the eukaryotic genome and

8/2/2019 The Eukaryotic Genome And

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The Eukaryotic Genome andIts Expression


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The Eukaryotic Genome and Its Expression

The Eukaryotic Genome

Repetitive Sequences in the Eukaryotic Genome

The Structures of Protein-Coding Genes

RNA Processing

Posttranscriptional Regulation

Translational and Posttranslational Regulation


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Eukaryotic genomes are larger than those of

prokaryotes.

Eukaryotic genomes have more regulatory

sequences and more regulatory proteins that bind

to them.

Much of eukaryotic DNA is noncoding.

Eukaryotes have multiple chromosomes.

In eukaryotes, transcription and translation arephysically separated.


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The genome of the yeast Saccharomyces

cerevisiae has has been sequenced and 5,600genes found.

By means of gene annotation, around 70 percent

have been assigned probable roles.

Yeast has become an important model for

eukaryotic cells.

The proportions of the yeast genome coding for

specific metabolic roles have been determined.


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Both E. coli(a prokaryote) and yeast (a

eukaryote) use about the same number of genesfor cell survival.

Yeast has many more genes for protein targeting.

Eukaryotes require a greater number of genesbecause of the compartmentalization of the cells,

confirming that eukaryote cells are structurally

more complex than prokaryote cells.


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Genes for other types of proteins that are present in

eukaryotes but have no homologs in prokaryotesinclude:

Genes encoding histones

Genes encoding cytoskeletal and motor proteinssuch as actin and tubulin

Genes encoding cyclin-dependent kinases that

control cell division

Genes encoding proteins involved in the

processing of RNA


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Caenorhabditis elegans, a small nematode, has

become a model for multicellular organisms.

The genome ofC. elegans has been sequenced

and contains about 19,000 protein-coding genes.

About 3,000 genes in the worm have homologs inyeast. These genes are the ones considered

essential to all eukaryotes.

Many of the remaining 16,000 genes perform

roles related to multicellularity.


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Table 14.3 C. elegans Genes Essential to Multicellularity


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Drosophila melanogasteris much larger than C.

elegans, having 10 times more cells, but the genomehas fewer protein-coding genes than C. elegans.

C. elegans has more copies of related genes than

Drosophila does.

About half of the fly genes have mammalian homologs.

The fly genome contains 177 genes whose sequences

are known to be directly involved in human diseases,

such as cancer.

The roles of such genes are often more easily studied

in the fly than in humans.


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The puffer fish, Fugu rubripes, has a very

compact genome consisting of about 30,000genes.

The human genome has about the same number

of genes in eight times the amount of DNA.

The human and puffer fish genomes have many

similar genes; the puffer fish genome is an

abridged version of the human genome.

Repetitive DNA sequences, which make up 40

percent of the human genome, are present in

much smaller proportions in the puffer fish

genome.


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The thale cress,Arabidopsis thaliana, has a small

genome and is a model organism for study byplant biologists.

The DNA sequence contains about 26,000

protein-coding genes, many of which are

duplicates of other genes.

Many of these genes have homologs in the fruit

fly and roundworm, suggesting that plants and

animals have a common ancestor.

Arabidopsis also has genes unique to plants, such

as those for cell walls and photosynthesis.


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Rice, Oryza sativa, has many genes similar to

Arabidopsis.

The genomes of different subspecies of rice have

been sequenced, and each has particular genes

that make it unique.

Analyses of these genes will lead to

improvements in this and other grain crops.


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Three types ofhighly repetitive sequences are

found in eukaryotes:

Satellites are 5 to 50 bp long, repeated side by

side up to a million times.

Minisatellites are 12 to 100 bp long and repeatedseveral thousand times. Individuals in a

population can vary in the number of copies.

Microsatellites are 1 to 5 bp and present in 10 to

50 copies per cluster. They are scattered all over

the genome.


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Telomeres are moderately repetitive sequences

at the end of the chromosomes. They are nottranscribed into RNA.

However, some moderately repetitive DNA

sequences code for tRNAs and rRNAs.

The genome has multiple copies of these coding

regions so that tRNAs and mRNAs can be

produced in amounts needed by most cells.


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In mammals there are four different rRNA

molecules that make up the ribosome: 18S, 5.8S,28S, and 5S.

The 18S, 5.8S, and 28S rRNAs are transcribed as

a single precursor RNA, which is twice the size of

all three ultimate products.

