BIS 1A Handout 19 - Eukaryotic Genetics and Genomes

How Eukaryotic Genomes Work and Evolve

Two features of eukaryotic genomes present a major information-processing challenge.

First, the typical multicellular eukaryotic genome is much larger than that of a prokaryotic cell.

Second, cell specialization limits the expression of many genes to specific cells.

This DNA is elaborately organized.

Not only is the DNA associated with protein, but also this DNA-protein complex called chromatin is organized into higher structural levels than the DNA-protein complex in prokaryotes.

Chromatin structure is based on successive levels of DNA packing

While the single circular chromosome of bacteria is coiled and looped in a complex but orderly manner, eukaryotic chromatin is far more complex.

Eukaryotic DNA is precisely combined with large amounts of protein.

Eukaryotic chromosomes contain an enormous amount of DNA relative to their condensed length.

Each human chromosome averages about 1.5 × 108 nucleotide pairs.

If extended, each DNA molecule would be about 4 cm long, thousands of times longer than the cell diameter.

This chromosome and 45 other human chromosomes fit into the nucleus.

This occurs through an elaborate, multilevel system of DNA packing.

Histone proteins are responsible for the first level of DNA packaging.

The mass of histone in chromatin is approximately equal to the mass of DNA.

Their positively charged amino acids bind tightly to negatively charged DNA.

The five types of histones are very similar from one eukaryote to another, and similar proteins are found in prokaryotes.

The conservation of histone genes during evolution reflects their pivotal role in organizing DNA within cells.

Unfolded chromatin has the appearance of beads on a string.

In this configuration, a chromatin fiber is 10 nm in diameter (the 10-nm fiber).

Each bead of chromatin is a nucleosome, the basic unit of DNA packing.

The "string" between the beads is called linker DNA.

A nucleosome consists of DNA wound around a protein core composed of two molecules each of four types of histone: H2A, H2B, H3, and H4.

The amino acid (N-terminus) of each histone protein (the histone tail) extends outward from the nucleosome.

A molecule of a fifth histone, H1, attaches to the DNA near the nucleosome.

Histones leave the DNA only transiently during DNA replication.

They stay with the DNA during transcription.

By changing shape and position, nucleosomes allow RNA-synthesizing polymerases to move along the DNA.

The next level of packing is due to the interactions between the histone tails of one nucleosome and the linker DNA and nucleosomes to either side.

With the aid of histone H1, these interactions cause the 10-nm to coil to form the 30-nm chromatin fiber.

This fiber forms looped domains attached to a scaffold of nonhistone proteins to make up a 300-nm fiber.

In a mitotic chromosome, the looped domains coil and fold to produce the characteristic metaphase chromosome.

Much of the interphase chromatin is present as a 10-nm fiber, and some is compacted into a 30-nm fiber, which in some regions is folded into looped domains.

Interphase chromosomes have highly condensed areas, heterochromatin, and less compacted areas, euchromatin.

Heterochromatin DNA is largely inaccessible to transcription enzymes.

Looser packing of euchromatin makes its DNA accessible to enzymes and available for transcription.

Gene expression can be regulated at any stage, but the key step is transcription

Like unicellular organisms, the tens of thousands of genes in the cells of multicellular eukaryotes are continually turned on and off in response to signals from their internal and external environments.

Gene expression must be controlled on a long-term basis during cellular differentiation, the divergence in form and function as cells in a multicellular organism specialize.

A typical human cell probably expresses about 20% of its genes at any given time.

Highly specialized cells, such as nerves or muscles, express only a tiny fraction of their genes.

The differences between cell types are due to differential gene expression, the expression of different genes by cells with the same genome.

The genomes of eukaryotes may contain tens of thousands of genes.

For quite a few species, only a small amount of the DNA-1.5% in humans-codes for protein.

Of the remaining DNA, a very small fraction consists of genes for rRNA and tRNA.

Most of the rest of the DNA seems to be largely noncoding, although researchers have found that a significant amount of it is transcribed into RNAs of unknown function.

Problems with gene expression and control can lead to imbalance and diseases, including cancers.

In all organisms, the expression of specific genes is most commonly regulated at transcription, often in response to signals coming from outside the cell.

The term gene expression is often equated with transcription.

Each stage in the entire process of gene expression provides a potential control point where gene expression can be turned on or off, sped up or slowed down.

A network of control connects different genes and their products.

These levels of control include chromatin packing, transcription, RNA processing, translation, and various alterations to the protein product.

Chromatin modifications affect the availability of genes for transcription.

In addition to its role in packing DNA inside the nucleus, chromatin organization regulates gene expression.

