Lectures related to Chromatin structure (Anna-Lisa Paul)
From HOS 3370 Introduction to Plant Molecular Biology; University of Florida, Robert J. Ferl
HOS 3370 Introduction to Plant Molecular Biology
The structure of plant genomes
Packaging
Packaging the genome into the nucleus.
The genome size of corn (an average, but delightful plant) is about 10 9 base pairs. 109 base pairs of DNA is
about 1 meter long, but only 2nm (2 x 10-9 meter!) thick. So the trick is getting a meter’s worth of a very
thin, actually kind of stiff, molecule into a nucleus less than 10 um in diameter. This would be like packing
100km of 6lb test mono-filament fishing line (0.2mm thick) into a large beach ball. Unwind a 1050 yard
spool (just 1 km) with a four-year-old in your living room and you will appreciate the magnitude of the
problem...
How is this packaging accomplished?
The DNA of the genome is associated with proteins, and this complex is referred to as “chromatin”.
Chromatin proteins (which exist in great variety) do three things: neutralize the highly negative charged
DNA molecule rendering it more flexible, facilitate condensation by creating structures that coil, fold and
loop the genome, and creating localized structures in and around genes that influence transcriptional
readiness.
The compaction of the genome proceeds through several “orders” of chromatin
structure
First order chromatin structure condenses the 2nm DNA molecule by wrapping around “beads” of basic
histone proteins (each wrapped bead is a “nucleosome”). This 11nm fiber is the nucleosome array and is
often referred to as “beads-on-a-string”.
Second order chromatin further condenses the genome by coiling the 11nm fiber into a 30nm fiber, or
solenoid structure, sort of a coiled, coil that has 6 nucleosomes per wrap of the coil.
Everything else is lumped under the heading “higher order” chromatin. All higher order structures start
with a coiling, or looping of the 30nm fiber anchored by specialized chromatin proteins. This coiling,
looping and folding of structures composed of the 30nm fiber can proceed all the way to a fully condensed
metaphase chromosome that can approach 1um in thickness!
Specialized structures facilitate genome organization
The folding, coiling and looping of the 30nm chromatin fiber that further compacts the genome within the
nucleus is mediated by specialized groups of proteins that play a variety of roles. Higher order chromatin
structure is the least well understood feature of the genome, but it is generally agreed that the genome is
organized into topologically independent supercoiled loops of the 30nm fiber. The anchor points, or “loop
basements” that partition the genome into loops are often specialized regions of DNA/protein interactions
that tack the base of the loop to a protein “matrix” that fills much of the interior of the nucleus. There
appears to be two types of matrix attachment points; those that serve to package and organize the genome
on a large scale (loop basement attachment sites - LBARs) and those that partition the genome into smaller,
often gene-sized loops that appear to play a role in the regulation of the genome (matrix attachment regions
- MARs).
LBARs anchor large sections of the genome.
Genomic DNA seems to naturally “fragment” into large, but consistent in size, sections during isolation.
This suggests that there is a feature of the genome that is a little more fragile every 50kb or so...
Experiments in nuclei with specialized reagents that preferentially cleave chromatin at the point where it is
attached to the nuclear matrix indicate that the genome is structurally anchored to the matrix every 50kb or
Chromatin lectures for HOS 3370 – A-L. Paul
Lectures related to Chromatin structure (Anna-Lisa Paul)
From HOS 3370 Introduction to Plant Molecular Biology; University of Florida, Robert J. Ferl
so... Recent experiments in plants indicate two interesting features of these large structural loops: first, the
median loop size can be variable among plant species (e.g. median size for maize is about 45kb and for
arabidopsis is about 25kb) and second, the genome is not packaged by means of a random gathering into
indiscriminate length loops, but rather, that the genome is gathered into specific domains and that a gene
consistently occupies a discrete physical section of the genome.
MARs anchor “functional” domains around genes.
Matrix Attachment Regions (you will also see “Scaffold Attachment Region” or SAR in the literature) are
specialize sequences in the genome that are recognized by nuclear matrix proteins and associated matrix
attachment factors. The MAR sequence is typically AT rich, several hundred bp long and contains
topoisomerase II recognition sequences embedded within it. The latter is interesting as topo II is an
enzyme that functions in relieving torsional stress in DNA by nicking it, then rejoining the strand after.
