PLNT 3140 Introductory Cytogenetics - 2023

Changes in Chromosome Structure I

Changes to chromosome structure are often from the cell's machinery repairing broken chromosomes

Sometimes, usually when the cell is performing regular activities, DNA will experience a break. Most of the time, the cell is able to repair the DNA without any problems, and go about its business. However, some of the time the repair process is completed erroneously, and this gives rise to some kind of mutation. It's hard to talk about each of these classes of mutations separately because all are the result of the cell's machinery. There are four main classes:

• Deletions (deficiencies)
  
• Duplications
• Inversions
• Translocations

What's important to know is that often, a single event produces more than one effect. For example, duplications and deletions often occur together.

Deletions (deficiencies are the irreversible loss of loci

The loss of a chromosome segment from a normal chromosome is called a deletion or deficiency (Df). Deletions can be either intercalary or terminal. Terminal deletions : deletion from the end of a chromosome, not including the centromere. Intercalary deletions - the loss of intercalary or interstitial segments requires two chromosome breaks and rejoining of the flanking chromosomal fragments.

Detection of deletions

FISH with a chromosome 22-specific probe shows that two loci hybridize in a complete copy of chromosome 22, but only one locus lights up in a DiGeorge chromosome 22.

Consequences of deletions

The size of the deletion can vary from the loss of a nucleotide to loss of large chromosome segments, and effects become more serious the more genetic material that is lost. The genetic proof which can distinguish deletions from point mutations is the failure to mutate back to the original form. If the deletion contains a centromere, the resultant acentric fragment will be lost.

Duplications create additional copies of genes, which can be advantageous

A duplication is an extra piece of chromosome segment on the same homologous chromosome (intra-chromosomal) or transposed to one of a nonhomologous chromosomes in the genome (inter-chromosomal). The size of the doubled segment can vary considerably. Duplications are generally more readily tolerated than deletions, often because the cell still has the minimum genetic information.

Types of duplicationsDeletions and duplications are identified both cytologically and molecularly

Depending on the size of the structural change, duplications and deletions may be visible as an unpaired segment at pachytene or by pairing patterns on banded chromosomes. As we have seen above, the deleted segment of chromosome 22 in DiGeorge Syndrome is large enough to be detected under a microscope.

In plants, transmission genetics can also be used to detect changes. The frequency of transmission is variable depending on the viability of the megaspores containing the duplication or deletion. (Remember that megaspores and pollen are haploid, so a deletion means that some genes aren't present at all in gametes carrying the deletion.) The frequency of transmission through the pollen is very low. This is either due to the nonviability of the pollen containing the duplication or deletion or the failure to compete with the normal pollen in fertilization. This is another example of the value of the haploid gametophyte generation in flowering plants. In haploids, deletion of critical genes can greatly reduce viability of gametes. In animals this is of little consequence because little gene expression occurs in gametes, particularly sperm. In maize, dup-del kernels borne on dup-del plants often make up less than 1/3 of the kernels as the dup-del gametes are transmitted almost exclusively through the female- egg cell.

Many chromosomal changes can be detected using FISH.

c) Loss of chromosomes 3 (red arrows) and chrom. 12 (yellow arrows) in colorectal cancer cells.  Interphase chromosomes were hybridized with centromeric probes for chrom. 3 (red spots) and chrom. 12 (yellow spots). Normal diploid nuclei contain two red and two yellow spots each. d) Chromosome translocation. Metaphase plate from neuroblastoma cells was hybridized with chromosome-painting probes specific for chromosome 1 (red) and chromosome 17 (yellow)  revealing a t(1;17) translocation. (Note that chromosome 17 is small, compared to chrom. 1) e) Gene amplification. N-myc oncogene probe (yellow); chromosome 1 (red). Figure shows N-myc amplification (arrow) within chromosome 1.

Gross chromosomal structural change can be the result of many mechanisms

Unequal crossing over creates duplication and deletion combinations

If unequal crossing over occurs between two homologous chromosomes, one chromosome will gain a duplication and the other chromosome will contain a deletion. This is sometimes referred to in the literature as a dup/del for short.

One example is the Bar locus in Drosophila a duplication of 16A segment from region 16A1 to 16A6 of chromosome X which contains 5 bands.   Origin of Bar-double by unequal crossing over in the Bar-locus of the salivary gland X chromosome of Drosophila melanogaster (Redrawn from Morgan et al., 1935. Figure 12. Cold Spring Harbor Press, New York.) If this region is duplicated (Bar mutation), the facets in the eye are reduced in number (homozygous Bar average number of facets is 68) which narrows the normal round eye to a bar-shaped eye. If unequal crossing over occurs between two chromosomes with the Bar duplication, a Bar double is produced which in heterozygous condition reduces the eye facets to 45. Gene expression is stronger when the duplicated genes are in tandem than when they are on separate chromosomes (position effect).

