N. Steele Scott

Homologies between nuclear and plastid DNA in spinach

SummaryHomologies between spinach nuclear (n) DNA and Chloroplast (pt) DNA, have been detected with a clone bank of spinach ptDNA as hybridization probes to restriction fragments of nDNA prepared from purified root nuclei. Every cloned fragment of ptDNA showed homologies to discrete restriction fragments of nDNA, different from those of ptDNA, indicating integration of these homologies into nDNA. While most ptDNA clones were relatively large and probably contained several genes, sequence homologies were also found to the cloned plastid gene for RuBP carboxylase and the β subunit of ptATPase. Many of the homologies in nDNA occur in regions of the genome that are highly methylated and are not digested by the methylation sensitive restriction endonucleases HpaII and MspI. In contrast these enzymes cleave ptDNA into small fragments which allows the nDNA homologies to be distinguished in total root DNA. The sequence homologies observed were not due to contaminating non nuclear sequences as shown by hybridization to mitochondrial (mt) and bacterial DNAs. The total amount of homology to ptDNA in nDNA is equivalent to about five copies of the plastome per haploid nuclear genome. The homologies generally appear to be in individual segments of less than 2 kbp in length, integrated into several different places in the genome.

Organelle biosynthesis: the chloroplast.

The cells of higher organisms contain highly differentiated bodies or organelles, each of which is delimited from the surrounding cytoplasm by an outer membrane. Organelles carry out specialized cellular functions; they possess complex and unique internal structures and contain a characteristic complement of enzymes and other constitutents. Clearly, knowledge of the genetic and metabolic regulatory systems involved in the differentiation of these cellular structures is crucial to our comprehension of the biochemistry of growth and differentiation of the cells of higher organisms.

Genetic transformation of grapevine cells

Biovar 1 strains ofAgrobacterium tumefaciens have been used to transform a cell suspension culture ofVitis vinifera cv. Cabernet Sauvignon. Cocultivation of cultures withAgrobacterium strains bearing either the cointegrate pGV3850::1103neo, or the binary vector pGA474-68, each gave rise to kanamycin resistant tissue. The stable integration and expression of the neomycin phosphotransferase gene was confirmed by Southern blotting and enzymic assay, respectively.

Plastid-DNA levels in the different tissues of potato.

The plastid-(pt) DNA levels in the different tissues of potato (Solanum tuberosum L.), including tubers of differing ages, have been studied. The DNA could be detected as a single nucleoid in amyloplasts of cells from young potato tubers by fluorescence microscopy, following staining of glutaraldehyde-fixed tissue with 4,6-diamidino-2-phenyl indole (DAPI). The renaturation kinetics of spinach ptDNA in the presence of total DNA from potato tissues and the fragments generated by restriction-enzyme digestion of potato-tuber DNA and chloroplast DNA indicated that the ptDNA of potato-tuber amyloplasts and of potato-leaf chloroplasts is essentially the same. Expressed as a percentage of the total DNA the level of ptDNA (5.2%) found in tubers, while less than that found in leaves (7.6%) was more than that found in petioles (3.4%), stems (3.0%) and roots (1.0%). There was a high level of both nuclear and plastid ploidy in mature potato-tuber cells and, on average, nuclei contained 32 pg of DNA (equivalent to 14C) and the 40 amyloplasts per cell contained DNA equivalent to 7800 copies of ptDNA, or 195 copies per amyloplast.

Nuclear and mitochondrial DNA have sequence homology with a chloroplast gene.

SummaryA PstI 7.7 kbp fragment from chloroplast (ct) DNA of spinach shows homology to an EcoRI 8.3 kbp fragment of mitochondrial (mt) DNA and in turn, both are homologous to a number of common regions of nuclear (n) DNA. The common area of homology between the chloroplast and mitochondrial fragments is between a KpnI 1.8 segment internal to the PstI sites in the ctDNA and an EcoRI/BamHI 2.9 kbp fragment at one end of the mitochondrial 8.3 kbp fragment. The KpnI 1.8 kbp ctDNA fragment is within a structural gene for the P700 chlorophyll a apoprotein. Further analysis of this KpnI 1.8 kbp fragment confined the homologous region in mtDNA to a ct 0.8 kbp HpaII fragment. These smaller pieces of the organellar genomes share homologies with nuclear DNA as well as displaying unique hybridization sites. The observations reported here demonstrate that there is a common or closely related sequence in all three genetic compartments of the cell.

