Chapter 6 Genomic Architecture

Molecular Structure of Genes and Chromosomes

Overview: structure of genes and chromosomes

10nm fiber

Transcriptionally active form

Nucleosome:

147 bp DNA-helix wound 2.66 times around 8 histone proteins (2 of each of H2A, H2B, H3, and H4)

Nucleosome: + histone H1 capping the entry and exit of the DNA strand into the nucleosome

6 Molecular definition of a gene

A gene is the entire nucleic acid sequence that is necessary for the controlled production of its final product (RNA or Protein)

In eukaryotes, genes lie amidst a large expanse of noncoding DNA with unknown function and genes may also span regions of DNA unrelated to the gene

If a gene is incapable of producing a final gene-product = pseudogene

Exons =coding region (ORF)

(Introns)

Basal Promoter

Regulatory region (enhancer/prepressor)

PolyA site

Splice site

TSS

Bacterial operons produce polycistronic mRNAs while most eukaryotic mRNAs are monocistronic and contain introns

Transcriped into 5 separate proteins

Simple and complex transcription units are found in eukaryotic genomes

The final mRNA contains a continous ORF flanked by a 3’UTR (incl polyA site) and a 5’UTR (incl Cap and Kozak sequence)

Only eucaryotic genes can contain introns

Many genes encode several different variations = isoforms (~60% of all genes)

Alternative splicing of one pre-mRNA

Alternative termination giving 2 different pre- mRNA

Alternative TSS giving 2 different pre-mRNA

There are several types of eukaryotic DNA, much of which is never transcribed

Protein coding genes can be Solitairy or duplicated

Solitary genes: occur only once in the genomeDuplicated genes:* Genefamilies: very similar (not identical) genes (example: containing very similar globular modules in the protein structure)

* Multicopy: identical (or nearly identical) copies of genes encoding products needed in high quantity (example: histones)

Genomes of higher eukaryotes contain much nonfunctional DNA

Amongst eukaryotes, cellular DNA content does not correlate with phylogeny.

Terminology: Gene-cluster, gene-desertThe beta –globins is an example of a gene family

Known nonprotein-coding RNAs and their functions

Repetitious DNA - Simple

Microsatellites

Tandem repeats of up to 150 repeats of 1-13 bp

The number of repeats within a microsatellite is very variable between individuals

Occurrence of microsatellites in vital genes (insertions) can be detrimental (even fatal)

They are often plentifull in centromere regions (see the FISH image to the left)

Microsattelites most likely occur by daughterstrand slippage during replication (giving small repeated regions)FISH= flourescent in-situ hybridization (same principle as Southern blot)

Mobile DNA

Moderately-repeated, mobile DNA sequences are interspersed throughout the genomes of prokaryotes, higher plants and animalsThese sequences range in size from hundreds to a few thousand base pairsThe sequences are copied and inserted into a new site in the genome by the process of transpositionOnce mobile DNA was termed “selfish” DNA, but actually it may have contributed to our genediversity through “exon-shuffling”

Classes of Mobile DNA* DNA-transposons* Retrotransposons

*LTR-retrotransposons (similar to retroviral provirus, but without coat-protein coding genes, most common: ERV)

*Non-LTR-retrotransposons (LINEs AND SINEs)

Long interspersed element (LINE)Short interspersed element (SINE)

Repetitious DNA - Mobile

Cut’n paste Copy and paste

6-9 General structure of bacterial IS elements (cut’n paste)

Transposase= an enzyme that cuts the element out of one genome site (exit), and cuts a whole at another site (entry)

Host genome insertion site (pale blue) becomes copied as part of the IS element insertion process (there fore “repeat” structure)

General structure of bacterial transposons -actively partaking in gene-recombination

Through genomic recombination (using the inverted repeats at both ends of the IS element) genes can become “trapped” witin Transposons and transported to other locations or exchanged between cells (through naturally occurring plasmids)

6-12 Retrotransposons containing LTRs behave like retroviruses in the genome Copy-paste

Reverse transcriptase + special primer (copies RNA into cDNA)

Integrase (integrates the cDNA into a new genomic site)

LTR: contains promoter region to initate transcription of the retrotransposal genes

6-13 Retroviral mechanism of multiplication and integration into genomic site Copy-paste

cDNA copy of the RNA

Reverse transcriptase + special primer

Integrase mediates integration of the copy into new genome location

6-13 Retroviral mechanism of multiplication and integration into genomic site Copy-paste

Most endogenous LTR retrotransposons have lost their genes and consist of only LTR sequences.

