Maize Chromosome Abnormalities and Breakage-Fusion-Bridge Cycles in Callus Cultures

The maize karyotype was first characterized by the observation of pachytene chromosomes. The somatic chromosomes were identified by C-banding and FISH with repetitive DNA sequences. C-banding was useful for the identification of chromosome abnormalities in callus cultures. In the present review, we focus on the involvement of heterochromatic knobs on the occurrence of chromosome abnormalities in callus cultures. In a previous work we detected anaphase bridges resulting from delayed chromatid separation at knob regions and typical bridges derived from dicentric chromatids in cultures. The analysis of altered chromosomes showed they were derived from a chromatid-type breakage-fusion-bridge (BFB) cycle. Fluorescent in situ hybridization (FISH) showed signals of telomere sequences in the broken chromosome arm, thus giving evidence of de novo telomere formation on the broken chromosome end. Further observations of long- and short-term cultures have shown the presence of chromosome alterations derived from BFB cycles followed by chromosome healing. Additionally, the occurrence of unequal crossing over in a knob region was observed in callus culture. These results are of interest for studies on the mechanisms of chromosome alterations during evolution.

Mechanisms generating cancer genome complexity from a single cell division error

The chromosome breakage-fusion-bridge (BFB) cycle is a mutational process that produces gene amplification and genome instability. Signatures of BFB cycles can be observed in cancer genomes alongside chromothripsis, another catastrophic mutational phenomenon. We explain this association by elucidating a mutational cascade that is triggered by a single cell division error—chromosome bridge formation—that rapidly increases genomic complexity. We show that actomyosin forces are required for initial bridge breakage. Chromothripsis accumulates, beginning with aberrant interphase replication of bridge DNA. A subsequent burst of DNA replication in the next mitosis generates extensive DNA damage. During this second cell division, broken bridge chromosomes frequently missegregate and form micronuclei, promoting additional chromothripsis. We propose that iterations of this mutational cascade generate the continuing evolution and subclonal heterogeneity characteristic of many human cancers.

Mechanisms Generating Cancer Genome Complexity From A Single Cell Division Error

ABSTRACTThe chromosome breakage-fusion-bridge (BFB) cycle is a mutational process that produces gene amplification and genome instability. Signatures of BFB cycles can be observed in cancer genomes with chromothripsis, another catastrophic mutational process. Here, we explain this association by identifying a mutational cascade downstream of chromosome bridge formation that generates increasing amounts of chromothripsis. We uncover a new role for actomyosin forces in bridge breakage and mutagenesis. Chromothripsis then accumulates starting with aberrant interphase replication of bridge DNA, followed by an unexpected burst of mitotic DNA replication, generating extensive DNA damage. Bridge formation also disrupts the centromeric epigenetic mark, leading to micronucleus formation that itself promotes chromothripsis. We show that this mutational cascade generates the continuing evolution and sub-clonal heterogeneity characteristic of many human cancers.

Alterations in Heterochromatic Knobs in Maize Callus Culture by Breakage-Fusion-Bridge Cycle and Unequal Crossing Over

The meiotic and mitotic behavior of regenerated plants derived from a long-term callus culture, designated 12-F, was analyzed. This culture was heterozygous for an amplification of the heterochromatic knob on the long arm of chromosome 7 (K7L). We aimed to investigate if the amplification resulted from a breakage-fusion-bridge (BFB) cycle or from unequal sister chromatid recombination. Therefore, C-banded mitotic metaphases and pachytene, diakinesis, and anaphase I of regenerated plants were analyzed. Additionally, the occurrence of alterations in K7L was investigated in C-banded metaphases from short-term callus cultures derived from lines related to the donor genotype of the 12-F culture. As a result, plants homozygous and heterozygous for the amplification were detected. Meiosis was normal with few abnormalities, such as a low frequency of univalents at diakinesis. In the callus cultures a chromosome 7 with knobs of different sizes in the sister chromatids was detected and interpreted as a result of unequal crossing over. Other chromosomal alterations were consistent with the occurrence of BFB cycles. The finding of unequal crossing over in the cultures supports the conclusion that the amplification in the culture 12-F would be derived from this mechanism. If the amplification was derived from a BFB cycle, the terminal euchromatic segment between knob and the telomere would be deleted, and possibly, homozygous plants would not be viable.

A Novel Mechanism for Intrachromosomal Gene Amplification in Multiple Myeloma: 1q12 Pericentromeric Heterochromatin Mediates Breakage- Fusion-Bridge Cycles of the 1q12~23 Amplicon

