Gene expression

Mutations

gene mutation = change in DNA base sequence of a gene

  • mainly occurs in DNA replication

  • occur spontaneously - frequency increased by exposure to mutagenic agents

  • can result in different amino acid sequence in primary structure = hydrogen/ionic bonds form in different locations = different tertiary structure (3D shape) = different function or nonfunctioning protein

  • mutation in genes that control cell cycle = cancer

6 types of gene mutations:

  • addition

    • one extra base added to sequence

    • all subsequent codons are altered - frame shift to right

    • bases may now code for different amino acids = different sequence of amino acids (polypeptide chain) = non functioning protein

  • deletion

    • removal of a base in a sequence

    • frame shift to left

    • bases may now code for different amino acids = different polypeptide chain = non functioning protein

  • substitution

    • one base changed for different base

    • only 1 codon changes - due to genetic code being degenerate it may still code for same amino acid and have no impact, or if mutation is in introns amino acids coded for would not change

  • inversion

    • section of bases detach from DNA sequence and when they rejoin they are inverted (back to front)

    • different amino acids coded for

    • e.g. original: TAC TTC AGG TGG mutation: TAC GGA CTT TGG

  • duplication

    • one base duplicated at least once in sequence

    • frame shift to right

    • different sequence of amino acids coded for

  • translocation of bases

    • section of bases on one chromosome detaches and attaches onto completely different chromosome

    • substantial alteration that can cause significant impacts on gene expression and resulting phenotype

stem cells

  • stem cells are undifferentiated cells that continually divide and become specialised by differentiation

  • different types of stem cells: different differentitation abilities

    • totipotent - can divide and produce any type of body cell, occur in early embryos (limited time)

    • pluripotent - found in embryos, can divide into almost every cell minus placenta and used to treat human disorders

    • multipotent - found in mature mammals - divide to form limited number of different cell types

    • unipotent - found in mature mammals - divide to form one cell type - used to make cardiomyocytes

  • ethical issues of pluripotent stem cells

  • induced pluripotent stem cells:

    • iPS cells can be produced from adult somatic cells using appropriate protein transcription factors to overcome some ethical issues with using embryonic stem cells

    • genes that were switched off to make cells specialised are switched back on using transcriptional factors - turned back into pluripotent state

Controlling gene expression:

transcription control

  • transcription of target genes can be stimulated or inhobited when specific transcriptional factors move from cytoplasm into nucleus

  • this can turn on/off genes so only certain proteins are produced in a particular cell

  • turning on/off particular genes in a cell is what enables them to become specialised

  • transcriptional factors:

    • transcription of a gene will only occur when a molecule from cytoplasm enters nucleus and binds to DNA in nucleus

    • molecules are proteins called transcription factors

    • each TF can bind to different base sequences on DNA - initiating transcription of genes

    • once bound transcription begins, creating mRNA molecule for that gene which can then be translated into cytoplasm to create protein

    • without binding of TF, the gene is inactive and protein won’t be made

oestrogen

  • steroid hormone that can initiate transcription

  • lipid based so diffuse through cell surface membrane into cell and binds to a receptor site on TF

  • this causes DNA binding site on TF to slightly change shape making it complementary and able to bind to DNA to initiate transcription - creates mRNA to be used in translation

Epigenetics (controls gene expression - protein synthesis)

  • epigenetics = heritable change in gene function without changing DNA base sequence - changes caused by changes in environment and can inhibit transcription

  • impacts:

    • increased methylation of DNA - inhibits transcription. When methyl groups added to DNA, they attach to cytosine base = prevents transcriptional factors from binding and attracts proteins that condense DNA-histone complex - therefore prevents section of DNA being transcribed

    • decreased acetylation oh histone proteins - inhibits transcription. If acetyl groups removed then histones become more positive and attracted more to phosphate group on DNA = DNA and histones more strongly associated so harder for TF to bind

RNA interference

  • translation of mRNA produced from target genes can be inhibited by RNAi

  • RNAi = mRNA molecules already transcribed gets destroyed before its translated to create polypeptide chain

  • done using small interfering RNA (siRNA)

