Journal of Heredity Advance Access originally published online on July 12, 2006
Journal of Heredity 2006 97(4):417-422; doi:10.1093/jhered/esl014
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PRINS-Labeled Knobs Are Not Associated with Increased Chromosomal Stickiness in the Maize st1 Mutant
From the Department of Crop Sciences, University of Illinois, 1201 W. Gregory Dr., 360 ERML, Urbana, IL 61801
Address correspondence to A. L. Rayburn at the address above, or e-mail: arayburn{at}uiuc.edu.
In maize, the st1 mutant has been observed to result in chromosomes that stick together during both mitotic and meiotic anaphase. These sticky chromosomes result in abnormal chromosome separation at anaphase. Although the mechanism producing the st1 mutant phenotype is unknown, delayed replication of knob heterochromatin has been implicated in similar phenomena that result in sticky chromosomes. Primed in situ labeling (PRINS) was used to locate the 180-bp knob DNA sequences on mitotic metaphase chromosomes of several maize lines. The chromosomal regions labeled by PRINS corresponded to the reported C bands found in these lines. Additionally, PRINS was used to identify knob-bearing regions in anaphase spreads of a line carrying the st1 mutant and a nonmutant line having a similar number of chromosome knobs. The increase in abnormal anaphase figures in the st1 mutant was not accompanied by an increase in association of knob DNA with abnormal anaphases. Thus, the increase in chromosomal stickiness appears to be due to an increase in stickiness of knob and nonknob chromosomal regions. The mechanism responsible for the st1 mutant, therefore, is hypothesized to be different from that implicated in the other previously described sticky chromosomes situations.
Beadle (1932) first described the st1 mutant in maize. The various characteristics of plants homozygous for st1 include small plant, striated leaves, scattered seed set, and pitted kernels. All these phenotypes, as well as others, result from sticky chromosomes both in meiosis and mitosis (Neuffer and others 1997; Rayburn and Wetzel 2002). In both mitosis and meiosis, the chromosomes seem to stick together during anaphase. The result of this stickiness is the formation of anaphase bridges. As the chromosome centromeres continue to separate, the bridge is broken, resulting in daughter cells with duplicated and deleted chromosomal segments (Rayburn and Wetzel 2002). Although the phenomenon of sticky chromosomes has been well established, the mechanism responsible for chromosomal stickiness in the st1 mutant is as of yet unknown.
Sticky chromosomes and anaphase bridges are not restricted to plants carrying the st1 mutation. One of the classic examples of mitotic anaphase bridges requires the interaction of the 2 most prominent heterochromatic elements in the maize genome, knobs and B chromosomes (Rhoades 1978). In the second mitosis of the microspore in one particular genotype, B chromosomes appear to delay replication of the heterochromatic knobs in both A and B chromosomes. This delayed replication results in an anaphase bridge being formed as the chromatids separate, and the centromeres move to opposite poles. The chromatids are held together by the unreplicated knob heterochromatin.
Knob heterochromatin has also been associated with anaphase bridges observed in cell culture. Edallo and others (1981) and McCoy and Phillips (1982) observed instability of maize chromosomes in culture. Lee and Phillips (1987) noted that chromosomal rearrangements occurring in cell culture were associated with chromosome arms containing knobs. de Agular-Perecin and others (2000) observed that anaphase bridges occurred during the mitotic division of maize cell culture. On C-banding anaphase spreads, they also noted that knob sites appeared to hold the sister chromatids together to form the resulting bridge. The late-replicating nature of the knob heterochromatin was hypothesized to be responsible for the formation of the anaphase bridges.
Because knob heterochromatin has been implicated in 2 different instances of chromosomal stickiness, one could hypothesize that knob heterochromatin is involved in the increased frequency of sticky chromosomes in the st1 mutant. One way of observing knob heterochromatin in mitotic figures is by C-banding. Positive-staining C bands in maize mitotic chromosome are mitotic manifestations of meiotic chromosome knobs (Ward 1980; Rayburn and others 1985). Although C-banding has been used successfully in observing knob heterochromatin, there is, however, a problem with using C-banding to identity knobs. Longley (1939) proposed that knobs were large chromomeres located on specific chromosome regions. Knobs are not only defined by size and location but also by several families of repetitive DNA located within the maize knob (Peacock and others 1981; Ananiev and others 1998). Therefore, the possibility exists that positive C bands could be observed on mitotic chromosomes that are not representing the classic maize knob. Alternatively, the C-banding may not reveal all the potential knob regions (Adawy and others 2004). To be precise about the presence of knobs, an alternative method of observing knobs is needed.