Therefore they can only move through recombination

Endogenous= occurring within a cell (as opposed to entering through viral infection)

6-16 Structure of non- LTR retrotranposons Different Copy-paste

SINEs (~600bp) are similar to LINEs but have lost the ORFs and can only transpose if they can use LINEs enzymes.

There are ~ 1,6 million SINEs in the average genome

~1.1 million are Alu sites (= cleaved by the AluI restriction enzyme)

6-17 Non-LTR retrotransposons (LINEs and SINEs) move by an unusual mechanism Different Copy-paste

Mobile DNA elements probably had a significant influence on evolution

Spontaneous mutations may result from the insertion of a mobile DNA element into or near a transcription unitHomologous recombination between mobile DNA elements may contribute to gene duplication and other rearrangements, including duplication of exons (generating gene-families), recombination of exons to create new genes “exon shuffling”, and altered control of gene expression (copying generegulatory elements between different promoters)

Biotechnical evolution ☺:mobile DNA elements are a possible tool for inserting therapeutic genes into patients (gene-therapy)

It has recently been discovered that some of these mobile element still play an active role in the human genome

6.6 Structural genome organization

Procaryotic: Most bacterial genomes are carried in one circular chromosomeStable replication requires one replication origin (ORI)the Genome is packed with polyamines (stabilizing proteins).

Eukaryotic: the genome is distributed over several linear chromosomes.Stable replication occurs from several replication origins within each chromosome, and additionally requires:Centromeres (for equal distribution between daughter cells during mitosis)Telomeres (to protect the chromosome ends against shortening during replicationEukaryotic DNA associates with many different proteins to form chromatin

6.28 Chromatin exists in extended and condensed forms

Nucleosome

10 nM fiber (beads-on-a-string) 30 nM fiber (condensed bead structure)

9.5 Nucleosomes are complexes of histones

9.5 The solenoid model of condensed chromatin

Morphology and functional elements of eukaryotic chromosomes

Half the mass of chromatin is made up by proteins, which are determinant for the condensation (-> heterochromatin) and opening (-> euchromatin) of the chromatin structureMicroscopic observations on the number and size of chromosomes and their staining pattern has revealed important aspects of chromosome structure

Chromosome number, size and shape at metaphase are species specific

Two species of Indian deer....

At metaphase the chromosomes become very condensed. This is required for a proper separation via the mitotic spindles before mitosis (Anaphase)

Heterochromatin consists of chromosome regions that do not uncoil

Electron micrograph: the heterochromatin is very condensed (dark appearance)

Nonhistone proteins provide a structural scaffold for long chromatin loops

Figure 9-34

A model for chromatin packing in metaphase chromosomes

Metaphase chromosome (electron micrograph)

6-41 Stained chromosomes have characteristic banding patterns

Giemsa staining of metaphase chromosomes reveal the DNA dense areas as dark bands

Stained chromosomes have characteristic banding patterns

Figure 9-38

6-36 Experimental demonstration of chromatin loops in interphase chromosomes

Sites A-F are separated by millions of bp of linear sequence but are physically close to each other in interphase nucleiSpecial DNA sequences associate with the scaffold protein in an ordered fashion

SARs

Newest research: “chromosome kissing” = the scaffold brings two gene areas from different chromosomes into proximity (so that they can be co-regulated)SARS: scaffold associated Regions

9.6 Chromosome painting distinguishes each homologous pair by color

Reading the histone code

The histones can be modified (like many other proteins)

The histone ends are acidic and link the nucleosomes together in a tighter structure

Acetylation neutralizes the ends -> looser structure -> gene activation

Methylation -> prohibits acetylation -> gene silencing

Additionally histones can be phosphorylated or ubiquitilated

The study of the histone code and how it determines which genes are actively transcriped in a cell is called Epigenetics

Organelle genomes: The “Kraftwerke” of eucaryotic cells carry their own genomes

Dual staining shows multiple mt DNA molecules in eucaryotic cell

Red= ethidium bromide staining, binds dsDNA and emmits lightGreen= diOC6, which is incorpoated into mitochondria

Human mtDNA

Mitochondrial DNA is circular, multicopy, and has its own genetic triplet-code

It encodes a fraction of the genes especially needed in mitochondrial functions

Genotype is almost 100% maternal inheritance (!)

