3.8 The control of gene expression

gene mutation

a change in the base sequence of a gene

addition mutation

One base is added to the DNA sequence. Causes reading frame shift.

deletion mutation

frame-shift mutation where a nucleotide is deleted from the genetic material

substitution mutation

A mutation in which a nucleotide or a codon in DNA is replaced with a different nucleotide

inversion mutation

A sequence of bases is reversed

duplication mutation

One or more bases are repeated

translocation mutation

A sequence of bases is moved from one location in the genome to another

Mutagenic agents

Increase rate of mutation by:

Acting as a base - chemicals can substitute for a base during DNA replication so change the sequence of amino acids

Altering bases- some chemicals can delete or alter bases

Changing structure of DNA - UV radiation changes structure of DNA

degenerate genetic code

more codons (64) than amino acids (20)

universal genetic code

refers to the fact that particular codons specify the same amino acids in almost all organisms

non-overlapping genetic code

A term used to describe the fact that each base is only part of one codon and that each codon is read one at a time in order.

Micropropagation

cloning method of a plant that involves taking tissue samples and growing them in labs on a specially selected media (contain carbohydrates, mineral salts, agar and sometimes vitamins, amino acids and growth regulators)

stem cells

have the ability to differentiate into specialised cells, regenerate an infinite number of times, relocate and differentiate when needed.

what causes differentiation in stem cells

genes switch off

totipotent stem cells

Stem cells that can differentiate into all types of specialised cells needed during embryo development

pluripotent stem cells

stem cells that can differentiate into any type except cells that make up placenta

multipotent stem cells

stem cells that can differentiate into a few types of specialised cells

unipotent stem cells

Stem cells that can only differentiate into one type of specialised cell

cardiomyocytes

cardiac muscle cells

induced pluripotent stem cells (iPS)

produced from a unipotent cell using appropriate protein transcription factors that control genes being switched on/off

Obtaining stem cells

inner cell mass of the blastocyst of an embryo contains pluripotent cells. With permission, obtain excess embryos from IVF clinics and isolate the cells. Or primordial germ cells from terminated pregnancies.

arguments for stem cell research

• cell at 3 days is not living

• cures for many diseases

• less lab animals & money wasted

• no waiting lists for organ transplants

• no problems with rejection

arguments against stem cell research

• considered living

• those with stem cells develop problems

• unstable - tumour growth

• against religion

• drugs to produce embryos are dangerous

Transcription

1. DNA helicase separates DNA by breaking H bonds

2. DNA strand acts as template

3. RNA nucleotides align along complementary bases (a-u, c-g)

4. RNA polymerase joins phosphodiester bonds

5. pre-mRNA produced

6. spliced to remove introns to form mRNA

Translation

1. mRNA leaves nucleus and binds to ribosome

2. tRNA carrying a specific amino acid binds by anticodon to complementary mRNA codon

3. More tRNA bind to next codon

4. amino acids bind via peptide bonds

5. tRNA leaves

6. process repeats forming polypeptide chain until a stop codon is reached and chain detaches.

transcriptional factors

Proteins that bind to complementary sites the promoter on DNA.

2 groups:

activators - stimulate or increase transcription and help RNA polymerase to bind to target gene

repressors - inhibits or decreases transcription by preventing RNA polymerase from binding to target gene

Oestrogen as a transcription factor

steroid hormone secreted by ovaries and binds to a transcription factor called an oestrogen receptor. Forms an oestrogen-oestrogen receptor complex. Complex moves to start of the target gene and either acts as an activator or repressor.

post-transcriptional factors

RNA interference (RNAi):

small interfering RNA (siRNA) stop mRNA from target genes being translated into proteins.

1. double stranded RNA cut by dicer enzyme

2. siRNA formed which unwinds and binds to protein

3. RISC is formed (siRNA protein complex)

4. mRNA target recognition and RISC bind

5. mRNA cleavage occurs

6. no protein formed

siRNA uses

antiviral tools

inhibit cancerous cells

treat neurodegenerative diseases

micro-RNA (miRNA)

small single stranded RNA molecules that bind to mRNA and can degrade mRNA or block its translation.

