| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
Corresponding author: Bor-Yaw Lin, National Chung Hsing University, Taichung 402, Taiwan, Republic of China., bylin{at}dragon.nchu.edu.tw (E-mail)
Communicating editor: J. A. BIRCHLER
 | ABSTRACT |
|---|
 TOP ABSTRACT MATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
|---|
The supernumerary B chromosome has no apparent effects on plant growth, and its molecular makeup is difficult to unravel, due to its high homology to the normal complement, which prevents conventional cloning. This difficulty was overcome previously by microdissecting the B chromosome under the microscope to result in 19 B clones, one of which is B specific and highly repetitive, dispersing over one-third of the B long arm and most regions of the centromeric knob. To gain insights into the molecular structure of the B chromosome, this sequence was used to screen a genomic library constructed from W22 carrying 16 B's. Five clones (>10 kb each) were isolated, and all were repetitive, showing homology with A chromosomes in Southern and FISH analyses. Two of them were further characterized and sequenced. Each is composed of several restriction fragments with variable degrees of repetitiveness. Some of these are B specific and others have variable degrees of homology with the A chromosomes. The order of each characteristic group is not contiguous; they intersperse within those of other groups. Sequence analysis reveals that their sequences (
26 kb) have no homology with any published gene other than sequences of transposable elements (retrotransposons and MITEs) and the B as well as the A centromeres. We uncovered a 1.6-kb CL-repeat sequence, seven units of which were present in the two clones in defective forms. Those repeats mostly arrange in tandem array in the B chromosome. Moreover, we detected transposition of a retrotransposon and a MITE element involved in the genesis of these two sequences.
THE maize B chromosome was originally identified by 
In this article, we took advantage of the unusual properties of this clone—its B-specific and repetitive nature—to screen large fragments of the B chromosome from a 
-library constructed from genomic DNA carrying 16 B chromosomes. Five clones were obtained, two of which were further sequenced and characterized. We found retrotransposons, a miniature inverted-repeat transposable element (MITE), CentC, and a 1.6-kb CL-repeat family, which is mostly arranged in tandem array in the two clones as well as in the B chromosome.
| MATERIALS AND METHODS |
|---|
| TOPABSTRACTMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
|---|
Plant material:
Two maize inbred lines (W22 and L289) were used in this study. The W22 plant bearing 16 B chromosomes (W22 + 16B) was used to construct a -library. L289 carrying 2 B chromosomes (L289 + 2B) was employed to prepare mitotic and meiotic chromosome spreads for FISH analysis, and the L289 containing 3 B's (L289 + 3B) and no B (L289 + 0B), respectively, were used for Southern analysis.
-library construction and screening:
The maize genomic library was constructed by S.-F. PENG (unpublished results), following the instructions of the supplier, using DASH II/EcoRI as vector (Stratagene, La Jolla, CA). Briefly, genomic DNA of W22 + 16B was partially digested with Tsp509I (New England Biolabs, Beverly, MA) and fractionated in sucrose gradient centrifugation at 154,000 x g for 24 hr. The 9- to 23-kb fraction was ligated to the arms of the -phage DASH/EcoRI, packaged (using Gigapack III Gold-11 packaging extract), used to infect host cells [XL1-Blue MRA (P2)], amplified, and plated at a low titer (200 plaques/100-mm plate on the same host cells). Plaques were lifted and hybridized with the B-specific clone pBPC51 (DNA preparation was carried out as described by
Genomic DNA isolation and Southern analysis:
Isolation of maize genomic DNA was previously described by
Polymerase chain reaction:
Two primers were designed to amplify CL-repeat sequences from the five B clones and the B chromosome: BP3 (5'-GATTCTTGGTTATGGACAACAATGC-3') from the 3' terminus of the CL repeat and BP5 (5'-GCACTACATATGGTTTAAGATAGC-3') from the 5' terminus. The polymerase chain reaction (PCR) was performed for 30 cycles with the following conditions: 94° for 30 sec, 55° for 30 sec, and 72° for 3 min.
Fluorescent in situ hybridization:
Using DIG-11-dUTP, large B fragments and CL repeats (pCLa1 and PCR products of pB51-12) were labeled by nick translation of the DIG-nick translation labeling system (Roche). The pCLa1 was cloned from PCR products of L289 + 3B, amplified by BP3 and BP5 primers. Chromosome spreads of root tips and pachytene chromosomes were prepared as outlined by
Sequence analysis:
DNA sequences similar to the two B clones, pB51-12 and pB51-15, were searched in the GenBank database using BLASTN of the National Center for Biotechnology Information website. Multiple alignments were made using the program PILEUP of the Genetics Computer Group program (Wisconsin, version 10.0) and displayed using the BoxShade server (http://www.ch.embnet.org/software/BOX_form.html). The sequences have been submitted to GenBank with accession nos.