  • siRNA disrupts mRNA created and prevents translation

  • enzyme cuts mRNA into siRNA - 1 strand of siRNA combines with another enzyme (RISC) = siRNA-enzyme complex will bind via complementary base pairing to another mRNA molecule. Once bound enzyme will cut up mRNA so cannot be translated

Cancer

  • results from mutations in genes that regulate mitosis - if these genes mutate and non-functioning proteins are made then mitosis not regulated = uncontrollable cell division = tumour

  • benign = slow growing, non cancerous (produce adhesive molecule which means in cannot move), surrounded by capsule so remain compact and can’t spread, localised impact - removed by surgery

  • malignant = grow large rapidly, cancerous, cell nucleus becomes large and cell can become unspecialised again, don’t produce adhesive so can undergo metastasise = tumour can break off and spread to other tissues in body (secondary tumours), not encapsulate so can grow into surrounding tissues and develop own blood supply, life threatening, removal of tumour and treatment e.g. chemotherapy needed

  • tumour develop due to gene mutation in either tumour suppressor gene or oncogene (can link to epigenetics e.g. methylation, oestrogen etc.)

  • oncogene = mutated version of proto-oncogene (which code for protein involved in initiation of DNA replication and mitosis cell division). Oncogene mutation results in process being permanently activated to make cells divide continually

  • tumour suppressor gene = genes produce proteins to slow down cell division and cause cell death if DNA copying errors detected. Mutation in TSG could cause not producing proteins to carry out this function = cell division could continue and mutated cells not identified and destroyed

  • epigenetics:

    • abnormal methylation - TSG could become hypermethylated = gene inactivated = turned off = protein not produced. Oncogenes = hypomethylated = gene permanently switched on = many proteins produced to initiate constant cell division

  • oestrogen

    • produced by ovaries to regulate menstrual cycle - after menopause this stops

    • fat cells in breast tissues can produce oestrogen (causing breast cancer in women post menopause = age is a risk factor)

    • oestrogen can activate a gene by binding to a gene that initiates transcription - if this is a proto oncogene = permanently turned on = cell division

    • tumour then results in even more oestrogen production = increases tumour size = attracts white blood cells = increases tumour size (positive feedback)

Genome

  • entire genetic material of organisms in nucleus of a cell

  • sequencing genome = working out DNA base sequence for all DNA in a cell

  • human genome project 2003

  • sequencing methods continuously improved and now automated

simpler organisms

  • do not contain introns in DNA = genome can be used directly to sequence proteins that derive from genetic code of organism (proteome)

  • useful for identifying potential antigens to use in vaccine

complex organisms

  • have introns and regulatory genes in DNA

  • genome cannot easily be used to translate proteome

  • proteome = entire set on proteins within a particular cell

Gene technologies

Recombinant DNA technologies = recombining DNA of 2 different organisms = allows to manipulate and alter genes to improve medical treatment

  1. creating DNA fragments - first step which produces or isolates fragments of DNA to be recombined

  • reverse transcriptase

    • enzyme makes DNA copies from mRNA (RT naturally occurs in viruses e.g. HIV).

    • find a cell that naturally produces protein of interest

    • cells should have large amounts of mRNA for protein

    • reverse transcriptase enzyme joins DNA nucleotides with complementary bases to mRNA sequence

    • single stranded dna made (cDNA) (+cDNA intron free as copied from mRNA - already spliced)

    • DNA polymerase makes dna fragment double stranded

  • restriction endonuclease

    • enzymes cut up DNA (naturally occur in bacteria)

    • active site complementary to range of DNA base sequences = each enzyme cuts DNA at specific location

    • some enzymes cut at same location in double strand = blunt end

    • staggered ends = exposed dna bases = sticky ends (palindromic) - ability to join to DNA with complementary base pairs

  • gene machine

  1. fragments cloned to amplify sample

    • in vivo cloning (clone inside living organism) - needs restriction endonuclease with sticky ends

      • dna fragments must be modified to ensure transcription of genes can occur - promoter region added at start of DNA fragment (sequence of DNA which is binding site for RNA polymerase to enable transcription to occur), terminator region added at end of a gene (causes RNA polymerase to detach and stop transcription so only one gene at a time copied into mRNA)

