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45S rDNA regions are chromosome fragile sites expressed as gaps in vitro on metaphase chromosomes of root-tip meristematic cells in Lolium spp.

Huang J, Ma L, Yang F, Fei SZ, Li L - PLoS ONE (2008)

Bottom Line: During the course of cytological mapping of rDNA on ryegrass chromosomes, we found that the number of chromosomes plus chromosome fragments was often more than the expected 14 in most cells for Lolium perenne L. cv.Top One.The chromosome lesions observed in this study are very similar cytologically to that of fragile sites observed in human chromosomes, and thus we conclude that the high frequency of chromosome lesions in vitro in Lolium species is the result of the expression of 45S rDNA fragile sites.

View Article: PubMed Central - PubMed

Affiliation: Key Laboratory of Ministry of Education for Plant Development Biology, College of Life Sciences, Wuhan University, Wuhan, China.

ABSTRACT

Background: In humans, chromosome fragile sites are regions that are especially prone to forming non-staining gaps, constrictions or breaks in one or both of the chromatids on metaphase chromosomes either spontaneously or following partial inhibition of DNA synthesis and have been well identified. So far, no plant chromosome fragile sites similar to those in human chromosomes have been reported.

Methods and results: During the course of cytological mapping of rDNA on ryegrass chromosomes, we found that the number of chromosomes plus chromosome fragments was often more than the expected 14 in most cells for Lolium perenne L. cv. Player by close cytological examination using a routine chromosome preparation procedure. Further fluorescent in situ hybridization (FISH) using 45S rDNA as a probe indicated that the root-tip cells having more than a 14-chromosome plus chromosome fragment count were a result of chromosome breakage or gap formation in vitro (referred to as chromosome lesions) at 45S rDNA sites, and 86% of the cells exhibited chromosome breaks or gaps and all occurred at the sites of 45S rDNA in Lolium perenne L. cv. Player, as well as in L. multiflorum Lam. cv. Top One. Chromatin depletion or decondensation occurred at various locations within the 45S rDNA regions, suggesting heterogeneity of lesions of 45S rDNA sites with respect to their position within the rDNA region.

Conclusions: The chromosome lesions observed in this study are very similar cytologically to that of fragile sites observed in human chromosomes, and thus we conclude that the high frequency of chromosome lesions in vitro in Lolium species is the result of the expression of 45S rDNA fragile sites. Possible causes for the spontaneous expression of fragile sites and their potential biological significance are discussed.

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Fluorescence in situ hybridization with 45S rDNA as a probe shows that 45S (green) rDNAs are the sites of chromosome lesions in meristematic cells of root tips in tetraploid Lolium multiflorum cv. Top One.A: black layer; B: color image by merging red layers and green layers. Arrows indicate sites. Bar = 5 µm.
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pone-0002167-g003: Fluorescence in situ hybridization with 45S rDNA as a probe shows that 45S (green) rDNAs are the sites of chromosome lesions in meristematic cells of root tips in tetraploid Lolium multiflorum cv. Top One.A: black layer; B: color image by merging red layers and green layers. Arrows indicate sites. Bar = 5 µm.

