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and Gene Expression
Firman Heru K
Alfiani Umi Farkha
• The nuclear genom contains most of the gene
required for the plant’s physiological functions.
• The first plant genome to be fully sequenced,
in 2000 was that of a small dicotyledonous
angiosperm called thale cress or Arabidopsis
• The genom of A. thaliana is made up of only
about 157 million base pairs (157 Mbp) which
are distributed over five chromosome
• Within its nuclear genome, A. thaliana holds
some 27.400 protein-coding genes and almost
another 5000 genes that are either
pseudogenes (nonfunctional genes) or parts of
The nuclear genome is packaged
• The nuclear genom consists of DNA molecules
that are wrapped around histone proteins to
form beadlike structure called nucleosomes.
• DNA and histone, together with other proteins
that bind to the DNA, are reffered to as
• Two types of chromatin can be distinguished :
euchromatin and heterochromatin
• Heterochromatin is usually more tightly
packaged and thus appears darker than the less
known as the subtelomeric • Heterochromatic structure often consist of highly repetitive DNA sequence.these genes are inactive or silent • Compared with euchromatin. or chromosome ends. heterochromatin is relatively gene poor.• Most genes that are actively transcribed in a plant are located within the euchromatic regions of a chromosome. several socalled knobs. or tandem repeats : blocks of nucleotide motifs of about 150 to 180 bp that are repeatd over and over . while many genes located in heterochromatic regions are not transcribed. and the regions immmadiately adjacent to the telomers. Heterochromatic regions include the centromeres.
One types of dispersed repeats is known as simple sequence repeats ( SSR) or microsatellites • These repeats consist of sequence motifs as short as two nucleotides that are repeated hundreds or even thousands of times. • Another dominant group of dispersed repeat found in heterochromatin is the “jumping genes” or transposons .• A second class of repeats is the dispered repeats.
telomeres. and nucleolar organizers. a protein complex surrounding the centromere • Centromere consist of highly repetitive DNA regions and inactive transposable elements . and nucleolar organizers contain repetitive sequence • The most prominent structural landmark on chromosomes are centromeres. • Centomers are constrictions of the chromosomes where sister chromatids adhere to each other and where spindle fibers attach during cell division • The attachment of fibers to the centromeres is mediated by the kinetochore. telomeres.Centromeres.
Telomeres act as caps on the chromosome ends and prevents loss of DNA during DNA replication • The DNA molecules that make up ribosomes (rRNA) are transcribed from nucleolar organizer (NO) regions. along with proteins that transcripts for assembly into ribosomes. it is not surprising that Nos contain hundreds of repeated copies of each rRNA gene • Depending on the plants species. Because ribosomes are needed for translation.• Telomeres are sequence located at the ends of each chromosome. one or several nucleolar organizers are presents within the genome • Due to their repetitive nature and their high GC content. forms a prominent nuclear structure called the nucleolus . Nos can be seen through a light microscope and thus can serve as chromosome-specific markers • The rDNA of the nucleolar organizer.
Transposons are mobile sequence within the genome • One dominant type of repetitive DNA within the heterochromatic regions of the genome is the transposon. • Transposon or transposable elements are alson known as “jumping genes” because some of them have the ability to insert a copy of themselves in a new location within genome • There are two large classes of transposons : the retroelements or retrotransposon and the DNA transposons • These two classes are distinguished by their mode of replication and insertion into a new location • Retransposons make an RNA copy of themselves. which is then reserve-transcribed into DNA before it is inserted elsewhere in the genom .
Insertion od a transposon close to a gene can also alter that gene’s expression pattern . in most cases keeping the total transposon copy number the same • Transposition into a gene can result in mutations. If a transposon lands within a coding region. by contrast. transposase. move from one position to another using a cut-and-paste mechanism catalyzed by an enzyme that is encoded within the transposon sequence • This enzymes.• DNA transposons. splices out the transposon and insertsit elsewhere in the genome. the gene may be inactivated.
