Lecture 3 .ppt
- 1. Keep it up!! Switch off your mobiles
- 2. Important points from Last lecture • Prokaryotic genomes may be single molecules or multipartite • The Extent of Lateral Gene Transfer revealed by sequencing is high • A plasmid is extra-chromosomal DNA Eukarytoic nuclear genomes • DNA is always linear, arranged into chromatin, forming chromosomes, packed in nucleus • Heterochromatin is transcriptionally inactive • Genome Size is highly variable (total number of nucleotides) • Chromosome number is not linked to genome size • The lack of precise correlation between the complexity of an organism and the size of its genome is termed as C-value paradox • More complex organisms have less compact genomes • There are gene poor and gene rich regions on a chromosome
- 4. 3D structure of Genome • Determined by Hi-C DNA is cross-linked, cut by RE, filled, biotinylated, Ligated ,sheared and sequenced
- 5. Hi-C Rao, SSP et al. 2014 Dec; 159,( 7),1665 1680,doi: http://dx.doi.org/10.1016/j.cell.2014.11.021 A 3D Map of the Human Genome at Kilobase Resolution Reveals Principles of Chromatin Looping Domains, 6 subcompartments, ~10,000 Loops
- 6. Transposable Elements (are very much part of the genomes) Transposable elements have been shaping the genome throughout evolution, contributing to the creation of new genes and sophisticated regulatory network systems
- 7. By type Retroelements (Retrotransposons and Retroposons) Class I transposons DNA transposons (No RNA intermediate of the whole transposon) Class II transposons By Mode of Transport Cut and Paste (DNA transposons) Copy and Paste (DNA transposons and Retroelements) By Autonomy Autonomous (DNA transposons and Retroelements) Non-Autonomous (DNA transposons and Retroelements) Classification
- 8. General Principle • A break in recipient DNA has to occur (cutting DNA) • DNA has to be ligated so that integration will occur (Joining DNA) • Short sequences at site of insertion in the recipient DNA may be important for integration of a mobile genetic element (of specific sequence or random) • Depending on the transposon, specific sites at site of insertion in the recipient DNA become duplicated at either end of the newly inserted transposon
- 10. DNA transposons • Are common in Prokaryotes. Less common in eukaryotes. (Tn family, Tc1/mariner family, MITES, Ac/Ds, Helitrons, Helentrons) • They have inverted repeats (ITR) at both ends and may have additional genes for transposition enzymes and antibiotic resistance (exceptions are the helitrons and helentrons) • Transpose conservatively (DNA cut out of the present site and pasted into next) Or • Replicative transposition (A DNA copy is made on a new site in genome by action of several enzymes)
- 11. Types of DNA transposons • Insertion sequences (Inverted repeats (ITR) and transposase) IS=Insertion Sequence • Composite transposons (Two insertion sequences and antibiotic resistance genes) Composite transposons • Non-composite transposons (Inverted repeats, transposase, resolvase, and antibiotic resistance genes) Non-composite transposons A new group of DNA transposons found in plants, nematodes, flies, fish and fungi that do not have inverted repeats • Some transposons encode a replicase and a helicase (The helitrons) (Use rolling circle mechanism to replicate) • Some helitrons also encode an endonuclease as found in class I transposons (In helentrons) (Use rolling circle mechanism to replicate)
- 13. A cornucopia of Helitrons shapes the maize genome Cédric Feschotte and Ellen J. Pritham Proc Natl Acad Sci U S A. 2009 November 24; 106(47): 19747–19748 Extra (For information only)
- 14. A class II typical transposon
- 15. Ac/Ds system in Maize • Most famous example is that of Ac/Ds elements of maize (Corn) • Ac=Activator, Ds=Disassociation • Ac is a functional transposon, Ds is dependant on Ac for mobility • Ac encodes Transposase which can excise Ac or Ds • Ds is a defective transposon and does not encode transposase • Once Ac excises Ds, it can insert it into other regions in the genome • Transposase is expressed by general transcription which then cuts and pastes the transposon
- 16. Miniature Inverted-repeat Transposable Elements (MITEs) • Almost identical DNA sequences of about 400 base pairs • Both ends have characteristic inverted repeats of 15 bp 5' GGCCAGTCACAATGG..~400 nt..CCATTGTGACTGGCC 3' 3' CCGGTCAGTGTTACC..~400 nt..GGTAACACTGACCGG 5‘ • 100,000 MITEs in the rice genome (representing 6% of the total genome). • Depend on mariner-like elements (MLE) which encode transposase (fewer copies -in 10’s)
