5 research outputs found
Complementation of mutant phenotypes and genotypes of cultured mammalian cells
This dissertation describes experiments aimed at the complementation
of a genetic mutation in cultured mammalian cells in order to investigate
several aspects of the structure and functioning of the human genome.
Complementation is indicated by the correction of a biochemical function
in which the mutant eel ls are deficient. Where appropriate in this text, a
synonymous use of the terms 1 complementation 1 and 1correction 1 is made.
Complementation at the level of the cellular phenotype was studied as well
as complementation at the level of the cellular genotype.
The phenotype of a cultured mammal ian cell can be changed by the introduction
of protein molecules which are not normally produced by that
cell or messenger RNA molecules which direct the intracellular synthesis
of such molecules. Since both protein and RNA molecules are not self perpetuating,
a transient change in cell phenotype is usually observed. We
have used phenotypic correction to investigate proteins for their ability
to correct the deficiency in DNA repair displayed by human excision deficient
xeroderma pigmentosum (XP) cells. For this purpose we developed an
assay procedure in which prokaryotic DNA repair enzymes of known specificity
were introduced into 1 iving XP cells by microinjection (Ml) via glass
microneedles and the complementation to a repair proficient phenotype was
investigated (Appendix paper 1). In addition, extracts of repair proficient
human cells were assayed for activities that are able to complement the
deficiency in XP cells. Appendix paper II describes the identification of
a protein factor which specifically corrects the deficiency in one class
of XP cells but not in others. These papers demonstrate the use of the
living cell as part of a microinjection assay system to investigate the
biological activity of proteins. The genotype of a cultured mammalian cell can be supplemented by the
introduction of genetic material (gene transfer). For DNA-mediated gene
transfer (DMGT) using genomic DNA and chromosome-mediated gene transfer
(CMGT) using metaphase chromosomes isolated from eukaryotic cells, the genetic
material is usually administered as a co-precipitate with calcium
phosphate. For DMGT via viral or plasmid DNA molecules, Ml has also been
found useful. Gene transfer can be detected as a transient or a more or
less permanent change in cell phenotype if the genetic material is expressed
correctly. The transfer and continued expression of genes generally occurs at such a low frequency that it is necessary to use marker genes
which confer viability on complemented cells in selective culture conditions.
Although transient genotypic complementation has been studied occasionally,
the more permanent mode of correction has been investigated extensively
and used in a number of eel genetic studies.
We have used the genotypic complementation of cultured mammalian cells
to compare the DMGT and CMGT processes. In addition, two aspects of the
structure and expression of the human genome were investigated. Firstly,
a contribution was made to the mapping of genes to human chromosomes by
the regional localization of the human gene for acid alpha glucosidase
(a lysosomal enzyme) on human chromosome 17, as deduced from the pattern
of co-transfer with syntenic genes (Appendix paper I I I). Secondly, the nature
of X-chromosome inactivation was investigated in OMGT experiments. It
is demonstrated in Appendix paper IV that DNA isolated from inactive human
X-chromosomes can be expressed efficiently after gene transfer.
Various aspects and appiications of Ml, OMGT and CMGT, including the
experimental work, wi 11 be discussed in chapters I I and I l I of this dissertation.
For such a discussion, a distinction can conveniently be made between
the donor cell providing the material transferred, the recipient
cell which receives the donor material and -in the case of gene transferthe
resulting transformant cell containing the recipient cell genome plus
a variable amount of donor genetic material (usually referred to as the
transgenome). Part of this text has appeared in a review of chromosome and
DNA-mediated gene transfer (de Jonge and Bootsma, 1984)
Transient correction of excision repair defects in fibroblasts of 9 xeroderma pigmentosum complementation groups by microinjection of crude human cell extract.
Crude extracts from human cells were microinjected into the cytoplasm of cultured fibroblasts from 9 excision-deficient xeroderma pigmentosum (XP) complementation groups. The level of UV-induced unscheduled DNA synthesis (UDS) was measured to determine the effect of the extract on the repair capacity of the injected cells. With a sensitive UDS assay procedure a (transient) increase in UV-induced UDS level was found in fibroblasts from all complementation groups after injection of extracts from repair-proficient (HeLa) or complementing XP cells (except in the case of XP-G), but not after introduction of extracts from cells belonging to the same complementation group. This indicates that the phenotypic correction is exerted by complementation-group-specific factors in the extract, a conclusion that is in agreement with the observation that different levels of correction are found for different complementation groups. The XP-G-correcting factor was shown to be sensitive to proteolytic degradation, suggesting that it is a protein like the XP-A factor
Microinjection of Micrococcus luteus UV-endonuclease restores UV-induced unscheduled DNA synthesis in cells of 9 xeroderma pigmentosum complementation groups.
The UV-induced unscheduled DNA synthesis (UDS) in cultured cells of excision-deficient xeroderma pigmentosum (XP) complementation groups A through I was assayed after injection of Micrococcus luteus UV-endonuclease using glass microneedles. In all complementation groups a restoration of the UV-induced UDS, in some cells to the repair-proficient human level, was observed. Another prokaryotic DNA-repair enzyme, T4 endonuclease V, restored the UV-induced UDS in a similar way after microinjection into XP cells. Since both enzymes specifically catalyse only the incision of UV-irradiated DNA, we conclude that this activity is impaired in cells of all 9 excision-deficient XP complementation groups tested