In situ hybridization of various forms has been used to localize the knob sequence on maize chromosomes (Peacock and others 1981; Bashir and others 1995; Kato and others 2004). Adawy and others (2004) demonstrated that in situ hybridization revealed cryptic knobs that had not been documented either by pachytene or C-band analysis. Thus, in situ hybridization has the potential to allow the more accurate identification of knobbed regions on maize chromosomes. Although in situ hybridization has been used very successfully, another molecular cytogenetic technique has been developed that has several advantages over in situ hybridization.
Primed in situ labeling (PRINS) has several advantages over typical hybridization. These advantages include speed of the assay, higher specificity, and lower background (Kubalakova and others 2001). PRINS have been successfully used to localize DNA sequences on both animal (Musio and Rainaldi 1997; Reiter and others 1999; Freeman and Rayburn 2005) and plant (Kubalakova and others 2001) chromosomes.
In this study, several maize lines of known knob composition were analyzed to determine if PRINS could be used to localize knob sequences on maize chromosomes. After the PRINS technique was optimized, mitotic anaphase cells of the st1 mutant and Pa91 were analyzed by PRINS. The total number of abnormal cells was determined. In addition, the occurrences of the knob sequence in anaphase bridges, chromosomal fragments, and lagging chromosomes were recorded.
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Maize Lines and Slide Preparation
The maize inbred lines used were H95, Mo17, Pa91 and Va26. Two additional lines, Abnormal K10 and the st1 mutant obtained from the Maize Genetics Cooperation Stock Center (Urbana, IL), were also analyzed. The corn kernels were placed on blotter paper in a germination box. After 24 h of continuous light, the boxes were placed at 4 °C in the dark for 24 h. The germination boxes were placed back under continuous light, and the roots were harvested when they obtained a length of 1–2 cm. For metaphase chromosome analysis, the roots were placed in 0.05% 8-hydroxyquinoline at 24 °C. After 2 h, the roots were fixed in 3:1 ethanol:glacial acetic acid at 4 °C for 24 h. The roots were then stored in 70% ethanol at 4 °C. For anaphase analysis, the 8-hydroxyquinoline step was omitted, and the root tips were immediately fixed in 3:1 ethanol:glacial acetic acid and then stored in 70% ethanol.
The root squashes were made according to methods of Bashir and others (1995). The root tips were stained in 1% acetocarmine. Each root tip was squashed on a microscope slide with a 18 x 18 coverslip. The slides were then placed at –70 °C for at least 48 h. The coverslip was removed just prior to the PRINS procedure.