Genes of densely condensed heterochromatin are usually not expressed, presumably because transcription proteins cannot reach the DNA.

Chemical modifications of histones play a direct role in the regulation of gene transcription.

The N-terminus of each histone molecule in a nucleosome protrudes outward from the nucleosome.

These histone tails are accessible to various modifying enzymes, which catalyze the addition or removal of specific chemical groups.

Histone acetylation (addition of an acetyl group -COCH3) and deacetylation appear to play a direct role in the regulation of gene transcription.

Acetylated histones grip DNA less tightly, providing easier access for transcription proteins in this region.

DNA methylation is the attachment by specific enzymes of methyl groups (-CH3) to DNA bases after DNA synthesis.

Inactive DNA is generally highly methylated compared to DNA that is actively transcribed.

For example, the inactivated mammalian X chromosome in females is heavily methylated.

Demethylating certain inactive genes turns them on.

However, there are exceptions to this pattern.

In some species, DNA methylation is responsible for long-term inactivation of genes during cellular differentiation.

Once methylated, genes usually stay that way through successive cell divisions.

Methylation enzymes recognize sites on one strand that are already methylated and correctly methylate the daughter strand after each round of DNA replication.

This methylation patterns accounts for genomic imprinting in which methylation turns off either the maternal or paternal alleles of certain genes at the start of development.

The chromatin modifications just discussed do not alter DNA sequence, and yet they may be passed along to future generations of cells.

Inheritance of traits by mechanisms not directly involving the nucleotide sequence is called epigenetic inheritance.

Transcription initiation is controlled by proteins that interact with DNA and with each other.

Multiple control elements are associated with most eukaryotic genes.

Control elements are noncoding DNA segments that regulate transcription by binding certain proteins.

These control elements and the proteins they bind are critical to the precise regulation of gene expression in different cell types.

To initiate transcription, eukaryotic RNA polymerase requires the assistance of proteins called transcription factors.

General transcription factors are essential for the transcription of all protein-coding genes.

Only a few general transcription factors independently bind to DNA.

Others in the initiation complex are involved in protein-protein interactions, binding each other and RNA polymerase II.

The interaction of general transcription factors and RNA polymerase II with a promoter usually leads to only a low rate of initiation and production of few RNA transcripts.

In eukaryotes, high levels of transcription of particular genes depend on the interaction of control elements with specific transcription factors.

Some control elements, named proximal control elements, are located close to the promoter.

Distant control elements, enhancers, may be thousands of nucleotides away from the promoter or even downstream of the gene or within an intron.

An activator is a protein that binds to an enhancer to stimulate transcription of a gene.

Protein-mediated bending of DNA brings bound activators in contact with a group of mediator proteins that interact with proteins at the promoter.

This helps assemble and position the initiation complex on the promoter.

Eukaryotic genes also have repressor proteins to inhibit expression of a gene.

Eukaryotic repressors can cause inhibition of gene expression by blocking the binding of activators to their control elements or to components of the transcription machinery or by turning off transcription even in the presence of activators.

Recent studies of the genomes of several eukaryotic species have found that some coexpressed genes are clustered near each other on the same chromosome.

Each eukaryotic gene in these clusters has its own promoter and is individually transcribed.

The coordinate regulation of clustered genes in eukaryotic cells is thought to involve changes in the chromatin structure that makes the entire group of genes either available or unavailable for transcription.

More commonly, genes coding for the enzymes of a metabolic pathway are scattered over different chromosomes.

Coordinate gene expression in eukaryotes depends on the association of a specific control element or combination of control elements with every gene of a dispersed group.

A common group of transcription factors binds to all the genes in the group, promoting simultaneous gene transcription.

For example, a steroid hormone enters a cell and binds to a specific receptor protein in the cytoplasm or nucleus, forming a hormone-receptor complex that serves as a transcription activator.

Every gene whose transcription is stimulated by that steroid hormone has a control element recognized by that hormone-receptor complex.

Systems for coordinating gene regulation probably arose early in evolutionary history and evolved by the duplication and distribution of control elements within the genome.

Post-transcriptional mechanisms play supporting roles in the control of gene expression.

Gene expression may be blocked or stimulated by any posttranscriptional step.

By using regulatory mechanisms that operate after transcription, a cell can rapidly fine-tune gene expression in response to environmental changes without altering its transcriptional patterns.

In alternative RNA splicing, different mRNA molecules are produced from the same primary transcript, depending on which RNA segments are treated as exons and which as introns.

The life span of an mRNA molecule is an important factor in determining the pattern of protein synthesis.

