2. Rules of the Class
1. Switch off your mobiles
2. Be in class on time
3. Attend all classes--for your own benefit
3. Course
Throughout and after the course, self study is important
• In depth understanding is absolutely necessary
4. DNA composition
Chargaff’s rules (In double stranded DNA only)
A=T, G=C
Chemical Formulae of Sugars in DNA and RNA, A, T, G, C, U bases,
Phosphate groups, Nucleosides, Nucleotides, phosphodiester bond,
DNA polymers and antiparallel orientations, different bonds present
inter and intramolecularly, bases per turn in different DNA forms
Basics of DNA
What is a 5’ end? 3’ end? Why is DNA negatively charged?
How do two strands of DNA form a double helix?
The two strands of DNA are linked only by hydrogen bonds (and not
covalent bonds)
The pitch, bases per turn, major and minor grooves of the different
forms of DNA
Single stranded DNA
Supercoiling in circular genomes and arrangement in higher order
Histones, nucleosomes, chromatin and DNA arrangement for
chromosomes in eukaryotes
You are expected to know
5. Figure Watson-Crick base pairing of nucleotides in DNA. The AT base pairs form two hydrogen bonds and the
GC base pairs form three hydrogen bonds. Thus, GC-rich regions are more stable than AT-rich regions.
6.
7. Basics
• In double stranded DNA
A=T and G=C (Exception: single stranded DNA)
• 5’ end of DNA is negatively charged. 3’end of DNA has free OH
• A covalent bond between OH and PO4 is termed as a phosphodiester
bond (present within a strand)
• The two antiparallel strands are held together by Hydrogen bonds only
Forms of DNA
• B and A are right handed, Z is left handed. B is the familiar form
• A in conditions of dehydration (humidity less than 75%) or if
polypurine tracts. In A form base pairs are tilted 20 degrees relative to
the helical axis, so the diameter of helix increases
• Z forms in regions of alternating purine and pyrimidine stretches. Also
at high ionic concentrations and due to DNA methylation
• In Z DNA base pairs flip 180 degrees relative to sugar-nucleotide bond
8. Figure The structures of different forms of DNA include the B-, A-, and Z-forms. The sugar phosphate backbone
of the DNA strands is colored blue. The nucleotide bases forming the internal base pairs are yellow for
pyrimidines (thymine and cytosine) and red for purines (adenine and guanine).
The positioning of the three types of DNA, B,
A, Z throughout the genome participates in
the regulation of gene expression
See
http://www.whatislife.com/reader/dna-
rna/dna-rna.html
9. What is Genomics?
• Sequence, analysis and assembly of structure and function of
genomes
• A Genome contains all information required for life and reproduction
(total DNA of haploid cell)
• The determination of the order of nucleotides or bases of ALL
DNA or RNA genome in an organism
The order of As, Cs, Gs, and Ts that make up an organism's DNA
The human genome is made up of over 3.2 billion of these genetic
letters
• Analyses used to discover functional and structural gene
information on all the genes of an organism. Includes the Methods
and Results of analysis on a genome-wide scale
11. Types of genomes
• Circular (Typically in prokaryotes, some viruses, mitochondrial &
chloroplast genomes)
• Linear (Typically in eukaryotes-arranged in chromosomes)
• DNA genomes: Most life forms
• RNA genomes: Certain viruses (Will be covered in Virology)
We will be focusing on DNA genomes
Composition
Structure
14. Inheritance
• Mitochondrial DNA and Chloroplast DNA are usually transmitted to
the next generation through only the maternal or paternal line and
rarely from both.
• The pattern is fixed for each species
• In most animals, plants and fungi, inheritance of mitochondrial DNA
is predominantly maternal. In plants, mitochondrial DNA can
occasionally be inherited from the paternal parent, e.g. in bananas
and some Conifers
• Chloroplast DNA is usually passed through the maternal line in
angiosperms
• Paternal line inheritance takes place in pines, biparental in some
angiosperms
16. Mitochondrial Genomes
• Mitochodrial genomes are usually circular and double stranded
Exception: Some microbial eukaryotes (Paramecium, Chlamydomonas
and several yeasts) have linear and double stranded
mitochondrial genomes (Nosek et al., 1998) i.e lower eukaryotes
• The number of identical chromosomes varies in each
mitochondrion (humans may have 10 molecules, while yeast may
have 100 in a single mitochondrion)
• There are usually 100-10,000 mitochondrial DNA in one human cell
• Heteroplasmy: Mitochondrial genomes in an organism may acquire
mutations, some DNA molecules are wild type and some have
mutations. it is termed as heteroplasmy. Contrast Homoplasmy
17. Mitochondrial Genomes
• Genes encode tRNA, rRNA and few proteins important in
respiratory chain
• Different genetic code in human mitochondrial genome than
nuclear genetic code (but not all codons are different) ,other
eukaryotes, including most plants, use standard code
• Usually 50-70kb but huge variation (6kb to 2MB)
• Variable in SIZE in different organisms (being much smaller in
complex multicellular organisms including humans—only 16.569 Kb
in humans) It can be up to 2MB in plants!