There are 280 copies of sequences coding for the

transcript located in clusters on five different

chromosomes.

Figure 14 2 A Moderately Repetitive Sequence Codes for rRNA


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Figure 14.2 A Moderately Repetitive Sequence Codes for rRNA


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Some moderately repetitive DNA sequences are

transposons of which there are four main types.

SINEs are short interspersed elements up to 500 bp

long. They are transcribed but not translated.

LINEs are long interspersed elements up to 7,000bp long. Some are transcribed and translated into

proteins.

Retrotransposons, constituting about 17 percent of

the human genome, also make an RNA copy when

they move.

DNA transposons do not use an RNA intermediate,

but actually move to a new spot without replicating.

Figure 14 3 DNA Transposons and Transposition


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Figure 14.3 DNA Transposons and Transposition


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Beneficial roles for transposons are unknown.

They may be cellular parasites that simplyreplicate themselves.

Insertion of a transposon into a functional genecan disable it or alter its transcription rate.

Insertions in a germ cell line can result in newmutations.

If insertion occurs in a somatic cell, cancer mayresult.

Transposition increases genetic variation byshuffling genetic material and creating new genes.

Transposons may have played a role in the

evolution of cell organelles.


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Many protein-coding genes in eukaryotes are

single-copy DNA sequences.

Unlike most prokaryotes, however, eukaryotes

have genes with noncoding internal sequences.

Eukaryotes also form gene families withstructurally and functionally related cousins in

the genome.


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Genes have three types ofnoncoding sequences:

The promoteroccurs at the beginning of the gene

and is the site where RNA polymerase begins

transcription.

The terminatoroccurs at the end of the gene andsignals the end of transcription.

Noncoding sequences called introns are

interspersed with the coding regions, called

exons.

Figure 14 4 The Structure and Transcription of a Eukaryotic Gene


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Figure 14.4 The Structure and Transcription of a Eukaryotic Gene


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The entire sequence, including introns, is

transcribed. The resulting RNA is the primarytranscript, orpre-mRNA.

The transcripts of the introns are removed from

the pre-RNA and the transcripts of the exons are

spliced together, resulting in mature mRNA.

Nucleic acid hybridization can be used to

determine the location of introns in DNA. This

method was also used in the initial discovery ofintrons.

Figure 14 5 Nucleic Acid Hybridization


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Figure 14.5 Nucleic Acid Hybridization

Figure 14.6 Nucleic Acid Hybridization Revealed Noncoding DNA (Part 1)


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About half of all eukaryotic protein-coding genes

have a single copy in the haploid genome. Therest have multiple copies.

Pseudogenes () are inexact, nonfunctional

copies of genes, often found near the functional

copy.


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Sometimes copies of genes are functional, but

slightly different. A set of duplicated or relatedgenes is called a gene family.

DNA sequences in gene families vary, but as long

as one member retains the original DNA sequence,

the other members can mutate without negative

effects.

These extra genes provide material for evolution. If

the mutated gene is useful, it will be selected for insucceeding generations.


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The gene family encoding the globins is an

example.

Humans have three -globins and five -globins.

During development, different members of the -

globin gene family are expressed at differenttimes and in different tissues.

Figure 14.8 Differential Expression in the Globin Gene Family


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g p y


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The globin gene family also includes nonfunctional

pseudogenes.

These black sheep family members result from

mutations that cause loss of function.

As long as some members of a gene family arefunctional and pseudogenes are not actively

detrimental, there appears to be little selective

pressure to eliminate the pseudogenes.

Figure 14.7 The Globin Gene Family


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RNA Processing

The first two steps of processing pre-mRNA take

place in the nucleus:

The G cap, a modified GTP, is added to the 5

end. It facilitates the binding of mRNA to the

ribosome and protects the mRNA from being

digested by ribonucleases.

A poly A tail is added to the 3 end. It is 100 to

300 residues of adenine (poly A) in length.

Figure 14.9 Processing the Ends of Eukaryotic Pre-mRNA


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RNA Processing

RNA splicing removes the introns and splices the

exons together:

At the boundaries between introns and exons areconsensus sequences.

A small ribonucleoprotein particle (snRNP) binds tothe consensus sequence at the 5 exonintronboundary.

Another snRNP binds near the 3 exonintron

boundary. Then other proteins bind, forming a large RNA

protein complex called a spliceosome. Thiscomplex cuts the RNA, releases the introns, andjoins the ends of the exons.

