Chapter 12. Gene Transfer from Chloroplast to Nucleus

Introduction

To determine whether gene transfer from an organelle genome to the nucleus can be observed in the laboratory, researchers constructed a chloroplast transformation vector that contained two selectable antibiotic-resistance markers, each with its own promoter: a spectinomycin-resistance gene and a kanamycin-resistance gene (see S. Stegemann et al., 2003, P. Natl. Acad. Sci. USA 100:8828–8833). The spectinomycin-resistance gene was controlled by a chloroplast promoter, yielding a chloroplast-specific selectable marker. Plants grown on spectinomycin are white unless they express the spectinomycin-resistance gene in the chloroplast. The kanamycin-resistance gene, inserted into the plasmid vector adjacent to the spectinomycin-resistance gene, was under the control of a strong nuclear promoter.

The researchers selected transgenic, spectinomycin-resistant tobacco plants following transformation with the plasmid by identifying green plants grown on medium with spectinomycin. These plants contained the two antibiotic-resistance genes inserted into the chloroplast genome by a recombination event; however, kanamycin resistance was not expressed because it was under the control of a nuclear promoter. These spectinomycin-resistant plants were grown for multiple generations and used in the following studies.

a. Leaves from the spectinomycin-resistant transgenic plants were placed in a plant-regeneration medium containing kanamycin. Some of the leaf cells were resistant to kanamycin, and grew into kanamycin-resistant plants. Pollen from kanamycin-resistant plants was used to pollinate wild-type (nontransgenic) plants. In tobacco, no chloroplasts are inherited from pollen. The resulting seeds were germinated on media with and without kanamycin. Half of the resulting seedlings were kanamycin-resistant. When these kanamycin-resistant plants were allowed to self- pollinate, the offspring exhibited a 3:1 ratio of kanamycin-resistant to sensitive phenotypes. What can be deduced from these data about the location of the kanamycin-resistance gene?

If the kanamycin-resistance gene were localized solely in the chloroplast DNA of leaf cells of the spectinomycin-resistant plants, no kanamycin-resistant plants could be generated from the leaves since the kanamycin-resistance gene is under the control of a nuclear promoter. However, kanamycin-resistant offspring were obtained from leaves of the original spectinomycin-resistant transgenic plants. Thus, these data suggest that the kanamycin-resistance gene was transferred to the nucleus of the leaf cells that grew into kanamycin-resistant plants. A 3:1 ratio of kanamycin-resistant offspring to sensitive off-spring among self-pollinating kanamycin-resistant plants indicates a Mendelian (i.e., nuclear) pattern of inheritance. A cross between two resistant plants in which a single copy of the kanamycin-resistance gene had been integrated into one autosomal chromosome would give a 3:1 ratio of kanamycin-resistant to sensitive offspring.

b. To determine whether transfer of the kanamycin-resistance gene to the nucleus was mediated via DNA or an RNA intermediate, DNA was extracted from 10 seedling plants germinated from seeds produced by a wild-type plant pollinated with a kanamycin-resistant plant. These 10 seedling plants, numbered 1–10 in the corresponding gel lanes in the figure below, consisted of 5 kanamycinresistant (1) and 5 kanamycin-sensitive (2) plants. Each DNA sample was subjected to PCR analysis using primers to amplify the kanamycin-resistance gene (gel at left) or the spectinomycin-resistance gene (gel at right). The lane marked M shows molecular weight markers. What does the correspondence between the presence or absence of PCR products generated in the same plant with both sets of primers suggest about the mode of transfer of the kanamycin gene to the nucleus?

_feedback: Because no chloroplasts are inherited from the paternal (pollen-producing, transgenic plant), the only source of a kanamycin-resistance gene in the progeny seedlings was the haploid pollen nucleus from the transgenic, kanamycin- and spectinomycin-resistant parent. Each kanamycin-resistant seedling contained the kanamycin-resistance gene, as expected. But every one of the kana-mycin-resistant progeny seedlings also contained the spectinomycin-resistance gene (seedlings 2, 3, 4, 7, 9). These findings suggest that the two antibiotic-resistance genes were transferred to the nucleus together in each case. Because only the spectinomycin-resistance gene was transcribed in chloroplasts and not the kanamycin-resistance gene, it is unlikely that the original mode of transfer of the kanamycin-resistance gene from a chloroplast to the nucleus was via an RNA intermediate. It is much more likely that a fragment of chloroplast DNA containing both the kanamycin- and spectinomycin-resistance genes was transferred from chloroplast DNA into the nuclear DNA of some of the leaf cells (i.e., the leaf cells that generated kanamycin-resistant plants).

c. When the original transgenic plants, which were selected on spectinomycin but not on kanamycin, were used to pollinate wild-type plants, none of the offspring were kanamycin resistant. What can be deduced from these observations?

_feedback: Most of the original, spectinomycin-resistant plants had incorporated the spectinomycin- and kanamycin-resistance genes into their chloroplast DNA molecules, making them spectinomycin resistant. However, most of these plants did not integrate the kanamycin-resistance gene into one of their nuclear chromosomes. Only rare leaf cells selected for resistance to kanamycin had transferred the kanamycin-resistance gene into a nuclear chromo-some. In the plants generated from these rare kanamycin-resistant leaf cells, kanamycin-resistance was inherited like any other nuclear autosomal gene, exhibiting Mendelian segregation patterns.