Summary of Paper: Comparative Genome Organization in Plants: From Sequence
and Markers to Chromatin and Chromosomes
I.
The Linear DNA Sequence
Complete sequencing of various plant and animal genomes has affirmed the
following: 1) the double helical DNA model is continuous from telomere to
telomere, and 2) no other bases beside adenine, guanine, thymine, and cytosine are
present.
Arabidopsis was the first plant genome to be completely sequenced because of
its small size (130-140Mbp). Its genome contained nearly 25,000 genes. However,
the small size of the genome does not correlate to a smaller number of genes. In fact,
most of the genes found in plant species with much larger genomes (pines have
~23,000Mbp) were also found in Arabidopsis. This is because the amount of highly
repetitive sequences (non coding regions) found in Arabidopsis was much less than
what is found in the genomes of larger plants.
II.
Repetitive DNA Sequences and the Large-Scale Organization of the
Chromosome
Most of the DNA code in humans and Arabidopsis that has yet to be
sequenced lies within regions of highly repeated DNA. These repeated stretches
make it hard for accurate reconstruction of the genetic sequence since they are over
several hundred base pairs long and fall outside the limits of typical sequencing
methods. Therefore, the logical ordering of sequenced contigs is very difficult due to
the high degree of repetition. It is interesting to note that some genes have been
discovered in the highly repetitive regions around the centromere. However, there is
no data to support that these repeated sequences play a role in gene expression or
whether they simply contribute to the overall size of the genome.
Many of the repeated sequences are highly conserved in all eukaryotic
species. Conversely, some repeated sequences show a great deal of variation and can
be different within two individuals of the same species. Therefore, the study of
differences between these highly conserved sequences between species can offer
information on the evolution of their respective genomes. Some more common
classes of repeated sequences are tandem repeats, retroelements, and telomeric or
rDNA units.
In situ hybridization can be used to provide information about the location of
these repeated sequences along the chromosome. Using denatured chromosomes and
labeled probes complementary to the repeated sequence in question, hybridization
can be conducted on the surface of a slide and the pairing can be visualized with a
microscope. The article shows some very nice pictures of this procedure that provide
information on where certain repeated sequences can be found on the chromosomes.
Various studies using in situ hybridization have been able to show where the majority
of these repeat groups occur on chromosomes. Other advantages of this technique are
its ability to scan for DNA from viruses and mitochondria without prior sequence
knowledge of the flanking regions.
III.
R-DNA
The r-DNA subunits are repeated hundreds or thousands of times and may
make up as much as 10% of the genome. The 45S rDNA loci consist of the 18S,
5.8S, and 26S rRNA genes (the S nomenclature is based on the unit’s sedimentation
rate in a centrifuge) that are reach approximately 10kb long in plants. These units
assume specific positions on chromosomes and can be subsequently used as markers
for chromosome identification. Due to the high degree of conservation of these
sequences, probes that have been isolated from one species can be used to identify the
similar unit in most other eukaryotic species.
Looking at changes in the chromosomal distributions of these subunits can
provide incite about rates of speciation and evolutionary trends.
IV.
Telomeres and Centromeres
Telomeres are highly conserved regions of DNA near the ends of
chromosomes. It is thought that conservation of this region is vital to the DNA
replication process. The region is constructed from a highly repeated short DNA
sequence (TTTAGGG). It is very species specific, but these repeats can be hundreds
of units long in many eukaryotes. Even within species, the number of telomeric
repeats can be different from one chromosome to another. Telomeres require a
special telomerase enzyme to aid in their replication. This telomerase not only
provides the RNA template for telomere replication, but it also serves as a substrate
to stabilize and repair damaged chromosomes.
Centromeres are also very highly conserved regions of DNA within the
chromosome. Centromeres provide the location for proper spindle attachment during
mitosis and meiosis. Therefore, it is vital for this region to remain conserved for
proper cell replication and gamete formation. It is believed that the centromeres are
made up in large part by a repeated 180bp sequence. This repeat is very prevalent
and can account for a large portion of the total genomic DNA of a given species
(0.3% in humans and 3% in Arabidopsis). Sequencing within the centromeric region
of Arabidopsis has revealed a large number of inactive mobile elements. One study
has been conducted to reveal the DNA sequences responsible for proper centromere
function. It was found that centromeres contain a central repetitive core, flanked by
moderately repetitive DNA with a low recombination rate, which is then flanked by
regions of mobile elements with normal rates of recombination. However, it is not
believed that the repeats alone are responsible for centromere function since some of
them are even more abundant in DNA outside of the centromere.
V.
Transposable Elements and Retroelements
Class I transposable elements (retroelements) are pieces of DNA which can
replicate and move from one region of the genome to another. These elements can
makeup half of the genome’s nuclear DNA and usually include 2-3 open reading
frames (ORFs) and are nearly 5kb in length. Depending on the species, transposable
elements can be found pretty much anywhere in the genome. In general, they are not
as prevalent in the rDNA and centromeric regions of the genome where there is a lot
of tandemly repeated DNA sequences. However, in Arabidopsis there is a high
proportion of transposons in the centromeric region of the chromosomes, where a
very few genes are located for disruption by retrotransposons.
Retroelements also play an active role in gene regulation and mutation.
