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Operon Like Gene Clusters In Plants
Kumar A
PhD Candidate, UofC
M.Sc (Agriculture), Dal. Univ
M.Sc (Biotechnology), CPMB TNAU
Definition of a gene
• Segment of DNA specifying production of a polypeptide
chain
• Regions preceding and following the coding region (leader
and trailer)
• Intervening sequences (introns) between individual coding
segments (exons)
What Is An Operon ?
• Unit of bacterial gene expression and regulation
• Structural genes and control elements in DNA recognized
by regulator gene product(s)
• Group of genes that encodes functionally linked proteins
The Most Common Operon
The lac operon
Expression of The Operons: Prokaryotes
Genes under negative control are expressed unless they are
switched off by a repressor protein
Prokaryotic operon
An operon of C. elegans and the production of monogenic
mRNAs from a polygenic mRNA by trans-spicing and
polyadenylation/cleavage
Eukaryotic operon
Expression of The Operons: Eukaryotes
• Clusters of functionally related genes include genes within
the same pathway, genes encoding interacting proteins, or
genes that affect the same trait
• Not all functionally related genes are clustered within or
among species
• In humans, it has been observed that genes expressed in a
given tissue, highly expressed genes, and broadly
expressed (“housekeeping”) genes map to clusters
Nat. Genet. 2002, 31: 180–183
Saccharomyces cerevisiae
• Genomic clustering of co-expressed genes was first
identified in studies of S. cerevisiae genes with cell-cycle
dependent expression patterns
(Mol. Cell., 1998, 2: 65-73.)
• About 25% of the genes expressed during the same
phase of the cell cycle are arranged as pairs(Nat. Genet., 2000, 26: 183-186.)
• 15 percent of the genes organized into operons, which
are transcribed as polycistronic messages. These
messages are subsequently processed into monocistronic
mRNAs by trans-splicing
( Nature 1994, 372: 270-272.)
• Tandem duplication of genes results in co-expression of
many paralogues
• Operons do not account for coexpression of all
neighboring genes in C. elegans
Caenorhabditis elegans
Drosophila
• 20% of genes are arranged into clusters spanning 20 to
200 kb and containing 10 to 30 genes each
(J. Biol. 2002, 1: 5)
• Gene duplications account for many two-gene clusters
but are not characteristic of clusters containing three or
more coexpressed genes
(Nature 2002, 420: 666-669)
Evidences From Plant Kingdom
• The Arabidopsis genome is the most widely studied one
• Local clusters of up to 20 genes that are coexpressed,
with an overall median cluster size of 100 kb
Genome Res. 2004, 14: 1060–1067.
• significant negative correlation between intergenic
distance and coexpression
• The degree of coexpression for genes from the same
pathway
• Data suggests that 5 to 9% of all nonduplicated Arabidopsis
gene pairs consist of coexpressed neighboring genes and
this number is 1.3 times more than expected by chance
alone
Plant Physiol. 2005,138: 923–934.
• In Arabidopsis thaliana, genes involved in root development
and mitochondrial functions tend to form distinct clusters
Plant Cell 2003,15:1619–1631
Science 2003, 302: 1956–1960
• Genes from the same pathways, however, were not a sole
source of coexpression of neighboring genes in Arabidopsis,
as shown by an analysis omitting these genes
Genome Res. 2004,14: 1060–1067
One Of The Earliest Paper citing Gene
Clustering
A Pioneering Research Article Establishing The
Concept Of Gene Clustering And Coregulation
A Critical Review
Article on coexpression and coregulation
published till 25th October 2009
Case study
Based on the above works these
Scientists formulated hypothesis for
clustering of triterpene metabolic
genes in Arabidopsis
Salient Points in this Research Paper
• Research has been carried out in Arabidopsis thaliana
• Done on the thalianol pathway, (triterpene synthesis)
• Reported that the genes are clustred and coexpressed
• Assembly of operon-like gene clusters for triterpene
synthesis has occurred independently in divergent
plant lineages (Arabidopsis and oat).
• Concluded that selection pressure may drive certain
plant metabolic pathway for gene clustering and
coexpression.
Brief introduction
• Triterpene against for pest and diseases, anticancer
agent
• Synthesized through isoprenoid pathway
• Basic step includes.
2, 3- oxidosqualene triterpene
oxidosqulene cyclase (OSC)
• 13 genes- predicted for this OSC
– Cycloartenol synthase(1) for sterol biosynthesis
– lanosterol synthase(1) function unknown but conserved
across the eudicots
– Remaining (11)
Rem
ainin
g 11
Candidate
metabolic gene
clusters
Neighborjoining tree of Arabidopsis and oat OSC enzymes
On what basis they hypothesized ?
