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Genetics, DNA, Problem! DNA synthesis can never go in 3’-5’ direction -…
Genetics
From the Ancient Greek " genesis" (creation/origin) - Study of inheritance, variation & genes.
- Molecular basis of gene structure and function (expression)
Bacteria -
are the majority of living organisms. – Approx. 4-6 x1030 cells = 5 thousand billion metric tonnes of biomass.Clinical problems caused by bacteria.
- Development of resistance to microbial drugs.
Industrial exploitation of bacteria.
- vitamin production, industrial enzyme production, biopharmaceuticals, bioplastics, biogas (developing world).
- can be optimised via genetic manipulation.
Bioremediation using bacteria
Horizontal gene transfer between bacteria.
- Spread of antibiotic resistance
- Safety of GMOs
Pre-molecular definition of a gene.
Discrete
- Genetic unit that control hereditary characteristics - e.g. ABO blood group, cystic fibrosis, cleft chin,
antibiotic resistance etc. etc...)
Alleles
An alternative forms of gene.
- e.g. wild-type vs
mutant – that may (or may not) change the phenotype
Experiments conducted in the mid 19th century proved that's genes are DNA. Griffith transformation experiment.
two types of S. pneumoniae:
- R strain produced small colonies- none pathogenic.
- S strain produced large smooth colonies- cells coated in hick capsule that elicits the immune response- Fatal in mice.
- Fred Griffith had noted that pathogenic phenotype ( and gene) could be transmitted from the S--> R strains via " Transformation"
IT analogy; DNA is like a computer hard drive carrying operating system & software that encodes structure & function of cells.DNA is relatively inert (stable) , but codes for every function of the cell. Structure of DNA:
- replicating (copying)
- how information stored as DNA is Transcribed (→mRNA) & Translated ( Protein) by cellular machinery.
Nucleotides; the building blocks of DNA. composed of 4 types of nucleotides:
- Nitrogenous base.
- Five-carbon sugar (deoxyribose)
- Phosphate group. (PO4)
Bases
- Adenine.
- cytosine
- **thymine or
guanine.**
Amounts of A = T, G = C, purines = pyrimidines [Chargaff’s
Rule]
Structure of DNA
Published by Watson & Crick in 1953.
- DNA is a double- stranded helix.
- Antiparallel strands: each strand has a PO4 group at 5' end: OH group at 3' end.
Nucleotides in each strand are linked by 3’ - 5’ (“3-prime
to 5-prime”) phosphodiester (C-PO4-C) bonds
Sugar- phosphate backbone is negatively charge and hydrophilic - water soluble
- Negatively charged used by proteins that bind to DNA ( which tend to carry a positive charge).
- bases on opposite strands are linked by hydrogen bonding: A with T; G with C ( Chargaff's rule)
DNA
Watson & Crick, 1953
- Information carrying DNA content to cell ( genome) has to be copied before every division
- One strand acts as a template for synthesis of other (complementary) strand.
Central Dogma:
- information flows unidirectionally from DNA ( genetic template to RNA ("expression" of gene -i.e. switched on) to protein.
An exception is retroviruses; convert RNA genome back to DNA.
Genes are made up of DNA (genotype)
- They are expressed to make polypeptides (proteins) that determine observable characteristics of organism (phenotype)
Prokaryotes (e.g. Bacteria) are simpler genetic systems
- No nucleus: “free” DNA
- Transcription & translation are concurrent (happen together)
Eukaryotes (you!) are more complex
- Transcription takes place within nucleus & mRNA transported out to
cytoplasm to be translated into protein
Three alternative models of DNA Replication:
- Semiconservative replication
- Conservative replication.
- Dispersive replication.
- Experiments of Meselson & Stahl proved that DNA is semiconservative.
Clinical Importance of DNA Replication
- 160 different proteins are involved in replicating
human genome.
- At least 80 genetic diseases result from mutations in these
proteins or from errors in DNA replication or repair.
- Infectious diseases resulting from DNA replication of DNA viruses.
- many therapeutic (antiviral, antibacterial) drugs are targeted to DNA replication proteins.
Basic " ingredients" needed for DNA synthesis are :check:
- DNA template.
- Deoxynucleoside tri-phosphates
(dNTPs)
- Protein complex involving DNA polymerase (DNA Pol III is primary replicate polymerase)
- DNA or RNA ' primer'
- Divalent metal cations (Mg2+)
Mechanism of DNA Replication
Catalysed by DNA polymerase enzyme.
- not a single protein - protein (enzyme) complex
- multiple versions (DNA polymerase I, DNA polymerase III)
DNA polymerase needs DNA/RNA primer to start. Nucleotides are added - by complementary base pairing with template strand. Substrate- deoxyribonucleoside triphosphates (dNTPs) - are hydrolysed as added, releasing energy for DNA synthesis.Enzymatic synthesis of DNA is ALWAYS in 5 ---> 3 direction - DNA polymerase adds nucleotides to the 3' end of the growing strand.
DNA polymerases require a primer to
synthesize DNA from a template.
- DNA polymerases: they can only extend** pre-
existing DNA (or RNA) chain by using a primer**
- requiring a double stranded template to begin.
For RNA polymerase to transcribe to DNA, they start scratch and do not need a primer.DNA replication needs a primer;
extension is in 5’ to 3’ directionTranscription does not need a primer;
extension is also in 5’ to 3’ direction
Mechanism of DNA Replication.
- DNA helicase unwinds the double helix
- Template strands are stabilized (e.g. held apart) by other proteins – single-stranded DNA binding proteins make template available.
- RNA primase catalyses synthesis of short RNA primers, to which nucleotides are added
- DNA polymerase III extends strand in 5’-to-3’ direction
RNA Primase
- Enzymes uses single DNA as template.
- to synthesise short complimentary RNA molecules ' primer'
- providing a double stranded template for DNA pol to extend.
Primer recognised by DNA pol III which then catalyses synthesis of new DNA strand.
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