AY426742 (pB51-12) and
AY426743 (pB51-15).
| RESULTS |
|---|
| TOPABSTRACTMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
|---|
Isolation of large B chromosome fragments:
Using pBPC51 as a probe to screen 2000 -clones from an amplified library, constructed from genomic DNA with 16 B chromosomes, nine positive phages were identified, five of which were further characterized. Following digestion with NotI, the inserts (>10 kb) were ligated into pBlueScript SK– and designated as pB51-11, pB51-12, pB51-13, pB51-14, and pB51-15, respectively. The NotI plus XbaI digests of these clones revealed one to six fragments of insert DNA (Fig 1A). When the blot was probed with pBPC51, one or two fragments of each clone displayed positive signals (Fig 1B). Hybridization of the same blot with genomic DNA of L289 + 3B as probe revealed signals on all insert fragments of pB51-11, pB51-12, pB51-13, and pB51-14 and on three of the six fragments of pB51-15 (Fig 1C). The remaining three fragments of pB51-15 without hybridization signal represented low-copy-number sequences in the L289 + 3B probe, which were not visible due to insufficient exposure under regular repetitive protocol. When probed with L289 + 0B, the same blot gave a similar hybridization pattern except the intensity of the largest fragment of pB51-13 was slightly reduced (Fig 1D). Taken together, these five clones were proven to be derived from the B chromosome, containing repetitive sequences common to the A and B chromosomes.
|
Hybridization of the BamHI digests of L289 + 3B and L289 + 0B with each of the five clones as probe resulted in a smeared pattern as well as discrete bands (Fig 2). The result indicates that the sequences of these five clones are highly or medium repetitive.
|
FISH localization of the five B clones:
To analyze the B location of the five clones, pachytene B chromosome was hybridized with the inserts as probes in FISH analysis (Fig 3). By and large, FISH signals appeared in all regions of the B chromosome. Fig 3A shows hybridization of pB51-11, where major signals are present on four distal heterochromatic regions. The hybridization pattern of pB51-12 is similar to that of pB51-11 (Fig 3B). FISH signals of pB51-13 were more concentrated on the distal heterochromatic region H3 (Fig 3C), and those of pB51-14 dispersed more evenly over almost the entire B chromosome (Fig 3D). Clone pB51-15 hybridized strongly on the distal heterochromatic regions, but with the centromeric knob and the proximal euchromatic region in lesser degrees (Fig 3E). Thus, the data verified the B origin of these sequences.
|
To study the distribution of the B clones on the A chromosomes, the five B clones were also used to probe root-tip chromosomes. The results are shown in Fig 4. FISH signals of the five clones dispersed on all A chromosomes, with the signals of pB51-11 and pB51-12 being the weakest (Fig 4A and Fig B) and that of pB51-13 and pB51-14 the strongest (Fig 4C and Fig D). Particularly interesting is the last clone (pB51-15), whose signals concentrated mostly around the centromeric regions (Fig 4E), although some signals dispersed on one or both arms. These results indicate that the five B clones contain repetitive sequences homologous to all A chromosomes. As would be expected, FISH signals of the five B clones were also present on the B chromosome of root-tip cells. The signals of pB51-11 and pB51-12 on the B chromosome displayed greater intensity than those on the A chromosomes (Fig 4A and Fig B). The pattern of pB51-13 and pB51-14 signals on the B chromosome is similar to that of the A chromosomes (Fig 4C and Fig D). Although the signals of pB51-15 appeared densely in the vicinity of the A centromeres, it did not do so on the B centromere (Fig 3E and Fig 4E).
