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The hormone 1,25-dihydroxyvitamin D3 (1,25(OH)2D3) elicits the programmed pattern of differentiation in epidermal keratinocytes. Based on data indicating a potential role of phospholipase D (PLD) in mediating keratinocyte differentiation, we investigated the effect of 1,25(OH)2D3 on PLD expression. A 24-h exposure to 1, 25(OH)2D3 stimulated PLD-1, but not PLD-2, mRNA expression. This 1, 25(OH)2D3-enhanced expression was accompanied by increased total PLD and PLD-1 activity. Time course studies indicated that 1,25(OH)2D3 induced PLD-1 expression by 8 h, with a maximal increase at 20-24 h. Exposure to 1,25(OH)2D3 inhibited proliferation over the same time period with similar kinetics. Expression of the early (spinous) differentiation marker keratin 1 decreased in response to 1, 25(OH)2D3 over 12-24 h. Treatment with 1,25(OH)2D3 enhanced the activity of transglutaminase, a late (granular) differentiation marker, by 12 h with a maximal increase after 24 h. In situ hybridization studies demonstrated that the highest levels of PLD-1 expression are in the more differentiated (spinous and granular) layers of the epidermis, with little expression in basal keratinocytes. Our results suggest a role for PLD expression/activity during keratinocyte differentiation.

We have derived Chinese hamster ovary (CHO) cell hybrids containing herpes simplex virus thymidine kinase (tk) heteroalleles for the study of spontaneous and restriction enzyme-induced interchromosomal recombination. These lines allowed us to make a direct comparison between spontaneous intrachromosomal and interchromosomal recombination using the same tk heteroalleles at the same genomic insertion site. We find that the frequency of interchromosomal recombination is less by a factor of at least 5000 than that of intrachromosomal recombination. Our results with mammalian cells differ markedly from results with Saccharomyces cerevisiae, with which similar studies typically give only a 10-to 30-fold difference. Next, to inquire into the fate of double-strand breaks at either of the two different Xho I linker insertion mutations, we electroporated PaeR7I enzyme, an isoschizomer of Xho I, into these hybrids. A priori, these breaks can be repaired either by recombination from the homology or by end-joining. Despite a predicted bias against recovering end-joining products in our system, all cells characterized by enzyme-induced resistance to hypoxanthine/aminopterin/thymidine were, in fact, due to nonhomologous recombination or end-joining. These results are in agreement with other studies that used extrachromosomal sequences to examine the relative efficiencies of end-joining and homologous recombination in mammalian cells, but are in sharp contrast to results of analogous studies in S. cerevisiae, wherein only products of homologous events are detected.

Homologous intrachromosomal recombination between linked genes can involve interactions that are either intramolecular (intrachromatid) or intermolecular (sister chromatid). To assess the relative proportions of chromatid interactions, we report studies of intrachromosomal recombination in mouse L cells containing herpes simplex virus thymidine kinase genes in two alternative configurations of direct repeats. By comparing products of reciprocal exchanges between these two configurations, we conclude that the majority of interactions that give rise to crossover products involve unequally paired sister chromatids after DNA replication. Analyses of an additional class of crossover products that involve discontinuous associated gene conversion suggest that these recombination events involve a heteroduplex DNA intermediate.

We have studied intrachromosomal gene conversion in mouse Ltk- cells with a substrate designed to provide genetic evidence for heteroduplex DNA. Our recombination substrate consists of two defective chicken thymidine kinase genes arranged so as to favor the selection of gene conversion products. The gene intended to serve as the recipient in gene conversion differs from the donor sequence by virtue of a palindromic insertion that creates silent restriction site polymorphisms between the two genes. While selection for gene conversion at a XhoI linker insertion within the recipient gene results in coconversion of the nearby palindromic site in more than half of the convertants, 4% of convertant colonies show both parental and nonparental genotypes at the polymorphic site. We consider these mixed colonies to be the result of genotypic sectoring and interpret this sectoring to be a consequence of unrepaired heteroduplex DNA at the polymorphic palindromic site. DNA replication through the heteroduplex recombination intermediate generates genetically distinct daughter cells that comprise a single colony. We believe that the data provide the first compelling genetic evidence for the presence of heteroduplex DNA during chromosomal gene conversion in mammalian cells.