Depending on where they insert themselves in a specific gene sequence, the
expression of that gene may be mutated or knocked out completely. In Drosophila,
retroelements are believed to account for nearly 80% of the mutations uncovered.
Retroelements may also serve in species evolution by altering gene functions. For
example, a retroelement may carry a promoter which, when inserted, activates a gene
that was previously unexpressed. Similarly, the element may alter the function of an
existing gene that leads to varied expression.
VI.
Simple Sequence Repeats
SSRs are series of repeated sequences. Allelic discrimination can be achieved if
the number of SSRs at a given site is different between alleles of a given gene.
Primers that flank both sides of the SSR can be synthesized for PCR amplification of
the repeated region. Variation can be visualized through agarose gel separation and
ethidium bromide staining.
Similarity within SSR sites of related species can provide a tool for estimating the
genetic distance between the two. This type of information is very valuable to those
studying the evolution of plant and animal species.
VII.
Methylation
DNA methylation is a process in which cytosine residues, of CG doublets in
particular, are methylated in the 5-position of the cytidine ring. Heavily methylated
regions of DNA are usually transcriptionally inactive. Methylation near genes or
gene promoter regions has resulted in decreased levels of gene expression.
Therefore, methylation has been shown to be an effective method of gene regulation.
Methylation occurs following DNA replication on the newly synthesized
DNA strand at specific nucleotide sequence sites. Methylation patterns are copied
from the parent DNA strand using maintenance methylases. However, in plants, the
methylation process does not seem to mimic that of the parent strand. Instead,
methylation appears to be conducted completely anew with no regard to the parent
strand. Studies have also shown that the amount of methylation appears to decrease
after repeated cycles of DNA replication. This results in regions of hemimethylated
DNA and ultimately in regions of unmethylated DNA. Different DNA methylase
enzymes have also been found which could be tissue specific or specific to a pattern
of methylation (symmetrical or asymmetrical). A demthylase enzyme has been
discovered which may have implications as a new mechanism for gene
activation/regulation.
VIII. Structure and Packaging of Linear DNA into Chromosomes
The higher order packaging of inactive DNA in the nucleosomes is not a static
system. This organization is found to be highly variable in both plants and animals.
In fact, in two closely related species such as rye and wheat, significant variation has
been seen in the length of linker DNA between telomeres and expected repetitive
DNA sequences in the chromatin material. These differences were uncovered using
micrococcal nuclease (MNase) to digest the linker DNA that connects nucleosome
particles together. In fact the sheer difference in sensitivity to MNase between these
closely related species also denotes a difference in DNA packaging.
Repetitive DNA sequences such as tandem arrays show a characteristic
arrangement around the nucleosomes and most likely play a stabilization role in DNA
packaging and chromatin condensation. Repeated adenine sequences of 4-6 bases in
phase with the helix promote bending that subsequently aids in nucleosome assembly.
Any DNA remaining after the repeats have been packaged can easily be fit into the
nucleosome.
IX.
Chromatin Remodeling and Histone Acetylation
Histone acetylation and chromatin remodeling play a similar role in gene
regulation as the DNA methylation of cytosine residues. Expression of gene products
may be altered or silenced depending on the extent of remodeling and acetylation. It
has been proposed that regions in the human genome where chromatin remodeling
occurs at a high frequency between mitosis and meiosis could be hot spots for
recombination.
X.
Packaging of Nuclear DNA
In contrast to earlier claims that the nucleus was an unorganized mess, there is
a preponderance of evidence to prove that the nucleus is actually highly organized
and functional. It has been shown that centromeres and telomeres occupy specific
parts of the nucleus in many species. Furthermore, the nuclear material can be
dissected into unique parts including chromosomes, euchromatic and heterochromatic
regions, the nucleolus, and regions of active DNA synthesis. However, the presence
of nuclear matrices, like nuclear scaffolds, cages, and compartments is
argumentative. Most of these documented structures have appeared in laboratory
situations that do not reflect cellular conditions in vivo.
The haploid chromosome consists of unique transcription subunits that
assume a specific formation that only the homologous chromosome could recognize
and pair with during meiosis. This “best fit” mechanism ensures proper alignment of
all homologous chromosomes.
The strongest evidence for nuclear compartmentalization comes from the
nucleoli. These are spherical units of the nucleus that have no boundaries. The
composition of nucleoli is very different from other nuclear components and
depending on cell type, may assume different locations within the nucleus.
XI.
Genomics, Chromosome, Evolution, and the Nucleus
The current depth of understanding concerning the nucleus and chromosomes
is far greater than it was only a few years ago. This will aid biologists in developing
new methods for gene cloning, evolutionary studies, gene transfer, and gene
expression. Future plant breeding efforts can focus on controlling plant evolution in a
way that conserves both biodiversity and the environment while still introducing new
traits of agronomic importance. Studies have shown that advantageous genetic traits
quickly move towards fixation within a species. Increased understanding of the
processes that lead to genetic variation will provide more avenues for researchers to
use. Furthermore, understanding the evolutionary processes that have already
occurred within the plant genome will enable breeders to understand the
consequences of any future changes. Finally, new technologies such as in silico
analyses, high-throughput DNA and protein studies, digital imaging methods, and in
vivo methods for studying the dynamic chromosome structures and interactions will
make sure that our understanding of the genome continues to increase.