Overview: How Eukaryotic Genomes Work and Evolve Two features of eukaryotic genomes are a major information-processing challenge: First, the typical eukaryotic genome is much larger than that of a prokaryotic cell Second, cell specialization limits the expression of many genes to specific cells The DNA-protein complex, called chromatin, is ordered into higher structural levels than the DNA-protein complex in prokaryotes

Concept 19.1: Chromatin structure is based on successive levels of DNA packing Eukaryotic DNA is precisely combined with a large amount of protein Eukaryotic chromosomes contain an enormous amount of DNA relative to their condensed length

Nucleosomes, or “Beads on a String” Proteins called histones are responsible for the first level of DNA packing in chromatin The association of DNA and histones seems to remain intact throughout the cell cycle In electron micrographs, unfolded chromatin has the appearance of beads on a string Each “bead” is a nucleosome, the basic unit of DNA packing Animation: DNA Packing

LE 19-2a 2 nm DNA double helix His- Histone tones tails 10 nm Histone H1 10 nm Linker DNA (“string”) Nucleosome (“bead”) Nucleosomes (10-nm fiber)

Higher Levels of DNA Packing The next level of packing forms the 30-nm chromatin fiber

LE 19-2b 30 nm Nucleosome 30-nm fiber

In turn, the 30-nm fiber forms looped domains, making up a 300-nm fiber

LE 19-2c Protein scaffold Loops 300 nm Scaffold Looped domains (300-nm fiber)

In a mitotic chromosome, the looped domains coil and fold, forming the metaphase chromosome

LE 19-2d 700 nm 1,400 nm Metaphase chromosome

Interphase chromatin is usually much less condensed than that of mitotic chromosomes Much of the interphase chromatin is present as a 10-nm fiber, and some is 30-nm fiber, which in some regions is folded into looped domains Interphase chromosomes have highly condensed areas, called heterochromatin, and less compacted areas, called euchromatin

Concept 19.2: Gene expression can be regulated at any stage, but the key step is transcription All organisms must regulate which genes are expressed at any given time A multicellular organism’s cells undergo cell differentiation, specialization in form and function

Differential Gene Expression Differences between cell types result from differential gene expression, the expression of different genes by cells within the same genome In each type of differentiated cell, a unique subset of genes is expressed Many key stages of gene expression can be regulated in eukaryotic cells

Chemical modification Degradation of protein LE 19-3 Signal NUCLEUS Chromatin DNA Gene available for transcription Gene Transcription RNA Exon Primary transcript Intro RNA processing Tail Cap mRNA in nucleus Transport to cytoplasm CYTOPLASM mRNA in cytoplasm Degradation of mRNA Translation Polypeptide Cleavage Chemical modification Transport to cellular destination Active protein Degradation of protein Degraded protein

Regulation of Chromatin Structure Genes within highly packed heterochromatin are usually not expressed Chemical modifications to histones and DNA of chromatin influence both chromatin structure and gene expression

Histone Modification In histone acetylation, acetyl groups are attached to positively charged lysines in histone tails This process seems to loosen chromatin structure, thereby promoting the initiation of transcription

LE 19-4 Histone tails DNA double helix Amino acids available for chemical modification Histone tails protrude outward from a nucleosome Unacetylated histones Acetylated histones Acetylation of histone tails promotes loose chromatin structure that permits transcription

DNA Methylation DNA methylation, the addition of methyl groups to certain bases in DNA, is associated with reduced transcription in some species In some species, DNA methylation causes long- term inactivation of genes in cellular differentiation In genomic imprinting, methylation turns off either the maternal or paternal alleles of certain genes at the start of development

Regulation of Transcription Initiation Chromatin-modifying enzymes provide initial control of gene expression by making a region of DNA either more or less able to bind the transcription machinery

Organization of a Typical Eukaryotic Gene Associated with most eukaryotic genes are control elements, segments of noncoding DNA that help regulate transcription by binding certain proteins Control elements and the proteins they bind are critical to the precise regulation of gene expression in different cell types

(distal control elements) Enhancer (distal control elements) Proximal control elements Poly-A signal sequence Termination region Exon Intron Exon Intron Exon DNA Upstream Downstream Promoter Transcription Poly-A signal Primary RNA transcript (pre-mRNA) Exon Intron Exon Intron Exon Cleaved 3¢ end of primary transcript 5¢ RNA processing: Cap and tail added; introns excised and exons spliced together Intron RNA Coding segment mRNA 3¢ Start codon Stop codon 5¢ Cap 5¢ UTR (untranslated region) 3¢ UTR (untranslated region) Poly-A tail

