DNA Complementary to Ribosomal RNA: Relation between Genomic Proportion and Ploidy

Siegel, Albert; Lightfoot, Donald; Ward, Oscar G.; Keener, Sherry L.

doi:10.1126/science.179.4074.682

Cited by 47 publications

(7 citation statements)

References 17 publications

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“…The deviations from the expected 2-fold increase may have been the result of either aneuploidy in the octoploid population as discussed by Meyers et aL (10) or the observed lack of doubling in the total cellular DNA. In nonisogenic diploid and tetraploid species of tobacco, Siegel et al (15) reported that the proportion of ribosomal DNA is lower in tetraploids than in diploids even though tetraploid species contained double the amount of DNA of diploids. Because the chromosome number of protoplasts isolated from the alfalfa octoploid population could not be verified, these cells may have contained less than a full complement of chromosomes.…”

Section: Discussionmentioning

confidence: 99%

Ploidy Effects in Isogenic Populations of Alfalfa

Molin¹,

Meyers²,

Baer³

et al. 1982

Plant Physiol.

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Photosynthetically-active protoplasts isolated from isogenic sets of diploid-tetraploid and tetraplold-octoplold alfalfa (Medicago sativa L.) leaves were used to investigate the consequences of polyploidization on several aspects related to photosynthesis at the cellular level. Protoplasts from the tetraploid population contained twice the amount of DNA, ribulose-1,5-bisphosphate carboxylase (RuBPCase), chlorophyll (Chi), and chloroplasts per cell compared to protoplasts from the diploid population. Although protoplasts from the octoploid population contained nearly twice the number ofchloroplasts and amount of ChW per cell as tetraploid protoplasts, the amount of DNA and RuBPCase per octoploid cell was only 50% higher than in protoplasts from the tetraploWd population. The rate of C02-dependent 02 evolution in protoplasts nearly doubled with an increase in ploidy from the diploid to tetraploid level, but increased only 67% with an increase in ploidy from the tetraplold to octoploid level. Whereas leaves and-protoplasts had simflar increases in RuBPCase, DNA, and Chi with increase in ploidy level, it was concluded that increased cell volume rather than increased cell number per leaf is responsible for the increase in leaf size with ploidy.A better understanding of the physiological consequences of polyploidization should facilitate the development of agronomically useful genotypes from experimental polyploids. A principal difficulty inherent in physiological comparisons of polyploids is that cell volume (18), chloroplast number per cell (2), and the amount of DNA per nucleus (5) have been shown, in some cases, not to increase in exact proportion to an increase in genome size. In these instances, data expressed on a fresh weight, leaf area, or Chl basis may be misleading if the increase in these characteristics in relation to ploidy level has not been determined. The preferred method of comparing polyploids is to isolate protoplasts so that the amount of DNA can be correlated with genome size, and physiological data can be expressed on a per cell basis. Protoplast isolation also eliminates variability resulting from differences such as stomatal density (14) or anatomical characteristics (3).Allopolyploids (3,7,12) and colchicine-induced polyploids (14, 18) have been used to study the effects of polyploidization on biochemical and physiological processes. Results from these studies are confounded by genetic differences in the case of allopolyploids and problems associated with inbreeding depression in colchicine-induced and spontaneously appearing polyploids. 'To whom correspondence should be addressed. topolyploids of alfalfa (11) avoid these influences and allow a more direct study of polyploidization. These polyploids consist of an isogenic diploid-tetraploid (DDC 2X-4X) population of alfalfa to which maximum heterozygosity was restored and an isogenic tetraploid-octoploid (IC 4X-8X) population that was passed through a single sexual cycle, thus restoring some heterozygosity to the plants. These isogeni...

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Section: Discussionmentioning

confidence: 99%

Ploidy Effects in Isogenic Populations of Alfalfa

Molin¹,

Meyers²,

Baer³

et al. 1982

Plant Physiol.