|
Southern analysis:
Since pB51-12 contains the B exclusive sequence (pBPC51) and its hybridization patterns to genomic DNA with and without B are similar (Fig 2), it was expected to contain sequences unique to B and those in common with the A chromosomes. To determine its detailed molecular organization, the clone was digested with five enzymes (BamHI, HindIII, PstI, XbaI, and XhoI). The resulting 18 fragments were subcloned into pBlueScript, and each (from 5' to 3' end, pB51-12-1 to pB51-12-18, respectively) was used to probe the BamHI and XbaI digests of L289 + 3B and L289 + 0B. The results are presented in Fig 5. On the basis of their signal patterns, the 18 subclones could be divided into three groups. The first group (pB51-12-1 and pB51-12-18) is a B-specific sequence, hybridizing with L289 + 3B but not with L289 + 0B. The second group, including 5 subclones, has two types of signals, either specific or not specific to the B chromosome. Clones pB51-12-2, pB51-12-16, and pB51-12-17 show strong hybridizations to L289 + 3B but weak ones to L289 + 0B, and 2 others display multiple signals, one of which is present in the XbaI digest of L289 + 3B but either not present (pB51-12-5) or weakly present (pB51-12-10) in that of L289 + 0B. The remaining 11 subclones formed the third group, which are of high copy number and show a similar hybridization pattern as well as intensity between DNAs with and without the B chromosome. It is notable that fragments of each group were not contiguous—they interspersed between fragments of other groups. For example, while the two B-specific fragments occupied the two termini, two fragments of the second group were inserted in two different locations within the third group.
|
The same analysis was applied to pB51-15, and results are shown in Fig 6. Fourteen subclones (from 5' to 3' end, pB51-15-1 to pB51-15-14, respectively), generated by cloning the 14 restriction fragments resulting from digestion of pB51-15 by the same five enzymes, were used to probe the BamHI and XbaI digests of L289 + 3B and L289 + 0B, and none of them displayed a signal pattern indicative of a B-exclusive nature. Seven of them yielded B-specific as well as not-B-specific signals; signals of pB51-15-3, pB51-15-4, pB51-15-5, pB51-15-6, and pB51-15-13 in L289 + 3B were stronger than those in L289 + 0B. The two other subclones displayed multiple bands, and each had a single signal either B specific (pB51-15-14) or much more intense (pB51-15-10) in the BamHI and XbaI digests of L289 + 3B, respectively. The remaining seven subclones hybridized equally between DNAs with and without the B chromosome.
|
Sequence analysis:
The sequences of pB51-12 were compared with those deposited in the GenBank database. Homology of pB51-12 (13,385 bp) to various sequences from GenBank is summarized in Fig 7. First, pBPC51 (GenBank accession no.
BH814977, nt 1–324) was present in two terminal regions of pB51-12 (nt 791–1116 and nt 12,461–12,783, respectively), having an overall similarity of 90%. Second, three regions (nt 1141–1263, nt 11,490–11,598, and nt 12,808–13,101, respectively) were homologous to the B centromere clones, exemplified by B4a with 81–89% identity. Similarity of these regions to B4a included four segments with an overall similarity of 83%: nt 1141–1263, nt 11,490–11,535, nt 11,525–11,598, and nt 12,808–13,101 (nt 361–481, nt 362–407, nt 578–653, and nt 361–653 in GenBank accession no.
U61992, respectively). Finally, nine regions (total 4766 bp) had homology to a maize genomic clone (226 kb) carrying bz1 in the short arm of chromosome 9 (nt 16,447–19,891, GenBank accession no.
AF391808). Four of these (nt 4598–5158, nt 8410–9075, nt 9103–9626, and nt 9627–10,626) were homologous to the coding region of RIRE2 gag/pol (nt 19,332–19,891, nt 19,529–18,873, nt 18,848–18,325, and nt 18,303–17,303 in accession, respectively) and the remainder (nt 1315–1637, nt 1669–2481, nt 10,658–11,002, nt 11,010–11,243, and nt 11,261–11,473) to its upstream sequences (nt 17,626–17,303, nt 17,264–16,453, nt 17,264–16,991, nt 16,688–16,921, and nt 16,659–16,447 in accession, respectively).
|
Clone pB51-15 was sequenced and analyzed with the same program. The clone had a sequence of 12,955 bp, and the result is presented in Fig 8. There were four regions (nt 3232–3562, nt 4018–4064, nt 4696–5022, and nt 6232–6399) in common with pBPC51 (nt 1–329, nt 21–67, nt 1–324, and nt 1–167 in GenBank accession no.
BH814977, respectively; 89% identity), five regions (nt 2297–2384, nt 3518–3558, nt 3636–3804, nt 4983–5022, and nt 5067–5345) with the K11 clone of maize B centromere (nt 577–662, nt 477–517, nt 485–651, nt 477–516, and nt 381–662 in GenBank accession no.