The Roles of Transcription Factors 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 In eukaryotes, high levels of transcription of particular genes depend on control elements interacting with specific transcription factors

Enhancers and Specific Transcription Factors Proximal control elements are located close to the promoter Distal control elements, groups of which are called enhancers, may be far away from a gene or even in an intron An activator is a protein that binds to an enhancer and stimulates transcription of a gene Animation: Initiation of Transcription

LE 19-6 Distal control element Activators Promoter Gene DNA Enhancer TATA box General transcription factors DNA-bending protein Group of mediator proteins RNA polymerase II RNA polymerase II Transcription Initiation complex RNA synthesis

Some transcription factors function as repressors, inhibiting expression of a particular gene Some activators and repressors act indirectly by influencing chromatin structure

LE 19-7 Liver cell nucleus Lens cell nucleus Available activators Enhancer Promoter Control elements Albumin gene Albumin gene not expressed Crystallin gene Albumin gene expressed Crystallin gene not expressed Crystallin gene expressed Liver cell Lens cell

Coordinately Controlled Genes Unlike the genes of a prokaryotic operon, coordinately controlled eukaryotic genes each have a promoter and control elements The same regulatory sequences are common to all the genes of a group, enabling recognition by the same specific transcription factors

Mechanisms of Post-Transcriptional Regulation Transcription alone does not account for gene expression More and more examples are being found of regulatory mechanisms that operate at various stages after transcription Such mechanisms allow a cell to fine-tune gene expression rapidly in response to environmental changes

Animation: RNA Processing 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 Animation: RNA Processing

LE 19-8 Exons DNA Primary RNA transcript RNA splicing or mRNA

mRNA Degradation The life span of mRNA molecules in the cytoplasm is a key to determining the protein synthesis The mRNA life span is determined in part by sequences in the leader and trailer regions

Animation: Blocking Translation Animation: mRNA Degradation RNA interference by single-stranded microRNAs (miRNAs) can lead to degradation of an mRNA or block its translation The phenomenon of inhibition of gene expression by RNA molecules is called RNA interference (RNAi) Animation: Blocking Translation Animation: mRNA Degradation

LE 19-9 Protein complex Degradation of mRNA Dicer OR miRNA Target mRNA Hydrogen bond Blockage of translation

Initiation of Translation The initiation of translation of selected mRNAs can be blocked by regulatory proteins that bind to sequences or structures of the mRNA Alternatively, translation of all mRNAs in a cell may be regulated simultaneously

Protein Processing and Degradation After translation, various types of protein processing, including cleavage and the addition of chemical groups, are subject to control Proteasomes are giant protein complexes that bind protein molecules and degrade them Animation: Protein Processing Animation: Protein Degradation

LE 19-10 Proteasome and ubiquitin to be recycled Ubiquitin Proteasome Protein to be degraded Ubiquitinated protein Protein fragments (peptides) Protein entering a proteasome

Concept 19.3: Cancer results from genetic changes that affect cell cycle control The gene regulation systems that go wrong during cancer are very same systems that play important roles in embryonic development Thus, research into the molecular basis of cancer has benefited from and informed many other fields of biology

Types of Genes Associated with Cancer Genes that normally regulate cell growth and division during the cell cycle include: Genes for growth factors Their receptors Intracellular molecules of signaling pathways Mutations altering any of these genes in somatic cells can lead to cancer

Oncogenes and Proto-Oncogenes Oncogenes are cancer-causing genes Proto-oncogenes are normal cellular genes that code for proteins that stimulate normal cell growth and division A DNA change that makes a proto-oncogene excessively active converts it to an oncogene, which may promote excessive cell division and cancer

LE 19-11 Proto-oncogene DNA Point mutation within a control element Translocation or transposition: gene moved to new locus, under new controls Point mutation within the gene Gene amplification: multiple copies of the gene New promoter Oncogene Oncogene Normal growth-stimulating protein in excess Normal growth-stimulating protein in excess Normal growth-stimulating protein in excess Hyperactive or degradation- resistant protein

Tumor-Suppressor Genes Tumor-suppressor genes encode proteins that inhibit abnormal cell division Any decrease in the normal activity of a tumor- suppressor protein may contribute to cancer

Interference with Normal Cell-Signaling Pathways Many proto-oncogenes and tumor suppressor genes encode components of growth-stimulating and growth-inhibiting pathways, respectively We will focus on products of two genes, the ras proto-oncogene and p53 tumor-suppressor gene