• Studies of mutants that are unable to maintain genome methylation have shown that slow loss of proper methylation over generations can activate dormant transposons and increase the frequency of transpositional mutations • Therefore. methylation and the formationof hererochromatin appear to play important roles in the stability of the genom .• Plants and other organisms seem to be able to regulate the activity of transposons through the methylation of DNA and histones • As more genomic noticed large numbers of highly methylated transposons in heterochromatic region.
the entire diploid (2n) genome can undergo one or more additional rounds of replication without undergoing cytokinesis to become polyploid • Two forms of polyploidy are distinguished : autopolyploidy and allopolyploidy.the number of copies of the entire genome in a cell-is another important aspect of genome structure that may have implications for both physiology and evolution • In many organism.Polyploids contain multiple copies of the entire genome • Ploidy level. but especially in palnts. while allopolypoids contain multiple complete genomes derived from two or more separate species . • Autopolyploids contain multiple complete genomes of a single species.
if a diploid egg is fertilized by diploid sperm.• Both types of polyploidy can result from incomplete meiosis during gametogenesis. the resulting zygote contains four copies of each chromosome and is said to be autotetraploid . diploid gametes result • Within a species or in a self-fertilizing individual. the chromosomes of a diploid germ cell undergo DNA replication followed by two rounds of division • If chromosome duplication is not followed by cell devision during meiosis. During meiosis.
Allopolyploids usually form in one of
two ways :
1.A haploid sperm from one species
and a haploid egg from another
species may form a diploid
2.Diploid gametes from two different
species may join to form a tetraploid
• Diploid interspecies hybrids occur naturally, but they are
frequently sterile because their chromosome cannot pair
properly during prophase 1 of meiosis
• The lack of fertility in interspecies hybrids is in stark
contast to the phenomenon known as hybrid vigor or
heterosis : the increase vigor often observed in the
offspring of crosses between two inbred varieties of the
same plants species
• Heterosis can contributed to larger plants, greater
biomass, and higher yields in agricultural crops
• Polyploidy can also be induced artificially by treatment
with the natural cell toxin colchicine, which is derived
from the autumn cronus (Colchicum autumnale)
They are hybrid between two different species Allopolyploids are frequently more vigorous or higher yielding than their parent species and are very common among agriculturally important plantssuch allopolyploids includecanola and collars. cotton. and sugarcane . oat. coffee. rye. wheat.Their genomes. like those of autopolyploids.Phenotypic and physiological responses to polyploidy are unpredictable Allopolyploids differ from their parental diploid progenitor species in two major ways : 1. are duplicated 2.
Some of the genetic changes that have been observed in newly formed allopolyploids compared with their parent species are the following : • Restructing of the chromosome. including loss of DNA sequence • Changes in epigenetic modifications • Changes in gene transcriptional activity • Activation of previously dormant transposable elements through loss of gene silencing .
• Polyploidy is in striking contrast to a condition called aneuploidy. . • Aneuploids are organisms whose genomes contain more or fewer individual chromosomes (not entire chromosome sets) than normal • Such states are known as trisomies if one type of chromosome is tripled or monosomies if only one chromosome of a given type is presents.
• The endosymbiotic theory. • Cytoplasmic genome are probably the evolutionary remnants the genomes of bacterial cells that were engulfed by another cell. which they share with animal cells. postulates that the original mitochondrion was an oxygen-using bacterium that was absorbed by another prokaryotic organism. .Plant Cytoplasmic Genomes : Mitochondria and Chloroplasts • In addition to the nuclear genome. plant cells contain two additional genome. and the chloroplast genome.
Both organella genomes show sequence similarity to procaryotic genomes. The organelar genomes. .• Two main lines of evidence are often cited in support of the endocymbiotic theory : 1. Both mitochondria and chloroplasts are enclosed by an outer and an inner membrane. and the inner membrane is continuous with additional membranebound compartments inside the organelle. like those of procaryotes are not enclosed in a nuclear envelope and are called nucleoids. 2. •.