U61997, respectively; 84% identity), and seven regions (nt 256–331, nt 458–641, nt 731–2044, nt 3–43, nt 256–337, nt 518–1099, and nt 1580–2043) with the LTRs of the maize gypsy/Ty3-type retrotransposon Tekay (nt 1662–1737, nt 1858–2047, nt 2136–3442, nt 10,533–10,573, nt 10,803–10,884, nt 11,062–11,638, and nt 11,657–12,118 in GenBank accession no.
AF050455, respectively; 89% identity). Also observed was a region (nt 2043–2275) having 91% identity with the maize clone mPIF381 (nt 239–6 in GenBank accession no.
AF416324;
|
Five regions (totaling 2.5 kb) showed similarity to the maize centromeric bacterial artificial chromosome (BAC) clone 15C5 (GenBank accession no.
AC116033;
Sequences that are unique to the B chromosome are of special interest for studying the molecular structure of the B chromosome. Accordingly, particular effort was made to examine each of the two regions found on pB51-12 and pB51-15 that do not hybridize to genomic DNA lacking a B chromosome. On pB51-12 there were two subclones (pB51-12-1 and pB51-12-2) at the 5' terminus and three (pB51-12-16, pB51-12-17, and pB51-12-18) at the 3' terminus (Fig 5). On pB51-15 four subclones from the internal region (pB51-15-3, pB51-15-4, pB51-15-5, and pB51-15-6) and one subclone located near the 3' terminus (pB51-15-13) had B-specific sequences (Fig 6). In terms of length, the B-specific region associated with pB51-12 is 3.6 kb and that related to pB51-15 is 5 kb. To determine whether the four regions were different segments of a long B-specific region or a repeat unit that appears more than four times in the two clones, the 5' terminal fragment of pB51-12 (pB51-12-1; 904 bp), the B-specific fragment based on Southern analysis, was used to compare with other regions by the Blast Two program (
Characterization of CL repeat:
To determine the 5' and 3' ends as well as the full length of this repeat unit (the CL repeat), sequences of the three matched regions (mentioned above) with their flanking sequences were aligned by the GCG program PILEUP, and the result is shown in Fig 9. Seven regions (four in pB51-12 and three others in pB51-15) were found to be associated with the CL repeat. For convenience of discussion, the four regions of pB51-12 (nt 1–1295, nt 1296–1314, nt 11,474–12,962, and nt 12,963–13,385) were designated as BR12-1, BR12-2, BR12-3, and BR12-4, respectively, and the three regions of pB51-15 (nt 2276–3662, nt 3663–5193, and nt 5194–6570) as BR15-1, BR15-2, and BR15-3, respectively. Particularly interesting is the finding that the last base of the 3' terminus of BR12-1 was in direct contact with the first base of the 5' terminus of BR12-2 (Fig 10). Furthermore, the former matched perfectly with the 3' terminus of BR12-3 and the latter with the 5' terminus of BR12-4, BR15-2, and BR15-3 (Fig 9), suggesting that the contact point between BR12-1 and BR12-2 is the junction of two CL repeats (a similar junction occurred between BR12-3 and BR12-4; Fig 10). In other words, the 3' terminus of BR12-1 represents the 3' end of the CL repeat, and the 5' terminus of BR12-2 the 5' end. Accordingly, the sequence extending from the 3' to the 5' end, 1552 bp, is the length of the CL repeat. In addition, since four junctions (two in pB51-12 as described above and two distorted ones in pB51-15, carrying deletions of 75 and 5 bp at the 3' end, respectively) of the seven repeat regions possessed a junction structure similar to that of BR12-1 and BR12-2 (Fig 16), most CL repeats in the chromosome appeared to be in the structure of tandem array.
|
|
|
|
|
|
|
|
To determine if the proposed CL repeat is valid, pBPC51 was used to probe the HindIII—cutting singly in the repeat (Fig 9)—digests of the genomic DNA of L289 + 3B and L289 + 0B. According to the estimation above, a predominant 1.6-kb signal would be expected, if the CL repeat is present in tandem array in the B chromosome. Results confirmed this expectation. A major signal of this size and several minor ones were shown only in L289 + 3B (Fig 11), indicating that the genomic distribution of the B-specific sequence is mostly in the form of tandem repeats and that each unit is 1.6 kb. The minor signals with variable intensity independent of molecular size might arise from either incomplete or isolated CL repeats. This result was further confirmed by PCR analysis, using primers derived from two end sequences of the CL repeat (primers BP3 and BP5, Fig 9). A major 1.6-kb product was amplified from DNAs of L289 + 3B but not of L289 + 0B (Fig 12A, lanes 7 and 8). Also produced from the former were two smaller (0.8 and 1.0 kb) smeared products. Likewise, the 1.6-kb product was produced when pB51-12 and pB51-15 were used as templates, apparently from BR12-3 and BR15-2, respectively (Fig 12A, lanes 3 and 6). However, no product appeared in the reactions of other B clones (pB51-11, pB51-13, and pB51-14; Fig 12Alanes 2, 4, and 5, respectively), suggesting that the CL repeats in these clones were missing one of the two primer-binding sites.
