Overexpression of an unstably inherited gene in cultured mouse cells.

Abstract

The specific activity of hypoxanthine-guanine phosphoribosyltransferase (IMP:pyrophosphate phosphoribosyltransferase, EC 2.4.2.8) is increased up to 58-fold in unstable gene transferents produced by the transfer of cell-free chromosomal material from one mouse L cell line to another; the specific activity of this enzyme returns to normal levels when the transferred gene becomes stabilized. This phenomenon, which is not observed 'in comparable heterospecific transfers, may be an effect of gene dosage (multiple copies of the transferred genetic fragment in the unstable gene transferents), or it may represent an escape of the unstably inherited gene from the normal regulatory mechanisms of the recipient cell. Previously we reported the transfer of a gene from one mouse L cell to another, the donor material being a mixture of metaphase chromosomes, nuclear fragments, and nuclei (1). The presence of the transferred gene, hprt, enables progeny cells to synthesize active hypoxanthine-guanosine phosphoribosyltransferase (HPRTase; IMP:pyrophosphate phosphoribosyltransferase, EC 2.4.2.8) and thus to grow in medium containing hypoxanthine, aminopterin, and thymidine (HAT). The transferred gene was an unusual mutant allele present in a strain (H29) isolated by Sharp et al. (2); it encodes a protein with reduced affinity for the toxic substrate analog, 8-azaguanine (AG). Gene transferents* carrying this allele are able to grow in AG medium and yield extracts containing AG-resistant HPRTase activity. In contrast, all spontaneous HPRT+ revertants of strains carrying our hprt-allele possess the wild-type enzyme, rendering them unable to grow in AG medium and yielding extracts containing AG-inhibitable HPRTase activity (1).Detailed study of the gene transferents showed that (i) the initial transfer event leads to an unstably inherited gene, lost at about 0.1 per cell per generation; (ii) under selective conditions the cells can be carried for at least 200 generations in continuous culture in the unstable state; and (iii) stabilized subclones can be isolated from each unstable subelone. The stabilization process is rare, occurring on the order of 1 X 10-5 per cell per generation. Similar phenomena have also been observed in heterologous crosses between mouse and Chinese hamster and between human and rodent cells (1, 3, 4).In addition, we have found that the transferred gene in our homologous system shows a striking feature not reported in any of the heterologous systems: a pronounced overproduction of gene product (HPRTase) when the gene is unstably inherited, which is abolished when the gene becomes stable. This interrelationship between the heritable state of the hprt locus and the cellular level of HPRTase is the subject of this report. A preliminary report of this work has appeared (5). MATERIALS AND METHODSCell Lines. The derivation of the cell lines used in these studies is shown in Fig. 1. The original recipient strain, 501-1, is a chloramphenicol-resistant derivative of the HPRT-mouse L cell line, A9 (6); H29, as ...

Amplification of hypoxanthine-guanine phosphoribosyltransferase genes in chromosome-mediated gene transferents.

“…Two-dimensional gel electrophoresis analysis of unstable CMGT cell lines demonstrated that HPRT polypeptides were overexpressed, corroborating previous findings of increased HPRT enzyme activity in these cells (5). Dot blot hybridization experiments showed that HPRT mRNA levels were elevated 10-to 15-fold in the GT427C line.…”

“…In the present study, we were unable to detect increased levels of expression of any other polypeptides in addition to HPRT by two-dimensional gel electrophoresis analysis. Previous experiments have shown that the levels of expression of the linked glucose-6-phosphate dehydrogenase gene were normal in GT427C and GT427D (5 …”

“…The origins of the cell lines used in this study have been described previously (5) (25). Filters were hybridized with nick-translated pHPT-2 probe.…”

Co-amplification of double minute chromosomes, multiple drug resistance, and cell surface P-glycoprotein in DNA-mediated transformants of mouse cells.

“…In this paper, the initial drug resistance was obtained by DNA-mediated gene transfer. Observations of initial instability of such resistance (33,49,61), suggestions of amplification of the transferred genes during this period (18,50), probable extrachromosomal maintenance of the transferred DNA during the unstable phase (33,44), and evidence for stabilization of the resistance by chromosomal integration of the transferred DNA (33,47,49) mirror the results obtained when cell mutants are selected in vitro for increasing drug resistance (1, 8, 14, 30, 41, 51, 58, 60). By applying an initial mild selection pressure after exposure to DNA, followed quickly by stepwise increases in the selection stringency ( (42).…”

The Right Place at the Right Time

“…On my return to Yale, Jody and I carried out a number of experiments on the cell fusion process (59) and I collaborated with Irv Miller, Jerry Eisenstadt, and Jerry Degnen on gene transfer between lines of cultured mammalian cells (22,23). While this work was going on, I was following with interest a small but growing literature on membrane transport systems in established lines of cultured mammalian cells.…”