DNA rearrangement is a phenomenon that involves the alteration of the basic genome sequences and results in diverse activities such as the regulation of gene expression, development, generation of antibody diversity etc. The role of DNA rearrangements is observed to be especially predominant in lower eukaryotes, which usually takes place in the somatic cells and the germlines remain unaltered. However, even the reproductive cycles (germlines) of some organisms have been shown to involve the loss of whole chromosomes or sets of chromosomes (such as observed in ciliated protozoans, nematodes, crustaceans etc). There may be two possibilities by which the gene rearrangements can control the gene expression; either new genes may be formed by the rearrangement that are needed for the expression in particular circumstances such as in the case of immunoglobulins or the rearrangement may switch the expression from a pre-existing gene to another. The following are some of the examples where the DNA rearrangement plays a major role-
Mating type switching in yeast is a gene conversion event:
Saccharomyces cerevisiae is a unicellular eukaryote that shows a simple type of sexual differentiation, where haploid and diploid states are maintained and the haploid cell can be either of a’ or ‘ mating type. Mating takes place only between the haploid cells of opposite mating type i.e the haploid cell with mating type-a can mate only with mating type- or vice versa. The -cells produce a pheromone called -factor’ that gives the signal, indicating the presence of an -cell to the neighboring a-cells. In the same way, the a-cells produce an a-factor to which the -cells repsond. Therefore, this type of response shown by the haploid cells to the pheromones of only the opposite mating type brings about the selective mating between a and cells, but not between the same mating types. These differences between a and cells are because of a set of genes that are actively expressed or repressed. a-cells express genes that encode a-factor and produce Ste2 (a cell surface receptor for cells) and at the same time the gene that governs for -type is repressed. The cells express the genes that encode for -factor and produce Ste3 (a cell surface receptor for a-cells) and at the same time represses the expression of the gene that governs the a-type trait.
The transcriptional activation and repression that gives rise to the mating types (a and alpha) is because of the two alleles of a locus called MAT. The two alleles of MAT locus are called MATa and MAT. MATa encodes a1 and a2 genes that define the a-cells by triggering the transcription of a-specific transcriptional event that include Ste2 expression and Ste3 repression. MAT allele encodes 1 and 2 genes which define the -cells by promoting the Ste3 expression and at the same time Ste2 repression (-speicific event).
The switching between the mating types is attained by the replacement of the information present at the MAT locus. The a-cell is switched to an -cell by the replacement of MATa allele with the MAT allele and vice versa. The presence of HML (Hidden MAT Left) and HMR (Hidden MAT Right), which are the extra silenced copies of MAT and MATa respectively, make the replacement of one allele for the other. These HML and HMR are also referred as the silent mating casettes, as the information present in these sequences is read into the active MAT locus. Since these additional copies (HML and HMR) of mating type are not expressed, they do not interfere with the function of MAT locus (MATa and MAT) thereby, allowing a haploid cell with MATa and a silenced copy of MAT (present at HML) to still remain as an a-cell and vice versa. Therefore, only the allele at the active MAT locus is expressed influencing the cell behaviour.
The mechanism of mating type switching is a gene conversion event which is brought about by HO gene. This HO gene is a haploid specific gene that is tightly regulated during the G1 phase of the cell cycle. HO gene encodes a DNA endonuclease that cleaves only the MAT locus because of the sequence specificity of the HO endonuclease. The cleavage in the MAT locus by HO endonuclease attracts exonucleases that can start cutting the DNA from the ends and degrade the DNA on both the sides of the HO endonuclease cut site thereby, eliminating the DNA which encoded the MAT allele. The gap resulted from this degradation is repaired by copying the sequence information present in either HML or HMR, thus filling in a new allele of either MATa or MAT gene.
In nematodes, the DNA rearrangement controls the development:
In parascaris and Ascaris, a developmentally controlled genome rearrangement called chromatin diminution has been reported that results in quantitative and qualitative differences in the DNA content between the somatic and the germline cells. Chromatin diminution is a complex mechanism involving chromosomal breakage, addition of new telomere and degradation of DNA in all the presomatic cells. The whole process is specific in terms of the developmental timing and elimination of chromosomal regions and serves as an alternative way of gene regulation. Some studies have been shown a link between the partial genome duplication and the process of chromatin diminution, which in turn maintains the genetic balance in the somatic cells and at the same time, allows a selective advantage to the germline cells of nematodes.
Parascaris shows only two large chromosomes in the germline cells, which are distributed to the daughter cells designated as S1 and P1 during the first cleavage division of the fertilized egg. This process is again repeated in the ventral P1 cells during the second cleavage division to give rise to the two four-cell staged cells called S2 and P2. The central portions of the two large chromosomes in the dorsal S1 cells disintegrate into many small chromosomes which are eventually distributed to the two daughter nuclei S1a and S1b, while the distal heterochromatic ends remain in the cytoplasm where they ultimately degrade. This results in less chromatin in the nuclei of S1a and S1b cells than the nuclei of S2 and P2 cells. Chromatin diminution is repeated further four more times during the subsequent rounds of cell divisions (S2 to S5 cells).