Think about what happens when you twist up a rubber band - it gets all knotted up and condensed with
respect to its original shape - you can return it to its original, unsupercoiled state by releasing it at its anchor
(your fingers). Topo II presumably does this to supercoiled loops in chromatin. MARs often flank genes,
and appear play a role in “preparing” genes for transcription by creating a structure that is more accessible
to the transcriptional machinery (transcriptional machinery - the suite of enzymes and other proteins
required for transcription). MARs also appear to set genes apart from one another by creating independent
topological domains. This means that although one section of the genome might be somewhat decondensed and ready for transcription, the adjacent section of the genome can remain condensed and
quiescent.
Specialized structures facilitate gene regulation
First order chromatin structures plays a direct role in gene regulation by creating surface features that are
recognized by the transcriptional machinery, by bringing distant sequence elements together with localized
bends and by managing the local sections of decondensed chromatin necessary for transcriptional
activation.
Nucleosome free regions of a gene are hypersensitive to nucleases.
Before a gene can be transcribed, certain features of the regulatory portion of the gene (the gene promoter,
situated in the 5' flanking region of the gene - more about this in subsequent lectures..) must be
“uncovered” before the transcriptional machinery can be recruited to the gene and transcription can
proceed. These features are generally in the form of sequence elements, or as structural perturbations of the
promoter DNA. Open regions are typically nucleosome free, and are thereby more sensitive to digestion by
exogenous (exogenous - anything added from the outside) nucleases like DNase I . DNase I preferentially
nicks DNA anywhere the DNA is not protected by associated proteins. Thus, condensed chromatin is less
sensitive to digestion, whereas localized open regions are hypersensitive to digestion. evidence of DNase I
hypersensitive sites within a gene promoter is a hallmark of a transcriptionally active gene.
Non-nucleosomal proteins associated with chromatin also influence promoter
structure.
The two major types of non-nucleosomal (non-histone) proteins that also play a role in promoter structure
and in gene activation are the histone-modifying proteins and the transcription factors. Histone modifying
proteins interact with histones of the nucleosome to create more open structures. Transcription factors
interact with specific sequence elements in the promoter. Some transcription factors create localized
chromatin changes by introducing specialized bends, kinks and other structural features used for “flagging
down” RNA polymerase. Others are actually part of the transcriptional machinery. The varied roles of
transcription factors be explored in later lectures.
(see pages 250-254 in Alberts et al.; 96-97 in Foskett;
web sites: http://cellbio.utmb.edu/cellbio/nucleus.htm
http://www.average.org/~pruss/Nucleosomes/othersites.html
Chromatin lectures for HOS 3370 – A-L. Paul
Lectures related to Chromatin structure (Anna-Lisa Paul)
From HOS 3370 Introduction to Plant Molecular Biology; University of Florida, Robert J. Ferl
HOS 3370 Introduction to Plant Molecular Biology
The nuclear genome
Nuclear genome organization
Genome size
The size of the nuclear genome varies among organisms. The amount of DNA in a haploid cell is referred
to as the C value. When the genome size of an organism is given, it is usually the haploid C value that is
given. Plants have C values ranging from 107 to 1011 base pairs (bp) coding for 15 thousand to 60 thousand
genes.
The genome size is roughly correlated with organism
complexity.
In other words, humans have larger genomes than most insects and insects have larger genomes than fungi.
Although organism complexity roughly correlates with genome size, the correlation breaks down among
chordates. For example, some amphibians have genomes almost 50 times larger than that of humans.
The eukaryote with the largest genome on earth is a type of lily.
Plants have representatives throughout the genome size range. The smallest known plant genome belongs
to Arabidopsis thaliana at 7x107 (roughly the same size as yeast and the nematode Caenorhabditis elegans)
and the one of the largest is a member of the lily family, Fritillaria assyriaca, with 1x1011. The lack of a
direct relationship between genome size and organism complexity is called the C-value paradox.
The genomes of Arabidopsis and Fritillaria code for about the
same number of genes.
The average gene takes up about 4,000 bp of DNA (4 kb) when you include coding region (ca. 1.3 kb) plus
flanking regions like the promoter and the non-coding intervening sequences. Using these parameters, there
is just enough space in the Arabidopsis genome to accommodate about 15,000 genes.
In plants, the c-value paradox is due to repetitive DNA and
polyploidy.
