Topic 8: the control of gene expression

inversion mutation

inversion mutation

one region of the chromosome is flipped and reinserted

duplication mutation

region of the chromosome is repeated, resulting in increase dosage from genes in gene in regions?

translocation mutations

region from one chromosome is aberrantly attached to another chromosome

transcriptional factors

= simulate the region of DNA to begin transcription

binds to specific region of the DNA

can be blocked by inhibitor, DNA is not transcribed, gene is not expressed

oestrogen

steroid hormone, lipid soluble molecule

combine with receptor/transcription factor, changing its shape so inhibitor is released and DNA binding site is exposed; will now bind to promoter region of DNA, initiating transcription

totipotent

can divide and specialise into any type of body cell and the placenta

early stage of fertilised eggs/mammalian embryonic stage only

pluripotent

can specialise into any body cell, but not cells that make up the placenta

divide in unlimited numbers and used in treating human disorders

multipotent

differentiate into a few/limited number of different types of cells

unipotent

can only differentiate into a single type of cell eg. cardiomyocytes of the heart

induced pluripotent stem cells

pluripotent stem cells produced from unipotent stem cells (adult somatic cells)

they are genetically altered in lab by including genes and protein transcription factors to express themselves

how do totipotent cells initiate specialisation?

part of DNA is translated

translation factors: small interfering RNA (siRNA)

double stranded RNA

1. cut down to smaller pieces by dicer

2. RISC complex is formed and binds to mRNA, degrading it

translation factors: micro interfering RNA (miRNA)

hair-pin bend of RNA

1. forms RISC complex

2. binds to mRNA that is complementary to RISC

how translation is stopped, when mRNA binds to RISC

mRNA is hydrolysed by enzyme - RNA hydrolase

or, ribosomes are prevented from attaching to mRNA

epigenetics

= heritable change in gene function without changes to base sequence of DNA

how epigenetics work?

histones and DNA are covered in tags eg. methyl

forming second layer called epigenome, determines shape of DNA-histone complex

DNA code is fixed, however, epigenome is flexible to change by environment

DNA is exposed = expressed gene

tightly packed histones = not expressed gene

acetylation

bind to = (amino acid of) histones

effect on DNA = (more acetyl) loosely packed histones

effect on gene expression = expressed

methylation

bind to = (cytosine) DNA base

effect on DNA = (more methyl) tightly packed histones

effect on gene expression = not expressed

epigenetic treating disease

= epigenetic changes are reversible

eg. drugs can inhibit enzymes involved in DNA methylation

tumour

= uncontrolled cell division

difference between benign and malignant tumours

benign tumours are localised to one area of the body, whereas, malignant has effects across whole body as it can spread

benign does not cause cancer; malignant causes cancer

benign grow slowly; malignant grow rapidly

how do tumours cause harm?

damage organ concerned

cause blockage/obstruction

damage/exerting pressure on other organs

tumour suppressor gene function

codes for proteins that slow down cell division, repair mistakes in DNA, and tells cells to die

hypermethylation of tumour suppressor gene

1. hypermethylation occurs in specific, promoter region of TSG

2. histones become tightly packed and transcription factors cannot reach DNA, TSG is not expressed

3. stops transcription of TSG

4. proteins that slow down cell division not produced/translated

5. leading to increase of/uncontrolled cell division

proto-oncogenes

= stimulate a cell to divide when growth factors attach to a protein receptor on its cell surface membrane

activates DNA to replicate + cell to divide

hypermethylation of oncogenes

growth factors are produced in excess

permanent activation of proto-oncogenes

cell divides too rapidly and out of control

increased oestrogen concentration on proto-oncogenes

cause proto-oncogenes of cells in breast to develop into oncogenes

ie. oestrogen releases inhibitor, activating genes so uncontrolled cell division

genome project (spec)

sequencing projects have read the genomes of a wide range of organisms

determining the genome of simpler organisms allows the sequences of proteins that derive from genetic code (proteome) of organisms to be determined, applications such as identification of potential antigens for use in vaccination

in more complex organisms, presence of non-coding DNA and of regulatory genes means knowledge of the genome cannot be easily translated into proteome

sequencing methods are continuously updated and have become automated

recombinant DNA technology

= involves transfer of fragments of DNA from one organism/species to another

genetic code is universal, so translation and transcription are too

transferred DNA will be translated within recipient (transgenic) cells

producing DNA fragments

1. reverse transcriptase: conversion of mRNA to cDNA (complementary DNA)

2. restriction endonuclease: cut a fragment containing the desired gene from DNA

3. gene machine: create the gene

using reverse transcriptase

take cell that rapidly produces required protein

cells have large quantity of relevant mRNA which is extracted

reverse transcriptase (+ DNA polymerase) used to make cDNA

using restriction endonuclease

= cuts up the viral DNA

cuts at recognition site

producing sticky ends, known as palindromic sequence

using gene machine

determine nucleotide sequence for desired protein

use machine to produce gene with no introns and duplicated by PCR

advantage: very accurate in short time and free from introns (so prokaryotes can translate)

plasmid

= small circular piece of DNA found naturally in bacteria

separate from main bacterial DNA

contains only a few genes

have the ability to insert themselves into bacteria

vector

= carries gene from one organism to another

why use bacteria as recipient cell?