PRINS Reaction
Slides were first dehydrated in an ethanol series (70%, 90%, and 100%) for 3 min each and then denatured for 2 min at 70 °C in 70% formamide, 2x standard saline citrate (SSC) (pH 7.0). The ethanol dehydration series was repeated, and slides were allowed to air-dry. Fifty microliters of a reaction mix was added onto each slide. The reaction mix contains 1x Taq buffer (Eppendorf, Westbury, NY), 0.2 mM deoxyadenosine triphosphate, 0.2 mM deoxycytidine triphosphate, 0.2 mM deoxyguanosine triphosphate, 0.02 mM deoxythymidine triphosphate, 0.02 mM fluorescein 12-deoxyuridine triphosphate, 1x self-seal reagent (MJ Research, Reno, NV), 0.01% bovine serum albumin, 1 U Taq polymerase (Eppendorf, Westbury, NY), and 3 µg primer. For the labeling of the knobs, the degenerate primer sequence was ATGTGGGGTGTGTAYGAGSTCYGGTC. The primer was made by the W.M. Keck Center for Comparative and Functional Genomics (University of Illinois at Urbana–Champaign) based on variants of the 180-bp sequence located in all known heterochromatic knobs of maize and first described by Peacock and others (1981) as the 185-bp–repetitive sequence. Coverslips were placed on each slide, and the reaction ran on an Omnigene Thermocycler equipped with a hybridization block. The single PRINS program for the knob primer was 4 min at 94 °C for denaturation, 3 min at 60 °C for annealing, and 30 min at 65 °C for extension. On completion of the reaction, the coverslips were removed and slides placed in stop buffer (0.5 M NaCl and 0.05 M ethylenediaminetetraacetic acid) for 1 min at 65 °C. Slides were then washed 3 times in 4x SSC, 0.05% Tween 20 for 5 min each. Slides were counterstained with propidium iodide (PI) at 0.2 µg/ml for 30 min. After counterstaining, slides were drained, and Vectashield antifade solution (Vector Laboratory, Burlingame, CA) was added to the slides. A coverslip was then placed on each slide. Fluorescent labeling was detected on the chromosomes using an Olympus BX61 microscope with Omega fluorescent filters. The first filter combination had an excitation wavelength of 475 nm with a 40-nm wave half-height width and an emission signal of 535 nm with a 45-nm wave half-height width to observe the fluorescein signal. The second filter combination had an excitation wavelength of 525 nm with a 45-nm wave half-height width and an emission signal of 565-nm long-pass to observe the counterstaining of the chromosomes with PI. Pictures were taken using an Olympus Magnafire digital color camera and software. Pictures were produced by using the color-merge option. For the metaphase study, 5 plants per line were examined. Ten spreads per line were analyzed. For the anaphase study, 5 plants per each line were examined. A t-test was used to determine if the number of abnormal anaphase cells was different between the st1 mutant and Pa91. In addition, a t-test was used to determine if the number of abnormal anaphases demonstrating knob heterochromatin involvement differed between the st1 mutant and Pa91.
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PRINS Labeling
The 4 maize inbred lines were selected based on their known knob composition (Table 1). The PRINS technique revealed positive areas of labeling in all the maize lines (Table 1). These areas appear as yellow regions in both the PI-stained nuclei and chromosomes (Figure 1A–D). Both H95 and Va26 were observed to have multiple strong signals. H95 was observed to have 12 strongly labeled areas, whereas Va26 was observed to have 12 areas of differential labeling (Figure 1A, B). Two of Va26 chromosomes were only faintly labeled and, as such, are not easily visible. Mo17 was observed to have 2 lightly labeled regions. The line designated Abnormal K10 had one very heavily labeled region and 8 regions labeled in a similar fashion to H95 and Va26 (Figure 1D). Pa91 was observed to have 10 labeled regions, whereas the st1 mutant was observed to have 9 (Figure 2A, B).
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For anaphase analyses, 5 plants of both Pa91 and the st1 mutant were analyzed. A minimum of 56 cells per plant was analyzed, resulting in more than 300 anaphase cells per line. After labeling, the number of lagging chromosomes or anaphase bridges involving the knob sequence was observed in each line (Figure 2C, D; Table 2). In Pa91,
20% of the mitotic anaphase figures were observed to be abnormal. In the st1 mutant, 42% of the mitotic anaphase figures were observed to be abnormal. This difference was significant at P = 0.00007. Chromosomal regions carrying the 180-bp knob sequence were observed in 6% of the abnormal figures in Pa91, whereas in the st1 mutant the sequence was involved in 8% of the abnormal figures. This difference was not significant, P = 0.32.
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Initially, 4 maize lines were analyzed. PRINS was used to label regions on metaphase chromosomes containing the 180-bp sequence. H95, Va26, and Mo17 have been previously C banded. According to Rayburn and others (1997), H95 has 8 positive C bands, Va26 has 12 positive C bands, and Mo17 has 2 light positive C bands. On PRINS labeling using a primer sequence derived from the 180-bp knob sequence, H95 was observed to have 12 labeled regions. The number of labeled sites in H95 exceed the expected number inferred from the C-band data. Two possibilities exist to explain this discrepancy. One, the kernels of H95 examined in this study were from a different plant grown at least 5 years previous from the C-banded study (data not shown). Heterogeneity could exist within this line with respect to C-band number. Thus, the actual number of C bands may indeed be different between the 2 studies. Two, the PRINS-labeling technique could have revealed a region of a chromosome that contains the 180-bp knob sequence but is not C-band positive (Adawy and others 2004).
