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GENES, GENOMES AND BIOMEDICAL IMPLICATIONS (PROKARYOTES (PHAGE STRATEGIES,…

- - - - GENOME DNA LIBRARY
        
        STORE AS BITS IN VECTOR (PLASMID OR VIRUS)
        
        USE UP THE SAMPLE, CONTAIN EVERY SEQUENCE
        
        DIGEST SAMPLE GENOME
      - cDNA LIBRARY
        
        DO NOT USE UP THE SAMPLE, BUT ONLY CONTAIN CODING SEQUENCE
        
        STORE IN VECTORS
        
        REVERSE TRANSCRIPT mRNA TO GET cDNA AND CONVERT TO DOUBLE STRAND USING DNA POL
    - - KARYOTYPING
        
        INDUCE CELL DIVISION (ADD CHEM, INDUCE 2-3 DAYS)
        
        ARREST CELL IN METAPHASE
        
        SPLATTER THE NUCLEUS
        
        USE GIEMSA STAINING, Q, R, C BANDING OR SKY
      - NUCLEOSOME STRUCTURE
        
        ENDONUCLEASE DIGEST LINKER DNA TO OBTAIN NUCLEOSOME
        
        TREATED WITH HIGH [SALT] SOLUTION TO SEPARATE DNA & OCTAMER
        
        DISSOCIATE OCTAMER
    - - PHYSICAL EVIDENCE
        
        No gene amplification/deletion in most tissues
        
        No. of chromosomes stay the same throughout the body, the structure doesn't change
      - FUNCTIONAL EVIDENCE
        
        CLONING putting donor nucleus into mother's egg/embryo and put back in mother's worm --> same phenotype as donor
        
        TRANSPLANTATION - put frog epidermal cell nucleus into denucleated eggs give tadpoles
  - - - 3' POLYADENYLATION
        
        MECHANISM
        
        CstF and CSPF BIND TO AAUAAA SEQUENCE (10-30 NT UPSTREAM CLEAVAGE)
        
        RECRUIT PROTEIN TO CLEAVE AT CA SITE AND POLYADENYLATION
        
        G RICH OR GU RICH SEQUENCE DOWNSTREAM CLEAVE SITE (30 NT DOWN)
        
        CONSEQUENCE
        
        POLY-A TURNOVER
        
        ACTIVELY TRANSLATED mRNA HAVE LONGER HALF LIFE
        
        CAN BE LENGTHENED
        
        FUNCTION
        
        PREVENT DEGRADATION
        
        TRANSLATION - PABP 1
        
        EXPORT PATHWAY
        
        SPLICE THE LAST INTRON
      - SPLICING
        
        CONSEQUENCE
        
        ALTERNATIVE SPLICING
        
        VARIANT CAN BE TISSUE-SPECIFIC
        
        ONE TRANSCRIPT = MANY PROTEINS
        
        INTERNAL SPLICE SITE/ MUTUALLY EXCLUSIVE EXONS/OPTIONAL INTRONS
        
        DISEASES
        
        SPINAL MUSCLE ATROPHY
        
        BETA-THALASSEMIA
        
        MECHANISM
        
        CHECK-RECHECK REARRANGEMENT BY U1 & U6 AT 5'
        
        U2 AND U6 SPLICEOSOME BRING 5' AND 3' END TOGETHER (5' - GU, 3' - AG)
        
        YURAC BRANCH POINT - UNPAIRED A OH GROUP ATTACK 5' END, CUT SUGAR PHOSPHATE BACKBONE
        
        FORM LARIAT LOOP, FREE EXON 1 ATTACK 3' END, JOIN EXON 1 & 2
        
        INTRONS DEGRADED
      - 5' CAPPING
        
        FUNCTION
        
        GUIDE EXPORT - CAP BINDING COMPLEX
        
        TRANSLATION START SITE (BINDING OF RIBOSOME SMALL SUBUNIT)
        
        PREVENT DEGRADATION
        
        MECHANISM
        
        BEGIN WHEN TRANSCRIPT ABOUT 25 NT
        
        P REMOVE FROM PRIMARY NUCLEOTIDE
        
        5' TO 5' ADDITION OF GMP (FROM GTP) USING TRIPHOSPHATE BRIDGE
        
        METHYLATION OF GUANINE AND PRIMARY NUCLEOTIDE
    - - NOT IN SYLLABUS
    - - INITIATION
        
        DNA
        
        PROMOTER
        
        TATA box
        
        INR (+1)
        