= small and easy to manipulate

reproduce quickly

have plasmids which can be used as vectors

in vivo: function and process

function = to clone DNA (in organism/bacteria)

processes = restriction endonuclease and ligase used to insert a gene into vectors, which are then transferred to host cells

genetic markers are used to identify transformed cells

in vivo cloning

1. restriction endonuclease used to cut desired gene

2. cuts at recognition site; both plasmid and gene

3. fragment of DNA has promoter and terminator region added to it

4. promoter region attaches RNA polymerase and transcription factor; terminator releases RNA polymerase (transcription starts and ends)

5. DNA ligase inserts foreign DNA into plasmid by catalysing the formation of phosphodiester bonds between gene and plasmid

6. plasmid is mixed with bacteria: cold calcium chloride used to shock bacteria so it takes up plasmid (known as transformation)

in vivo cloning: why are there more than one transformations of plasmid/gene

all cut DNA have the same complementary sticky ends/base sequence

process of sticky ends joining is random

so, may form plasmid + gene, plasmid + no gene, and no plasmid + gene

in vivo cloning: replica plating

circular, velvet sterile pad pressed on agar plate

bacteria stick to it so it can be transferred to another plate

pressed onto new plate so colonies will grow in exactly same position

in vivo cloning: industrial

on large scale:

must contain all nutrients for growth and replication;

supply oxygen;

temperature and pH must be controlled for optimum growing conditions

in vitro cloning: function and process

function = to clone DNA (in test tube) (aka DNA amplification)

processes = heating to separate DNA strands

cooling - allow primers to attach

heating DNA polymerase binds free nucleotides

in vitro cloning: PCR

polymerase chain reaction

1. DNA heated to 90-95°C

2. DNA is denature and strands separate

3. cooled to temperatures below 70°C

4. primers added (annealation)

5. (Taq) polymerase and nucleotides added and attach by complementary base pairing

6. increase temperature to 70-75°C

7. polymerase joins nucleotides together to form new strand

8. cycle repeated (20-30 times)

primer

short length of double stranded DNA with complementary bases

= attach to starting point of gene, starting point for polymerase to begin the new DNA strand

gene therapy: function and process

function = replace defective gene/treat genetic disease with (healthy) genes

process = use a vector (harmless virus or liposome) to carry gene into human cell

DNA probes: function and process

function = to locate specific gene

process = probe is complementary to gene/part of gene of interest

probe mixed with single stranded DNA

probe binds to DNA (DNA hybridisation)

DNA probe

= short piece of single stranded DNA, complementary to known base sequence/gene

radioactive probes identified by x-ray photographic film

fluorescent probes emit fluorescence/light under UV light

DNA hybridisation

probe is complementary to part of gene of interest

DNA of interest is treated to separate the 2 strands

DNA is mixed with probe which binds to complementary bases

genetic screening

for health risk, drug response, and heritable conditions

eg. notice mutation of a gene, family history of genetic disease

genetic counselling

give advice and information for people to make decisions about themselves and offsprings (eg. the likelihood of the child being born with a disease)

research family history of inheritable disease

make people aware of further medical tests

genetic fingerprinting: function and process

function = determines with genetic variability within a population

processes = DNA cuts using VNTRs and cloned by PCR

separated by gel electrophoresis

radioactive (or fluorescent) DNA probes are used to bind to core sequences to identify strands

gel electrophoresis: function and process

function = (gel electrophoresis) separate DNA according to size/mass

process = fragments placed into a well at one end of agarose gel

electric current passes through the gel

pieces of DNA are attracted to positive charge

smaller section of DNA travel further

how gel electrophoresis works?

DNA is negatively charged

VNTRs of DNA are cut by restriction endonuclease, after the quantity is increased by PCR

placed in the wells of agarose gel

current induced at negative electrode, repelling DNA to the positive DNA

smallest DNA travel furthest

Southern Blotting

DNA separated into single strands by alkaline solution

thin nylon membrane is placed over the gel, after electrophoresis

draws gel containing DNA up, in to nylon membrane by capillary action: same exact place as on electrophoresis

DNA probes added to membrane so they attach to DNA: UV light used to view fluorescent probe, or X-ray film used to view radioactive label

VNTRs

variable numbers tandem repeat

genome of organism contains many repetitive intron bases eg. GCGCGCGCGC

number and length of VNTRs are unique to everyone; more similar = more closely related

gel electrophoresis (mark scheme)

1. DNA is cut by restriction endonuclease

2. use gel electrophoresis

3. separate according to mass/length

4. Southern Blotting (nylon membrane)

5. alkaline conditions (make single stranded)

6. apply probe

7. radioactive/fluorescent

8. reference to VNTRs

9. autoradiography

DNA sequencing: function and process

function = determine the base sequence of a gene

process = automated process and continually updated