Perturbation of chloroplast DNA amounts and chloroplast gene transmission in Chlamydomonas reinhardtii by 5-fluorodeoxyuridine

Abstract

5-Fluorodeoxyuridine selectively decreases the rate of chloroplast DNA replication in Chlamydomonas resulting after several generations of growth in equilibrium levels as low as one-seventh of normal. When the maternal parent is treated prior to mating, the decrease of chloroplast DNA alppears to perturb the normal maternal transmission of chloroplast genes, dramatically increasing the proportion of exceptional zygotes transmitting chloroplast genes from the paternal parent. Uniparental inheritance in the isogamous green alga Chlamydomonas reinhardtil was discovered more than 20 years ago by Sager (1). Much information about this genetic system, now thought to reside in chloroplast DNA, has been obtained since (2-6). Multiple copies of this DNA are believed by most workers to be present in the single chloroplast of C. reinhardtfl because the absolute amount of chloroplast DNA per cell is 25-50 times the molecular weight of an individual chloroplast DNA molecule. This is true whether molecular weight is determined by kinetic complexity (7)(8)(9), by size measurements of intact molecules observed in the electron microscope (10), or by summation of the molecular weights of EcoRl restriction fragments (11, 12). However, the mechanisms responsible for the uniparental inheritance of chloroplast DNA and chloroplast markers as well as the number of genetic copies transmitted to meiotic progeny are still in dispute (2-4, 6, 13-15).Sager has proposed a mechanism of uniparental inheritance modeled after the restriction-modification systems in bacteria (13,14). In most zygotes from a cross, paternal chloroplast DNA is destroyed by a restriction enzyme while maternal chloroplast DNA is modified by methylation and hence is protected from restriction. All progeny receive two copies of the chloroplast genome. In contrast, our laboratory (3, 4) has proposed a multicopy model with certain analogies to a phage cross. The input of maternal and paternal chloroplast genomes is variable and the output of genomes from the two parents depends upon their relative success in competing for attachment sites in the single zygote chloroplast that results from fusion of the two gametic chloroplasts (16). Normally, these sites are occupied by maternal chloroplast genomes.To test directly which of the two foregoing models is correct we have used the thymidine analogue 5-fluorodeoxyuridine (FdUrd) to perturb the amount of chloroplast DNA and then observed the effect of this analogue on the transmission of chloroplast genes. Our rationale was as follows. In Chlamydomonas, exogenous radioactive thymidine labels chloroplast, but not nuclear, DNA in vegetative cells and cells undergoing gametogenesis (17,18) MATERIALS AND METHODS C. reinhardtll wild-type strain 137c+ (GB-125) and antibiotic-resistant strains carrying the chloroplast markers spr-u-1-6-2, mt-(GB-146, paternal parent) and er-u-37, sr-u-2-60, mt + (GB-148, maternal parent) isolated from this strain (3,4) were grown either phototrophically in high-salt medium bubbled with 5% CO...

Origins of Eukaryotic Sexual Reproduction

“…The usual pattern, described in detail below, is that chloroplast genomes from the minus parent are destroyed by nucleases in the early zygote, leading to UP inheritance of plus genomes. However, if the zygotic input of plus chloroplast genomes is reduced, either by growth in FuDR (Wurtz et al 1977;Matagne and Beckers 1983;Armbrust et al 1995) or by the mat3 mutation (Gillham et al 1987;Armbrust et al 1995;Umen and Goodenough 2001), destruction of minus genomes is aborted and chloroplast DNA inheritance is biparental (BP).…”

Chloroplast DNA methylation and inheritance in Chlamydomonas

“…Decreased cpDNA copy number in mt + gametes is known to affect cpDNA inheritance (Wurtz et al 1977;Armbrust et al 1995). Therefore, we also measured the levels of cpDNA in cells treated with 5adc to determine whether the drug blocks cpDNA replication and decreases cpDNA copy number in vegetative cells and gametes.…”

A 3' stem/loop structure of the Chlamydomonas chloroplast atpB gene regulates mRNA accumulation in vivo.

“…CC373 cultures were grown in reduced light by partially covering the flask with aluminum foil. Chloroplast transformatíon was performed using the particle delivery system of Du Pont, with cells that had grown for approximately 5 to 6 doublings in 0.5 mM 5-fluoro-2'-deoxyuridine to reduce chloroplast DNA copy number (Wurtz et al, 1977;). Approximately 5 x 10' cells were plated onto selective plates and bombarded with 1 W g of plasmid DNA that had been precipitated onto tungsten particles .…”