For the most part, <2% of the genome codes for necessary products, much of the rest comprises huge sets
of repetitive DNA sequences of similar but not necessarily identical repeated sequences. Repetitive DNA
can be subdivided into two classes based on its organization in the genome, tandem repeats (one sequence
motif repeated right after another)and dispersed repeats (repeated sequence motif scattered throughout the
genome). Another source of “genome inflation” is due to polyploidy. “Ploidy” refers to the number of sets
of chromosomes in the nucleus (recall haploid = half the set, as in gametes and diploid = the normal
compliment of two sets in somatic cells). Many plants have duplicated genomes and have multiple sets of
chromosomes
Chromatin lectures for HOS 3370 – A-L. Paul
Lectures related to Chromatin structure (Anna-Lisa Paul)
From HOS 3370 Introduction to Plant Molecular Biology; University of Florida, Robert J. Ferl
Repetitive DNA contributes to the character and function of
specialized structures in chromosomes and plays a role in
genome organization.
Non-coding, tandem repetitive DNA is referred to as satellite DNA. Satellite DNA is primarily associated
with either the centromere or the telomeres in plants and is usually heterochromatic, that is, it remains
condensed and is not transcribed. Dispersed repeat sequences make up a significant portion of the
genome. These sequences differ from the tandem repeats of centromeres and telomeres in that copies are
dispersed throughout the genome rather than lying adjacent to one another.
C0t analyses can be used to determine the relative amount of
repetitive DNA in a genome.
C0t = concentration of DNA (in moles of nucleotides per liter) x time (in seconds)
The percentage of single copy DNA in a genome may be approximated biochemically by reassociation
kinetics and construction of “C0t curves” that plots the conversion of singled stranded DNA molecules to
double-stranded (reassociated) DNA over time. The slower the DNA reassociates, the higher the percent of
repetitive sequences.
(see pages 95-113 in Foskett)
Providing gene products
We think of the role of the genome as one of providing gene products, but typically < 2% of the DNA
in many genomes is transcribed and translated during normal cellular activities. Striking evidence that the
actual coding capacity is likely to be relatively constant among plants is seen in the comparison of the
genomes of Arabidopsis and maize. Both genomes code for essentially the same number of genes, but the
genome sizes differ by two orders of magnitude.
Not all repetitive DNAs are non-coding sequences.
Large multigene families that are evolutionarily conserved are often clustered within the genome.
Gene families consist of genes tandemly repeated numerous times. Even though they are arranged as
tandem repeats, each gene is individually regulated. Such repeated genes typically code for gene products
that are in great demand. Ribosomal genes are repeated thousands of times in a region of the genome
known as the nucleolar organizer region (NOR) and represent one of the largest families of repetitive
sequences in eukaryotes. Histone proteins are also needed in great abundance within the cell as they
comprise a major component of the chromatin proteins.
Smaller multigene family members share extended DNA sequence
homology and code for functionally related proteins.
Some genes occur in families containing 20 to 25 repeats. Although these genes may be clustered or linked,
they are not tandemly repeated. One example is the maize zein family, which encodes seed storage
proteins.
Single-copy DNA is present only once in the haploid genome.
Most “routine” genes are present once (or perhaps twice, in a slightly modified form) in the genome, and
are referred to as single copy genes.
Chromatin lectures for HOS 3370 – A-L. Paul
Lectures related to Chromatin structure (Anna-Lisa Paul)
From HOS 3370 Introduction to Plant Molecular Biology; University of Florida, Robert J. Ferl
Not all single copy DNA encodes genes.
In tobacco, (C=1.7x109), only 2% of the genome is transcribed into mRNA. Biochemical analyses,
however, indicate that as much as 40% of the tobacco genome is composed of single-copy DNA. It
appears, therefore, that the genome contains many single-copy sequences that are not transcribed.
The organization and arrangement of single-copy genes is evolutionarily
conserved among related plant species.
Genome mapping projects have revealed that segments of chromosomes are conserved among species.
Maize and sorghum, for example, contain many of the same genes and linkage groups residing at the same
loci (physical locations). This colinearity of loci is called synteny.
(see p. 95-113 in Foskett and p. 291-296 in Alberts et al.)
Transposable elements
Transposable elements are mobile DNA sequences that can make up a
significant portion of the nuclear genome.
Transposable elements are sections of DNA (sequence elements) that move, or transpose, from one site
in the genome to another. These mobile DNA elements carry genetic information with them as they
transpose, making them important features of genome organization. Transposable elements from organisms
as diverse as Drosophila, yeast and maize show a substantial conservation in organization and the mode of
transposition.