      • insert DNA fragment into vector

        • vector = something to carry isolated DNA fragment into host cells (often plasmids)

        • cut open plasmid using same restriction endonuclease = creates same sticky ends

        • DNA fragment sticky ends are complementary to sticky ends on plasmid

        • combined and DNA ligase sticks them together in condensation reaction to create phosphodiester bonds

      • transform a host cell with vector

        • vector needs to be inserted into host cell where gene expressed to create protein

        • cell membrane of host cell must be more permeable

        • increase permeability = mixed with calcium and heat shocked (sudden increase in temperature)

        • enables vector to enter host cells cytoplasm

      • check that plasmid took up DNA fragment and host cell took up plasmid using gene markers (identify if cell transformed)

        • 3 issues can occur: recombinant plasmid doesn’t get inside cell, plasmid re-joins before dna fragment entered, DNA fragment sticks to itself rather than inserting plasmid

        • marker genes - on plasmid. USed to identify which bacteria took up recombinant plasmid. 3 types of marker genes: antibiotic resistance genes (grow bacteria on agar and transfer bacteria to plate with antibiotic in agar and see which bacteria still there), genes coding for fluorescent proteins (green fluorescent protein inserted into plasmid - DNA fragment inserted into GFP gene - prevents GFP production = not glowing = successful DNA fragment), genes coding for enzymes (lactase turn substance from blue to colourless - gene for this enzyme into plasmid - dna fragment inserted in middle of gene = disrupts = bacteria grown on agar plate with colourless substance - remain blue = success)

      • grow host cell (clone)

    • in vitro cloning (not in living organism)

      • polymerase chain reaction (PCR)

      • amplify fragments of dna via PCR using automated machine

        • temperature increased to 95 degrees to break h bonds and split dna into single strands

        • temperature decreased to 55 degrees so primers attach

        • dna polymerase attaches complimentary free nucleotides and makes new strand to align next to each template (synthesis) - temperature increased to 72 degrees for this stage (optimum for taq Dna polymerase)

        • repeats mant times

      • advantages = automated so more efficient, rapid, doesn’t require living cells (quicker and less complex)

DNA probe = short single stranded piece of DNA - labelled radioacively or fluorescently so can be identified - used to locate specific allels of genes and to screen patients for heritable conditions etc.

  • sample of patient DNA removed and heated to single stranded

  • mixed with DNA probe complementary to range of different alleles

  • if patient has allele DNA will bind with probe - can be identified using x ray or UV light

DNA hybridisation = DNA heated to separate double helix into single strand, the mixed with complementary sequences of single stranded DNA - once cooled complementary strands anneal (stick together)

  • used in medical diagnosis to identify is someone has copy of particular allele within DNA

personalised medicine and genetic counselling

  • screening for presence of particular alleles allows doctors to select medicines and give health advice based on genotype

  • some drugs are more or less effective depending on alleles present = enables more effective and cost effective treatment

  • genetic counseling = type of social work giving people advice and information following screening of disease causing alleles

Genetic fingerprinting = way to examine DNA (analysis of VNTR dna fragments)

  • uses VNTRs

  • most of DNA made up of introns which consists of many VNTRS

  • more closely related individuals = more similar VNTRS

  • used to determine genetic relationships and genetic variability within a population

  • collection - collecting sample of DNA (PCR can amplify small amounts of DNA)

  • extraction

  • digestion - restriction endonuclease added to cut DNA into smaller fragment = sticky ends

  • separation - DNA samples loaded into wells in agar gel - gel placed in buffer liquid within electrical voltage applied. DNA negatively charged so DNA samples move through gel towards positive end of gel. Gel electrophoresis - agar gel created resistance for DNA - smaller pieces of DNA can move faster and further along gell - different lengths of DNA separated - alkaline added to separate double strands of dna

  • hybridisation - DNA probes added - bind

  • development - DNA probes and VNTRs transferred to nylon sheet - exposed to xrays or UV light to visualise position of probes

  • analysis - position of DNA bands compared to identify genetic relationships, presence of disease causing genes, match samples etc.