• The OSCs in clade I have close homologs in other eudicots
• Clade II appear to be restricted to the Brassicaceae
• Oat (Avena spp.), a monocot diverged from the eudicots.
• Produces defense- related triterpenes known as avenacins
• Avenacin synthesis is catalyzed by the OSC beta-amyrin
synthase (encoded by Sad1), mediates 1st step
• Sad2 newly described monocot-specific CYP51H subfamily
of cytochrome P450 enzymes (CYP450s), mediates 2nd step
• Sad1 and Sad2 are embedded in a gene cluster that
includes genes required for acylation, glucosylation, and
other steps in the pathway
• The avenacin biosynthesis genes are tightly regulated
and expressed only in the root epidermis, the site of
accumulation of the pathway end product
• The avenacin gene cluster lies within a region of the oat
genome lacking synteny with rice and other cereals
• These OSCs produce various triterpenes when
expressed in yeast but their couldn't be generalized to
Aarabidopsis
Map of the triterpene gene cluster
on Arabidopsis chromosome 5
flankingflanking
At5g47970 At5g48020
Not involved in the triterpene metabolism
Immediately
thalianol synthase
Thalianol hydroxylase
Thalianol-diol desaturase
acyltransferase
Brassicaceae-specific enzyme subgroup
Microarray expression profiles of the genes
Two immediately flanking genes At5g47970 and At5g48020
(neither of which are implicated in secondary metabolism)
flanking flanking(THAS)(THAH)(THAD)(BADH)
T-DNA insertion mutants for the hypothesized gene
To test the hypothesis on the coregulation and
coexpression: Scientists produced T-DNA insertion
mutants for their hypothesized genes and then
performed GC-MS and analyzed the data
GC-MS Analyses
Automated Mass Spectral Deconvolution and Identification
System (AMDIS)
GC-MS Analyses
Thalianol (1), Thalian-diol (2a, 2b, and 2c), Desaturated thalian-diol (3a and 3b)
The data showed that THAS, THAH, and THAD are
contiguous coexpressed genes encoding biosynthetic
enzymes required for three consecutive steps in the synthesis
and modification of thalianol
Overexpression studies
(A) Plants overexpressing thalianol synthase
(B) Root length of 7 days old plants
Other studies carried out by the research
group
• The avenacin gene cluster in oat (Avena spp.) confers
broad-spectrum resistance to fungal pathogens
• Defense related test were carried out
• Challenged to strains of fungal and bacterial plant
pathogens (Alternaria brassicicola, Botrytis cinerea, and
Pseudomonas syringae pv tomato DC3000)
• No significant observations could be concluded
Pathogenicity assays.
Colonization of wild type
Arabidopsis roots by
Atlernaria brassicicola
20μM.
Colonization of wild type and mutant Arabidopsis roots by Pseudomonas syringaepv. tomato DC3000 at 7 days post inoculation
Model Of Thalianol Cluster Evolution
Conclusions From The Study !
• Clustering facilitates the inheritance of beneficial
combinations of genes
• disruption of metabolic gene clusters can lead to
accumulation of deleterious intermediates
• Lead to severe dwarfing in Arabidopsis
• Suggests distinct and organ-specific effects of
thalianol and thalian-diol on plant growth
• Clustering may also facilitate coordinate regulation
of the gene cluster at the chromatin level
• Sequential rearrangements, duplications, and
gene loss presumably led to formation of the
present-day thalianol cluster
• Thalianol and avenacin gene clusters - products of
separate and recent evolutionary events
• Eukaryotic genomes- capable of remarkable
plasticity and can assemble operon-like gene
clusters de novo
• Other examples of gene clusters for plant defense
compounds (for rice diterpenes and maize
benzoxazinoids)
Question Remained Unanswered??
Why genes for some metabolic pathways
are clustered, whereas others are not ?
When Things Are So Simple, Why So Large
Genomes….?
• No good correlation between genome size and genetic
complexity
• An increase in the minimum genome size required to
make organisms of increasing complexity
• Wide variations in the genome sizes of organisms within
many phyla
MOLECULAR INSIGHT OF
COREGULATION AND COEXPRESSION
The number of genes in
bacterial and archaeal
genomes is proportional
to genome size
• A representative 65 kb region of DNA is illustrated for
each organism
• The region that encodes the largest subunit of RNA
polymerase (RNA Pol II for eukaryotic cells) is indicated
in red
Genome analysis shows that many genes belong to families;
the 30,000 genes identified in the human genome fall into
~15,000 families, so the average gene has a couple of
relatives in the genome.
