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  Variable amounts of DNA related to the size of chloroplasts III. Biochemical determinations of DNA amounts per organelle

Rauwolf, U., Golczyk, H., Greiner, S., & Herrmann, R. G. (2010). Variable amounts of DNA related to the size of chloroplasts III. Biochemical determinations of DNA amounts per organelle. Molecular Genetics and Genomics, 283(1), 35-47. doi:10.1007/s00438-009-0491-1.

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Item Permalink: http://hdl.handle.net/11858/00-001M-0000-0014-32FB-C Version Permalink: http://hdl.handle.net/11858/00-001M-0000-0014-32FC-A
Genre: Journal Article

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Rauwolf, U.1, Author
Golczyk, H.1, Author
Greiner, S.1, Author              
Herrmann, R. G.1, Author
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1External Organizations, ou_persistent22              

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Free keywords: quantification and arrangement of plastid DNA chloroplast development cellular genome-plastome ratios nuclear and plastid ploidy beta-vulgaris l higher-plants nuclear-DNA leaf development copy number plastid nucleoids spinach leaves genome transcription arabidopsis
 Abstract: Plastid genomes (plastomes) are part of the integrated compartmentalised genetic system of photo-autotrophic eukaryotes. They are highly redundant and generally dispersed in several regions (nucleoids) within organelles. DNA quantities and number of DNA-containing regions per plastid vary and are developmentally regulated in a way not yet understood. Reliable quantitative data describing these patterns are scarce. We present a protocol to isolate fractions of pure plastids with varying average sizes from leaflets (<= 1 mm) and leaves of different developmental stages continuously up to maturity (25 cm) from Beta vulgaris L. (sugar beet) to determine DNA amounts per organelle. The approach is based on plastid purification from homogenates of moderately fixed tissue by differential and isopycnic gradient centrifugations and on application of two different DNA specific colorimetric reactions after removing potentially interfering compounds. The sensitive fluorochrome DAPI (4',6-diamidino-2-phenylindole) was used to estimate numbers and emission intensity of nucleoids per plastid. The amounts determined ranged from 0.15 to 4.9 x 10(-2) pg DNA for plastids of 1 -> 8 mu m average diameter, corresponding from approximately a dozen to 330 genome equivalents per organelle and on average four to seven copies per nucleoid. The ratio of plastid/nuclear DNA changed continuously during leaf development from as little as 0.4% to about 20% in fully developed leaves. On the other hand, mesophyll cells of mature leaves differing in ploidy (di-, tri- and tetraploid) appeared to maintain a relatively constant nuclear genome/plastome ratio, equivalent to about 1,700 copies per C-value.

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Language(s): eng - English
 Dates: 2009-11-172010
 Publication Status: Published in print
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 Identifiers: ISI: ISI:000273626100003
DOI: 10.1007/s00438-009-0491-1
ISSN: 1617-4623 (Electronic) 1617-4623 (Linking)
URI: ://000273626100003 http://www.springerlink.com/content/fx7108987781x020/fulltext.pdf
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Title: Molecular Genetics and Genomics
Source Genre: Journal
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Pages: - Volume / Issue: 283 (1) Sequence Number: - Start / End Page: 35 - 47 Identifier: -