The Ras protein, encoded by the ras gene, is a G protein that relays a signal from a growth factor receptor to a cascade of protein kinases Many ras oncogenes have a mutation that leads to a hyperactive Ras protein that issues signals on its own, resulting in excessive cell division

LE 19-12_1 Growth factor G protein Cell cycle-stimulating pathway MUTATION Hyperactive Ras protein (product of oncogene issues signals on its own. G protein Cell cycle-stimulating pathway Receptor Protein kinases (phosphorylation cascade) NUCLEUS Transcription factor (activator) DNA Gene expression Protein that stimulates the cell cycle

The p53 gene encodes a tumor-suppressor protein that is a specific transcription factor that promotes synthesis of cell cycle–inhibiting proteins Named for its 53,000-dalton protein product, the p53 gene is often called the “guardian angel of the genome” Mutations that knock out the p53 gene can lead to excessive cell growth and cancer

LE 19-12_2 MUTATION Cell cycle-stimulating pathway NUCLEUS Growth factor MUTATION Hyperactive Ras protein (product of oncogene) issues signals on its own G protein Cell cycle-stimulating pathway Receptor Protein kinases (phosphorylation cascade) NUCLEUS Transcription factor (activator) DNA Gene expression Protein that stimulates the cell cycle Cell cycle-inhibiting pathway Protein kinases MUTATION Defective or missing transcription factor, such as p53, cannot activate Active form of p53 UV light DNA damage in genome DNA Protein that inhibits the cell cycle

Increased cell division, possibly leading to cancer, can result if the cell cycle is overstimulated (as in Figure 19.12a) or not inhibited when it normally would be (as in Figure 19.12b)

LE 19-12_3 Growth MUTATION factor G protein Cell cycle-stimulating Hyperactive Ras protein (product of oncogene) issues signals on its own G protein Cell cycle-stimulating pathway Receptor Protein kinases (phosphorylation cascade) NUCLEUS Transcription factor (activator) DNA Gene expression Protein that stimulates the cell cycle Cell cycle-inhibiting pathway Protein kinases MUTATION Defective or missing transcription factor, such as p53, cannot activate Active form of p53 UV light DNA damage in genome DNA Protein that inhibits the cell cycle Effects of mutations EFFECTS OF MUTATIONS Protein overexpressed Protein absent Cell cycle overstimulate Increased cell division Cell cycle not inhibited

The Multistep Model of Cancer Development More than one somatic mutation is generally needed to produce a full-fledged cancer cell About a half dozen DNA changes must occur for a cell to become fully cancerous These changes usually include at least one active oncogene and mutation or loss of several tumor- suppressor genes Colorectal cancer, with 135,000 new cases and 60,000 deaths in the United States each year, illustrates a multistep cancer path

LE 19-13 Colon Loss of tumor- suppressor gene APC (or other) Activation of ras oncogene Loss of tumor- suppressor gene p53 Colon wall Loss of tumor- suppressor gene DCC Additional mutations Normal colon epithelial cells Small benign growth (polyp) Larger benign growth (adenoma) Malignant tumor (carcinoma)

Certain viruses promote cancer by integration of viral DNA into a cell’s genome By this process, a retrovirus may donate an oncogene to the cell Viruses seem to play a role in about 15% of human cancer cases worldwide

Inherited Predisposition to Cancer The fact that multiple genetic changes are required to produce a cancer cell helps explain the predispositions to cancer that run in some families Individuals who inherit a mutant oncogene or tumor-suppressor allele have an increased risk of developing certain types of cancer

Concept 19.4: Eukaryotic genomes can have many noncoding DNA sequences in addition to genes The bulk of most eukaryotic genomes consists of noncoding DNA sequences, often described in the past as “junk DNA” However, much evidence is accumulating that noncoding DNA plays important roles in the cell

The Relationship Between Genomic Composition and Organismal Complexity Compared with prokaryotic genomes, the genomes of eukaryotes: Generally are larger Have longer genes Contain much more noncoding DNA

The sequencing of the human genome reveals what makes up most of the 98.5% of the genome that does not code for proteins, rRNAs, or tRNAs Most intergenic DNA is repetitive DNA, present in multiple copies in the genome About three-fourths of repetitive DNA is made up of transposable elements and sequences related to them

Exons (regions of genes coding for protein, rRNA, or tRNA) (1.5%) LE 19-14 Exons (regions of genes coding for protein, rRNA, or tRNA) (1.5%) Repetitive DNA that includes transposable elements and related sequences (44%) Introns and regulatory sequences (24%) Unique noncoding DNA (15%) Repetitive DNA unrelated to transposable elements (about 15%) Alu elements (10%) Simple sequence DNA (3%) Large-segment duplications (5–6%)