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“…Ribonuclease TI (EC 3.1.4.8); ribonuclease A (EC 3.1.4.22); a-amylase(EC 3.2.1.1); fi-amylase (EC 3.2.1.2); pronase (EC 3.4.21.4 + 3.4.24.4). similar absolute numbers of rRNA genes despite a two-fold variation in chromosome number [7]. On the other hand, in a polyploid series of plants (haploid to hexaploid) of Datura innoxia produced by culture of pollen grains in vitro, the proportion of rDNA has been shown to be constant [8].…”

mentioning

confidence: 99%

Ribosomal RNA Genes in the Amoebal and Plasmodial Forms of the Slime Mould Physarum polycephalum

Hall¹,

Turnock²,

Cox³

1975

Eur J Biochem

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1. The degree of homology between ribosomal RNA isolated from microplasmodia and amoebae of the slime mould Physarum polycephalum has been determined by competitive hybridisation of the RNA from the two sources to homologous DNA in solution. The extent of competition was measured both by hybridisation to saturation and by following the kinetics of hybrid formation.In each case competition was found to be 100 %, indicating that the ribosomal RNAs from the two, quite different vegetative forms of the organism exhibit a high degree of homology and are probably transcribed from the same genes.2. The relationship between the amount of nuclear DNA that codes for ribosomal RNA (rDNA) and ploidy has been investigated in three strains of P. polycephalum which exhibit a 1 : 2 : 5 variation in the amount of DNA per nucleus. Ribosomal RNA saturation values were determined by hybridisation to DNA isolated from prophase nuclei of plasmodia. The proportion of rDNA was found to be constant at 0.16-0.18% of the total genome in the three strains.The genes coding for ribosomal RNA in the slime mould Physarum polycephalum have been shown to be located in the nucleolus by cell fractionation [l], light microscopic autoradiography [2] and electron microscopic autoradiography [3]. Unlike bulk DNA, rDNA is replicated throughout the mitotic cycle [l, 4,5] with the exception of the first hour after mitosis [l]. Measurements of the amount of rDNA have varied from 0.1 % to 1.3 % [5,6,41] of the nuclear DNA.In this paper we have applied RNA-DNA hybridisation techniques to investigate the following two basic questions concerning rDNA in P. polycephalum. a) Are the same genes involved in rRNA transcription in the plasmodia1 and the amoeba1 stages of the life cycle? b) Does ploidy have any effect on rRNA gene dosage in plasmodia?The latter question has been investigated in several other organisms. Species of the genus Nicotiana have Abbreviations. rDNA, that fraction of DNA which codes for rRNA; standard saline citrate, 0.15 M NaCl plus 0.015 M sodium citrate, 5 mM EDTA, pH 7.0; Napi, equimolar mixture of NaH,P04 and Na2HP04, e.g. 0.24 M Napi is equivalent to 0.12 M NaH2P04, 0.12 M Na,HPO,; T,, the mean temperature of dissociation measured optically; Tm,i, the mean temperature of strand separation.Enzymes. Ribonuclease TI (EC 3.1.4.8); ribonuclease A (EC 3.1.4.22); a-amylase(EC 3.2.1.1); fi-amylase (EC 3.2.1.2); pronase (EC 3.4.21.4 + 3.4.24.4). similar absolute numbers of rRNA genes despite a two-fold variation in chromosome number [7]. On the other hand, in a polyploid series of plants (haploid to hexaploid) of Datura innoxia produced by culture of pollen grains in vitro, the proportion of rDNA has been shown to be constant [8]. Investigations of this kind, however, are only strictly meaningful if either (a) the proportion of rDNA is constant throughout the cell cycle, i.e. bulk DNA and rDNA are replicated at the same time, or (b) determinations of gene dosage are carried out at a fixed point in the cell cycle necessitating synchronous cultu...

