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Operon Like Gene Clusters In Plants
Kumar A
PhD Candidate, UofC
M.Sc (Agriculture), Dal. Univ
M.Sc (Biotechnology), CPMB T...
Definition of a gene
• Segment of DNA specifying production of a polypeptide
chain
• Regions preceding and following the c...
What Is An Operon ?
• Unit of bacterial gene expression and regulation
• Structural genes and control elements in DNA reco...
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 repres...
An operon of C. elegans and the production of monogenic
mRNAs from a polygenic mRNA by trans-spicing and
polyadenylation/c...
• Clusters of functionally related genes include genes within
the same pathway, genes encoding interacting proteins, or
ge...
Saccharomyces cerevisiae
• Genomic clustering of co-expressed genes was first
identified in studies of S. cerevisiae genes...
• 15 percent of the genes organized into operons, which
are transcribed as polycistronic messages. These
messages are subs...
Drosophila
• 20% of genes are arranged into clusters spanning 20 to
200 kb and containing 10 to 30 genes each
(J. Biol. 20...
Evidences From Plant Kingdom
• The Arabidopsis genome is the most widely studied one
• Local clusters of up to 20 genes th...
• Data suggests that 5 to 9% of all nonduplicated Arabidopsis
gene pairs consist of coexpressed neighboring genes and
this...
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 path...
Brief introduction
• Triterpene against for pest and diseases, anticancer
agent
• Synthesized through isoprenoid pathway
•...
Remaining11
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 rest...
• Sad1 and Sad2 are embedded in a gene cluster that
includes genes required for acylation, glucosylation, and
other steps ...
Map of the triterpene gene cluster
on Arabidopsis chromosome 5
flankingflanking
At5g47970 At5g48020
Not involved in the tr...
Microarray expression profiles of the genes
Two immediately flanking genes At5g47970 and At5g48020
(neither of which are i...
T-DNA insertion mutants for the hypothesized gene
To test the hypothesis on the coregulation and
coexpression: Scientists ...
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 TH...
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 res...
Pathogenicity assays.
Colonization of wild type
Arabidopsis roots by
Atlernaria brassicicola
20μM.
Colonization of wild ty...
Model Of Thalianol Cluster Evolution
Conclusions From The Study !
• Clustering facilitates the inheritance of beneficial
combinations of genes
• disruption of ...
• Sequential rearrangements, duplications, and
gene loss presumably led to formation of the
present-day thalianol cluster
...
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 i...
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 R...
Genome analysis shows that many genes belong to families;
the 30,000 genes identified in the human genome fall into
~15,00...
Clusters And Repeats:
How Did They Appear In The Course Of Evolution?
• The initial event that creates related exons or ge...
Duplicated genes may
diverge to generate different
genes or one copy may
become inactive.
After a gene has been
duplicated...
Functional Divergence after Gene Duplication
(i) Neofunctionalization - One copy acquires an entirely
new function whereas...
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 inclu...
Genome Reorganization:
Another Mechanism in Evolution
• There are two types of circumstances in which gene
rearrangement i...
Unequal crossing-over (also known as nonreciprocal
recombination) can occur as the result of pairing between
two sites tha...
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
M...
Chromosome Territories: Unit of Nuclear Organization
• Chromosomes have preferred position with respect to the
center or p...
Spatial Organization Of Chromosomes Affects
Gene Expression
(O’Brien, et al, 2003)
• Association of gene loci with NPC, nu...
Model Of Dynamic Association Of Genes
With Transcription Factories
(Osborne et al., 2004)
Chromosome
territory
RNA
Polymer...
Colocalization Of Genes In The Nucleus For
Expression Or Coregulation
(Fraser & Bickmore, 2007)
Correlation between chromo...
Models of the chromosome territory
(Heard & Bickmore, 2007)
Interchromosome domain
Interchromatin compartment
The lattice ...
Models Of The Chromosome Territory:
Interchromosome Domain
(Heard & Bickmore, 2007)
• Interchromosome domain:
-Boundary be...
Models Of The Chromosome Territory:
Interchromatin Compartment
(Heard & Bickmore, 2007)
• Interchromatin compartment:
-Loo...
Models Of The Chromosome Territory: Lattice
Model
(Heard & Bickmore, 2007)
• Lattice Model:
-Extensive intermingling of ch...
Discussion
Operon like gene clusters in plants
Operon like gene clusters in plants
Operon like gene clusters in plants
Operon like gene clusters in plants
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Operon like gene clusters in plants