Gene amplification is a marked copy number (CN) increase in a restricted region of a chromosome arm, and is a mechanism for acquired drug resistance and oncogene activation. In multiple myeloma (MM), recent studies utilizing gene expression profiling, high-resolution array comparative genomic hybridization (aCGH), and global single nucleotide polymorphism arrays have all provided molecular evidence for the importance of genes in the proximal 1q. In fact, the aCGH studies have defined a particularly notable region of proximal 1q with a marked enrichment of genes which spans approximately 143–158 Mb corresponding to a 1q21-23 amplicon. The finding of high CNs of CKS1B and other genes in the 1q21~23 amplicon have been associated with disease progression and poor prognosis in MM. To investigate the possible mechanisms for focal gene amplification in this region, we identified 70 patients showing gain of 1q by G-banding. We then performed a comprehensive metaphase analysis utilizing fluorescence in situ hybridization (FISH) and spectral karyotyping to further characterize the karyotypic aberrations. Six FISH probes spanning the 1q12~23 region were used, including among others, probes for satII/III at 1q12 in the pericentromeric region to demark a proximal boundary, CKS1B at 1q21 to demark a point near the center of the amplicon, and RP11-57D16 at 1q25.2 to demark a distal boundary. In seven patients (10%) evidence for at least one breakage-fusion-bridge (BFB) cycle involving 1q12~23 in an inverted duplication was found. Strikingly, in three patients (4%) extended ladder-like structures of 1q12~23 inverted duplications were identified with up to 18 copies of CKS1B in contiguous duplicated regions. In these patients, the “amplicon ladders” showed the progression from two, to four, to eight copies, of CKS1B in different cells. Several key structures that are predicted intermediates in BFB cycles were observed in these patients, including equally spaced organization of amplicons, isodicentric chromosomes 1 with a clustering of breakpoints in the duplicated 1q12 pericentromeric regions, inverted repeat organization of amplicons along the same chromosome arm, and deletion of sequences distal to the amplified region. In these patients, site-specific breakage in the 1q12 pericentromeric heterochromatin mediated the organization of the BFB cycles by ultimately bracketing both the proximal and distal boundaries of the amplicon. Two chromosomal mechanisms have been described for the initiation of BFB cycles in tumor cells: the telomere fusion model in which chromosome breakage is induced by shortened or dysfunctional telomeres, and the alternative mechanism whereby replication stress and/or delay induces chromosome breakage in common fragile sites (CFS). The site-specific chromosome breakage in the satII/III sequences in the different phases of the intermediate chromosome structures identified here strongly support the concept that the BFB cycles were initiated by a CFS mechanism. A possible candidate fragile site in the 1q12 pericentromeric region is FRA1J, a known 5-azacytidine fragile site. It is well established that 5-azacytidine is a methyl transferase inhibitor which induces hypomethylation of the 1q12 pericentromeric DNA in metaphase chromosomes resulting in a characteristic pattern of pericentromeric decondensation and chromosome breakage. Our findings provide the first evidence for the BFB cycle mechanism of gene amplification in MM, and that the amplification process is induced by the secondary activation of a CFS in the 1q12 pericentromeric heterochromatin.

Construction of midget chromosomes in wheat

To test the usefulness of breakage–fusion–bridge (BFB) cycles in generating new chromosome aberrations in bread wheat (Triticum aestivum L.) and to extend the range of aberrations available, a series of midget chromosomes was produced from the long arm of chromosome 1B. Using a reverse tandem duplication initiated chromatid type BFB cycle, the 1BL arm was broken and fused with centromeres of either chromosome 5BL or 1RS to form dicentric chromosomes. The 1R and 5B centromeres were broken by centric misdivision. Among the progenies of plants with dicentric chromosomes, two classes of monocentric chromosomes were selected: deficient chromosomes 1B and chromosomes that had 1RS or 5BL for one arm and various fragments of 1BL for the other arm. Following centric misdivision of these monocentrics, midget chromosomes 1BL were isolated: deficient and deletion telocentrics and telocentrics derived from interstitial regions of 1BL. By chance, one deficient chromosome 1BS and one deletion chromosome 1BS were identified in unrelated lines of the same wheat. Following centric misdivision of these chromosomes, two midget chromosomes covering the whole of 1BS were added to the set.Key words: breakage–fusion–bridge cycle, centric misdivision, chromosome aberrations.

Chromatid and chromosome type breakage-fusion-bridge cycles in wheat (Triticum aestivum L.).

During the development of disomic additions of rye (Secale cereale L.) chromosomes to wheat (Triticum aestivum L.), two reverse tandem duplications on wheat chromosomes 3D and 4A were isolated. By virtue of their meiotic pairing, the reverse tandem duplications initiated the chromatid type of the breakage-fusion-bridge (BFB) cycle. This BFB cycle continued through pollen mitoses and in the early endosperm divisions, but no clear evidence of its presence in embryo mitoses was found. The chromosome type of BFB cycle was initiated by fusion of two broken chromosome ends resulting in a dicentric or a ring chromosome. Chromosome type BFB cycles were detected in embryo mitoses and in root tips, but they did not persist until the next meiosis and were not transmitted to the progeny. Active BFB cycles induced breakage of other wheat chromosomes that resulted in additional reverse tandem duplications and dicentric and ring chromosomes. Four loci, on chromosome arms 2BS, 3DS, 4AL, and most likely on 7DL, were particularly susceptible to breakage. The BFB cycles produced high frequency of variegation for pigmentation of the aleurone layer of kernels and somatic chimeras for a morphological marker. With the exception of low mutation rate, the observed phenomena are consistent with the activity of a Ds-like element. However, it is not clear whether such an element, if indeed present, was of wheat or rye origin.