Plastid DNA Levels in Albino and Green Leaves of the «albostrians» Mutant of Hordeum vulgare

Summary The levels of plastid DNA (pt DNA) in both the green and albino leaves of the barley mutant «albostrians» (Hordeum vulgare, Mutant 4205) are the same. The albino leaves of this plant are similar in size to green leaves and contain numerous undifferentiated plastid structures. They have previously been shown to contain no plastid ribosomes, (Borner et al., 1976). These plastids contain a single pt DNA nucleoid while fully differentiated chloroplasts in green leaves each contain a number of small, peripherally located nucleoids. We conclude that both the replication of pt DNA and the formation of simple undifferentiated plastids does not require plastid protein synthesis, is controlled by nuclear DNA, and is mediated by the cytoplasmic protein synthesizing system. We suggest that during differentiation pt DNA is redistributed through the plastid matrix. The implications of these observations in relation to the standing of the concept that plastids are semi-autonomous, self-replicating organelles is discussed.

A hypervariable middle repetitive DNA sequence from citrus

The use of hypervariable sequences for DNA typing of plants is focussed on microsatellites and on amplification of regions defined by random (RAPD) or defined (AFLP) primers for PCR analysis of genomes. A hypervariable length of middle repetitive DNA has been isolated from citrus that contains no obvious hypervariable structures. The fingerprinting probe was shown to have an important commercial application in the separation of zygotic from nucellar progeny. A somatic variant of the sequence within one orange tree suggests that somatic variation in hypervariable markers may be a common event.

Homologies to chloroplast DNA in the nuclear DNA of a number of Chenopod species

SummarySequences homologous to chloroplast (ct)DNA have been found in nuclear DNA in five species of the Chenopodiaceae, extending the earlier observations of ‘promiscuous’ DNA in Spinacia oleracea (Timmis and Scott 1983). Using the 7.7 kbp spinach ctDNA Pst I fragment as a hybridization probe, several separately located homologies to ctDNA were resolved in the nuclear DNA of Beta vulgaris, Chenopodium quinoa, and Enchylaena tomentosa. In Chenopodium album and Atriplex cinerea the major region of homology was to a nuclear Eco RI fragment (6 kbp) indistinguishable from that in ctDNA. These homologies may therefore involve larger tracts of ctDNA because the same restriction sites are apparently retained in the nucleus. This suggests that in these latter two species there is a contrasting, more homogeneous arrangement of ctDNA transpositions in the nucleus.

RNA synthesis during chloroplast development in Euglena gracilis

Abstract Changes in chloroplast and cytoplasmic RNA were measured during chloroplast development in Euglena gracilis over a 3-day period. The cells were grown in the dark and then illuminated by 1500 lx of white light at a cell density which was sufficiently high to prevent any cell division in illuminated cultures. Light stimulated incorporation of 14 C-orotic acid into both chloroplast and cytoplasmic RNA. The most rapid rate of light-dependent incorporation occurred during the first 8 hr of illumination and before the main period of chlorophyll synthesis. The specific activity of chloroplast RNA was higher than that of cytoplasmic RNA, particularly during the first 24 hr of illumination. By 32 hr the rate of incorporation into total cell RNA had decreased to that of dark control cells. White there was no significant change in the amount of RNA per cell during chloroplast development, the amount of chloroplast RNA increased to a maximum of 7 per cent of the total cellular RNA by 16 hr. In growing autotrophic cultures, or in cultures in which chloroplast development was followed at low cell densities (3 × 10 6 cells per ml), chloroplast RNA accounted for about one-fifth of the total cellular RNA.

Movement of Genetic Information Between the Chloroplast and Nucleus

The discovery of cytoplasmic inheritance in plants at the turn of the century (Correns and Baur as described in Kirk and Tilney-Basset, 1978), culminated in the demonstration of plastid (pt) DNA in the late 1960’s. At the same time there was mounting biochemical and genetic evidence which showed that most of plastid biogenesis and function was controlled by nuclear genes and involved proteins synthesized on cytoplasmic ribosomes (see reviews by Kirck and Tilney-Bassett, 1978; Ellis, 1983). The expression of plastid DNA and the use of plastid ribosomes to synthesize large amounts of particular plastid proteins has only been described in the special case of the photosynthetically competent plastid, the chloroplast (Scott and Possingham, 1980). We wish to distinguish between the general term plastid which describes a family of related plant cell organelles of which the most commonly studied and perhaps the most numerous and important are chloroplasts. In general we will use the name plastid and only use the term chloroplast in specific instances. It appears however that chloroplasts and all other plastids carry an identical subgenome which has been called the plastome. In this article we will briefly describe the interaction of the genetic information from nucleus and plastid that is involved in the formation of chloroplasts and other plastid forms and go on to discuss in more detail the recent observations which indicate that plastids and nuclei share extensive DNA sequence homology (Timmis and Scott, 1983).