There are two basic types of transposable elements.
The first type of transposable elements are the Ac/Ds type described by Barbara McClintock in the
1940’s. The elements in this category code for one or two gene products necessary for the transposition of
the element. The second category consists mainly of retrotransposons, which are almost certainly viral in
origin. They resemble the structure left by the integrated form of RNA tumor viruses, and transpose by way
of an RNA intermediate.
(see p. 113-119 in Foskett and p. 289-298 in Alberts et al.)
Chromosomes
Chromosomes are not just “fat X’s”
Probably everyone has seen chromosomes drawn as fat X’s. Keep in mind that they only look this for a
narrow window of the cell cycle at metaphase (we’ll go into cell cycle in more detail in later lectures).
Metaphase is the stage of the cell cycle where the chromosomes are the most condensed (50,000x more
condensed than the original length of the DNA molecule). However, in Interphase, the stage where the cell
normally “lives” until replication and division, the chromosomes are organized as 30nm fibers gathered
into loops and coils in varying degrees of condensation along its length. Chromosomes are not just floating
around free in the nucleus, they are associated with the 3-dimensional nuclear matrix, as well as being
anchored at their ends (telomeres) to proteins on the inner surface of the nucleus (the nuclear lamina).
Genes reside on chromosomes
That meter’s worth of corn genome is actually partitioned into ten chromosomes. Each chromosome is a
single molecule of DNA complexed with proteins as described above. Chromosomes are found as
homologous pairs in diploid cells, each thereby containing one allele of each gene pair in a diploid
Chromatin lectures for HOS 3370 – A-L. Paul
Lectures related to Chromatin structure (Anna-Lisa Paul)
From HOS 3370 Introduction to Plant Molecular Biology; University of Florida, Robert J. Ferl
genome. If the alleles are the same, the same form of that gene is found on each member of the
chromosome pair, and the organism is homozygous for that gene. If the alleles are different, the organism
is heterozygous. Round and Wrinkled of Mendel’s peas are alleles of the same gene. For instance, if you
have brown eyes like your Dad, but Mom has blue eyes, you are heterozygous, and one chromosome has
the Brown allele and the other in the pair has the blue allele.
How was the connection made that chromosomes contained genes?
Mendel's pea experiments were the first to correlate physical traits (phenotype) with heritable components
(genotype), and represent only one example of how plants have played a central role in the foundations of
genetics. The term “gene” was not used until 1909, when it was coined by W.L. Johannsen. On the basis of
his findings, Mendel proposed two laws of genetics, which hold true for any unlinked gene:
1) The principle of segregation: The two alleles of a gene segregate during the formation of gametes, i.e.
one gamete gets one allele, the other gets the other allele.
2) The principle of independent assortment: The factors (genes) for different traits assort independently
from one another.
Walter Sutton and Theodor Boveri postulated the chromosome theory of heredity in 1903 based on the
cytological observation that chromosomes were consistently transmitted from one generation to the next, as
were certain traits. They concluded that Mendelian factors (the term “gene” was still not used) were found
on chromosomes. In 1916, Calvin B. Bridges correlated the non-disjunction of the X chromosome in
Drosophila with specific heritable traits. (Nondisjunction - when homologous chromosomes fail to separate
during meiosis, leaving some daughter cells with two copies of the chromosome, while others have no
copies).
How was the connection made that genes were made of DNA?
A hundred years after Mendel, the question still raged as to the composition of these factors, which we now
know as genes. It was known that chromosomes consisted mostly of two types of molecules: protein and
DNA. Whatever its composition, a molecule responsible for transmitting genetic information must be able
to accomplish three tasks: First, it must encode all of the information needed for cell growth, development,
structure, and reproduction. Second, it must replicate accurately to ensure that daughter cells contain the
same information as the parent and finally, it must be capable of variation to accommodate the changes and
adaptations evidenced by evolution. Although DNA seemed too simple to carry so much information, other
features made a compelling argument for it being the genetic molecule. First, DNA is very stable. It does
not undergo the metabolic turnover seen for many proteins. Second, the amount of DNA is roughly
associated with the complexity of the organism (i.e. bacteria have less than humans). Another clue: diploid
cells contain twice as much DNA as the haploid gametes in the same organism.