Figure 14.10 The Spliceosome, an RNA Splicing Machine (Part 1)


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Figure 14.10 The Spliceosome, an RNA Splicing Machine (Part 2)


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Transcriptional Regulation of Gene Expression

Each cell in a multicellular organism contains all

the genes of the organisms genome.

For normal development, the expression of genes

must be regulated.

Regulation of gene expression can occur at manypoints during development.

Some mechanisms result in the selective

transcription of specific genes.

Figure 14.11 Potential Points for the Regulation of Gene Expression in Eukaryotes (Part 1)


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Figure 14.11 Potential Points for the Regulation of Gene Expression in Eukaryotes (Part 2)


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With few exceptions, all cells in an organism have

the same genes, but they express them differently.

For example, both brain and liver cells transcribe

housekeeping genes that code for enzymes and

other molecules essential to the survival of all cells.

However, liver cells transcribe some genes for liver-

specific proteins, and brain cells transcribe some

genes for brain-specific proteins.

The difference in the production of proteins is dueto differential transcription.


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Unlike prokaryotes, in which related genes are

transcribed in units called operons, eukaryotes tendto have solitary genes.

Eukaryotes have three different RNA polymerases:

RNA polymerase II transcribes protein-codinggenes to mRNA.

RNA polymerase I transcribes rRNA coding

sequences.

RNA polymerase III transcribes tRNA and small

nuclear RNAs.


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Most eukaryotic genes have other DNA

sequences that regulate transcription.

In prokaryotes, a single peptide subunit can cause

RNA polymerase to recognize the promoter; in

eukaryotes many different proteins are involved in

initiating transcription.


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Transcription factors are regulatory proteins

required for transcription in eukaryotes.

RNA polymerase II does not bind until several

other proteins, such as TFIID, have already bound

the proteinDNA complex.

Some DNA sequences, such as the TATA box,

are common to most promoters; others are unique

to only a few genes.

Transcription factors play an important role in celldifferentiation during development.

Figure 14.12 The Initiation of Transcription in Eukaryotes (Part 1)


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Figure 14.12 The Initiation of Transcription in Eukaryotes (Part 2)


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In addition to the promoter, nearby regulator

sequences also affect transcription by bindingregulator proteins that activate RNA polymerase.

Much farther away are enhancer regions, which

bind activator proteins and strongly stimulate the

transcription complex.

Negative regulatory regions of DNA called

silencers bind proteins called repressors and turn

off transcription. Thus they have the oppositeeffect of enhancers.

Figure 14.13 The Roles of Transcription Factors, Regulators, and Activators (Part 1)


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Figure 14.13 The Roles of Transcription Factors, Regulators, and Activators (Part 2)


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In eukaryotes, genes on different chromosomes

may require coordination.

Regulation of various genes can be coordinated if all

have the same regulatory sequences that bind to

the same activators and regulators.

One example is the stress response element in

plants.

Stress response elements near each of the

scattered genes stimulate RNA synthesis.

RNA then codes for proteins needed for water

conservation.

Figure 14.14 Coordinating Gene Expression


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Key to transcription regulation in eukaryotes is the

binding of protein to specific DNA sequences.

Proteins need to recognize and bind appropriate

sites.

There are four different structural themes ormotifs for proteinDNA interactions:

Helix-turn-helix

Zinc finger

Leucine zipper

Helix-loop-helix

Figure 14.15 Protein-DNA Interactions (Part 1)


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Figure 14.15 Protein-DNA Interactions (Part 2)


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T i ti l R l ti f G E i


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Other mechanisms that regulate transcription act

on the structure of chromatin and chromosomes.

The packaging of DNA by the nuclear proteins in

chromatin can make DNA physically inaccessible

to RNA polymerase and associated components.



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Nucleosomes inhibit initiation and elongation of

transcription.

Nucleosomes are inactivated by two protein

complexes in a process called chromatin

remodeling.

Nucleosome disaggregation occurs by acetylation of

amino groups on the histones, and is associated

with the activation of genes.

Nucleosomes reform by deacetylation of the aminogroups, and is associated with gene deactivation.

Figure 14.16 Local Remodeling of Chromatin for Transcription


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Two different kinds of chromatin can be

distinguished by staining the interphase nucleus.

Euchromatin stains lightly. It contains DNA that is

transcribed into mRNA.

Heterochromatin stains densely and is generallynot transcribed. Any genes in heterochromatin are

thus inactivated.