        ENHANCERS/SILENCERS
        
        PROTEINS
        
        GENERAL TRANSCRIPTION FACTOR
        
        TFIID
        
        TATA-binding protein, binds to TATA box
        
        TAF150
        
        TAF250
        
        Distortion (80* bending)
        
        Landmark to locate promoter
        
        Signal binding of other protein
        
        TFIIH
        
        Helicase - unwind DNA
        
        Kinase - phosphorylate RNAPII for elongation
        
        TFIIA
        
        Recruited by TFIID
        
        Enhance TFIID binding to TATA box
        
        TFIIB
        
        Bind to BRE
        
        Help selecting the correct INR
        
        Recruited by TFIID
        
        TFIIE
        
        Regulate TFIIH
        
        TFIIF
        
        Follow RNAPII for about 30 nt
        
        Act like sigma factor, ensure correct assembly of RNAPII to GTF
        
        CO-REGULATORS
        
        CO-REPRESSOR
        
        RECRUIT DEACETYLASE PROTEIN (NO INTRINSIC ENZYMAE)
        
        INVOLVE IN DEACETYLATION SOMEHOW
        
        RETURN NUCLEOSOME TO PRE-TRANSCRIPTION CONFORMATION
        
        CO-ACTIVATOR
        
        MECHANISM
        
        HISTONE MODIFICATION
        
        2 more items...
        
        IDENTITY
        
        DISTINCT FUNCTION CO-ACTIVATORS
        
        TFIIA, B
        
        DOMAIN ON GTF BIND TO TF
        
        FUNCTION
        
        STABILIZE PRE-INITIATION COMPLEX
        
        INTRINSIC HISTONE MODIFICATION (ACETYLATION) ACTIVITY
        
        BRIDGE/ MEDIATOR OR ADAPTOR BETWEEN SEQUENCE SPECIFIC TF AND GTF
        
        SEQUENCE-SPECIFIC REGULATORS
        
        REPRESSOR
        
        BIND TO AD TO PREVENT BIND TO GTF
        
        BIND TO ACTIVATOR BINDING SITE ON GTF
        
        BIND TO DNA SEQUENCE SAME AS ACTIVATORS
        
        ACTIVATORS
        
        DNA-BINDING DOMAIN
        
        GIVE SPECIFICITY TO GENE
        
        LIGAND BINDING DOMAIN (nuclear receptors)
        
        Binding can lead to conformational change
        
        3 more items...
        
        Ligand can be hormones, molecules...
        
        ACTIVATION DOMAIN
        
        BIND TO GTF
      - EPIGENETICS
        
        DNA
        
        LOCUS CONTROL REGION
        
        CONTROL A CLUSTER OF GENES
        
        RECRUIT HISTONE MODIFYING PROTEINS
        
        MATRIX ATTACHMENT REGION
        
        SEQUENCE CAN BIND TO MATRIX PROTEINS
        
        KEEP DNA AS LOOP FOR TRANSCRIPTION
        
        PROTEIN
        
        INSULATOR
        
        INDEPENDENT DOMAIN WITH FUNCTIONAL STRUCTURE
        
        CAN STOP HETEROCHROMATIN PROPAGATION
    - - TINY RNA INTERFERENCE
        
        piRNA
        
        ASSOCIATED WITH PIWI PROTEIN
        
        26-30 NT
        
        NOT FORMED BY DICERS
        
        TARGETING TRANSPOSONS
        
        siRNA
        
        FEATURES
        
        DOUBLE-STRANDED
        
        FOUND IN PLANTS, ANIMALS WITH NO IMMUNE SYSTEM
        
        FUNCTION
        
        PREVENT SPREAD OF TRANSPOSABLE ELEMENTS
        
        KEEP CENTROMERIC DNA IN HETEROCHROMATIN
        
        WITH RITS, BIND TO NASCENT mRNA, RECRUIT HISTONE MODIFYING ENZYMES
        
        miRNA
        
        FEATURES
        
        FOUND IN MAMMALS
        
        19-22 NT
        
        SINGALE STRANDED
        
        FORMATION
        
        dsRNA SPLICED BY DICERS, BIND TO RISC
        
        ARGONAUTE DEGRADE 1 STRAND, VIABLE STRAND BIND TO RISC
        
        ARGONAUTE GUIDE TO TARGET mRNA BY EXPOSING 5' END TO 5' UTR
        
        ARGONAUTE CLEAVES POLY-A TAIL AND EXPOSE mRNA TO ENDONUCLEASES
        
        crRNA
        
        FORMED FROM CRISPR LOCUS (CONTAIN VIRAL DNA)
        
