Larger genomes are not generated by increasing the number of copies of the same sequences present in smaller genomes. It is due to the presence of more repetitive DNA Six major types of noncoding human DNA have been described Noncoding DNA in Eukaryotes noncoding introns Segmental duplications Simple sequence repeats (SSRs Structural DNA Transposable elements Long interspersed elements Dead transposons Long terminal repeats Short interspersed elements Pseudogenes 5 I. Noncoding DNA within genes -Protein-encoding exons are embedded within much larger noncoding introns II. Structural DNA -Called constitutive heterochromatin -Localized to centromeres and telomeres III. Simple sequence repeats (SSRs) -One- to six-nucleotide sequences repeated thousands of times 6 IV. Segmental duplications -Consist of 10,000 to 300,000 bp that have duplicated and moved V. Pseudogenes -Inactive genes Transposable elements (transposons) -Mobile genetic elements -Four types: -Long interspersed elements (LINEs) -Short interspersed elements (SINEs) -Long terminal repeats (LTRs) -Dead transposons 7 Introns--Untranslated intervening sequences in mRNA Exons Translated sequences Process-RNA splicing Heterogeneous nuclear RNA (hnRNA)-Transcript before splicing is complete Introns contain invariant 5-GU and 3-AG sequences at their borders (GU-AG Rule) Recognized by small nuclear ribonucleoprotein particles (snRNPs) that catalyze the cutting and splicing reactions. Internal intron sequences are highly variable even between closely related homologous genes. Distribution of Uninterrupted and Interrupted Genes in Various Eukaryotes While majority of the genes in yeast are uninterrupted, most of genes in flies are interrupted by one or two introns and most genes in mammals are interrupted by many introns Sizes of Exons and Introns Exons coding for proteins usually are short, but introns usually range from very short to very long ExonsIntrons II. Structural DNA Prokaryotic Prokaryotic genes that are turned on and off together are often clustered into operons which are transcribed into one mRNA molecule and translated together Eukaryotic Eukaryotic genes coding for enzymes of a metabolic pathway are often scattered over different chromosomes and are individually transcribed There is an increasing complexity of regulatory sequences from a simple bacterial gene controlled by a repressor to a human gene controlled by multiple activators and repressors. Major types of regulatory DNA elements in eukaryotes S silencer P promoter I insulator E enhancer TF transcrition factor heterochromatin 2010, 11, o Promoters recognition sequences for binding of RNA polymerase o Enhancers increase transcription of a related gene o Silencers decrease transcription of a related gene o Insulators or boundary elements block undesirable influences on genes: 1. enhancer blockers prevent communication between enhancers and unrelated promoters 2. barrier sequences prevent spread of heterochromatin 3. combined o LCR locus control regions activate some gene clusters Each cell in our bodies has about 6 feet of DNA stuffed into it-However, less than one inch is devoted to genes! Complex genomes have roughly 10x to 30x more DNA than is required to encode all the RNAs or proteins in the organism or have any apparent regulatory function 17 Non-Protein Coding Genes Encode functional RNAs There are non- protein genes in the genome that encode functional RNAs. These RNAs are important in regulating the expression of genes Assigned Reading: The functional genomics of noncoding RNA. Mattick et al. (2005), Science 309: By fluorescence in situ hybridization (FISH),the simple- sequence DNAs are localized near the centromers and telomeres of mouse chromosome Types of repited dna Two types Tandemly repetitive Interspersed repetitive The tandem repeat DNA are usually identical Proportions of DNA 10-15% Length of each repeated unit10-10 million bp Length of repeated per site in bp 1- regular satellite 100,0000 to 1 million 2- mini satellite microsatellite The interspread repeat DNA Are very similar but not identical Proportions of DNA 25-40% Length of each repeated unit100-10,000bp No. of repetition of each genome 10-1 million 1- Satellite DNAs When eukaryotic DNA is centrifuged, fragmented and centrifuged to equilibrium in a Cesium chloride (CsCl) density gradient (CsCl gradient), two components are observed: Main band: most of the genomic DNA Satellite band: one or multiple miner bands; they could be heavier or lighter than the main band The main band DNA has density of g/cm with a G-C content of 42%, and minor band DNA has the buoyant density of g/cm with a G-C content of 30% Satellite DNAs Satellite DNA highly repetitive DNA consisting of short unusual nucleotide sequences that are tandemly repeated 1000s of times It is found at the tips of chromosomes and the centromere Its function is not known, perhaps it plays a structural role during chromosome replication and separation. Highly repetitive DNA (simple sequence DNA): Satellite DNA is characterized by rapid rate of hybridization, consists of very short sequences repeated many times in tandem in large clusters. In addition, multi-cellular eukaryotes have complex satellites with longer repeat units mainly in heterochromatic region (Centromeric heterchromatin--- necessary for separation of chromosome to daughter cells Satellite DNA Simple-sequence DNA (6% of the human genome), size 14 to 500 bp Microsatellite, (also called as transposable elements) 1-13 bp. Interspersed repetitive