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Heterochromatin is in found in the inactive X

chromosome of mammals.

One of the X chromosomes in each cell of a

female is inactivated early in development.

The chromosome remains condensed andappears as a Barr body under the microscope.

Condensation physically prevents DNA from being

transcribed.

Methylation of cytosine on DNA may be involvedwith the inactivation.



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The inactive X has one gene that is only lightly

methylated and transcriptionally active, calledXist.

The RNA transcribed fromXistis not an mRNA and

remains in the nucleus.

It binds the X chromosome that transcribes it andtriggers inactivation.

This RNA transcript is called interference RNA

(RNAi).

Figure 14.18 A Model for X Chromosome Inactivation


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Some gene expression is regulated by DNA

rearrangement.

Saccharomyces cerevisiae has two mating types,

a and . All cells have alleles for both types, but

only one is expressed at at time.

The alleles have separate locations on the

chromosomes, and are separate from the MAT

locus.

The mating type of a given yeast cell depends onwhich copy, a or, exists at the MAT site. Alleles

at the MAT site can be moved in and out.



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One cell can make more proteins than another

cell by making more copies of a gene, a processcalled gene amplification.

Mature frog and fish eggs have up to a trillionribosomes, which are used for massive protein

synthesis following fertilization. To make this number, ribosomal rRNA gene

clusters are selectively amplified and copied untilthere are a million copies in just one cell.

Later, after cell division begins, the number ofcopies returns to normal.

The mechanism for this selective amplification ofa single gene is not clearly understood.

Figure 14.19 Transcription from Multiple Genes for rRNA


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There are many ways in which gene expression

can be regulated after transcription.

Pre-mRNA can be processed in the nucleus by

cutting and splicing.

The longevity of mRNA in the cytoplasm can alsobe regulated.



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Alternative splicing of a specific pre-mRNA can

generate different proteins from a single gene.

For example, cells in five different tissues splice

the pre-mRNA for the structural protein

tropomyosin into five different mRNAs.

As a result, each of the five tissues in mammals

(skeletal muscle, smooth muscle, fibroblast, liver,

and brain) has a different form of tropomyosin.

Figure 14.20 Alternative Splicing Results in Different mRNAs and Proteins


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RNA has no repair mechanisms.

Different mRNAs have different life spans, and the

less time an mRNA spends in the cytoplasm, the

less of its protein can be translated.

Specific AU-rich nucleotide sequences withinsome mRNAs mark them for rapid breakdown by

a ribonuclease complex called the exosome.

Signaling molecules, such as growth factors, are

made only when needed and then break downrapidly.



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RNA editing can be used to change the

sequence of mRNA after transcription.

This editing can take place by either the insertion

of nucleotides to the mRNA sequence or the

alteration of nucleotides in the mRNA.

Figure 14.21 RNA Editing


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Proteins can regulate translation by binding to

mRNA in the cytoplasm.

This is important for long-lived mRNAs. It

prevents the production of unnecessary proteins.

For example, cyclin, which stimulates the cellcycle, must be shut off after it has done its job. If

not, inappropriate cell division may lead to a

tumor.



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The translation of mRNA can be regulated to

control levels of certain proteins.

1. Regulation by the G cap: An mRNA capped

with an unmodified GTP is not translated. These

mRNAs can be stored and modified later when

the proteins are needed.



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2. Regulation of ferritin, an iron storage protein:

When excess iron is present, ferritin synthesisincreases, but the amount of ferritin mRNA

remains constant.

When iron is low, a translational repressor

protein binds to ferritin mRNA and preventstranslation.

When iron levels rise, excess iron binds to the

repressor and alters its structure, causing it to

detach from the mRNA. Translation thenproceeds.



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3. Regulation of hemoglobin:

Hemoglobin consists of four globin units andfour heme pigments.

If globin synthesis does not equal heme

synthesis, some heme stays free in the cell.

Excess heme in the cell increases the rate oftranslation of globin mRNA by removing a

block to initiation of translation at the

ribosome.



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Regulating the lifetime of a protein is a way to

control its actions.

Proteins identified for breakdown are often linked

to the protein ubiquitin.

The proteinubiquitin complex then binds to acomplex called a proteasome, nicknamed the

molecular chamber of doom.

The protein is cleaved from the ubiquitin and three

different proteases digest it.

Overall, concentrations of proteins depend on

rates of synthesis and rates of digestion.

Figure 14.22 The Proteasome Breaks Down Proteins


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