Transfer of methotrexate and 5-methyltryptophan resistance from carrot (Daucus carota) to tobacco (Nicotiana tabacum) was achieved by fusion between leaf mesophyll protoplasts of tobacco and irradiated cell culture protoplasts of carrot. Some of the regenerated somatic hybrids exhibited normal tobacco morphology with coexpression and independent segregation of the transferred resistance markers. Chromosomal instability resulted in aneuploid somatic hybrids with significantly lower chromosome number than predicted by simple addition of parental chromosome number. The methotrexate resistance phenotype was correlated with the expression of carrot-specific dihydrofolate reductase as judged by isozyme and immunological characteristics of the enzyme. The genomic construct of these somatic hybrids made the transmission of the resistance character into the next sexual generation possible.protoplasts (11). In some of the resulting nitrate reductasepositive tobacco plants, barley enzyme was detected with immunological methods. Because of leakiness of selection techniques, based on a single recessive and unstable mutation, the authors did not conclude that cell fusion-mediated gene transfer had occurred. At present, available experimental data are insufficient to explore the potential of asymmetric cell fusion for widening genetic variability.In this paper we describe a fusion system that allowed the monitoring of two dominantly acting selectable markers in tobacco + carrot fusion products. Molecular evidence confirming the expression of carrot-specific dihydrofolate reductase (DHFR; EC 1.5.1.3) in regenerated hybrid plants with normal tobacco morphology is presented. Furthermore, sexual progenies are analyzed to test the inheritance of transferred genetic markers.Gene flow between incompatible species is strongly restricted by evolutionary boundaries in sexual crossings. The use of somatic cells as targets in genetic manipulation experiments, based on DNA transformation or cell hybridization, has opened new horizons for combining diverse genes from unrelated species. If the gene of interest has been cloned, advanced transformation systems may provide the most efficient way of introducing it into a foreign host genome (1, 2). When isolated genes are lacking, asymmetric hybridization mediated by protoplast fusion offers an alternative approach for alien gene transfer. The asymmetric nature of nuclear genomes in cell hybrids originates from spontaneous or induced genome instability, with preferential loss of chromosomes belonging to one of the parental species. Enforced chromosome elimination can be a prerequisite of hybrid plant production in several wide fusion combinations in which somatic incompatibility prohibits hybrid development (3-5). The transient coexistence of two diverse genomes in hybrid cells may provide the opportunity for recombination of chromosomes or a few genes from the eliminated partner. To test this hypothesis, experimental methods are needed to control chromosome elimination from fusion produ...

Chromosomes formed de novo which originated from the centromeric region of mouse chromosome 7, have been analysed. These new chromosomes were formed by apparently similar large-scale amplification processes, and are organized into amplicons of approximately 30 Mb. Centromeric satellite DNA was found to be the constant component of all amplicons. Satellite DNA sequences either bordered the large euchromatic amplicons (E-type amplification), or made up the bulk of the constitutive heterochromatic amplicons (H-type amplification). Detailed analysis of a heterochromatic megachromosome formed de novo by an H-type amplification revealed that it is composed of a tandem array of 10-12 large (approximately 30 Mb) amplicons each marked with integrated "foreign' DNA sequences at both ends. Each amplicon is a giant palindrome, consisting of two inverted doublets of approximately 7.5-Mb blocks of satellite DNA. Our results indicate that the building units of the pericentric heterochromatin of mouse chromosomes are approximately 7.5-Mb blocks of satellite DNA flanked by non-satellite sequences. We suggest that the formation de novo of various chromosome segments and chromosomes seen in different cell lines may be the result of large-scale E- and H-type amplification initiated in the pericentric region of chromosomes.

The optical alignment of biological samples is of great relevance to microspectrometry and to the micromanipulation of single particles. Recently, Bayoudh et al. (J. Mod. Opt. 50:1581-1590 have shown that isolated, disk-shaped chloroplasts can be aligned in a controlled manner using an in-plane-polarized Gaussian beam trap, and suggested that this is due to their nonspherical shape. Here we demonstrate that the orientation of various micrometer-sized isolated biological particles, trapped by optical tweezers, can be altered in a controlled way by changing the plane of linear polarization of the tweezers. In addition to chloroplasts, we show that subchloroplast particles of small size and irregular overall shape, aggregated photosynthetic lightharvesting protein complexes as well as chromosomes can be oriented with the linearly polarized beam of the tweezers. By using a laser scanning confocal microscope equipped with a differential polarization attachment, we also measured the birefringence of magnetically oriented granal chloroplasts, and found that they exhibit strong birefringence with large local variations, which appears to originate from stacked membranes. The size and sign of the birefringence are such that the resulting anisotropic interaction with the linearly polarized laser beam significantly contributes to the torque orienting the chloroplasts.