNAhistone promoted much less RNA synthesis in the incubation mixture
than did the DNA without histone. This was not due to a denaturing of
the DNA by the histone as can happen under certain conditions since
the complexed nucleo-histone had a higher sedimentation constant and
melting point temperature than did the isolated DNA. Direct evidence
that the original state of gene repression is to some extent conserved in
the isolated nucleo-histone or chromatin comes from the remarkable
finding that isolated pea seed chromatin makes the characteristically
large amount of pea seed globulin—a protein which is made only in very
small amounts by other cell types and by chromatin extracted from
these cells (Bonner et al., 1963).
Some comparable work by Allfrey et al. (1963) has shown that the
amount of RNA synthesized by isolated thymus nuclei can be increased by
trypsin removal of histones from the nuclei and decreased again by adding histone. Furthermore the RNA made appeared to be messenger-RNA.