The Va26 and Mo17 data were identical to the previously reported C-band data. The 10 heavily labeled regions and 2 lightly labeled regions observed in Va26 were consistent with the 12 reported C bands. With respect to Mo17, the 2 faintly labeled regions directly correspond to the very light C bands observed by Rayburn and others (1997). Thus, the PRINS technique using the 180-bp knob primer is labeling the knobbed regions of the maize chromosomes. To ensure that this is indeed the case, a maize line heterozygous for the Abnormal K10 knob was also analyzed. As expected, the large K10 knob was intensely labeled. PRINS with the 180-bp sequence was, therefore, determined to reveal the location of chromosomal regions that are composed of the knob sequence.
Having established that the PRINS-labeling technique did indeed reveal the 180-bp knob sequence, PRINS was used to reveal the knob region in mitotic metaphase of st1 mutant and Pa91. The st1 mutant, containing the st1 gene, was observed to have 9 sites that labeled with the 180-bp knob primer sequence. Because an odd number of sites were observed, this line is polymorphic with respect to knob number. This polymorphism appears due to the fact that the st1 mutant was not maintained at the Maize Genetics Cooperative Stock Center in a specific homogeneous inbred line but in a heterogeneous background. Pa91 was observed to have 10 labeled regions which correspond to the number of C bands (Rayburn and others 1997). The 7 sites of high intensity and 2 of light intensity of the st1 plants were similar to the 8 high-intensity regions and 2 light-intensity regions of Pa91. In order to determine if the st1 mutant did indeed have an increase in the amount of knob heterochromatin associated with chromosomal stickiness, it was important that a line not containing the st1 gene yet having about the same number of knob regions be compared. Because Pa91 and st1 had similar knob regions, anaphase chromosomes were analyzed.
The percentage of sticky chromosome anaphases observed in the st1 mutant line of this study was nearly identical to that observed by Rayburn and Wetzel (2002). In addition, the normal controls in both studies were also very similar. This observation was important because in Wetzel and Rayburn (2002) the control was from a heterogeneous population. Having a consistent homogeneous control will allow comparisons to be made in future studies. The heterogeneity of the control used in the previous study precludes such comparisons. Given these results between the 2 studies, the use of Pa91 as a homogeneous control was substantiated. The statistically significant increase of abnormal anaphase spreads in the st1 mutant (42%) versus Pa91 (20%) was not due to increased stickiness of the knob region. When observing the number of abnormal spreads involving the knob region, the st1 mutant and Pa91 had a nonsignificant difference in percentage of knob regions involved (8% vs. 6%). This observation indicates that both knob and nonknob regions of the chromosome increased in chromosomal stickiness. If the increase were due just to increased knob region stickiness, the percentage of abnormal anaphase cells with knob region involvement would have increased. The data suggests that the st1 mutant increases chromosomal stickiness in both knob and nonknob regions. Therefore, the mechanism for inducing sticky chromosomes in the st1 mutant is hypothesized to be different than the delayed chromosome separation due to late-replicating knob heterochromatin described in other sticky chromosome systems in maize.
In conclusion, this study demonstrates that PRINS labeling can be used to identify knob regions on maize chromosomes. In 3 of the 4 lines, the PRINS results were as expected from previously reported C-banding data. In the fourth line, the deviations from the expected results appeared due to potential heterogeneity in the inbred line or increased sensitivity of PRINS. By using PRINS, maize knob regions were not found to disproportionally increase in abnormal anaphase mitotic figures, suggesting that the mechanism behind the st1 mutation is not the same as the previously hypothesized mechanisms of induced chromosomal stickiness in other maize systems and may not involve knob heterochromatin.
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Acknowledgments |
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We thank Ms. Laura E. Guest of the W.M. Keck Center for Comparative and Functional Genomics, University of Illinois at Urbana–Champaign, for help with primer design.