In a series of experiments between 1928 and 1953, researchers combined the sciences of biochemistry and
genetics to confirm DNA as the genetic material. Frederick Griffith showed in 1928 that a component of
heat-killed virulent bacteria could transform avirulent bacteria into the virulent form. Oswald T. Avery,
Colin M. MacLeod and Maclyn McCarty utilized the same biological system to demonstrate in 1944 that
Griffith’s “transforming principle” was, in fact, DNA. In 1953, Alfred Hershey and Martha Chase used a
virus that infects bacteria (a bacteriophage) to show that the DNA component of the phage was the
infectious agent, not the protein. Then in 1956, A. Gierer and G. Schramm discovered that purified RNA
(the nucleic acid component of Tobacco Mosaic Virus) alone could initiate an infection of TMV in tobacco
leaves, suggesting that the RNA carried all of the genetic information necessary for the synthesis of new
viruses. The next year, H. Fraenkel-Conrat and B. Singer confirmed the suggestion using hybrids of two
distinct strains of TMV - each hybrid carrying the RNA of one strain and the protein of the other. The
progeny of the hybrid viruses in infected tobacco leaves always matched the type represented by the RNA
component.
Chromatin lectures for HOS 3370 – A-L. Paul
Lectures related to Chromatin structure (Anna-Lisa Paul)
From HOS 3370 Introduction to Plant Molecular Biology; University of Florida, Robert J. Ferl
Chromosomes contain specialized structures
Centromeres divide a chromosome into two, not necessarily equal, parts called
“arms”.
The centromere is a region of the chromosome to which the spindle fibers attach for the separation of the
replicated chromatids in mitosis and meiosis (the intersection of the “fat X). The centromere contains
certain repetitive sequence elements that can be repeated millions of times. These sequences appear to be
essential for the recognition of the centromere by the spindle apparatus, as in yeast, mutations in this region
disrupt centromere function.
The relative position of the centromere within the chromosome can be characteristic of a gene within an
organism; three distinctions are made: metacentric - the centromere is in the middle and creates
chromosome arms of equal length, acrocentric - the centromere is off center, creating arms of unequal
length, and telocentric - where the centromere is at the very tip of the chromosome.
Telomeres define the ends of chromosomes.
Remember that a chromosome is just a single, long linear piece of DNA associated with various proteins,
and therefore has two “ends”. The telomere is the structure that defines the end of a chromosome. This
specialized chromosomal cap offsets the tendency for DNA to shorten with each round of replication.
Telomeres contain repetitive sequence elements, but unlike those of centromeres, these sequences are
highly conserved among eukaryotes in both sequence and arrangement. For example, humans and
trypanosomes (a flagellated protozoan) have the same sequence repeated thousands of times, TTAGGG,
and Arabidopsis differs by one base with TTTAGGG. As with centromeres, telomeres play a crucial role in
the replication of the genome. At the end of the chromosome, after the several thousand copies of the
telomere sequence, lies a section of single-stranded DNA composed of only two or three copies of the
telomeric sequence. In eukaryotes, a specialized enzyme called telomerase maintains the single stranded
overhang between cell generations (so it is not shortened with each round of replication.
(see pages 246-256 Alberts et al. 49-55 Fosket)
web sites: http://util.ucsf.edu/sedat/sedat.html
http://bio.fsu.edu/~bass/images2.html
Chromatin lectures for HOS 3370 – A-L. Paul
Lectures related to Chromatin structure (Anna-Lisa Paul)
From HOS 3370 Introduction to Plant Molecular Biology; University of Florida, Robert J. Ferl
HOS 3370 Introduction to Plant Molecular Biology
Cytoplasmic (non-nuclear) genomes
The chloroplast genome
Content and structure of the chloroplast genome
Plastids have their own genomes. The genome itself and the machinery utilized in its replication and
regulation is very similar to prokaryotic systems.
There are more types of "plastids" than just chloroplasts developmental
variations of the chloroplast to fill specialized roles.
Etioplasts are an arrested developmental stage of a chloroplasts that occurs when a plant is grown in the
dark, when exposed to light, etioplasts develop into photosynthetic chloroplasts. Chromoplasts (function in
the synthesis and storage of carotenoids) and amyloplasts (starch storage) are derivatives of chloroplasts,
but do not develop any photosynthetic machinery.
All plastids within a plant contain exactly the same genome.
Variation exists only in the number of copies of the genome in each plastid, and which genes from the
genome are expressed; the genetic information is identical in chloroplasts, amyloplasts chromoplasts and
etioplasts.