DNA-fingerprint DNA profiling

For the coding part of genes a limited number of alleles exist (high conservation). The remaining ~98% of the human genome has very high allelic variation. -> each human has a unique genomic composition (although one-egged twins are almost identical)

Examples of variable regions: number of repeats in repeated DNA, single-nucleotide polymophisms (SNPs), variable number of gene-copies.

Identification of these classes of differences can be used to generate techniques for determining individual DNA profiles.

All organisms can be DNA profiled

DNA-profiling

* Determines relationships between the source of two samples (identical?, closely related? Distant related?) (forensic, paternity tests, phylogenetic studies, identification of infections, Dinosaur studies, etc.)

* Determines the presence/absence of disease genes (diagnostic)

Most commonly used techniques are based on

RFLP; Restriction fragment length polymorphism genetic variation causes Restriction sites to dissapear and appear at different distances in the genomes

VNTR; variable number of tandem repeats variable number of minisatellites (10-100bp sequences that are present in a variable number of head-to-tail copies (=repeats))

STR; short tandem repeats variable number of microsatellites (4-10bp)

SNPs; Single nucleotide polymorphismsMitochondrial DNA sequencing (when very little non-degraded cellular material is available)

Southern blotting, detection with radioactive probesPCR, detection with gelectrophoresis or capillary electrophoresis (very specific, and very little original input DNA required)

Simple-sequence DNAs are concentrated in specific chromosomal locations

DNA fingerprinting depends on differences in length of simple- sequence DNAs

PCR-based STR analysis

How?DNA is isolated and purified

PCR is performed on the DNA with primers flanking known microsatellite areas. (so that only these areas with a high size-variability are amplified)

The amplified DNA is size-separated with gel-electrophoresis

The resulting pattern of size-fractionated DNA-bands is compared to other patterns to determine similarity.

Usually each area is chosen on a separate chromosome. 13 primerpairs (microsatellite areas) are enough to ensure that the actual probability that 2 random persons have the same STR-pattern is only 1 in 3 trillions

Examples

A modern family tree

How are these 2 parents and 4 children related?

NB: a classic example of a “cheap” paternity test! Far to few markers have been applied for it to be significant.

Next lecture: 19.01-2010

Regulation of Transcription InitiationRNA Processing, Nuclear Transport, and Post-Transcriptional Control

After completion of this lecture you should be able to:

Know how eucaryotic DNA is packaged in the nucleus: doublehelix – nucleosomes (beads on a string) – condensation into 30 nm fibers (euchromatin) – further condensed fibers (heterocromatin)Define nucleosomes and mention their components Know that procaryotic DNA is circular and simply packed with polyamines (whereas eucaryotic DNA is packed with many different proteins including various histonesKnow the major types of classification of DNA sequences in the genomeKnow the definition of a gene and be able to sketch the major structure and place the various functional elements (introns, exons, TSS, enhancers, etc....)Know the definition of a pseudo-geneKnow that prokaryotic genes differ from eucaryotic genes in that they contains no introns and can be polycistronic.Define the terms polycistronic and monocistronicDefine the term protein isoform, and describe how several isoforms can arise from one gene Know that coding sequence (exons) only constitute about 1.1% of the entire human genome and be able to mention some of the other major classes of nuclear eucaryotic DNAUnderstand the term nonprotein-coding RNA, and give examplesDefine the term mobile DNA, and briefly describe the two classes; DNA-transposons and RetrotransposonsUnderstand why mobile DNA is important for continued evolutionary developmentKnow the basics of what LINEs and SINEs areKnow that eucaryotic chromatin is organised on scaffolds and define what SARs areDefine ther term EpigeneticsGive examples of the histone code, and its purposesKnow that mitochondria have their own circular DNA, reminiscent of ancient eubacterial DNADescribe the purpose os DNA-fingerprinting (DNA-profiling) and give examples of the techniques used for this