Prokaryotic mRNA molecules may be degraded after only a few minutes.

Eukaryotic mRNAs typically last for hours, days, or weeks.

In red blood cells, mRNAs for hemoglobin polypeptides are unusually stable and are translated repeatedly.

A common pathway of mRNA breakdown begins with enzymatic shortening of the poly-A tail.

This triggers the enzymatic removal of the 5' cap.

This is followed by rapid degradation of the mRNA by nucleases.

Nucleotide sequences in the untranslated region at the 3' end affect lifetime of mRNA.

During the past few years, researchers have found small single-stranded RNA molecules called microRNAs, or miRNAs, that bind to complementary sequences in mRNA molecules.

A miRNA associates with a protein complex and binds to target mRNA.

The miRNA-protein complex then degrades the target mRNA or blocks its translation.

The phenomenon of inhibition of gene expression by RNA molecules is called RNA interference (RNAi).

Translation of specific mRNAs can be blocked by regulatory proteins that bind to specific sequences or structures within the 5' leader region of mRNA.

This prevents attachment of ribosomes.

mRNAs may be stored in egg cells without poly-A tails of sufficient size to allow translation initiation.

At the appropriate time during development, a cytoplasmic enzyme adds more A residues, allowing translation to begin.

The cell limits the lifetimes of normal proteins by selective degradation.

Many proteins, like the cyclins in the cell cycle, must be short-lived to function appropriately.

Proteins intended for degradation are marked by the attachment of ubiquitin proteins.

Giant protein complexes called proteasomes recognize the ubiquitin and degrade the tagged protein.

Mutations making cell cycle proteins impervious to proteasome degradation can lead to cancer.

Eukaryotic genomes can have many noncoding DNA sequences in addition to genes

Several trends are evident when we compare the genomes of prokaryotes to those of eukaryotes.

There is a general trend from smaller to larger genomes, but with fewer genes in a given length of DNA.

Humans have 500 to 1,500 times as many base pairs in their genome as most prokaryotes, but only 5 to 15 times as many genes.

In eukaryotes, most of the DNA (98.5% in humans) does not code for protein or RNA.

Gene-related regulatory sequences and introns account for 24% of the human genome.

Introns account for most of the difference in average length of eukaryotic (27,000 base pairs) and prokaryotic genes (1,000 base pairs).

Most intergenic DNA is repetitive DNA, present in multiple copies in the genome.

Transposable elements and related sequences make up 44% of the entire human genome.

Eukaryotic transposable elements are of two types: transposons, which move within a genome by means of a DNA intermediate, and retrotransposons, which move by means of an RNA intermediate, a transcript of the retrotransposon DNA.

Transposons can either move to a new place or copy itself to a new location.

Retrotransposons always leave a copy at the original site, since they are initially transcribed into an RNA intermediate.

Most transposons are retrotransposons, in which the transcribed RNA includes the code for an enzyme that catalyzes the insertion of the retrotransposon and may include a gene for reverse transcriptase.

Reverse transcriptase uses the RNA molecule originally transcribed from the retrotransposon as a template to synthesize a double-stranded DNA copy.

Multiple copies of transposable elements and related sequences are scattered throughout eukaryotic genomes.

A single unit is hundreds or thousands of base pairs long, and the dispersed "copies" are similar but not identical to one another.

Some of the copies are transposable elements and some are related sequences that have lost the ability to move.

Repetitive DNA that is not related to transposable elements probably arose by mistakes that occurred during DNA replication or recombination.

Repetitive DNA accounts for about 15% of the human genome.

Five percent of the human genome consists of large-segment duplications in which 10,000 to 300,000 nucleotide pairs seem to have been copied from one chromosomal location to another.

Simple sequence DNA contains many copies of tandemly repeated short sequences of 15-500 nucleotides.

There may be as many as several hundred thousand repetitions of a nucleotide sequence.

Simple sequence DNA makes up 3% of the human genome.

Much of the genome's simple sequence DNA is located at chromosomal telomeres and centromeres, suggesting that it plays a structural role.

The DNA at centromeres is essential for the separation of chromatids in cell division and may also help to organize the chromatin within the interphase nucleus.

Telomeric DNA prevents gene loss as DNA shortens with each round of replication and also binds proteins that protect the ends of a chromosome from degradation or attachment to other chromosomes.

Gene families have evolved by duplication of ancestral genes.

In humans, solitary genes present in one copy per haploid set of chromosomes make up only half of the total coding DNA.

The rest occurs in multigene families, collections of identical or very similar genes.

Some multigene families consist of identical DNA sequences that may be clustered tandemly.