To clarify whether the two smaller products (0.8 and 1.0 kb; Fig 12A, lane 7) were amplified from different forms of the CL repeat in the B chromosome, the gel of PCR products (Fig 12A) was blotted and probed with pBPC51. The hybridization signals were evident not only in the 1.6-kb product, but also in the 0.8 and 1.0 kb as well as in the smeared products (Fig 12B). The 0.8-kb signal was much weaker than the 1.0-kb one (Fig 12B, lane 7), although it had more products, suggesting that it deleted most of the pBPC51 sequence. This explanation conforms to the result of the same analysis using the product of pB51-12 as probe, where the signal intensity is proportional to the amount of PCR products (Fig 13A). Since the product of pB51-12 was amplified from BR12-3, which is a defective CL repeat deficient in the 5' terminal 14 bp and two internal regions (box III and box IV, Fig 9), the 0.8- and 1.0-kb products of L289 + 3B should derive from the truncated versions of the CL repeat. The smeared products represented the CL-repeat-carrying sequences amplified from sequences that had the binding site of either BP3 or BP5 and a second binding site located in upstream or downstream regions of the CL repeat.
In addition to the 1.6-kb product, a second product was produced from pB51-12 and pB51-15, although it was not visible in the gel stained with ethidium bromide (Fig 12A, lanes 3 and 6). This product was revealed in Southern hybridization of the gel with the pBPC51 probe, where an intense 1.6-kb signal and a weaker 0.8-kb one were visible (Fig 12B, lanes 3 and 6). The 0.8-kb fragment resulted from a second binding site of primer BP5 located in box II (Fig 9), which had a 58% similarity to BP5. The last five nucleotides of the 3' end of BP5 had a perfect match with this site. The product amplified from BP5 binding to this site and BP3 is expected to be 738 bp in pB51-12 and 768 bp in pB51-15.
To determine the distribution of the CL repeat in the B chromosome, the CL-repeat clone (pCLa1; see MATERIALS AND METHODS) and PCR products, amplified by BP3 and BP5 from pB51-12 (Fig 12A, lane 3), were used as probe to hybridize the pachytene B chromosome. As shown in Fig 14, FISH signals of both probes appeared strongly on the distal heterochromatic regions H1, H2, and H3 and weakly on the centromeric knob. This result agrees with that of a similar hybridization using pBPC51 as probe (
To gain further insight into the nature of the CL-repeat PCR products, the BamHI and XbaI digests of L289 + 3B and L289 + 0B were probed by the PCR products of pB51-12, pB51-15, and L289 + 3B, primed by BP3 and BP5 (Fig 15). The product of pB51-12 hybridized with L289 + 3B but not with L289 + 0B, indicative of a B exclusive repeat (Fig 15A). Hybridization pattern of the same digests with the product of pB51-15 is identical with that of pB51-12 except for the appearance of slight, smeared signals associated with L289 + 0B (Fig 15B). These exceptional signals must be the results of the presence of two short stretches of sequences (Fig 9, box III and box IV), which were homologous to the A chromosomes and deleted from pB51-12. PCR products using L289 + 3B as a template yielded basically the same pattern as above but with three additional signals (0.4, 0.5, and 0.9 kb) in the XbaI digest of L289 + 3B (Fig 15C). These three signals represented either the repeat sequences of the B chromosome not present in pB51-12 and pB51-15 or the sequences unrelated to the two clones that were amplified by BP3 and BP5 primers.