– For many years organelar chromosomes had been thought to contain a genome-sized DNA molecule in circular form. similar to the circular plasmids of bacteria. – Organelar genomes are complex in structure and they usually consist of multiple copies of the genome on the same DNA molecul . – Most of the DNA in both plant mitochondria and chloroplasts is found in linear molecules that my contain more than one copy of the genome.• Organellar genomes consist mostly of linear chromosomes.
for Angisperms from maternal parent. 2. Organellar genetics do not obey Mendelian laws. Both chloroplasts and mitochondria can segregate vegetatively ( vegetative cell (as opposed to a gamete) can give rise to another vegetative cell via mitosis that is genetically different. Both mitochondria and plastid generally show uniparental inheritance ( sexual offspring (via pollen and eggs) only inherit organelles from one parent). but usually show uniparental inheritance and vegetative segragation.• Organellar genetics do not obey Mendelian laws. 1. . –. – The genetics of organellar gene are governed by two principles that distinguish them from Mendelian genetics. Among the Gymnosperms from paternal parent.
– This level of regulation. transcription. . determines when and whether an mRNA is made. • The path from gene to protein is a multistep process catalyzed by many enzymes. – Regulation of the first step. which is referred to as transcriptional regulation. and termination. maintenance. • Each step is subject to regulation by the plant to control the amount of protein that is produced by each gene. includes the control of trancription initiation.Transcriptional Regulation of Nuclear Gene Expression.
– Finally. . known as posttranscriptional regulation.– The next level in the regulation of gene expression. protein stability (posttranslational regulation) plays an important role in the overall activity of a gene or its product. occurs after transcription includes controls on mRNA stability. translation efficiency and degradation.
Core promoter or minimum promoter. – Gene transcription is facilitated by an enzyme called RNA polymerase.• RNA polymerase II binds to the promoter region of most protein-coding genes. which control the activity of the core promoter. Regulatory sequence. – The region of the gene that binds RNA polymerase is called the promotor. which binds to the DNA to the DNA to be transcribed and makes an mRNA transcript complementary to the DNA sequence. . – The structure of the eukaryotic promoter into two part : 1. consisting of the minimum upstream sequence required for gene expression. 2.
• These regulatory proteins bind to the DNA and become part of the transcription initiation complex. . most genes. especially those that play important roles in development. require specific transcription factors ( also often called gene regulatory proteins) for RNA polymerase to become active.• In addition to RNA polymerase and the general transcription factors.
. a process mediated by covalent modifications of both DNA and histones. its packaging has to be “looseened”. • Because these modifications can change a gene’s behavior without changing the DNA sequence of the gene its self. • To make the DNA accessible. they are referred to as epigenetic modifications.Epigenetic modifications help determine gene activity • Transcription can be initiated only if the DNA is accessible to the RNA polymerase and other required binding protein.
. help the determine gene activity.• Epigenetic modifications. such us methylation of DNA and methylation and acetylation of histone proteins.
Posttranscriptional Regulation of Nuclear Gene Expression • An organism often produceds mRNA in response to a specific situation. In order to remain useful as a specific respone to a specific situation. called cis-elements. – One mechanism by which mRNA stability is regulated depents on the presence of certain sequence within the mRNA molecule it sel. – These cis-elements can be bound by RNA-binding proteins. • RNA stability can be influenced by cis-elements. . which may either stabilize the mRNA or promote its degradation by nuclease. individual mRNAs must have a finite lifetime.
– The RNAi pathway is a set of cellular reactions to the presence of double-stranded RNA (dsRNA) molecules. – Recall that mRNA is usually a single-stranded molecule (ssRNA). – Another mechanism for regulating mRNA stability is the RNA Interference (RNAi) pathway. – In plant cells. dsRNA usually occur as a result of one of three types of events .• Noncoding RNAs regulate mRNA activity via the RNA interference (RNAi) pathway.