        ATTACK SUBSEQUENT VIRAL DNA INFECTION
        
        DO NOT ATTACK CRISPR DUE TO LACK OF UPSTREAM ELEMENT
      - UBIQUITINATION/DEGRADATION
        
        nURPs
        
        MONOUBIQUITINATION
        
        HISTONE CODE, BINDING SITE
        
        POLYUBIQUITINATION
        
        DEGRADATION
        
        MECHANISM
        
        E1 BIND TO E2-E3 COMPLEX, TRANSFER UBIQUITIN TO E2
        
        E2-E3 COMPLEX BIND TO TARGET/SIGNAL PROTEIN, TRANSFER UBIQUITIN FROM E2 TO PROTEIN
        
        ATP-DEPENDENT UBIQUITINATION OF E1 AT S RESIDUE
        
        PROTEASOME FEATURES
        
        20S CORE
        
        MULTIPLE SUBUNITS FORM 4 LAYERS OF HEPTAMERIC RING TO FORM INTERIOR ACTIVE SITE
        
        UNFOLD AND CUT PROTEIN AT MULTIPLE SIDE TO PEPTIDES
        
        2 19S HEADS
        
        20 SUBUNITS (>6 ATPase)
        
        RECOGNISE UBIQUITINATED PROTEIN
      - IRES
        
        CLEAVE eIF4 TO BIND TO IRES
        
        VIRUS COMPETE W HOST RNA AND PRODUCE >1 PROTEIN
        
        APOPTOSIS MECHANISM
        
        DOWNSTREAM TIS
    - - GENE LOSS
        
        Hemoglobin maturation
        
        Erythoblast - nucleus exude (in bone marrow) --> reticulocyte - exude ribosome & mitochondria (on way out, 1-2 days)--> erythrocyte
        
        Nucleus degrade by macrophages
        
        After all the required proteins have been synthesised
      - CHROMATIN REMODELING
        
        HAT/PHOSPHORYLATION
        
        HDAC/METHYLATION
      - GENE AMPLIFICATION
        
        Drosophila "puffs"
      - GENE REARRANGEMENT
        
        iMMUNOGLOBULIN (in preB cells upon infection)
        
        LIGHT CHAIN
        
        V arm
        
        J arm
        
        HEAVY CHAIN
        
        D arm
        
        J arm
        
        V arm
      - GENE (DE)ACTIVATION
        
        METHYLATION PATTERN IS DYNAMIC TO REGULATE DEVELOPMENT OF ORGANISM
        
        Genome wide demethylation/methylation during embyonic development
        
        Inhibit methyl transferase for demethylation
        
        Recruit demethylase for demethylation
        
        Hemoglobin
        
        METHYLATION OF CpG island at C lead to DEACTIVATION
  - - - TISSUE-SPECIFIC GENES
        
        SPECIALISED PROTEINS
        
        CELLULAR UNIQUE PHENOTYPE
      - HOUSEKEEPING GENES
        
        COMMON PROTEIN
        
        METABOLIC ENZYMES/ EXPRESSION REGULATOR/ REPLICATION PROTEINS
    - - REPEATS
        
        INTERSPERSED REPEATS
        
        LINEs
        
        SINEs
        
        TANDEM REPEATS
  - - - NUCLEAR PORES
        
        TRANSPORT MATERIALS
      - NUCLEOLUS
        
        WHERE MOST DNA GATHER
      - OUTER/INNER MEMBRANE
        
        COMPARTMENTALIZATION
      - MATRIX
        
        ORGANISE COMPOSITION
        
        GIVE SHAPE
        
        ANCHOR DNA AND MATERIALS
    - - CHROMOSOME STRUCTURE
        
        P ARM (ON TOP, SMALLER)
        
        Q ARM (BELOW, LONGER)
      - CHROMOSOME ARRANGEMENT
        
        EUCHROMATIN
        
        SUSCEPTIBLE TO DNAse 1
        
        ACTIVE, PROTEIN CODING GENE
        
        ALU RICH
        
        HETEROCHROMATIN
        
        RESISTANT TO DNAse 1
        
        INACTIVE, REPEATS
        
        ALU POOR