BASIC GENETICS IN RELATION TO DNA, GENE OGRANISATION, GENE EXPRESSION & POLYMORPHISM

what type of medicine is going to be used increasingly in the future 

personalised medicine

define transcription and translation

• transcription: process of copying a segment of DNA into RNA

• translation: process of synthesising new protein from messenger RNA

is the genome static/ fixed or dynamic

• the genome is static\ fixed

• most cells have a copy of the same genome

are cells static/ fixed or dynamic

• a cell is dynamic

• because they respond to external conditions

• most cells follow a cell cycle of division

when do cells differentiate

during development

define pseudogenes

pseudogenes: a DNA sequence/ section of chromosome that is an imperfect copy of a functional gene but is actually non-functional

what percentage of the genome encodes for proteins 

3% (21,000 genes)

outline non-coding DNA (‘junk’ DNA)

• landing spots for proteins that influence gene activity i.e. switch on or off

• strands of RNA with myriad roles

• places where chemical modifications silence stretches of chromosome

outline mRNA

• shorter than DNA

• only contains information coding for one protein or part of a protein

• has uracil instead of thymine

• single stranded

• has a ribose sugar

why does RNA contain uracil instead of thymine 

• uracil costs a lot less energy to make than thymine

• we make a lot of RNA, hence it contains U instead of T

outline transcription

• genetic information is only carried on the coding strand of DNA

• the other strand is the template strand

• strands of DNA that serves as a coding template for one gene may be non-coding for other genes in the same chromosome

• the coding strand is always the top strand of DNA

what does this image show

the mRNA sequence is complementary to the DNA template strand and identical to the coding strand (apart from T-U)

outline initiation

• initial step: find the start of the gene on the coding strand of DNA

• RNA polymerase must pick the correct strand and find the start of the gene

• RNA polymerase binds to one or more short sequences upstream of the start of the gene

  • closer to the 5’ end

  • promoter sequences

what are the types of RNA polymerases found in eukaryotes

• polymerase I - transcribes large ribosomal RNA

  • nucleolar region of nucleus

• polymerase II - produces mRNA precursors

• polymerase III - small RNAs (tRNA), 5S ribosomal RNA and other small DNA sequences

outline RNA polymerase II

• produces mRNA precursors

• composed of several subunits but requires several accessory proteins (transcription factors)

• all added to the complex in a defined order to initiate transcription

• RNA polymerase wraps around both strands

  • large enough to enclose the promoter and beginning of gene

what is TFIID

• TFIIID recognises specific region called TATA box

  • ensures correct placement of RNA polymerase

what is the function of the TATA box 

• affects transcription rate

• determines location of start site

where is the TATA box located

• the basal promoter contains the TATA box and is found in all protein-coding genes

define enhancers

enhancers: DNA sequences which control the efficiency and rate of transcription

• regulates expression of genes in specific cell types and controls timing of gene expression

why are promoters and enhancers ‘cis acting elements’

• they are found on the same molecule (strand) of DNA as the gene they regulate

what characteristic of DNA allows for DNA looping

flexibility of DNA allows for DNA looping

where are promoters located 

• promoter necessary to start transcription

  • usually on 5’ side of gene to be transcribed

where are enhancers located

• enhancers can affect transcription from afar

  • on the 5’ or 3’ of transcription start site

  • on introns or even on non-coding strand

• enhancers can be thousands of nucleotides away from interacting promoters

how are enhancers and promoters brought closer together

• by the looping of DNA

• this occurs due to interactions between proteins bound to enhancers and promoters

what is the name for the proteins facilitating DNA looping

activators 

what is the name for proteins that inhibit DNA looping

repressors

what type of promoters and enhancers are there 

‘strong’ or ‘weak’ promoters and enhancers 

what can changes in promoter strength result in

• disease

• can also have deleterious effects on a cell

—

e.g. tumour promoting viruses transform healthy cells by inserting strong promoters in vicinity of growth-stimulating genes

e.g. translocation in some cancer cells place genes that should be ‘turned off’ in proximity of strong promoters and enhancers

what do transcription factors bind to

TF bind to promoter and enhancer sequences and recruit RNA polymerase

outline basal transcription factors

• required at every promoter site for RNA polymerase interaction - TFIID

why are transcription factors ‘trans’ acting factors 

because they are proteins encoded by a different gene to that being regulated

outline transcription after RNA polymerase is attached

• RNA polymerase unwinds the double helix over a short length and splits them apart

  • this creates a bubble of 10 bases

• RNA polymerase catalyses sugar phosphate bond between 3’ -OH of ribose and and 5’ PO₄

• nucleotides are added to the 3’ -OH of growing chain

• DNA zips back up as polymerase moves along

• the bubble moves along the chain

• the growing RNA chain detaches from template

how does the structure of RNA allow it to fold into a 3D structure 

• RNA is single stranded but base pair interactions between complementary sequences found elsewhere on the same molecule allows 3D folding

• folding similarly to the way a polypeptide chain folds into a protein

what does the ability of RNA to fold into complex 3D shapes allow for

allows for some RNA molecules to have structural and catalytic functions (active sites can be formed by RNA folding)

outline pre-mRNA processing

as RNA polymerase moves down the gene

1. cap on 5’ end - this stabilises the mRNA which is essential for transporting RNA out of the nucleus