In view of sequence similarity between the CL repeat and the B centromeric sequences, these two possibilities were further characterized. According to sequence analysis, two terminal regions of the CL repeat had homology with B centromere sequences, including the sequence of BP3 and BP5 (Fig 9, box I and box V). To look into the possibility that the B centromere sequences were amplified by the primers of the CL repeat, the PCR products of L289 + 3B were probed by the B centromere sequence (see MATERIALS AND METHODS). Two intense (0.8 and 1.0 kb) and one very weak (1.6 kb) signal were observed after prolonged exposure (Fig 13B), suggesting the presence of an appreciable portion of B centromere sequences in the 0.8- and 1.0-kb products. The weak 1.6-kb signal reflected the small proportion of the CL-repeat sequence capable of annealing with the B centromere probe. This result is consistent with the fact that the size of the three additional signals of the XbaI digest of L289 + 3B (Fig 15C) corresponds to the three B-specific signals (0.4, 0.5, and 0.9 kb) noted by
All of the CL repeats present in the two clones were not complete units; each unit deleted either one (3' or 5') of the two ends or both (Fig 9 and Fig 16). Of the three regions matched with pB51-12-1, two were associated with pB51-12 and one with pB51-15. The 5' matched region of pB51-12 carried two units: the upstream unit (BR12-1; 1295 bp) missed the 5' end and the downstream unit (BR12-2; 19 bp) missed the 3' end. The 3' end of BR12-1 was closely followed by the 5' end of BR12-2, forming a junction of two tandem repeats as mentioned above (Fig 10). The second matched region, located in the 3' terminus of pB51-12, also contained two units (BR12-3 and BR12-4) and had the same structure as that of the 5' matched region (Fig 10). BR12-3 and BR12-4 had 1495 and 423 bp, respectively. The last matched region, situated in pB51-15, was composed of three repeat units: BR15-1, BR15-2, and BR15-3 in close association. BR15-1, the upstream unit (1387 bp), was deficient in both the 3' and 5' ends, and BR15-2 (1531 bp) contained only the 5' end. The last unit in the row, BR15-3 (1377 bp), likewise deleted the 3' end of the repeat unit.
Insertion of sequences into the CL repeat:
Sequence of pB51-12 is a complex structure, composed of CL repeats, portions of 9S bz clone (containing retrotransposon RIRE2), and two stretches of unknown element(s). As shown in Fig 16, the CL repeats are located at the 3' (BR12-1 and BR12-2) and 5' (BR12-3 and BR12-4) termini. The remaining elements lie in the central region delimited by BR12-2 and BR12-3. Particularly striking is the finding that the last five nucleotides of the 3' end of BR12-2 are identical to the first five nucleotides of the 5' end of BR12-3, forming a pair of direct repeats flanking the central region (Fig 9 and Fig 17A), which has a 6-bp inverted repeat (IR) at its two termini (Fig 17A). Moreover, the sequence of the 3' end of BR12-2, the 5-bp repeat mentioned above, and the sequence following the same 5-bp repeat of the 5' end of BR12-3 is contiguous in the CL-repeat sequence (Fig 9 and Fig 17A). It appears that BR12-2 and BR12-3 were originally two consecutive components of the same CL repeat and were separated by an insertion (Fig 17A). The inserted element is most likely a mobile element—a retrotransposon, MITE, or transposable element—since the two 5-bp direct repeats at the 3' and 5' ends of BR12-2 and BR12-3, respectively, may be its target sequences, and the 6-bp IR flanked by the direct repeats were the residual IR termini. To our knowledge, only three mobile elements have a 5-bp target site duplication (TSD): RIRE2, Dasheng, and RIRE8. Two additional facts suggest that this mobile element may be a member of RIRE2 family. First, the direct repeat fits the consensus TSD sequence of 194 RIRE2 elements: A (44%) T(40%) A(39%) T(21%) G(37%) as documented by
|
The sequence structure of pB51-15 is more complicated. From the 5' to the 3' terminus, it contains retrotransposon, MITE, CL repeats, a centromeric BAC, and an unknown element (Fig 16). Three CL repeats (BR15-1, BR15-2, and BR15-3) are located in the central region of the clone. The region upstream of these CL repeats is a MITE element (mPIF;
| DISCUSSION |
|---|
| TOPABSTRACTMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
|---|
Molecular composition and organization of the B chromosome has been a challenge to maize geneticists, because it resists cloning by conventional protocols, owing to its repetitive nature and high homology to the A chromosomes. In our previous study (
Clone pBPC51 provides a unique approach for studying the maize B chromosome. Two previously published maize B-specific sequences (pZmBs,
In this report, pBPC51 was used to screen a library, constructed from DNA carrying 16 B's, resulting in five B sequences (>10 kb each), which are repetitive and homologous to the A chromosomes in Southern and FISH analyses. Two of them were further characterized and sequenced. Each is composed of B-specific sequence intermingled with those in common with A chromosomes. Both have no similarity to any published gene other than transposable elements (retrotransposons and MITEs) and the B and A centromeres. And both carry a novel CL repeat.
