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Gene20002 Door Mind Map: Gene20002

1. Gene maintenance and expression

1.1. DNA replication

1.1.1. Prokaryotic DNA replication is semi conservative DNA polymerase synthesize from 5' to 3' DNA replication requires double stranded template Components Initiator protein Helicases SSB proteins DNA gyrase Primase DNA polymerase III DNA polymerase I DNA ligase

1.1.2. Eukaryotic DNA replication is semi conservative DNA polymerase synthesize 5' to 3' requires double stranded template Components Initiator protein Helicases SSB proteins DNA gyrase Primase complex (DNA polymerase, DNA pol alpha-primase) DNA polymerase delta DNA polymerase epsilon DNA polymerase gamma DNA ligase

1.2. Transcription

1.2.1. Prokaryotic Initiation: at -10 recognition (Pribnow box) and -35 recognition Elogation: All genes by single RNA polymerase with sigma factor Termination: Rho independent (GC rich) Rho dependent

1.2.2. Eukaryotic Initiation: 5 Cap Elogation: Multiple RNA polymerases Termination: Poly A tail Post-transcriptional modification Splicing:

1.2.3. Type of rRNA in eukaryotic rRNA required for translation final product of gene expression not translated stable protein coding (mRNA) translated into polypeptide intermediate of gene expression unstable tRNA required for translation wooble / nondegeneracy final product of gene expression not translated stable

1.3. Translation

1.3.1. Prokaryotic cells Initiation Starts with Shine Dalgarno sequence initiator tRNA fMET (AUG start site) Elogation E-P-A site Termination release factor (RF 1 and RF 2) recognizing stop codon

1.3.2. Eukaryotic cells Initiation Cap binding protein (eIF4F) initiator tRNA MET (AUG start site) Elogation E-P-A site Termination stop codon

2. Mutation

2.1. Location

2.1.1. Coding sequence

2.1.2. Promoter

2.1.3. Intron

2.1.4. Intergenic

2.2. Type

2.2.1. First mutation Large scale duplication inversion rearragement Single base pair Silent Nonsense (stop codon) Missense Indels Altered reading frame (frameshift)

2.2.2. Second mutation Supression mutation Reversion Intragenic Extragenic

2.3. Consequence

2.3.1. Lose of gene function

2.3.2. Gain of gene function

2.3.3. No effect

2.3.4. Dominant negative function

2.4. classification based on cause

2.4.1. Spontatenous mutation DNA replication error Base pair substitution Slippage during DNA replication Damage to DNA depurination deamination oxidation strand breakage

2.4.2. Induced mutation by mutagens Replace normal base pair Specific mispairing loss of pairing chromosome breaks

3. Repair

3.1. Mechanism

3.1.1. Reversal DNA damage phr gene (photoreactivation) ada gene (methyl transferase)

3.1.2. Removal DNA damage nucleotide excision repair uvr excision DNA glycosylase and AP endonuclease

3.1.3. Post replication repair mismatch repair Sos repair

3.1.4. Double stranded break repair Non homologous end joining (no template) Homologous end joining (need template)

4. Characteristic of DNA, Gene and Genome

4.1. Characteristic of DNA

4.1.1. double helix

4.1.2. anti parallel

4.1.3. phosphate sugar backbond

4.1.4. nucleotide inside

4.1.5. carries genetic code

4.2. Characteristic of genome

4.2.1. prokaryotic chromosomal DNA usually circular located in nucleoid (membraneless) supercoil low repetitive DNA high density protein coding gene extra chromosomal DNA (plasmid) low to high copies number (variable) generally dispensable for host function unlike chromosomal DNA transfer from cells to cells (cross species)

4.2.2. eukaryotic Nuclear DNA Located in nucleus Linear Present as one, two or multiple copy (haploid, diploid etc) Chromosome number does not relate to complexity Associated with protein (histone) Structure Mitochondrial DNA Located in mitochodnria Mostly circular DNA double stranded and supercoil Genetic variation in size Chroloplast DNA Located in chloroplast All genome circular DNA double strand and supercoil Generally similar in size

