The control of gene expression

What is a mutation?

An alteration to the DNA base sequence

When are mutations likely to occur?

During DNA replication (interphase)

Why might a mutation lead to a non-functioning protein?

• Changes sequence of base triplets in DNA so changes sequence of codons on mRNA

• This can result in a different amino acid sequence in the polypeptide

• When the protein is modified into the tertiary structure, it will form hydrogen and ionic bonds in different places and fold differently

• This results in a different 3D shape, and therefore a non-functioning protein

Explain why not all gene mutations affect the order of amino acids

• Some substitutions change only 1 triplet code/codon which could still code for the same amino acid - as genetic code is degenerate

• Some occur in introns which do not code for amino acids as they are removed during splicing

What is a mutagenic agent?

Factors that increase the rate of gene mutation

How does ionising radiation act as a mutagenic agent?

Radiation such as UV light and x-ray can cause damage and disrupt the structure of DNA

How do carcinogens act as mutagenic agents?

Chemicals alter the structure of DNA and interfere with transcription

What is a frameshift?

• Occurs when mutations change the number of nucleotides/bases by a number not divisible by 3

• Shifts the way the genetic code is read, so all the DNA triplets/mRNA codons downstream from the mutation change

• Significant effect on the polypeptide

What is an addition mutation?

When an extra base is added to the DNA sequence

What is the effect of an addition mutation?

All subsequent triplets are altered - frameshift produced

All of the altered triplets could potentially code for different amino acids, resulting in a non-functioning protein

What is a deletion mutation?

1 or more bases/nucleotides are lost from the DNA base sequence

What is the effect of a deletion mutation?

All subsequent triplets are altered - frame shift

Altered triplets could code for different amino acids, resulting in a non-functioning protein

What is a substitution mutation?

When one or more bases/nucleotides in the DNA sequence is replaced by another

What are the possible effects of a substitution mutation?

No frame shift

Number of bases stay the same

• One of 3 of the stop codons may be formed that mark the end of a polypeptide chain - production would be stopped prematurely

• The new triplet/codon could code for a different amino acid - changes structure of the polypeptide

• The new triplet/codon could code for the same amino acid as before as genetic code is degenerate - no effect

What is a duplication mutation?

When a sequence of bases/nucleotides duplicate and repeat

What is the effect of a duplication mutation?

Frame shift occurs

Amino acid sequence altered

3D shape is different so resulting protein is usually non-functional

What is an inversion mutation?

When a sequence of bases/nucleotides detaches from the DNA sequence, then rejoins at the same position in the reverse order

What is the effect of an inversion mutation?

The base sequence of the mutated portion is reversed which affects the amino acid sequence that results

What is a translocation mutation?

A sequence of DNA bases/nucleotides detaches and is inserted at a different location within the same or a different chromosome

What is the effect of a translocation mutation?

Produces a frameshift

Explain why a change in amino acid sequence is not always harmful

• May not change tertiary structure of protein (if position of ionic/disulphide/hydrogen bonds dont change)

• May positively change the properties of the protein, giving the organism a selective advantage

Describe cell differentiation

The process by which cells become specialised for different functions

What are stem cells?

Undifferentiated/unspecialised cells capable of:

• Dividing by mitosis to replace themselves indefinitely/can continually divide

• Differentiating into other types of specialised cells

They require constant replacement

Name the 4 types of stem cell

• Totipotent stem cells

• Pluripotent stem cells

• Multipotent stem cells

• Unipotent stem cells

What are totipotent stem cells?

• Found in early embryo

• Can divide and differentiate into any type of cell

What are pluripotent stem cells?

• Found in embryos

• Can divide and differentiate into almost any type of cell

What are multipotent stem cells? Give an example

• Found in mature mammals

• Can divide and differentiate into a limited number of cell types

• Eg multipotent cells in bone marrow can divide and differentiate into different types of blood cell

What a unipotent cells? Give an example

• Found in mature mammals

• Can divide and differentiate into just one cell type

Eg unipotent cells in the heart can divide and differentiate into cardiomyocytes (cardiac muscle cells)

Describe how stem cells become specialised during development

• Stimuli leads to the activation of some genes

• So mRNA is transcribed only from these genes and then translated to form proteins

• These proteins modify cells permanently and determine cell structure/function

Explain how induced pluripotent stem (iPS) cells are produced

• Obtain adult body cells from patient (unipotent cell)

• Add specific protein transcription factors associated with pluripotency to cells to turn on genes so they can be expressed

• Culture cells to allow them to divide by mitosis

Explain how stem cells can be used in the treatment of human disorders

• Transplanted into patients to divide in unlimited numbers

• Then differentiate into required healthy cells

Evaluate the use of stem cells in treating human disorders

For:

• Can divide and differentiate into required healthy cells, so could relieve human suffering

• Embryos are often left over from IVF and so would rather be destroyed

• iPS cells unlikely to be rejected by patients immune system as made with patients own cells

• iPS cells can be made without destruction of embryo and adult can give permission

Against:

• Ethical issues with embryonic stem cells as obtaining them requires destruction of an embryo and potential life

• Immune system could reject cells and immunosuppressant drugs required

• Cells could divide out of control, leading to formation of tumours/cancer

What is epigenetics?