Sequence characteristics of five large B inserts confirm the previous report that most B sequences are repetitive and share homology with the A chromosomes. This is evident in Southern analysis where each, as a probe, generated smeared signals in the 0B DNA as well as in the 3B DNA, and the signal intensity is unrelated to the B number (Fig 2). Also evident are the FISH signals of these clones, which dispersed over all root-tip A chromosomes (Fig 4) and most regions of the pachytene B chromosome (Fig 3). These observations are consistent with earlier reports.
The major B-specific sequences published to date are tandem repeats in plant species. Three maize B-specific sequences reported previously—pZmBs (
Three regions within the two clones did not have homology with any sequence in the GenBank database. Two (2 and 3 kb) of the regions are in the central region of pB51-12, and the third one (3.5 kb) is at the 3' terminus of pB51-15 (Fig 16). Six subclones of pB51-12 (pB51-12-4, pB51-12-5, pB51-12-7, pB51-12-8, pB51-12-9, and pB51-12-10) and four of pB51-15 (pB51-15-11, pB51-15-12, pB51-15-13, and pB51-15-14) contain exclusively sequences of these regions, and all, when used as probes in Southern analysis, show a highly repetitive signal pattern in DNAs with and without B's (Fig 5 and Fig 6), suggesting that these novel sequences might derive from degenerated mobile elements, which remain to be discovered.
Two popular views of the origin of the B chromosome have been promoted: A chromosomes of the current host or that of closely related species (
| FOOTNOTES |
|---|
Sequence data from this article have been deposited with the EMBL/GenBank Data Libraries under accession nos.
AY426742 and
AY426743. 
| ACKNOWLEDGMENTS |
|---|
We thank S.-F. Peng for sharing the genomic library, Y.-P. Lin for providing the B centromeric clone, and H.-H. Yang for technical assistance. This research was supported in part by grant NSC92-2311-B-005-024 from the National Science Council, Taiwan, Republic of China.
Manuscript received November 11, 2003; Accepted for publication December 31, 2003.
| LITERATURE CITED |
|---|
| TOPABSTRACTMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
|---|
ALFENITO, M. R. and J. A. BIRCHLER, 1993 Molecular characterization of a maize B chromosome centric sequence. Genetics 135:589-597.[Abstract]
ANANIEV, E. V., R. L. PHILLIPS, and H. W. RINES, 1998 Chromosome-specific molecular organization of maize (Zea mays L.) centromeric regions. Proc. Natl. Acad. Sci. USA 95:13073-13078.
AUSUBEL, F. M., R. BRENT, R. E. KINGSTON, D. D. MOORE, J. G. SEIDMAN et al., 1990 Current Protocol in Molecular Biology. John Wiley & Sons, New York.
BATTAGLIA, E., 1964 Cytogenetics of B-chromosome. Caryologia 17:245-299.
BLUNDEN, R., T. J. WILKES, J. W. FORSTER, M. M. JIMENES, and M. J. SANDERY et al., 1993 Identification of the E3900 family, a second family of rye B chromosome specific repeated sequences. Genome 36:706-711.
BURNHAM, C. R., 1982 Details of the smear technique for studying chromosomes in maize, pp. 107–118 in Maize for Biological Research, edited by W. F. SHERIDAN. Plant Molecular Biology Association, Charlottesville, VA.
CHENG, Y.-M. and B.-Y. LIN, 2003 Cloning and characterization of maize B chromosome sequences derived from microdissection. Genetics 164:299-310.
HSIA, A. P. and P. S. SCHNABLE, 1996 DNA sequence analyses support the role of interrupted gap repair in the origin of internal deletions of the maize transposon, MuDR.. Genetics 142:603-618.[Abstract]
HSU, F. C., C. J. WANG, C. M. CHEN, H. Y. HU, and C. C. CHEN, 2003 Molecular characterization of a family of tandemly repeated DNA sequences, TR-1, in heterochromatic knobs of maize and its relatives. Genetics 164:1087-1097.
JIANG, N., I. K. JORDAN, and S. R. WESSLER, 2002 Dasheng and RIRE2: a nonautonomous long terminal repeat element and its putative autonomous partner in the rice genome. Plant Physiol. 130:1697-1705.
JOHN, U. P., C. R. LEACH, and J. N. TIMMS, 1991 A sequence specific to B chromosomes of Brachycome dichromosomatica.. Genome 34:739-744.[Medline]
JONES, R. N., and H. REES, 1982 B Chromosome. Academic Press, New York.