1. The presence of microRNAs (miRNAs). which are involved innormal developmental processes. .
The production of short interfering RNAs (siRNAs). which silence certain genes .2.
The introduction of foreign RNAs. either by viral infection or via transformation by foreign gene .3.
mRNA stability plays an important role in the ability of the gene to produce a functional protein. . 76-amino acid polypeptide called ubiquitin. • The cytoplasmic pathways of protein turnover involves the ATP-dependent formation of a covalent bond between the protein that is to be degraded and a small. We turn next to the stability of proteins an the mechanisms that regulate a protein’s life span.Posttranslational regulation determines the life span of proteins • As we have seen.
• Ubiquitination is initiated when the ubiquitin activating enzyme (E1) catalyzes the ATP dependent adenylylation of the C terminus of ubiquitin. The adenylylated ubiquitin is then transferred to a cysteine residue on a second enzyme. Proteins destined for degradation are bound by a third type of protein. a ubiquitin ligase (E3) . the ubiquitin conjugating enzyme (E2).
Tools for Studying Gene Function • Individuals that contain specific changes in their DNA sequence are called mutants. . The analysis of mutants is an extremely powerful tool that can help scientists infer the function of a gene or map its location on the chromosoms.
Mutant analysis can help to elucidate gene function • The use of mutants for gene identification relies on the ability to distinguish a mutant from a normal individual. so the change in the mutant’s nucleotide sequence must result in an altered phenotype. .
• There are several ways to map a mutation to its chromosome and ultimately clone the affected gene. explain a method called map based cloning. . which uses crosses between a mutant and a wild type palnt and genetic analysis of the offspring to narrow down the location of the mutation to a short segment of the chromosome. which is then sequenced.
• All microarray techniques use a solid support. or only I vegetative ones.Molecular techniques can measure the activity of genes • Once a gene of interest has been identified. • For example. such as a glass slide. onto which DNA sequences are spotted that are representative of single genes of a given species. a gene may be expressed only in reproductive tissues. . scientists are usually interested in where and when the gene is expressed.
Gene fusions can introduse reporter genes • A gene fusions is an artificial construct that combines part of the gene of interest with another gene that produces a readily detectable protein • A gene’s expression is regulated by transcription factors that fine-tune its activity and allow it to be trancsribed only where and when its needed. .
Genetic modification of crop plants • In contrast to classical selective breeding. bioengineering allows the transfer of specific gene ora gened between spiceis that cannot be crossed succesfully .
• Plants produced in this way are commonly referred to as genetically modified organisms (GMOs) .• Biotechnological tools circumvent this problem by allowing insertion of only the desired genes into the recipient plant. most often either by Agrobacterium-mediated transformation or by biolistics.
not just those with which the recipient can be succesfully crossed • GMOs may carry gene constructs that are the product of splicing a variety of genetic components together to produce genes with altogether new functions (for example.There are three essesntial differences between GMOs and conventionally bred varieties of crops • Gene transfer into GMOs occurs in the laboratory and does not require crossbreeding • The donor genes of GMOs can be derived from any organism. the promoter-GFP fusion genes we described earlier .
which kills weeds but does not harm resistant crop plants. .Transgenes can confer resistance to herbicides or plant pets • Any gene articially tranferred into an organism is referred to as a transgene • Plants carrying a transgene for glyphosate resistance will survive a field application of glyphosate (the commercial herbicide Roundup).
• Another commonly used transgene encodes an inseticidal toxin from th soil bacterium Bacillus thuringiensis (Bt) .
In spite of their enoumous humanitarian potential.Genetically modified organisms are controversial • The development of GMOs has not been greeted with universal enthusiasm and support . . many individuals. as well as the government of some countries. look on GMOs with suspicion and cocern.