When chromosomal material from another organism is incorporated, it is called an alien chromosome

The presence of alien chromosomes in a genome increases the incidence of chromosome structural changes. This has been observed in wheat (Triticum) lines in which chromosomes from Aegilops were maintained (monosomic). The mechanism appears to be increased incidence of chromosome breakage. There is support for this in the higher number of deletions occurring in the chromosomes with the higher amount of heterochromatin. Endo and colleagues produced numerous deletions by introducing Aegilops cylindrica chromosomes into hexaploid wheat cv. Chinese Spring (see figure below). These deletions have been used for mapping genes on the wheat chromosomes. The number of deletions is greatest in the B-genome and these chromosomes are more heterochromatic than either the A or D genome chromosomes. A series of deletions were detected using Giemsa C-banding technique in chromosome 5B. Observations of deletions and translocations were made in almost half the progeny of a wheat line in which a single species have been added through crossing and selection.

Figure 6.2 A series of deletions in chromosome 5B (normal chromosome 5B extreme left) detected by Giemsa C banding technique. The horizontal line represents the kinetochore. (From Endo, T.R. 1990. Jpn. J. Genet. 65:135-152.)

 The ability of wheat to tolerate such drastic chromosomal deletions is probably due to the fact that it is hexaploid. Most genes are therefore present on three homologous chromosome pairs.

Double stranded breaks can also result in duplications and deletions

Just about any mechanism that can generate double-stranded breaks could result in duplications or deletions. Tandem, reverse tandem and duplications in a different arm are intrachromosomal while displaced duplications are interchromosomal. Diagram of a tandem chromosome duplication. (A) The first two breakpoints (B1 and B2) in the normal chromosome result in (B) a deleted centric chromosome (abc.ghi) (C) and an acentric fragment (def). If the third break occurs in a homologous chromosome (B3 ), the acentric fragment (def) could insert into the partner chromosome resulting in (D) a tandem duplication.

Induced deletions can be generated by irradiating pollen. The pollen is then applied to the stigmas of plants carrying recessive alleles at loci which may be in the region of the induced deficiency. In the progeny a few plants will show the recessive phenotype. (Normally, all F1's should have the dominant phenotype.) This phenotype is an indication that the recessive allele is carried in hemizygous condition due to a deletion in the corresponding loci on the chromosome from the irradiated pollen. Genetically, deletions are indistinguishable from point mutations. Further cytological analysis is required to verify a deficiency.

If these chromosomes are examined cytologically at pachytene, one region of chromosome may show a loop if the deficiency is long enough and located in an interstitial region. A terminal deficiency results in an unpaired end region. X-ray induced deletions occur in either heterochromatin or euchromatin.

Breakage-fusion bridge cycles are the result of inversion or translocation in earlier generations

Duplications and deletions can arise in the progeny of inversion or translocation heterozygotes in which crossing over produces dicentric bridges at meiosis.

Diagram of a reverse tandem duplication in the short arm of chromosome 9 of maize initiating a breakage-fusion-bridge cycle. A terminal inverted tandem repeat (54321) can recombine with the homologous region (12345) if the chromosome with the repeat folds back to allow homologous regions to pair. In this example, only two chromosomes participate in crossing over. Therefore, it is only necessary to pay attention to the two recombinant chromosomes,since the non-recombinants will be unchanged. A crossover between 2 and 3 yields one acentric chromosome containing the "345" region from the distal copy of the repeat.

At anaphase, the other recombinant chromosomes will be dicentric, and will break at anaphase. In this figure, the break is shown to occur between 3 and 4.

In the next cell cycle, the broken ends of each mutant chromosome will be joined, resulting in dicentric chromosomes that will break again at the next anaphase. (Modified from McClintock, 1941).