- 17. Sleeping Beauty • Resurrected transposon system of fish (Tc1/mariner family) from an inactive transposon • Molecular reconstruction was used to produce this transposon, Sleeping Beauty (SB) that transposes in higher eukaryotic cells • They have a small target site for integration (TA) and require few host cell factors for activity • Used as tools for creating mass scale insertional mutations in mice (More will be discussed later)
- 18. Retroelements
- 19. Retroelements (Retrotansposons and Retroposons) No retrotransposons have been found in Prokaryotes Retrotransposons are found in eukaryotes Retrotransposons usually have LTR at both ends, some virus related genes, Reverse Transcriptase and other DNA replication related genes may also be present Retroposons such as LINE, SINE have no LTR, but they have a Poly A tail at 3’. Very common in humans. About million copies of SINE SINE need other retroelements to supply the enzymes needed for transposition
- 20. Some abbreviations • LTR=Long terminal Repeats • LINE=Long Interspersed Nuclear Elements • SINE=Short Interspersed Nuclear Elements • Pol =DNA polymerase (Reverse Transcriptase) • Env =genes for envelope necessary for virus encapsulation • Gag = internal virion proteins (including a nucleic acid binding protein) They also may have genes encoding RNase H and endonuclease which is necessary for integration into host cells
- 21. Some retrotransposon genes may have introns
- 22. gag prot env RT RNAse H endo gag prot endo RT RNAse H gag RT RNAse H (TAA)n ITR or Inverted repeat DR or Direct Repeat LTR LTR LTR Prot=protease, RT=Reverse Transcriptase, endo=endonuclease ALV (virus) Ty1 (a yeast retrotransposon) Drosophila I factor (retroposon)
- 23. Retrotransposition • DNA is copied into RNA by process of cellular transcription • The RNA transcript is copied into DNA, which initially exists as an independent molecule outside of the genome. This conversion of RNA to DNA, the reverse of the normal transcription process, reverse transcriptase • Often the reverse transcriptase is coded by a gene within the retrotransposon and is translated from the RNA copy synthesized in step 1 • The DNA copy of the retrotransposon integrates into the genome • DNA is inserted into a new site on the chromosome or the same site
- 24. SUMMARY • The mobility of a Retrotransposon is through Transcription (to RNA) and then Integration of newly formed DNA into another site in the genome (Copy and paste) • The mobility of a DNA transposon is 1. Cut and paste (DNA is cut and pasted into a new site) or 2. Copy and paste (A DNA copy is made which is inserted into a new site) Replicative transposition increases DNA content of the organism
- 25. Self Study An inverted repeat (or IR) is a sequence of nucleotides that is the reversed complement of another sequence further downstream For example, 5'---GACTGCnnnnnnnnnnGCAGTC---3‘ (Note that in this sequence the second sequence after “n” is the reverse complement of GACTGC). “n” can be any nucleotide Inverted repeats define the boundaries in DNA transposons When no nucleotides intervene between the inverted repeat and its downstream complement, it is called a Palindrome For example, 5' GAATTC 3' (note that in this sequence TTC is reverse complement of GAA) Discover--Direct repeats
- 26. Transposition can also create new genes (We will discuss this later in the course)
- 27. Transposons and disease • Disable genes: A mobile element that inserts into a gene or If is excised from a gene, the resulting gap will probably not be repaired correctly • Unequal crossovers: Multiple copies of the same sequence, such as Alu sequences can hinder precise chromosomal pairing during mitosis and meiosis, resulting in unequal crossovers, one of the main reasons for chromosome duplication • Aberrant Expression: Many transposons contain promoters which can cause aberrant expression of linked genes, causing disease or mutant phenotypes Disorders that in some individuals are known to be caused by transposons include hemophilia A and B, severe combined immunodeficiency, porphyria, predisposition to cancer, and Duchenne muscular dystrophy Considered to be selfish DNA parasites similar to viruses Excessive transposon activity can destroy a genome. Mechanisms to reduce transposition to a manageable level have evolved (Theoretical) • Prokaryotes may undergo high rates of gene deletion as part of a mechanism to remove transposons and viruses from their genomes • Eukaryotic organisms may have developed the RNA interference (RNAi) mechanism as a way of reducing transposon activity