DNA dispersed throughout the genome Telomere is a series of short tandem repeats at the ends of eukaryotic chromosomes; prevents chromosomes from shortening with each replication cycle Repeat SequenceSpecies TTTAGGGArabidopsis TTAGGGHuman TTTTGGGGOxytricha TAGGGSlime Mold TTGGGGTetrahymena TAGGGTrypanosome Until recently, little was known about molecular structure of telomeres. However, during the last few years, telomeres have been isolated and characterized from several sp. Eukaryotic chromosomes are linear, not circular like prokaryotic chromosomes. Most eukaryotic chromosomes have short, species-specific sequences tandemly repeated at their telomeres Blackburn and Greider have shown that chromosome lengths are maintained by telomerase, which adds telomere repeats without using the cells regular replication machinery. The ends of eukaryotic chromosomes are formed by an enzyme called telomerase.Telomerase an enzyme adds repeats of 3 ends of eukaryotic chromosomes In the ciliate Tetrahymena, the telomere repeat sequence is 5` TTGGGG-3` The telomeres of this organism end in the sequence 5'-TTGGGG-3'. The telomerase adds a series of 5'-TTGGGG-3' repeats to the ends of the lagging strand. A hairpin occurs when unusual base pairs between guanine residues in the repeat form. Finally, the hairpin is removed at the 5'-TTGGGG- 3' repeat. Thus the end of the chromosome is faithfully replicated. Tetrahymena - protozoa organism. RNA Primer - Short stretches of ribonucleotides (RNA substrates) found on the lagging strand during DNA replication. Helps initiate lagging strand replication Interspersed repetitive ~1/2 of the human genome consists of interspersed repetitive sequences. 5- Transposable elements (transposons) Transposons jump and interrupt the normal functioning may increase or decrease production of one or more proteins can carry a gene that can be activated when inserted downstream from an active promoter and vice versa Retrotransposons transposable elements that move within a genome by means of an RNA intermediate, a transcript of the retrotransposon DNA to insert it must be converted back to DNA by reverse transcriptase 5- Transposable elements (transposons) -Mobile genetic elements -Four types: -Long interspersed elements (LINEs) -Short interspersed elements (SINEs) -Long terminal repeats (LTRs) -Dead transposons 36 38 Current definitions: LINEs = Active or degenerate descendants of transposable elements. SINEs = Non-autonomous transposable elements (lacking the ability to mediate their own transposition) and their degenerate descendents. LINEs & SINEs 39 interspersed repeats Original definitions: SINE short-interspersed repetitive elements (SINEs) Non-autonomous transposable elements (lacking the ability to mediate their own transposition) and their degenerate descendents. Most abundant repeated DNA, Short LINE long-interspersed repetitive elements (LINEs). Active or degenerate descendants of transposable elements Moderately Abundant 41 The reverse transcriptase has LINE specificity, i.e., a reverse transcriptase from one LINE will only recognize the 3 end of that LINE, and will be less efficient at recognizing and reverse transcribing other LINEs. Ex: LINE in Human 6 Kb in length Has a poly A tail Flanked by short repeats 5% of genome 95% of the sequence are truncated at the 5 end Contains two reading frames - OPR I ( 375 codons) - OPR II ( 1,300 codons) Does not posses long terminal repeats 43 SINEs are retrosequences that range in length from 75 to 500 bp. SINEs do not possess any reading frame. Thus, their retroposition must be aided by other genetic elements. 44 SINE 7SL - RNA derived tRNA - derived Primate Alu + Rodent B1 All others 45 Length = ~300 bp Repetitive: > 1,000,000 times in the human genome Constitute >10% of the human genome Found mostly in intergenic regions and introns Propagate in the genome through retroposition (RNA intermediates). 46 Alu elements are found only in primates. All the millions of Alu elements have accumulated in a mere ~65 million years. 47 Alu elements can be sorted into distinct families according to shared patterns of variation. At any given point in time, only one or several Alu master copies are capable of transposing. Early in primate evolution, Alu transposition rate was approximately one new jump in every live birth. Today, it is about one new jump in every 200 live births. 48 Evolution of Alu elements & Rodent BI DNA 49 GENETIC AND EVOLUTIONARY EFFECTS OF TRANSPOSITION 1. Duplicative transposition increases genome size. Lily Edible frogSunflower 50 2. Bacterial transposons often carry genes that confer antibiotic or other forms of resistance. Plasmids can carry such transposons from cell to cell, so that resistance can spread throughout a population or an ecosystem. 51 3. Gene expression may be altered by the presence of a transposable element. a. a. An insertion may eliminate the reading frame (phenotypic effects). b. b. A transposable element may contain regulatory elements (effects on transcription of nearby genes). c. c. Transposable elements may contain splice sites (effects on RNA processing even if the element is in an intron). 52 53 Caused by the insertion of a transposable element. 54 55 4. Transposable elements promote gross genomic rearrangements a. a. directly (moving a DNA sequence from one genomic location to another). b. b. indirectly (as a result of transposition, two sequences become similar to one another so that unequal crossing-over between them is possible). 56 57 That is the end