Ring chromosomes are formed from the fusion of broken chromosome ends

The cellular mechanisms for repairing double-stranded breaks will sometimes cause chromosomes with broken ends to fuse, forming ring chromosomes. Ring chromosomes have been found in several plant species: maize, tobacco, Antirrhinum, Petunia, barley, as well as Drosophila and Homo sapiens. McClintock in papers from 1931 - 1941 studied the behaviour of ring chromosomes in mitosis and meiosis. Ring chromosomes are not stable during cell division and are often eliminated. McClintock developed the breakage-fusion-bridge cycle hypothesis to explain the changes in chromosome size which occurred as a result of the ring chromosomes. If the ring reproduces itself in interphase with no sister strand cross-over in prophase, the ring chromatids can separate in anaphase without difficulty resulting in two equal sized ring-chromosomes, the same size as the original. If sister chromatid exchange occurs, a ring of twice the size is produced with two centromeres

Normally, ring chromosomes can segregate evenly during anaphase. However, if a crossover event occurs, the two daughter chromosomes effectively act as a single, circular, dicentric chromosome. The two centromeres move to opposite poles in anaphase and form an anaphase double bridge. As the centromeres get pulled to opposite poles, the chromosomes break at two places. During telophase, chromosome ends tend to recircularize, in the absence of telomeric sequences. If the break occus as shown below, balanced chromosomes will result, with neither deletions nor insertions.

Breaks are possible at different points along the ring chromosome. If breaks occur asymmetrically, deletions and duplications can result.

Transposable elements move from one chromosomal location to another

Structure

Transposases can cleave and rejoin double-stranded DNA at specific recognition sequences. When these sequences occur in an inverted repeat orientation, a transposase can remove the target sequence from its original location, and insert it elsewhere. The insertion is essentially a reversal of the excision. The Ac element of maize is illustrated at right. It contains a transposase gene flanked by  11bp Terminal Inverted Repeats that are recognized by the transposase. Thus, the Ac element can cause its own transposition.  5' end (C/TAGGGATGAAA)                  3' end (TTTCATCCCTA) Other transposeable elements, such as Ds in maize, are flanked by the required 11bp repeats, but do not encode a transposase. Therefore, Ac must be present at another locus to produce the transposase. In maize lines with Ds but no Ac, Ds elements do not transpose. When an Ac-bearing line is crossed with a Ds line, the Ds elements can transpose. This is referred to as "mobilization of Ds elements". Du C (2011) The complete Ac/Ds transposon family of maize. BMC Genomics 12:588. DOI:   [10.1186/1471-2164-12-588]

Discovery of transposition occurred early in the 20th century, in maize

Ds was first discovered by Barbara McClintock because one of the Ds elements on chromosome 9 contained a sequence that led to frequent chromosome breaks. These breaks always occurred at the same place on chromosome 9, leading to a breakage-fusion-bridge cycle as described above. Surprisingly, when McClintock crossed a Ds line with other maize lines, Ds was mobilized to other loci, causing breaks at those loci.

Activator (Ac) locus, was named by Barbara McClintock for its ability to activate chromosome breakage at another locus; Dissociation, or Ds (a). The two loci are shown here on the same chromosome, but they can be on different chromosomes. Ac is able to promote its own transposition (b), or that of Ds (c) to another site either on the same chromosome or on a different one. Ds cannot move unless Ac is present in the same cell. Ac is an autonomous transposable element and Ds is a nonautonomous element of the same family.

McClintock's Activator-Dissociator system in maize is a possible origin of chromosome structural changes. This is a two gene system: the Ac locus and the Ds locus. When both loci are present, chromosome breakage increases leading to an increase in chromosome structural changes: deletions, duplications, inversions, translocations and ring chromosomes. Ac and Ds are visualised as blocks of heterochromatin that move by transposition between different sites in the chromosomes. Ac does not have a mutating effect alone but promotes the movement of Ds (Ac= activator).

Transposon insertions can cause reversible mutations

Example: The C locus produces an anthocyanin pigment, resulting in colored aleurone ie. wild-type. Insertion of a transposon into C blocks pigment production, resulting in the colorless (c) phenotype. During seed development, excision of the Ds element can result in patches of cells (each progeny of a single cell) expressing the colored phenotype. This is referred to as variegation. If the Ac locus moves to a position adjacent to Ds, it promotes its movement away from the C locus during the development of the kernel and the locus reverts to normal expression. The larger the number of Ac factors present, the greater expression of variegation in the tissue. Larger coloured patches are formed when Ds is transposed early in development; smaller patches when Ds is transposed late.

MUTATION OF C LOCUS,a gene required for synthesis of a purple pigment in the aleurone (a), takes place when Ds moves into the locus (b). The mutation disables the gene, the pigment is not made and the aleurone is colorless. If Ac is present in the genome, however, it promotes the transposition of Ds away from the locus in some cells during kernel development (c). The mutation reverts when the element leaves, giving rise to cells in which the C locus is functional. Each such cell gives rise in turn to a pigmented sector in the aleurone.