• Yeast as well as flowering plants, have larger and less compact
mitochondrial genomes, with some genes containing introns
• Humans don’t have any introns in their genes in mitochondria
20. Huge variation in repetitive DNA in mtDNA depending on size
Some unusual gene structures
A few genes in some species are split into many modules and are
scrambled through the genome located on both strands of the
DNA
Separate transcription yields discrete rRNA which are then held
together by base pairing of complementary sequence
22. Chloroplast Genomes
• Usually circular and double stranded
• Exception: Genome is partitioned, extremely so in the marine
algae, dinoflagellates ---genome is many small circles, each
containing just a single gene
• Photosynthetic microorganisms (e.g. Chlamydomonas) have
approximately 1000 chloroplast genomes per cell
• About 5000 chloroplast genomes are present in a higher plant cell
• Chloroplast genomes are of almost the same size among different
green algae and plant species (120-200kb) (variable in chlorophyta)
• Most have a structure similar to that for the rice chloroplast
genome
• Encodes 70-90 proteins including ribosomal proteins, RNA
polymerase, proteins for photosynthesis and genes for rRNA, 30 or
more tRNA genes
24. ctDNA
Origin of Replication sequences are also present
10-24kb sequence is present in two identical copies but as an inverted
repeat
Length of introns can differ
Standard genetic code but RNA editing at 2nd nucleotide may take
place (Maier et al. 1995—extra information)
25. Ancestry/Evolution and Genome
• Euglena gracilis one of the simplest photosynthetic organisms
with about 140 kb of ctDNA. (is also a heterotroph)
• Astasia longa is a flagellate which is closely related to Euglena
However it is a heterotrophic organism (No photosynthesis)
• Astasia longa has a plastid DNA that is 73Kb
• Sequencing of this plastid DNA has shown that all photosynthesis
related genes except one are missing.
26. Origin of mitochondria and Chloroplast
Mitochondria and Chloroplast resemble bacteria
• It is thought that at some time bacteria were engulfed by eukaryotes
and mitochondria and chloroplasts are the remnants
The endosymbiotic theory: mitochondria and chloroplasts are relics
of free-living bacteria that formed a symbiotic association with the
precursor of the eukaryotic cell, early in evolution
The genomes of mitochondria and chloroplasts resemble prokaryotic
genomes, however the genomes are much smaller than any free
living prokaryote (Gene loss and transfer)
29. Prokaryotic Genomes
• Chromosome is an irregular DNA/protein complex in the cytosol
called the nucleiod which lacks a nuclear membrane
Exception: bacterial order Planctomycetes have a membrane around
their nucleoid and contain other membrane-bound cellular structures
• Circular in most prokaryotes, and linear in very few
Exception: Linear DNA in Borrelia burgdorferi, (causes Lyme
disease), Streptomyces and some other bacteria
• Genomes are usually smaller than eukaryotes (Mycoplasma has
only 580.073Kb) and most are smaller than 5 Mb
Exception: B. megaterium, has a huge genome of 30 Mb
30. The Genetic organization of the prokaryotic
genome
1. Genomes have compact genetic organizations with very little
space between genes
2. Very little non-coding DNA
• only 11% of the total DNA in E.Coli is non-coding, distributed around
the genome in small segments
3. Operons are characteristic features of prokaryotic genomes
• An operon is a group of related genes, located adjacent to one
another, with just one or two nucleotides between the genes
• 600 operons in the E. coli K12 genome, with two or more genes
• Methanococcus jannaschii (Archaeon) Aquifex aeolicus (bacterium)
genes in an individual operon rarely have any biochemical
relationship
4. Each Bacterial DNA molecule has only a single “Origin of
Replication” sequence
31. Packaging
• The bacterial DNA is attached to proteins including DNA gyrase,
DNA topoisomerase I, HU proteins
• Archaea do not possess HU but have proteins more similar to
histones which may help in packaging the DNA
In prokaryotes
• No chromatin is present. Compacting of DNA is by supercoiling
• DNA is not packaged into chromatin, but it is still referred to as a
chromosome. It is very different from a eukaryotic chromosome
32. Archaea
• Archaea are genetically distinct from bacteria and eukaryotes
• Some genes are similar to those of bacteria and others more similar
to those in eukaryotes.
• 15% of the proteins encoded by any one archaeal genome are
unique to the Archaea, although most of these unique genes have
no known function
• Some archaea have multiple origins of replication
• Use DNA polymerases that resemble the equivalent eukaryotic
enzymes
• However, many other proteins including those that direct cell
division are similar to their bacterial equivalents
• Some archaea haves self splicing introns in their tRNA and rRNA
genes
33. Prokaryotic Genomes have demonstrated
• Different strains of a single prokaryotic species can have very
different genome sequences, and may even have individual sets of
strain-specific genes
• Transfer has occurred between very different species, even
between bacteria and archaea and vice versa (Lateral transfer)