4.2.3. HUMAN genome Intergenic DNA (highest in composition) Interspersed repeats Other intergenic region Gene and related sequence Exon (lowest in composition) Related sequence

4.3. Variety in genome

4.3.1. Base composition (GC content) DNA melt curve

4.3.2. Complexity reassociation kinetics

4.3.3. Composition DNA hybridization and gene cloning

4.3.4. Size Quantification of phosphate, re association kinetic, flow cytometry

5. Genomic study and evolution

5.1. Identifying ORF

5.1.1. contain ATG

5.1.2. contain termination codon

5.1.3. codon bias

5.1.4. exon intron boundary

5.1.5. Tata box

5.2. Functional and comparative genomic

5.2.1. Homology Orthology Paralogy

5.2.2. How to study function of gene GEnome wide screen insertional inactivation knockdown using RNA interference Gene wide expression analysis

5.3. Evolution

6. Gene cloning

6.1. How to do gene cloning

6.1.1. In vitro PCR

6.1.2. Invivo recombinant DNA How?

6.2. Types of DNA library

6.2.1. genomic how to make? take genomic DNA, cut with Restriction enzyme (partial or complete) then clone it into vector

6.2.2. cDNA how to make? take RNA, copy mRNA to DNA, ligate into suitable vector

6.3. Types of Library vector

6.3.1. plasmid size of insert: up to 8 kb

6.3.2. bacterophage size insert up to 40 kb need packaging

6.3.3. Fosmid size insert up to 35 - 45 kb hybrid between phage and F factor plasmid need packaging

6.3.4. Bacterial arteficial chromosome size insert up to 100 to 150 kb no need packaging

6.3.5. yeast artificial chromosome size insert up to 500 kb make in yeast

6.4. Types of library screening

6.4.1. By function (phenotype)

6.4.2. By sequence homology (probe)

6.4.3. By protein recognition (antibody)

6.4.4. By sequencing Sanger sequencing

6.4.5. Mapped based cloning approach chromosomal walking chromosomal jumping

6.5. Types of blotting

6.5.1. Southern blotting - DNA

6.5.2. Northern blotting - RNA high strigency for mRNA abundance low strigency for mRNA of different species

6.5.3. Westren blotting - protein

6.6. Genetic maps

6.6.1. Molecular marker Single nucleotide polymorphism detect by sequencing detect as restriction fragment length polymorphism using DNA hybridization Simple sequence length polymorphism usually to detect microsatelite detect by PCR detect by DNA hybridization

7. Gene regulation

7.1. Types of gene regulation

7.1.1. Constitutive

7.1.2. Regulated Long term ex: human haemaglobin Short term ex: apoptotic genes

7.2. Transcriptional gene regulation by positive/negative control

7.2.1. Regulatory proteins for transcriptional gene regulation Negative (repressor) passive negative control active negative control Positive control (activator)

7.2.2. In Prokaryotic Example: lac operon in E.coli LacI repressor CRP gene (CAP protein) I* (initiator binding element) Promoter (RNA polymerase binding site) Operator (lacI repressor binding site) lac Z LacY lacA Example: cI repressor in lamba lysogeny cI repressor operator (cI binding site)

7.2.3. In Eukaryotic Example: galactose utilization in yeast GAL80 (repressor) GAL4 (activator) Example: in mamalian cells negative control is called silencer positive control is called enhancer

7.3. Transcriptional gene regulation by regulating chromatin

7.3.1. in prokaryotes ( none, no histone)

7.3.2. in eukaryotes Structure and key features of chromatin made of DNA wrapped around histone (octomer) chromatin structure is variable chromatin structure is dynamic chromatin structure is heritable

7.4. Translational gene regulation

7.4.1. In prokaryotes

7.4.2. In eukaryotes affecting global tranlational 5 cap recognition inhibition by 4eBP met-TRNA complex formation inhibition by GCN2 affecting gene specific translation specific binding protein bind to 3' UTR of mRNA post-translational modification many example but notable is