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