Mentions: Following FISH with 45S rDNA as a probe, we detected seven 45S rDNA hybridization sites in the diploid L. perenne cv. Player and our FISH results also revealed all of 45S rDNA signals occurred in the middle of the chromosomes when no lesions happened (e.g. 14 chromosomes) (Figure 2). These are agreement with the results reported in ryegrasses [23]. Chromosome preparations were examined for the presence or absence of lesions and the pattern of the lesions. A cell was considered to contain chromosomes with lesion sites in vitro if one or both of the chromatids of one or more chromosome were broken or had gaps on one or more chromosomes. If there is only one chromosome break at a single site or only one gap appears on one chromosome, there will be 14 chromosomes plus 1 chromosome fragment in a cell, resulting in a chromosome plus chromosome fragment count of 15. Accordingly cells with 2, 3, 4, 5 or 6 lesions would result in cells with chromosome plus chromosome fragment counts of 16, 17, 18, 19 and 20, respectively. In the 100 cells analyzed, 86 cells showed at least one chromosome lesion. FISH revealed that chromosome lesions occurred exclusively at the 45S rDNA sites and each one of the seven sites could be involved with a lesion, although the number of lesions varied among different cells in vitro. However, we are not sure that whether each of the seven 45S regions is as frequently involved in chromosome breaks as the other, e.g. whether some fragile sites are more prone to breakage than others because the identification of specific chromosomes is difficult. We also did not determine whether the broken chromosomes are homologous, however based on the fact that the number of breaks varies from cells to cells, we guess that breaks could occur randomly and could occur on either or both of a pair of homologous chromosomes when multiple breaks occur in a cell. In cells without any chromosome lesions at the 45S rDNA regions in vitro, metaphase chromosome number is always 14, confirming that lesions occurred exclusively at the 45S rDNA regions and there were no lesions at any other parts of the chromosomes. In L. multiflorum cv. Top One (2n = 28), chromosome lesions were also frequent at 45S rDNA sites (Figure 3). Furthermore, FISH analysis on the chromosome preparations in the other two cultivars of L. perenne L. and L. multiflorum Lam. showed that 45S rDNAs regions were the sites of chromosome lesions (data not shown). These results lead us to believe that 45S rDNA is a region of chromosome fragility in Lolium. Cytological appearance of lesions at 45S rDNA fragile sites in Lolium appears to be analogous to that of fragile sites observed in human chromosomes. Three different cytological appearances of lesions were observed at the 45S rDNA sites in Lolium: first, breakage or constriction occurred to a single chromatid within the 45S rDNA region (Figure 4, A1 and A2); second, one formed a gap within the rDNA between the two chromosome ends, with the chromosome still connected through one or a few thin DNA fibers (local despiralizations of the chromatid) (Figure 4, B1 and B2); and third, breakage occurred to both chromatids of a chromosome with no detectable DNA hybridization signals between the broken ends of the two chromosome fragments (Figure 4, C1 and C2).


45S rDNA regions are chromosome fragile sites expressed as gaps in vitro on metaphase chromosomes of root-tip meristematic cells in Lolium spp.

Huang J, Ma L, Yang F, Fei SZ, Li L - PLoS ONE (2008)

Fluorescence in situ hybridization with 45S rDNA as a probe shows that 45S (green) rDNAs are the sites of chromosome lesions in meristematic cells of root tips in tetraploid Lolium multiflorum cv. Top One.A: black layer; B: color image by merging red layers and green layers. Arrows indicate sites. Bar = 5 µm.
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Related In: Results  -  Collection