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Corresponding Editor: Prem Jauhar
Received November 28, 2005
Accepted May 26, 2006
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Adawy SSM, Stupar RM, Jiang J. (2004) Fluorescence in situ hybridization analysis reveals multiple loci of knob-associated DNA elements in one-knob and knobless maize lines. J Histochem Cytochem 52:1113–6.
Ananiev EV, Phillips RL, Rines HW. (1998) A knob-associated tandem repeat in maize capable of forming fold-back DNA segments: are chromosome knobs megatransposons? . Proc Natl Acad Sci USA 95:10785–90.
Bashir A, Biradar DP, Rayburn AL. (1995) Determining relative abundance of specific DNA sequences in flow cytometrically sorted maize nuclei. J Exp Bot 46:451–7.
Beadle GW. (1932) A gene for sticky chromosomes in Zea mays . Z Indukt Abstammungs- Vererbungsl 63:195–217.
de Agular-Perecin MLR, Fluminhan A, de Santos-Serefo JA, Garingo JR, Bertao MR, Decico MJU, Mondin M. (2000) Heterochromatin of maize chromosomes: structure and genetic effects. Genet Mol Biol 23:1015–9.
Edallo S, Zucchinali C, Perenzin M, Salamini F. (1981) Chromosomal variation and frequency of spontaneous mutation associated with in vitro culture and plant regeneration in maize. Maydica 26:39–56.
Freeman JL and Rayburn AL. (2005) Localization of repetitive DNA sequences on in vitro Xenopus laevis chromosomes by primed in situ labeling (PRINS). J Hered 93:603–8.
Kato A, Lamb JC, Birchler JA. (2004) Chromosome painting using repetitive DNA sequences as probes for somatic chromosome identification in maize. Proc Natl Acad Sci USA 101:13553–9.
Kubalakova M, Vrana J, Cihalikova J, Lysak MA, Dolezel J. (2001) Localization of DNA sequences on plant chromosomes using PRINS and C-PRINS. Methods Cell Sci 23:71–82.[CrossRef][Medline]
Lee M and Phillips RL. (1987) Genomic rearrangements in maize induced by tissue culture. Genome 29:122–8.
Longley AE. (1939) Knob positions on corn chromosomes. J Agric Res 59:475–90.
McCoy TJ and Phillips RL. (1982) Chromosomal stability in maize (Zea mays) tissue cultures and sectoring in some regenerated plants. Can J Genet Cytol 24:559–65.
Musio A and Rainaldi G. (1997) Cycling-PRINS a method to improve the accuracy of telomeric sequence detection in mammalian chromosomes. Mutat Res 390:1–4.[ISI][Medline]
Neuffer MG, Coe EH, Wessler SR. (1997) Mutants of maize (Cold Spring Harbor Laboratory Press, Cold Spring Harbor) pp. 468.
Peacock WJ, Dennis ES, Rhoades MM, Pyror A. (1981) Highly repeated DNA sequence limited to knob heterochromatin in maize. Proc Natl Acad Sci USA 78:4490–4.
Rayburn AL, Bashir A, Biradar DP. (1997) Chromatin composition in maize F1 hybrids. Maydica 42:393–9.
Rayburn AL, Price HJ, Smith JD, Gold JR. (1985) C-band heterochromatin and DNA content in Zea mays. Am J Bot 72:1610–7.[CrossRef]
Rayburn AL and Wetzel JB. (2002) Flow cytometric analysis of intraplant nuclear DNA content variation induced by sticky chromosomes. Cytometry 49:36–41.[CrossRef][ISI][Medline]
Reiter LT, Liehr T, Rautenstrauss B, Robertson HM, Lupski JR. (1999) Localization of mariner DNA transposons in the human genome by PRINS. Genome Res 9:839–43.
Rhoades MM. (1978) Genetic effects of heterochromatin in maize. In Walden BD (Ed.). Maize breeding and genetics (Wiley and Sons, New York) pp. 641–72.
Ward EJ. (1980) Banding patterns in maize mitotic chromosomes. Can J Genet Cytol 22:61–7.
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