Plastid genomes have a highly conserved structural organization.
Almost all plastid genomes range in size from 120kb – 160kb and consist of a single, circular chromosome
typically organized into three regions. First, there is a large region of single copy genes (LSC), second, a
small region of single copy genes (SSC) and third, two copies of an inverted repeat (IRA and IRB) that
separate the two sections of single copy genes in the circular genome.
Most of size variations in the plastid genomes of higher plants are due to
variations in the size of the inverted repeats.
Because the only real difference among plastid genomes is related to the repeated sequences, this is the
character used to classify plastid genomes. Group I plastid genomes lack inverted repeats (certain
legumes), group II genomes contain inverted repeats (almost all plants) and group III sort of oddball
genomes which have tandem repeats (Euglena – a photosynthetic protist).
The genetic contribution of the chloroplast genome
Gene content and organization of genes within the genome is also highly
conserved.
There are around 100-150 genes in the average plastid genome. The IRs contains the rRNA genes (four
genes in each IR) and some of the tRNA genes (5-7, the other 30 or so are coded on the SSC and LSC
regions), and an average 100 protein coding genes are found in the single copy regions. There is extreme
conservation among plant species in the identity, and relative positions, of plastid genes.
Chromatin lectures for HOS 3370 – A-L. Paul
Lectures related to Chromatin structure (Anna-Lisa Paul)
From HOS 3370 Introduction to Plant Molecular Biology; University of Florida, Robert J. Ferl
The genes required for photosynthesis are distributed between the plastid and
nuclear genome.
The major protein in chloroplasts is Ribulose-1,5-bisphosphate carboxylase/oxygenase (or Rubisco for
short, and it is also one of the most abundant proteins on the planet). Rubisco is made up of 8 copies each
of two types of subunits. The smaller one is encoded in the nuclear genome (rbcS) and the larger subunit
(rbcL) is encoded in the plastid genome. This pattern of dividing the location of genes for protein subunits
or complexes is found in the genes encoding the proteins of other photosynthetic systems as well.
Photosystems I and II (PSI, PSII) have genes encoded in both the nuclear and plastid genomes, as do the
proteins of the cytochrome b/f complex and ATP synthase.
Much of the plastid genome consists of multigene transcription units that
resemble bacterial operons.
Many of the genes that are contained in the plastid genome are organized into polycistronic transcription
units (polycistronic – more than one gene product contained in a single transcribed section of the genome).
Whereas most prokaryotic genes are polycistronic, all eukaryotic nuclear genes are monocistronic (only
one gene product from a single messenger RNA molecule). Note: an operon describes a group of coding
sequences under the control of the same promoter, usually subunits of a protein
The mitochondrial genome
Content and structure of the mitochondrial genome
Mitochondria also have their own genomes. Again, the genome itself, and the machinery utilized in its
replication and regulation, is very similar to that of prokaryotic systems.
The content of the mitochondrial genome is conserved among plants, but the
physical arrangement of the DNA is highly variable.
The genome size for plant mitochondria can range from around 200kb in Brassica (mustard) species to
2600kb in muskmelon. Part of the variability in size is due to the accumulation of non-coding sequences.
This is in stark contrast to animal mitochondrial genomes, which are very compact, having virtually no
noncoding sequences between genes, and whose gene organization and expression is conserved across
phylogenetic borders.
The physical arrangement of the genes in the mitochondrial genome of plants is
also highly variable.
This variability is mostly tied to the characteristic that the mitochondrial genome is multipartite, that is,
the mitochondrial genome consists of several subgenomic circular molecules that freely recombine with
each other. The mitochondrial genome is generally thought of as circular, and the "starting point" for the
subgenomic circles is referred to as the master chromosome.
There are certain general features of the mitochondrial genome that are
conserved among plants.
There are several sections of repeated sequences (repeats) distributed throughout the genome that function
in recombination events. Recombination results in the generation of subgenomic circles as well as isomeric
forms of the master chromosome.
The mitochondrial genome also contains smaller DNA molecules known as
Mitochondtrial plasmids.