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pone-0002167-g003: Fluorescence in situ hybridization with 45S rDNA as a probe shows that 45S (green) rDNAs are the sites of chromosome lesions in meristematic cells of root tips in tetraploid Lolium multiflorum cv. Top One.A: black layer; B: color image by merging red layers and green layers. Arrows indicate sites. Bar = 5 µm.
Mentions: Following FISH with 45S rDNA as a probe, we detected seven 45S rDNA hybridization sites in the diploid L. perenne cv. Player and our FISH results also revealed all of 45S rDNA signals occurred in the middle of the chromosomes when no lesions happened (e.g. 14 chromosomes) (Figure 2). These are agreement with the results reported in ryegrasses [23]. Chromosome preparations were examined for the presence or absence of lesions and the pattern of the lesions. A cell was considered to contain chromosomes with lesion sites in vitro if one or both of the chromatids of one or more chromosome were broken or had gaps on one or more chromosomes. If there is only one chromosome break at a single site or only one gap appears on one chromosome, there will be 14 chromosomes plus 1 chromosome fragment in a cell, resulting in a chromosome plus chromosome fragment count of 15. Accordingly cells with 2, 3, 4, 5 or 6 lesions would result in cells with chromosome plus chromosome fragment counts of 16, 17, 18, 19 and 20, respectively. In the 100 cells analyzed, 86 cells showed at least one chromosome lesion. FISH revealed that chromosome lesions occurred exclusively at the 45S rDNA sites and each one of the seven sites could be involved with a lesion, although the number of lesions varied among different cells in vitro. However, we are not sure that whether each of the seven 45S regions is as frequently involved in chromosome breaks as the other, e.g. whether some fragile sites are more prone to breakage than others because the identification of specific chromosomes is difficult. We also did not determine whether the broken chromosomes are homologous, however based on the fact that the number of breaks varies from cells to cells, we guess that breaks could occur randomly and could occur on either or both of a pair of homologous chromosomes when multiple breaks occur in a cell. In cells without any chromosome lesions at the 45S rDNA regions in vitro, metaphase chromosome number is always 14, confirming that lesions occurred exclusively at the 45S rDNA regions and there were no lesions at any other parts of the chromosomes. In L. multiflorum cv. Top One (2n = 28), chromosome lesions were also frequent at 45S rDNA sites (Figure 3). Furthermore, FISH analysis on the chromosome preparations in the other two cultivars of L. perenne L. and L. multiflorum Lam. showed that 45S rDNAs regions were the sites of chromosome lesions (data not shown). These results lead us to believe that 45S rDNA is a region of chromosome fragility in Lolium. Cytological appearance of lesions at 45S rDNA fragile sites in Lolium appears to be analogous to that of fragile sites observed in human chromosomes. Three different cytological appearances of lesions were observed at the 45S rDNA sites in Lolium: first, breakage or constriction occurred to a single chromatid within the 45S rDNA region (Figure 4, A1 and A2); second, one formed a gap within the rDNA between the two chromosome ends, with the chromosome still connected through one or a few thin DNA fibers (local despiralizations of the chromatid) (Figure 4, B1 and B2); and third, breakage occurred to both chromatids of a chromosome with no detectable DNA hybridization signals between the broken ends of the two chromosome fragments (Figure 4, C1 and C2).

Bottom Line: During the course of cytological mapping of rDNA on ryegrass chromosomes, we found that the number of chromosomes plus chromosome fragments was often more than the expected 14 in most cells for Lolium perenne L. cv.Top One.The chromosome lesions observed in this study are very similar cytologically to that of fragile sites observed in human chromosomes, and thus we conclude that the high frequency of chromosome lesions in vitro in Lolium species is the result of the expression of 45S rDNA fragile sites.

View Article: PubMed Central - PubMed

Affiliation: Key Laboratory of Ministry of Education for Plant Development Biology, College of Life Sciences, Wuhan University, Wuhan, China.

ABSTRACT

Background: In humans, chromosome fragile sites are regions that are especially prone to forming non-staining gaps, constrictions or breaks in one or both of the chromatids on metaphase chromosomes either spontaneously or following partial inhibition of DNA synthesis and have been well identified. So far, no plant chromosome fragile sites similar to those in human chromosomes have been reported.

Methods and results: During the course of cytological mapping of rDNA on ryegrass chromosomes, we found that the number of chromosomes plus chromosome fragments was often more than the expected 14 in most cells for Lolium perenne L. cv. Player by close cytological examination using a routine chromosome preparation procedure. Further fluorescent in situ hybridization (FISH) using 45S rDNA as a probe indicated that the root-tip cells having more than a 14-chromosome plus chromosome fragment count were a result of chromosome breakage or gap formation in vitro (referred to as chromosome lesions) at 45S rDNA sites, and 86% of the cells exhibited chromosome breaks or gaps and all occurred at the sites of 45S rDNA in Lolium perenne L. cv. Player, as well as in L. multiflorum Lam. cv. Top One. Chromatin depletion or decondensation occurred at various locations within the 45S rDNA regions, suggesting heterogeneity of lesions of 45S rDNA sites with respect to their position within the rDNA region.

Conclusions: The chromosome lesions observed in this study are very similar cytologically to that of fragile sites observed in human chromosomes, and thus we conclude that the high frequency of chromosome lesions in vitro in Lolium species is the result of the expression of 45S rDNA fragile sites. Possible causes for the spontaneous expression of fragile sites and their potential biological significance are discussed.

Show MeSH
Related in: MedlinePlus