2. alternative splicing

3. polyA tail on 3’ replaces 3’ UTR causes cleavage at stop codon (AAUAAA)

what is the stop codon needed for

• AAUAAA needed for release of polymerase from DNA template

how long is pre-mRNA

• pre-mRNA is only 20-40 nucleotides long

when does capping pre-mRNA take place

• takes place during transition from transcription initiation to elongation

why does capping pre-mRNA take place

• protects from degradation

• serves as assembly point for proteins needed to recruit small subunits of ribosomes to begin translation

outline alternative splicing

• several variants of the same protein are produced by one gene

• mutations at splice sites can result in aberrant or truncated proteins that do not function properly

  • this plasticity allows for disease development e.g. cancer

what is a spliceosome

spliceosome: exons defined by short, degenerate classical splice site sequences at intron/ exon borders

what are the types of spliceosome

• major spliceosome: removes 99.5% of introns

• minor spliceosome: removes remaining 0.5% of introns

what are the two main functions of spliceosomes

1. recognition of intron/ exon boundaries

2. catalysis of reactions which remove non-coding introns and stitch flanking exons back together

how many different proteins are associated with human spliceosomes

> 300 different proteins

what are mutations that can alter splice sites or spliceosome proteins and what do they result in

• mis-splicing » rapid degeneration of mRNA

• mis-regulation of splicing factor levels » cancer

list some types of RNA

• mRNA

• rRNA

• tRNA

• ncRNA

• small nuclear RNA (snRNA)

• small nucleolar RNA (snoRNA)

• microRNA (miRNA)

outline mRNA

• wide range of sizes reflecting size of polypeptide

• many common to most cells

  • ‘housekeeping’ proteins needed by all cells e.g. enzymes of glycolysis

• specific for only certain types of cells

  • proteins needed by ‘specialised’ cells e.g. haemoglobin in precursors of RBCs

outline rRNA

• builds ribosomes

• 4 kinds in eukaryotes

what are the 4 kinds of rRNA in eukaryotes 

• 18S - one of these plus other proteins make small subunit

• 28S, 5.8S, 5S - one each of these plus proteins make a large subunit

• S = Svedburg unit (sedimentation rate related to mass and shape)

  • basically how dense each RNA is

outline tRNA 

• 32 different kinds in typical eukaryotic cells

• each kind carries one of 20 amino acids at 3’ end (most amino acids have more than one tRNA)

outline ncRNA

• 97% of DNA is referred to as junk DNA

• 90% of genome is actively transcribed and thus more complex

• ncRNA can modify protein levels by a mechanism independent of transcription

• play major roles in cellular physiology, development, metabolism, implicated in disease process

outline long non-coding RNA

long intronic RNA are from cleaved introns from mRNA

outline short non-coding RNA

outline small nuclear RNA (snRNA)

• several members part of spliceosome

outline small nucleolar RNA (snoRNA)

• participate in making ribosomes 

  • cleave precursors of 28S, 18S, 5.8S

• implicated in alternative splicing

• template for synthesis of telomeres

• vertebrates: snoRNA made from cleaved introns

what are telomeres

telomeres: found at the end of chromosomes, areas of long RNA repeat regions thought to protect chromosomes from degradation

• gets shorter as we get older

outline microRNA (miRNA)

• tiny (18-25 nucleotides)

• regulate gene function post-transcriptionally

• binds to mRNA and causes degradation

  • inhibits protein synthesis

• regulation of developmentally timed events

• tissue-specific and/ or developmental stage-specific expression

  • can be transported to other cells

if miRNA is poorly regulated what can it lead to

cancer development

what type of RNA plays a critical role in tooth development

miRNA

in what areas of diagnostics are genetics involved in

• Polymerase Chain Reaction (PCR)

• quantitative reverse transcriptase PCR (qRT-PCR)

• whole genome sequencing

outline PCR

• form of DNA amplification

• starting material: DNA

• amplifies a specific section of DNA

• allows visualisation of gene presence or absence on a gel

what does PCR use

• thermostable DNA polymerase from bacteria (Thermus aquaticus)

• template DNA

• primers

• dNTPs (A, T, C, G)

• Mg²⁺

outline qRT-PCR

• starting material: mRNA

• convert to DNA (cDNA)

• detects the accumulation of the amplicon during reaction

• different amounts of mRNA in starting sample

  • high amount: amplifies quickly

  • low amount: takes longer

• quantifies amount of mRNA expression

what is qRT-PCR now used for

• heavily used during COVID to detect amount of pathogen

• now often used for cancer diagnostics in precision medicine

table comparing PCR and qRT-PCR

outline whole genome sequencing

• sequences, assembles, analyses human genome

• can analyse for presence or absence of genes or specific mutations in (vast computer processing):

  • neurological disorders

  • specific cancers

  • blood disorders

• cost and speed of this vastly improved

what is an issue with whole genome sequencing

data storage

outline salivomics

• able to monitor (uses computer programming):

  • genomics, transcriptomics, proteomics, metabolomics, microbiome

what is GWAS

Genome Wide Association Studies

• rapid scanning of genomes of many people to find genetic variations associated with a particular disease 

• precision medicine