Mitochondtrial plasmids can be either linear or circular, and their function is mostly unknown with the
exception of those associated with the cmsT genome in maize that confers male-sterility. The mitochondria
Chromatin lectures for HOS 3370 – A-L. Paul
Lectures related to Chromatin structure (Anna-Lisa Paul)
From HOS 3370 Introduction to Plant Molecular Biology; University of Florida, Robert J. Ferl
of all S-type cytoplasmic male sterile (cms) maize lines contain two linear plasmid-like DNAs that have
been named S1 and S2. These plasmids do not have any homology to any known mitochondrial or nuclear
sequences, suggesting an exogenous (external – like an infection from a virus) origin for the sequences.
The genetic contribution of the mitochondrial genome
The genetic content of the plant mitochondrial genome is more conserved than
any of the structural features
Mitochondrial genomes in general do not code for many genes, as most of the enzymes required for DNA
replication, transcription and translation are encoded by the nucleus. The genes that are encoded in the
mitochondrial genome are predominantly related to either respiration (inner membrane proteins for electron
transport and ATPase) or to mitochondrial translation (ribosomal RNA, ribosomal proteins etc), and in fact,
there is a great deal of sequence similarity between the mitochondrial genomes of plants and animals.
The mitochondrial genome has been referred to as an evolutionary mosaic as it
contains sequences from chloroplast, nuclear, and perhaps even viral genomes.
Examples of chloroplast genes that have been duplicated in the mitochondrial genome are the genes for 16S
rRNA, several tRNAs and segments from other genes such as the large subunit of RuBisCo, (rbcL). The
presence of these genes does not seem to disrupt mitochondrial function in any way.
The genetic code employed by mitochondrial genome differs from the universal
code defined by nuclear and chloroplast codons.
That is, the triplet code of DNA that codes for amino acids (three nucleotides = one amino acid) can be
different in mitochondria. Further, there are differences in codon usage between plant and animal
mitochrondrial genomes. In animal mitochondria, the standard TGG code for tryptophane is replaced by
TGA. Still another codon is used in plant mitochondria, where CGG codes for tryptophan, and TGA is the
stop codon.
The endosymbiont theory
Organelles as leftover prokaryotic symbionts
There are a number of features of the organelles of chloroplasts and mitochondria that suggest that they
have once been free-living organisms similar to living prokaryote such as cyanobacteria (the “bluegreen
algae”) and other bacteria.
Chloroplast and mitochondrial ribosomes are very similar to those of modern
bacteria.
Part of the coding sequences of chloroplast ribosomal RNA genes are very similar to those of the bacterium
E. coli, and chloroplast ribosomes are able to use bacterial tRNAs in protein synthesis. Further, chloroplast
ribosomes are sensitive to several bacterial antibiotics (like chloramphenicol, streptomycin, erythromycin
and tetracycline).
Protein synthesis starts at a modified methionine.
In eukaryotes, protein synthesis begins at a methionine residue. In chloroplasts, mitochondria and bacteria
it starts with N-methylmethionine.
Chromatin lectures for HOS 3370 – A-L. Paul
Lectures related to Chromatin structure (Anna-Lisa Paul)
From HOS 3370 Introduction to Plant Molecular Biology; University of Florida, Robert J. Ferl
Many chloroplast and mitochondrial genes are polycistronic.
The polycistronic nature of plastid genomes is very bacteria-like. For example, the maize rRNA genes are
organized into an operon consisting of 16S, 23S, 4.5S, and 5S. Further, most land plants, from the nonvascular Marchantia to the monocot maize had their rRNA genes organized in exactly the same way.
The plastid genomes of evolutionarily distant plants are highly conserved.
The similarity of the plastid genomes of a primitive liverwort (Marchantia) and a vascular plant (maize)
suggests that plastid genomes have evolved slowly, and was incorporated early in the evolutionary history
of plants.
Plant mitochondria provide an interesting perspective on the evolution of
eukaryotic genomes.
The ribosomal genes and genes encoding components of the respiratory chain of the mitochondrial genome
indicate a clear link with a eubacterial ancestor. However, an examination of the mitochondrial genomes
from green algae (the putative progenitor of higher plants) shows that the sequences of the rRNA genes are
more different than comparative sequences from the nuclear genome. Thus, it appears as if plants shared a
common nuclear ancestor more recently than a mitochondrial ancestor. It is possible then, that the
mitochondrial genomes of plants acquired rRNA genes in separate endosymbiotic events, creating an
evolutionary mosaic through a lateral transfer of genetic information as well as in an evolutionary
progression.
(130-136 in Foskett, 438 in Alberts and web material “The genomes of chloroplasts and mitochondria.)
Chromatin lectures for HOS 3370 – A-L. Paul