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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

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Operon like gene clusters in plants

  1. 1. Operon Like Gene Clusters In Plants Kumar A PhD Candidate, UofC M.Sc (Agriculture), Dal. Univ M.Sc (Biotechnology), CPMB TNAU
  2. 2. 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)
  3. 3. 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
  4. 4. The Most Common Operon The lac operon
  5. 5. Expression of The Operons: Prokaryotes Genes under negative control are expressed unless they are switched off by a repressor protein Prokaryotic operon
  6. 6. 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
  7. 7. • 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
  8. 8. 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.)
  9. 9. • 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
  10. 10. 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)
  11. 11. 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
  12. 12. • 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
  13. 13. One Of The Earliest Paper citing Gene Clustering
  14. 14. A Pioneering Research Article Establishing The Concept Of Gene Clustering And Coregulation
  15. 15. A Critical Review
  16. 16. Article on coexpression and coregulation published till 25th October 2009
  17. 17. Case study
  18. 18. Based on the above works these Scientists formulated hypothesis for clustering of triterpene metabolic genes in Arabidopsis
  19. 19. 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.
  20. 20. 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)
  21. 21. Remaining11 Candidate metabolic gene clusters Neighborjoining tree of Arabidopsis and oat OSC enzymes
  22. 22. 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
  23. 23. • 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
  24. 24. 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
  25. 25. 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)
  26. 26. 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
  27. 27. GC-MS Analyses
  28. 28. Automated Mass Spectral Deconvolution and Identification System (AMDIS) GC-MS Analyses
  29. 29. 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
  30. 30. Overexpression studies (A) Plants overexpressing thalianol synthase (B) Root length of 7 days old plants
  31. 31. 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
  32. 32. Pathogenicity assays. Colonization of wild type Arabidopsis roots by Atlernaria brassicicola 20μM. Colonization of wild type and mutant Arabidopsis roots by Pseudomonas syringae pv. tomato DC3000 at 7 days post inoculation
  33. 33. Model Of Thalianol Cluster Evolution
  34. 34. 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
  35. 35. • 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)
  36. 36. Question Remained Unanswered?? Why genes for some metabolic pathways are clustered, whereas others are not ?
  37. 37. 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
  38. 38. MOLECULAR INSIGHT OF COREGULATION AND COEXPRESSION
  39. 39. The number of genes in bacterial and archaeal genomes is proportional to genome size
  40. 40. • 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
  41. 41. 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.
  42. 42. 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)
  43. 43. 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
  44. 44. 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
  45. 45. All globin genes have evolved by a series of duplication, transpositions and mutations from a single ancestral gene
  46. 46. 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
  47. 47. 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
  48. 48. 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
  49. 49. histone acetylases nucleosome remodeling complexes Chromosome modeling Inside The Eukaryotic Nucleus
  50. 50. 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?
  51. 51. 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
  52. 52. 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
  53. 53. Model Of Dynamic Association Of Genes With Transcription Factories (Osborne et al., 2004) Chromosome territory RNA Polymerase II transcription factory Transcribed genes Potentiated genes
  54. 54. 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
  55. 55. Models of the chromosome territory (Heard & Bickmore, 2007) Interchromosome domain Interchromatin compartment The lattice model
  56. 56. 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)
  57. 57. 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)
  58. 58. 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)
  59. 59. Discussion

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