Epigenetics

study of heritable changes in gene expression that does not involve changes to the underlying DNA sequence. A change in phenotype rather than genotype which affects how cells read the genes

Chromatin

DNA + histones

How do epigenetics work?

alter how easy it is for enzymes and other proteins involved in transcription to interact with DNA and can also occur in response to the environment

Increased methylation of DNA

Attaching methyl groups to cytosine-guanine site and so it changes DNA structure so transcriptional machinery (RNA polymerase) find it harder to interact and bind to the gene so the gene is switched off

acetylation of histones

\-Histone acetyltransferases: enzymes lead to addition of acetyl groups makes chromatin structures less condense so transcriptional machinery can access DNA allowing it to be transcribed.

\-Histone deacetylase: enzymes lead to removal of acetyl group makes chromatin become highly condensed so genes in the DNA cannot be transcribed as transcriptional machinery cannot access them.

Treating epigenetic disease

Epigenetics is reversible so new drugs that inhibit methylation and reduce acetylation enzyme activity

Acquired mutations

Mutations that occur in an individual after fertilisation. If occur in genes that control rate of cell division (tumour suppressor genes and proto oncogenes) can causes tumours.

Tumour suppressor genes

\-Normal conditions: slows down cell division by producing proteins called tumour suppressor proteins which causes cells to stop dividing or causes apoptosis.

\-Mutated conditions: gene inactivated so tumour suppressor protein is not produced and allows uncontrollable division

Proto-oncogenes

\-Normal conditions: stimulates cell division by producing proteins that enable division

\-Mutated conditions: genes become overactive and becomes an oncogene that stimulate uncontrollable division

Tumour types

malignant: cancerous, grow rapidly and metastasis

benign: non-cancerous, slow growth, often covered in fibrous tissue but can cause blockages and put pressure on organs

Characteristics of tumour cell

• larger and darker nucleus

• may have more than 1 nucleus

• irregular shape

• doesn't produce proteins

• different antigens

contributing factors to tumour growth

• Hypermethylation of tumour suppressor gene: genes harder to transcribe so cell division is not suppressed.

• Hypomethylation of proto-oncogenes: genes become overactive causes uncontrollable division

• oestrogen: stimulates breast cells to divide. Possibility it can introduce mutations directly into DNA of breast cells

receptors for breast cancer

• hormone receptors: breast cancers with oestrogen receptor positive = 70% of breast cancers, respond well to hormonal therapies

• protein receptors: some breast cancers have receptor for protein HER2 = 15% of breast cancers

Recombinant DNA technology

• conversion of mRNA to complementary DNA (cDNA) which can form double DNA strand, using reverse transcriptase.

• using restriction enzymes to cut a fragment containing the desired gene from DNA via a hydrolysis reaction . at recognition sites. Straight cuts = blunt ends, staggered ends = complementary sticky ends

• creating the gene in a 'gene machine', sequence is designed, first nucleotide is fixed to a support, more nucleotides added step by step, short sections of DNA called oglionucleotides are produced and then removed so they can joined to more oglionucleotides

transgenic organism

Organisms that contain functional recombinant DNA from a different organism

Benefits of transgenic organisms

• agriculture: higher yields, more nutritious, higher resistance to pests and drought

• medicine: large quantities, cheap, quick

• industry: produce enzymes in larger quantities, cheaper

possible problems of transgenic organisms

• anti-globalisation activists

• less biodiversity - environmental effects

• introduce toxins into the food chain

• unethical use - designer babies

• ownership issues

gene therapy

\-gene supplementation: insert healthy dominant allele to work alongside defective recessive genes whilst masking its effects.

\-gene replacement: germline - replace defective gene in the gamete. somatic cells - targets affected tissues and needs to be repeated every few days as cells renew constantly. Future development is to target undifferentiated adult stem cells to offer life-long cure.