- 28. Transposons as tools • Transposon tagging, in which inactivation is achieved by the insertion of a transposable element into the gene • Under normal circumstances, transposition is a relatively rare event • Modified transposons that change their position in response to an external stimulus. Yeast retrotransposon Ty1, and fish sleeping beauty (SB one of the most efficient systems evolved) Drosophila transposon called the P element • Transposon tagging is more applicable to global studies of genome function, in which genes are inactivated at random and groups of genes with similar functions identified by examining the progeny for interesting phenotype changes
- 29. Mutations
- 30. Change in the base sequence of DNA can be termed as a mutation Usually a mutation is thought to be harmful or beneficial (It is not usually so) At nucleotide level a mutation may be classified as: 1. Insertion (One or more nucleotides may be inserted in a DNA sequence) 2. Deletion (One or more nucleotides may be deleted from DNA sequence) 3. Indels (insertions of some bases and deletions of others) 4. Transition (CT or TC ---or AG or GA substitution) 5. Transversion (GC or CG ---or AT or TA substitution) These mutations can be anywhere in the genome, coding or non- coding
- 31. Mutations in coding region by effect 1. Silent mutations (Nucleotide change does not result in amino acid change in the protein—due to degenerate genetic code) 2. Nonsense mutations (Nucleotide change results in Stop codon in DNA and truncation of protein (TAA oc, TAG op , TGA am) 3. Missense (change of a Single nucleotide results in substitution of a different amino acid) • Conservative substitutions (amino acid replaced with similar type) • Non-conservative substitutions (Very different a.a e.g. RD) Non-conservative substitution may be deleterious (depends) 4. Frameshift (deletion or insertion of one or more nucleotides which are not in multiples of 3—change reading frame and change sequence of amino acids after mutation and/or truncate protein 5. Insertion or deletion (One or more amino acids may be inserted or deleted from protein if 3 or more nucleotides in multiples of 3 are inserted or deleted from DNA) 6. Readthrough (Stop codon may change into a coding extending protein– can be deleterious)
- 32. Others • Gain-of-Function mutations • Loss-of-Function mutations • Conditional-lethal • Inhibitor-resistant mutants • Temperature sensitive • Regulatory
- 33. When is a mutation not a mutation? When it is sufficiently common in a population that it cannot be explained by recurring mutation. It is a polymorphism
- 34. SNP • Single nucleotide polymorphisms (SNP) ACGGGGGTTTCCC ACGGGGGTTTCCG Coding SNPs Non coding SNPs A SNP can change recognition sequence in a DNA for a Restriction enzyme, so that it can no longer be recognized by it A SNP can create a new site for Restriction Enzyme • Restriction fragment length polymophisms (RFLP) Length of a restriction fragment changes. Detected by Southern blotting and now typed by PCR
- 35. Where do mutations arise? Germ line Mutations Arise in reproductive cells during DNA replication (de novo mutations) Passed on to the next generation (hereditary) In humans about 100 such mutations are thought to be present in every individual These are absent from parents DNA samples from other cells since they were created at time of germ cells production Somatic Mutations Arise in DNA of any cells except the germ cells Produced during DNA replication before cell division Mostly, these have no phenotype for an organism (WHY?) In some cases they may cause Cancer Not passed on to the next generation
- 36. Somatic mutation and congenital disorder • Patients with Congenital Disorders of Glycosylation, or CDG, have mutations in one of the many enzymes the body uses to glycosylate proteins and cells • CDG children experience a wide variety of symptoms, including intellectual disability, digestive problems, seizures, and low blood sugar • Few children with CDG who are mosaics -- only some cells in some tissues have the mutation in SLC35A2 • Mosaicism of the UDP-Galactose Transporter SLC35A2 Causes a Congenital Disorder of Glycosylation. American Journal of Human Genetics, 2013; 92 (4): 632 (extra)