Clusters And Repeats:
How Did They Appear In The Course Of Evolution?
• The initial event that creates related exons or genes is
Duplication
• A set of genes descended by duplication and variation
from some ancestral gene is called a gene family
• Its members may be clustered together or dispersed on
different chromosomes (or a combination of both)
Duplicated genes may
diverge to generate different
genes or one copy may
become inactive.
After a gene has been
duplicated, differences may
accumulate between the
copies. The genes may
acquire different functions or
one of the copies may
become inactive
Functional Divergence after Gene Duplication
(i) Neofunctionalization - One copy acquires an entirely
new function whereas the alternative copy maintains
the original function
(ii) Subfunctionalization - Each descendant copy adopts
part of the tasks of the ancestral gene
All globin genes have evolved by a
series of duplication, transpositions
and mutations from a single
ancestral gene
The Rise Of Gene Clusters
Each of the α-like and β-like globin gene families is
organized into a single cluster that includes functional
genes and pseudogenes
Genome Reorganization:
Another Mechanism in Evolution
• There are two types of circumstances in which gene
rearrangement is used to control expression
• Rearrangement may create new genes, needed for
expression in particular circumstances, as in the case of
the immunoglobulins
• Rearrangement may be responsible for switching
expression from one preexisting gene to another. This
provides a mechanism for regulating gene expression
Unequal crossing-over (also known as nonreciprocal
recombination) can occur as the result of pairing between
two sites that are not homologous.
Unequal Crossing-over
histone acetylases nucleosome remodeling
complexes
Chromosome modeling
Inside The Eukaryotic Nucleus
About Chromosome Territories (CTs)
(Maeburn and Misteli, 2007)
Nucleoplasmic
channels within CT
plants Higher eukaryotes
Models of chromatin
structure within CT
• All cells have them, except lower eukaryotes
• Interior of CT are permeated by interconnected networks
of channels
• DNA structure within CT is non-random
• Folding of chromosome to a specific form: mechanism
still unkown?
Chromosome Territories: Unit of Nuclear Organization
• Chromosomes have preferred position with respect to the
center or periphery of the nucleus
• Non-random neighbors:
purpose is to facilitate
proper gene expression.
• Variability between
cell-types
• Complex folded surface with active genes(red)
extends (or loops) into the interchromatin space
Spatial Organization Of Chromosomes Affects
Gene Expression
(O’Brien, et al, 2003)
• Association of gene loci with NPC, nuclear periphery,
or specific nuclear bodies can all affect gene gene
expression
Model Of Dynamic Association Of Genes
With Transcription Factories
(Osborne et al., 2004)
Chromosome
territory
RNA
Polymerase II
transcription
factory
Transcribed
genes
Potentiated
genes
Colocalization Of Genes In The Nucleus For
Expression Or Coregulation
(Fraser & Bickmore, 2007)
Correlation between chromosome location and gene expression
Chromosome territory
Cis and trans
co-associationCis-interaction/trans
interaction
Speckle
Chromatin loopTranscription factory
Models of the chromosome territory
(Heard & Bickmore, 2007)
Interchromosome domain
Interchromatin compartment
The lattice model
Models Of The Chromosome Territory:
Interchromosome Domain
(Heard & Bickmore, 2007)
• Interchromosome domain:-Boundary between the surface of a CT and gene
expression machinery compartment
-Predict active genes are all located at the surface of CTs
Splicing-factor enriched
speckles (red)
RNAPII
(light blue)
Models Of The Chromosome Territory:
Interchromatin Compartment
(Heard & Bickmore, 2007)
• Interchromatin compartment:
-Loops of decondensed chromatin containing active genes
may loop out into this compartment
-Genes from different CTs can localize together with gene
expression factories or splicing-factor enriched speckles
Splicing-factor enriched
speckles (red)
RNAPII (light blue)
Models Of The Chromosome Territory: Lattice
Model
(Heard & Bickmore, 2007)
• Lattice Model:
-Extensive intermingling of chromatin fibres from
periphery and adjacent CTs
-Genes from different CTs can localize together with
gene expression factories or splicing-factor enriched
speckles
Splicing-factor enriched
speckles (red)
RNAPII (light blue)
Discussion