Use of liposomes

1. normal gene isolated from human cell and inserted into plasmids

2. plasmids placed back into bacteria & gene markers identify plasmids with the gene

3. bacteria cloned

4. plasmids taken and wrapped in lipid molecule to form liposome

5. liposome sprayed into patient's nostrils

use of viral vectors

1. modify virus gene so harmless, no infection

2. grow viruses in labs and add plasmids containing normal gene

3. gene taken up by virus

4. viruses isolated and purified

5. virus sprayed into patient's nostrils

6. DNA including normal gene injected into epithelial cells of lungs

in vivo cloning

Gene copies made within a living organism:

1. isolate DNA containing required gene

2. restricting gene by cutting using the same restriction endonucleases to form complementary sticky ends

3. Inserting DNA fragment into vector, usually plasmid using DNA ligase

4. transfer plasmid with DNA into suitable host, alter temperature and add calcium to increase permeability

5. identification of plasmid and DNA using gene markers such as antibiotic resistant, fluorescent or enzyme whose actions are identifiable

6. Culturing and cloning to mass produce DNA

Advantages of in vivo cloning

1. used to produce commercially/ medically important proteins

2. less risk of contamination

3. very accurate

4. specific genes can be isolated

5. speed and easy

6. sensitivity

7. used on a broad range

In vitro cloning (PCR)

Gene copies made outside a living organism:

1. select and isolate piece of DNA

2. raise temperature using a thermocycler to 95 degrees to separate strands

3. add primers, polymerase and nucleotides

4. reactants cooled to 53 degrees for 20 seconds to allow primers to bind to DNA

5. reactants heated to 75 degrees to allow polymerase to add nucleotides

6. cycle repeated

disadvantage of in vitro cloning

1. extreme sensitivity

2. specific primers needed

3. loss of efficiency

4. short sections of DNA

genome project

• Used to determine the sequence of bases in an organism for a wide range of species and allows the sequences of the proteins that derive from the genetic code (the proteome) of the organism to be determined.

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

DNA probe

a short piece of single stranded DNA, produced to be complementary to a known DNA sequence.

How probes are used

1. take a sample of DNA, digest into fragments using restriction enzymes and separate them using gel electrophoresis

2. separated fragments transferred onto a nylon membrane and incubated with a labelled DNA probe (radioactive or fluorescent). If target allele is present, the probe will hybridise (bind) to complementary part of gene

3. wash away excess probes and then expose membrane to UV lights or X rays and if present probe will show up.

screening multiple genes

• DNA microarray - glass slide with microscopic spots of different probes attached in rows

• labelled DNA added (fluorescent)

• wash

• expose to UV - any spot shows DNA contains allele

Uses of screening with DNA probes

• to help identify inherited conditions

• to help determine how a patient will respond to specific drugs

• to help identify health risks

• genetic counselling

• personalised medicine

genetic fingerprinting

compares DNA samples from different sources, works due to intron regions of DNA being variable and repeated after each other VNTRs (variable number tandem repeats).

gel electrophoresis

1. DNA sample amplified using PCR

2. restriction enzymes cut DNA

3. DNA separated using electrophoresis

4. DNA loaded into wells in gel between electrodes, electric current passed through buffer solution

5. DNA moves towards positively charged electrode, smaller molecules move further

6. produces genetic fingerprint

Uses of genetic fingerprinting

1. determining genetic relationships - each band on a DNA fingerprint should have a corresponding band in one of the parents' GF. can be used to establish whether someone is the genetic father of the child.

2. determining genetic variability within a population - more closely 2 individuals are related, the closer the resemblance of their GFs. similar GFs = little genetic diversity.

3. forensic science - DNA left at a crime scene can be analysed to help identify the suspect.

4. medical diagnosis - identify genetic disorders & cancers.

5. animal & plant breeding - identify plants that have a desirable allele