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“…Quantitation of the ribosomal genes by DNA/ RNA hybridization techniques requires that one be able to determine accurately both the percentage hybridization (1) and the amount of DNA per nucleus (6,11). We wish to report an additional parameter which influences the hybridization values obtained for higher plants and the interpretation of these results.…”

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confidence: 99%

Distribution of Ribosomal Deoxyribonucleic Acid in Subcellular Fractions of Higher Plants

Jaworski

Key²

1974

Plant Physiol.

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Methods are described by which ribosomal DNA can be enriched in subeeliular fractions of carrot and soybean. With both carrot and cucumber it was possible to obtain a distinct satellite DNA which contained the rDNA. Hybridization values greater than 0.49 % were necessary before a satellite component was observed. Saturation hybridization values for soybean, carrot, and cucumber DNA were 0.2, 0.49, and 1.14%, respectively. These values were increased 1.6-and 2-fold in soybean and carrot, respectively, but enrichment was not obtained for cucumber.Quantitation of the ribosomal genes by DNA/ RNA hybridization techniques requires that one be able to determine accurately both the percentage hybridization (1) and the amount of DNA per nucleus (6, 11). We wish to report an additional parameter which influences the hybridization values obtained for higher plants and the interpretation of these results. In this paper we show that it is possible to enrich artificially for rDNA! by techniques which are commonly used for the isolation of plant nuclei. Experiments with carrot, soybean, and cucumber show that enrichment occurs in nuclear pellets of carrot and soybean and not with cucumber. pellet. Further fractionation of the nuclei was accomplished by resuspending the nuclear pellets in 10 ml of sucrose buffer or, alternatively, in sucrose-citrate buffer (0.5 M sucrose; 0.1 X SSC). These nuclear suspensions were layered over 20 ml of 60% sucrose containing the resuspension buffer and then centrifuged for 1 hr at 24,000 rpm in a Spinco SW 25.1 rotor. All operations were carried out at 0 to 4 C. MATERIALS AND METHODSExtraction of DNA. Total tissue DNA was extracted by the following method. Tissue was homogenized in detergent buffer (6% p-amino salicylate, 1% triisopropyl naphthalene sulfonic acid, 0.05 M NaCl, 0.05 M tris, pH 7.5) with either a VirTis 45 homogenizer or a mortar and pestle. Homogenates were adjusted to 0.5 M NaCl followed by the addition of an equal volume of chloroform-isoamyl alcohol (24:1). The mixture was gently shaken, and the layers were separated by centrifugation. The aqueous layer was washed twice with phenol followed by precipitation of nucleic acids by the addition of 2 volumes of 95% ethanol. The precipitate was collected by centrifugation and dissolved in SSC. RNase (Worthington pancreatic, 100 ,Fg/ml) was added for 2 hr at 37 C followed by the addition of pronase (500 ,ug/ml, Calbiochem B grade predigested at 37 C for 1 hr) and further incubation for 2 to 4 hr. DNA and undigested RNA were pelleted by centrifugation at 36,000 rpm for 12 to 16 hr in a Spinco type 65 rotor. DNA was extracted from the 10OOg supernatant by a similar method except that 10-fold concentrated salts and detergent were added directly to the supernatant.DNA was extracted from the various subcellular pellet fractions by an alternative method. Pellets were resuspended in SSC-EDTA (1 X SSC-20 mm EDTA) followed by the addition of 0.05 volume of 20% SLS. This mixture was incubated at 50 C for 30 min after which predigested ...

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DNA Complementary to Ribosomal RNA: Relation between Genomic Proportion and Ploidy

Cited by 47 publications

References 17 publications

Ploidy Effects in Isogenic Populations of Alfalfa

Ploidy Effects in Isogenic Populations of Alfalfa

Ribosomal RNA Genes in the Amoebal and Plasmodial Forms of the Slime Mould Physarum polycephalum

Distribution of Ribosomal Deoxyribonucleic Acid in Subcellular Fractions of Higher Plants

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