Transient complementation of xeroderma pigmentosum cells by microinjection of poly(A)+ RNA.

Abstract

An assay has been developed in which excision repair deficiency of xeroderma pigmentosum cells is transiently complemented, as measured by unscheduled DNA synthesis, by microinjection of cytoplasmic poly(A)+ RNA derived from HeLa cells. Four different complementation groups of xeroderma pigmentosum have been assayed. Groups A and G showed complementation, whereas groups D and F did not. Survival for cells in each of the groups subsequent to microinjection was =75%. Approximately 10-25% of surviving cells from groups A and G were complemented, as judged by nearnormal unscheduled DNA synthesis. Fractionation of cytoplasnic poly(A)+ RNA on a 15-30% nondenaturing sucrose gradient and subsequent microinjection of the individual fractions indicate that repair mRNAs that complement xerbderma pigmentosum groups A and G sediment at approximately 11 S and 12 S, respectively. This assay should be of great utility in the cloning and biochemical analysis of DNA repair genes.Xeroderma pigmentosum (XP) is an autosomal, recessive disease of humans characterized by dermatological and ocular symptoms and a high predisposition to skin cancer after exposure to sunlight. Some forms of XP have associated neurological disorders (1) and possible immunological abnormalities. The primary biochemical defect in most XP cells is thought to be an inability to initiate excision repair synthesis of their DNA after exposure to UV irradiation (2, 3). Recently, the feasibility of transiently complementing mutant cell lines by direct microinjection of unenriched cytoplasmic poly(A)+ RNA has been demonstrated (4, 5). In this report, we show that microinjection of cytoplasmic poly(A)+ RNA derived from HeLa cells can transiently complement XP cells, as determined by a temporary increase in the level of unscheduled DNA synthesis (UDS). In addition, fractionation of poly(A)+ RNA on nondenaturing sucrose gradients and subsequent microinjection of the individual fractions indicated that the repair mRNAs that complement XP-A and -G cells sedimented at unique positions. MATERIALS AND METHODSCell Cultures. Fibroblast cell strains XP25RO (XP-A), XP3NE (XP-D), XP2YO (XP-F), and XP2BI (XP-G) were obtained from the Human Genetic Mutant Cell Repository. The cells were routinely grown in minimal essential medium (Flow Laboratories) containing 20% fetal calf serum (Irvine Scientific), penicillin G (100 units/ml), streptomycin (100 ,ug/ml), and glutamine (2 mM). HeLa S3 cells were grown in suspension culture containing minimal essential medium and 10% calf serum (Flow Laboratories).Isolation of Cytoplasmic Poly(A)+ RNA. The procedure for preparation of poly(A)+ RNA was a modification of the method described by Lin et al. (5). Approximately 5 x 109 cells were pelleted, washed twice with saline, and resuspended in 50 ml of ice-cold lysis buffer containing 30 mM Tris-HCl (pH 7.4), 30 mM KCI, 0.1 M NaCi, heparin (1 mg/ml), 0.15% Nonidet P-40, and 10 mM vanadyl ribonucleoside complex (Bethesda Research Laboratories). After 10 min on ice the sample was centrifuge...

Glass Needle-Mediated Microinjection of Macromolecules and Transgenes into Primary Human Mesenchymal Stem Cells

“…For example, glass needle-mediated microinjection of macromolecules, e.g. peptide inhibitors [1], purified active proteins [53], antibodies [16,25,30], RNA [32,52], and DNA [9], into living mammalian somatic cells has proven to be a powerful approach for studying cell biology at the molecular level. In addition, precise gene modification/repair can be accomplished with a variety of relatively small DNAs such as small fragment homologous replacement [12,20,21] and/or RNA/DNA hybrids [28,34,49].…”

Increased UV resistance in xeroderma pigmentosum group A cells after transformation with a human genomic DNA clone.

“…The design of this competition hybridization strategy was based on the observation that, when HeLa cell poly(A)+ RNA, -700 nucleotides long, was microinjected into an XP-A cell line and the cells were challenged with UV, these cells were transiently capable of repairing the DNA damage as measured by enhanced unscheduled DNA synthesis (UDS) (15). In this study we demonstrate that the human genomic DNA sequences corresponding to our cDNA clones are capable of partially restoring the UV resistance to two independent XP-A cell lines but not to an XP-F cell line.…”

Complementation of the UV-sensitive phenotype of a xeroderma pigmentosum human cell line by transfection with a cDNA clone library.

“…Stable corrections of the UV sensitivity of simian virus 40 (SV40)-transformed XP cell lines of complementation group A (XP-A cell lines) by transfection with normal human DNA (2) or following fusion with either x-ray-irradiated Chinese hamster ovary (CHO) cells (3) or microcells containing a single human chromosome (4) were reported. Transient complementation of excision-repair defects of XP primary cell lines was achieved either by microinjecting mRNA from normal human cells into XP-A and XP-G cells (5) or by introducing protein extracts from normal human cells into XP-A cells (6,7) or into cells of all other XP complementation groups (8). Another approach for isolation of human excision-repair genes has been the stable complementation of excision-deficient CHO cells produced in the laboratory.…”