These code for RNA products or for histone proteins.

For example, the three largest rRNA molecules are encoded in a single transcription unit that is repeated tandemly hundreds to thousands of times.

This transcript is cleaved to yield three rRNA molecules that combine with proteins and one other kind of rRNA to form ribosomal subunits.

Two related families of nonidentical genes encode globins, a group of proteins that include the ± (alpha) and ² (beta) polypeptide sequences of hemoglobin.

The different versions of each globin subunit are expressed at different times in development, allowing hemoglobin to function effectively in the changing environment of the developing animal.

Within both the ± and ² families are sequences that are expressed during the embryonic, fetal, and/or adult stage of development.

In humans, the embryonic and fetal hemoglobins have higher affinity for oxygen than do adult forms, ensuring transfer of oxygen from mother to developing fetus.

Also found in the globin gene family clusters are several pseudogenes, DNA sequences similar to real genes that do not yield functional proteins.

Duplications, rearrangements, and mutations of DNA contribute to genome evolution

The earliest forms of life likely had a minimal number of genes, including only those necessary for survival and reproduction.

The size of genomes has increased over evolutionary time, with the extra genetic material providing raw material for gene diversification.

An accident in meiosis can result in one or more extra sets of chromosomes, a condition known as polyploidy.

In a polyploid organism, one complete set of genes can provide essential functions for the organism.

The genes in the extra set may diverge by accumulating mutations.

These variations may persist if the organism carrying them survives and reproduces.

In this way, genes with novel functions may evolve.

Errors during meiosis due to unequal crossing over during Prophase I can lead to duplication of individual genes.

Slippage during DNA replication can result in deletion or duplication of DNA regions.

Such errors can lead to regions of repeats, such as simple sequence DNA.

Major rearrangements of at least one set of genes occur during immune system differentiation.

Duplication events can lead to the evolution of genes with related functions, such as the ±-globin and ²-globin gene families.

A comparison of gene sequences within a multigene family indicates that they all evolved from one common ancestral globin gene, which was duplicated and diverged about 450 500 million years ago.

After the duplication events, the differences between the genes in the globin family arose from mutations that accumulated in the gene copies over many generations.

The necessary function provided by an ±-globin protein was fulfilled by one gene, while other copies of the ±-globin gene accumulated random mutations.

Some mutations may have altered the function of the protein product in ways that were beneficial to the organism without changing its oxygen-carrying function.

The similarity in the amino acid sequences of the various ±-globin and ²-globin proteins supports this model of gene duplication and mutation.

In other gene families, one copy of a duplicated gene can undergo alterations that lead to a completely new function for the protein product.

The genes for lysozyme and ±-lactalbumin are good examples.

Lysozyme is an enzyme that helps prevent infection by hydrolyzing bacterial cell walls.

±-lactalbumin is a nonenzymatic protein that plays a role in mammalian milk production.

Both genes are found in mammals, while only lysozyme is found in birds.

The two proteins are similar in their amino acids sequences and 3-D structures.

These findings suggest that at some time after the bird and mammalian lineage had separated, the lysozyme gene underwent a duplication event in the mammalian lineage but not in the avian lineage.

Subsequently, one copy of the duplicated lysozyme gene evolved into a gene encoding ±-lactalbumin, a protein with a completely different function.

Rearrangement of existing DNA sequences has also contributed to genome evolution.

The presence of introns in eukaryotic genes may have promoted the evolution of new and potentially useful prote3ß è A

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Mixing and matching of different exons within or between genes owing to errors in meiotic recombination is called exon shuffling and could lead to new proteins with novel combinations of functions.

The persistence of transposable elements as a large percentage of eukaryotic genomes suggests that they play an important role in shaping a genome over evolutionary time.

These elements can contribute to evolution of the genome by promoting recombination, disrupting cellular genes or control elements, and carrying entire genes or individual exons to new locations.

The presence of homologous transposable element sequences scattered throughout the genome allows recombination to take place between different chromosomes.

Most of these alterations are likely detrimental, causing chromosomal translocations and other changes in the genome that may be lethal to the organism.

Over the course of evolutionary time, an occasional recombination may be advantageous.

The movement of transposable elements around the genome can have several direct consequences.

If a transposable element "jumps" into the middle of a coding sequence of a protein-coding gene, it prevents the normal functioning of that gene.

If a transposable element inserts within a regulatory sequence, it may increase or decrease protein production.

During transposition, a transposable element may transfer genes to a new position on the genome or may insert an exon from one gene into another gene.

Over long periods of time, the generation of genetic diversity provides more raw material for natural selection to work on during evolution.