Transposable Elements and Related Sequences The first evidence for wandering DNA segments came from geneticist Barbara McClintock’s breeding experiments with Indian corn McClintock identified changes in the color of corn kernels that made sense only by postulating that some genetic elements move from other genome locations into the genes for kernel color

Movement of Transposons and Retrotransposons Eukaryotic transposable elements are of two types: Transposons, which move within a genome by means of a DNA intermediate Retrotransposons, which move by means of an RNA intermediate

LE 19-16 New copy of transposon Transposon DNA of genome Transposon is copied Insertion Mobile transposon Transposon movement (“copy-and-paste” mechanism) New copy of retrotransposon Retrotransposon DNA of genome RNA Insertion Reverse transcriptase Retrotransposon movement

Sequences Related to Transposable Elements Multiple copies of transposable elements and related sequences are scattered throughout the eukaryotic genome In primates, a large portion of transposable element–related DNA consists of a family of similar sequences called Alu elements

Other Repetitive DNA, Including Simple Sequence DNA Simple sequence DNA contains many copies of repeated short sequences Simple sequence DNA is common in centromeres and telomeres, where it probably plays structural roles in the chromosome

Genes and Multigene Families Most eukaryotic genes are present in one copy per haploid set of chromosomes The rest of the genome occurs in multigene families, collections of identical or very similar genes

Some multigene families consist of identical DNA sequences, usually clustered tandemly, such as those that code for RNA products

Part of the ribosomal RNA gene family LE 19-17a DNA RNA transcripts Non-transcribed spacer Transcription unit DNA 18S 5.8S 28S rRNA 5.8S 28S 18S Part of the ribosomal RNA gene family

The classic examples of multigene families of nonidentical genes are two related families of genes that encode globins Globin gene family clusters also include pseudogenes, nonfunctional nucleotide sequences that are similar to the functional genes

The human a-globin and b-globin gene families LE 19-17b Heme -Globin Hemoglobin a-Globin a-Globin gene family -Globin gene family Chromosome 16 Chromosome 11    1 2 1  A     Fetus and adult Embryo Embryo Fetus Adult The human a-globin and b-globin gene families

Concept 19.5: Duplications, rearrangements, and mutations of DNA contribute to genome evolution The basis of change at the genomic level is mutation, underlying much of 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

Duplication of Chromosome Sets Accidents in meiosis can lead to one or more extra sets of chromosomes, a condition known as polyploidy The genes in one or more of the extra sets can diverge by accumulating mutations; these variations may persist if the organism carrying them survives and reproduces

Duplication and Divergence of DNA Segments Unequal crossing over during prophase I of meiosis can result in one chromosome with a deletion and another with a duplication of a particular region

LE 19-18 Gene Transposable element Nonsister chromatids Crossover Incorrect pairing of two homologues during meiosis and

Evolution of Genes with Related Functions: The Human Globin Genes The genes encoding the various globin proteins evolved from one common ancestral globin gene, which duplicated and diverged After the duplication events, differences between the genes in the globin family arose from mutations that accumulated in the gene copies over many generations

LE 19-19 Evolutionary time Ancestral globin gene Duplication of ancestral gene Mutation in both copies   Transposition to different chromosomes Evolutionary time   Further duplications and mutations         1 2 1    A    a-Globin gene family on chromosome 16 -Globin gene family on chromosome 11

Subsequent duplications of these genes and random mutations gave rise to the present globin genes, which code for oxygen-binding proteins

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

Evolution of Genes with Novel Functions The copies of some duplicated genes have diverged so much in evolution that the functions of their encoded proteins are now very different

Rearrangements of Parts of Genes: Exon Duplication and Exon Shuffling An exon can be duplicated on one chromosome and deleted from the homologous chromosome In exon shuffling, errors in meiotic recombination lead to some mixing and matching of exons, either within a gene or between two nonallelic genes

LE 19-20 EGF EGF EGF EGF Epidermal growth factor gene with multiple EGF exons (green) Exon shuffling Exon duplication F F F F Fibronectin gene with multiple “finger” exons (orange) F EGF K K K Plasminogen gene with a “kringle” exon (blue) Exon shuffling Portions of ancestral genes TPA gene as it exists today

How Transposable Elements Contribute to Genome Evolution Movement of transposable elements or recombination between copies of the same element may generate beneficial new sequence combinations Some mechanisms can alter functions of genes or their patterns of expression and regulation