KUWADA, Y., 1915 Ueber die Chromosomenzahl von Zea Mays L. Bot. Mag. Tokyo 29:83-89.
LEACH, C. R., T. M. DONALD, T. K. FRANKS, S. S. SPINIELLO, and C. F. HANRAHAN et al., 1995 Organisation and origin of a B chromosome centromeric sequence from Brachycome dichromosomatica.. Chromosoma 103:708-714.[Medline]
LIN, B.-Y., 1977 A squash technique for studying the cytology of maize endosperm and other tissues. Stain Technol. 52:197-201.[Medline]
LIN, B.-Y. and H.-P. CHOU, 1997 Physical mapping of four RAPDs in the B chromosome of maize. Theor. Appl. Genet. 94:534-538.[CrossRef]
LIN, B.-Y., S.-F. PENG, Y.-J. CHEN, H.-S. CHEN, and C.-F. KAO, 1997 Physical mapping of RFLP markers on four chromosome arms in maize using terminal deficiencies. Mol. Gen. Genet. 256:509-516.[CrossRef][Medline]
NAGAKI, K., J. SONG, R. M. STUPAR, A. S. PAROKONNY, and Q. YUAN et al., 2003 Molecular and cytological analyses of large tracks of centromeric DNA reveal the structure and evolutionary dynamics of maize centromeres. Genetics 163:759-770.
NASSIF, N., J. PENNEY, S. PAL, W. R. ENGELS, and G. B. GLOOR, 1994 Efficient copying of nonhomologous sequences from ectopic sites via P-element-induced gap repair. Mol. Cell. Biol. 14:1613-1625.
PAGE, B. T., M. K. WANOUS, and J. A. BIRCHLER, 2001 Characterization of a maize chromosome 4 centromeric sequence: evidence for an evolutionary relationship with the B chromosome centromere. Genetics 159:291-302.
PEACOCK, W. J., E. S. DENNIS, M. M. RHOADES, and A. J. PRYOR, 1981 Highly repeated DNA sequence limited to knob heterochromatin in maize. Proc. Natl. Acad. Sci. USA 78:4490-4494.
SANDERY, M. J., J. W. FORSTER, R. BLUNDEN, and R. N. JONES, 1990 Identification of a family of repeated sequences on the rye B chromosome. Genome 33:908-913.
STARK, E. A., I. CONNERTON, S. T. BENNETT, S. R. BARNES, and J. S. PARKER et al., 1996 Molecular analysis of the structure of the maize B-chromosome. Chromosome Res. 4:15-23.[CrossRef][Medline]
TATUSOVA, T. A. and T. L. MADDEN, 1999 Blast 2 sequences—a new tool for comparing protein and nucleotide sequences. FEMS Microbiol. Lett. 174:247-250.[CrossRef][Medline]
VIOTTI, A., E. PRIVITERA, E. SALA, and N. POGNA, 1985 Distribution and clustering of two highly repeated sequences in the A and B chromosomes of maize. Theor. Appl. Genet. 70:234-239.[CrossRef]
WARD, E. J., 1979 A postulated origin of the B chromosome. Maize Genet. Coop. News Lett. 53:100-102.
ZHANG, X., C. FESCHOTTE, Q. ZHANG, N. JIANG, and W. B. EGGLESTON et al., 2001 P instability factor: an active maize transposon system associated with the amplification of Tourist-like MITEs and a new superfamily of transposases. Proc. Natl. Acad. Sci. USA 98:12572-12577.
This article has been cited by other articles:
 |
|
 |
|
  V. V. Lia, V. A. Confalonieri, and L. Poggio B Chromosome Polymorphism in Maize Landraces: Adaptive vs. Demographic Hypothesis of Clinal Variation Genetics, October 1, 2007; 177(2): 895 - 904. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 S. Brunner, K. Fengler, M. Morgante, S. Tingey, and A. Rafalski Evolution of DNA Sequence Nonhomologies among Maize Inbreds PLANT CELL, February 1, 2005; 17(2): 343 - 360. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
S.-F. Peng, Y.-P. Lin, and B.-y. Lin Characterization of AFLP Sequences From Regions of Maize B Chromosome Defined by 12 B-10L Translocations Genetics, January 1, 2005; 169(1): 375 - 388. [Abstract] [Full Text] [PDF]
|
|
| ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
X174-HaeIII digest). Molecular weights are shown on the left, and the arrow indicates the position of the plasmid vector.