The chromosomal breakage takes place in the presomatic cells at specific regions on the chromosome called CBRs (Chromosomal Breakage Regions), which is followed by the addition of 2-4kb of telomeric TTAGGC sequences de novo. The addition of telomere occurs at many different sites which are scattered throughout the region of 2-3kb length. The CBRs represent unique sequences rather than cross-hybridization to each other or to any other genomic DNA fragment. 1-4bp of sequences that are homologous to the telomeric repeats at the junction regions of all the telomere addition sites are known to provide limited homology for telomerase priming. The role of telomerase-mediated healing has also been well documented in the programmed chromosomal rearrangements that occur during the development of the ciliate macronuclei.
In Parascaris, 79-88% of the total nuclear DNA is eliminated during the chromatin diminution and the eliminated DNA is observed to be enriched in repetitive sequences. Especially, the germ-line-speicific chromatin is enriched in highly repetitive satellite DNA sequences, which are mostly eliminated from the presomatic cells. In Ascaris, the middle-repititive retrotransposon-like elements called Tas, also become partially eliminated from the presomatic cells during the chromatin diminution. The 7.6kb long Tas element is present in appoximately 50 copies per haploid germ line genome and exists in two structural variants called Tas-1 and Tas-2. About 25% of the Tas-1 elements and almost all the Tas-2 elements are eliminated from the somatic genomes.
Role of DNA rearrangement in Antibody diversity:
In humans and other vertebrates, a specific DNA rearrangement phenomenon is documented that results in the antibody diversity for the recognition of diverse foreign antigens and is termed as V(D)J rearrangement. The immunoglobulin protein molecules (antibodies) in humans are made up of a heavy and a light chain, each showing two distinct domains called constant (C) and variable (V) regions that are encoded by three types of genes namely heavy chain gene, light chain kappa gene and a light chain lambda gene. The immunoglobulin heavy chain encoding region contains 200 Variable (V) genes, 12 Diversity (D) genes, and 4 Joining (J) genes. Similarly, the light chain encoding regions show numerous V and J genes, but no D genes. By the DNA rearrangement of these regional genes it is possible to generate an antibody diversity of greater than 107 possible combinations (200 x 12 x 4 = 9600, in addition to the 1000 possible light chain combinations).
In the developing B cell, the first recombination takes place between a D and a J gene of the heavy chain locus, thus the DNA that exists between these two genes will be deleted. This is termed as D-J recombination and is followed by the joining of one V gene, to the upstream of the newly formed DJ complex thereby resulting in the formation of a V(D)J rearranged gene. This leads to the deletion of all the other genes from the genome that are located in between the V and the D segments of the new V(D)J gene. The primary RNA transcript that is not yet spliced shows the regions containing the V(D)J region of the heavy chain and both the constant (C and C) chains which looks like V-D-J- C-C. Similarly, the kappa and the lambda chains of the immunoglobulin light chain loci rearrange, except the fact that light chains do not show a D segment. The translation of the spliced mRNA for kappa or lambda chains results in the formation of Ig and Ig light chain proteins. The formation of a complete immunoglobulin molecule is brought about by the assembly of these heavy and the light chains.
Specific sequence called the Recombinational Signal Sequences (RSS) flank the V,D and J genes and these signal sequences are recognized by a group of enzymes known as VDJ recombinase. An RSS consists of seven conserved nucleotides called a heptamer that occurs next to the gene encoding sequence, followed by a 12-23 unconserved stretch of nucleotides called space, followed by again a conserved 9 base pair sequence called a nonamer. The RSSs sequences flank on the downstream of a V region and the upstream of the J region, thus forming the sites that involve in the joining.
References:
1. Fritz Muller, Vincent Bernard and Heinz Tobler. Chromatin diminution in nematodes. BioEssays Vol.18 no.2 133.
2. Gene VIII by Benjamin Lewin, Pearson, Prentice Hall, NJ.
3. Matthew P Scott, Paul Matsudaira, Harvey Lodish, James Darnell, Lawrence Zipursky, Chris A Kaiser, Arnold Berk, Monty Krieger (2004). Molecular Cell Biology, Fifth Edition. WH Freeman and Col, NY
4. Leland H Hartwell, Leroy Hood, Michael L. Goldberg, Ann E. Reynolds, Lee M. Silver, Ruth C. Veres (Genetics: From Genes to Genomes, copyright 2000). Chapter 24, Evolution at the molecular level; pages 805-807.ISBN 0-07-540923-2