A heritable change in gene function without change to the base sequence of DNA

What causes epigenetics? Give examples

Changes in the environment eg diet stress and toxins, which chemical tags on the epigenome respond to

Define epigenome

A single layer of chemical tags on the DNA which impacts the shape of the DNA-histone complex and whether the DNA is tightly wound or unwound

Define epigenetic silencing

The epigenome causes parts of DNA to be tightly wound, which means it wont be expressed

What happens if DNA is tightly wound?

• Transcription factors cannot bind

• Therefore, the epigenome (which determines how tightly wound it is) can inhibit transcription

What are transcriptional factors?

• Molecules that move from the cytoplasm into the nucleus

• Bind to a specific DNA base sequence on a promoter region

• Regulate transcription of specific target genes

How do transcriptional factors cause transcription to begin?

• Transcription factors move from cytoplasm to nucleus

• Each transcriptional factor has a site that binds to a specific base sequence of DNA (promoter region)

• When it binds, it stimulates this region of DNA to begin the process of transcription by helping RNA polymerase bind

Explain how oestrogen affects transcription

• Oestrogen is a lipid-soluble steroid hormone so diffuses into cell across phospholipid bilayer

• In cytoplasm, oestrogen bind with receptor molecule, inactive transcription factors, forming an oestrogen-receptor complex

• This changes the shape of the DNA binding site on the inactive transcription factor, forming an active transcription factor which can now bind to DNA

• The transcriptional factor can now diffuse into the nucleus

• It binds to a specific DNA base sequence on the promoter region of a target gene

• Stimulates transcription of target genes by helping RNA polymerase to bind

Why does oestrogen only affect target cells?

Other cells do not have oestrogen receptors

How does increased methylation of DNA affect gene transcription?

• When methyl groups are added to DNA, they attach to the cytosine base

• This prevents transcriptional factors from binding and attracts proteins that condense the DNA-histone complex

• Prevents RNA polymerase binding to promoter

• Therefore transcription is inhibited

How does decreased acetylation of histones affect gene transcription?

• If acetyl groups are removed from the DNA then the histones becomes more positive and are attracted more to the phosphate group on DNA

• So DNA becomes more tightly wound

• DNA not accessible to transcription factors

• Prevents RNA polymerase from binding to promoter

• Inhibits transcription

Explain the relevance of epigenetics on disease development and treatment

• Environmental factors can leads to epigenetic changes

• These can stimulate or inhibit expression of certain genes that can lead to disease development

• Diagnostic tests can be developed that detect these epigenetic changes before symptoms present

• Drugs can be developed to reverse these epigenetic changes

What is RNA interface (RNAi)?

• Inhibition of translation of mRNA produced from target genes, by types of RNA molecules such as siRNA and miRNA

• This inhibits expression of a target gene (silences it)

Describe the regulation of translation by RNA interface involving siRNA

• An enzyme cuts large double-stranded molecules of RNA into smaller sections called small interfering RNA (siRNA)

• One of the 2 siRNA strands combines with an enzyme

• The siRNA molecule guides the enzyme to a mRNA molecule by paring up its bases with the complementary ones on mRNA

• The enzyme cuts the mRNA into smaller sections

• The mRNA is no longer capable of being translated into a polypeptide

• The gene cannot be expressed

What is cancer?

A result of mutations in genes that regulate mitosis, leading to uncontrolled cell division

Are benign tumours cancerous? Why?

No

• They grow at a slow rate

• Produce adhesion molecules, sticking them together and to a particular tissue and are surrounded by a capsule, so they remain compact and can be easily removed (impact is localised)

Are malignant tumours cancerous?

Yes

Explain how malignant tumours work

• The cell nucleus becomes large and the cell can become unspecialised again

• They do not reproduce the adhesive molecules, instead metastasis occurs - tumour breaks off and spreads to other parts of the body

• Tumour isn’t encapsulated and instead can grow projections into surrounding tissues and develop its own blood supply

Compare the main characteristics of benign and malignant tumours

• Benign tumours usually grow slowly (cells divide less often), malignant usually grow faster (cells divide more often)

• In benign tumours, cells are well differentiated/specialised, in malignant tumours, cells become poorly differentiated/unspecialised

• In benign tumours, cells have normal, regular nuclei, in malignant tumours, cells have irregular, darker nuclei

• Benign tumours have well defined borders and are often surrounded by a capsule so do not invade surrounding tissue, malignant tumours have poorly defined borders and are not encapsulated so can invade surrounding tissues (grow projections)

• Benign tumours do not spread by metastasis (due to cell adhesion molecules), malignant tumours spread by metastasis - cells break off and spread to other parts of body, forming secondary tumours (due to lack of adhesion molecules)

• Benign tumours can normally be removed by surgery and rarely return, malignant tumours can normally be removed by surgery combines with radiotherapy/chemotherapy but often return

Describe the role of proto-oncogenes

Control cell division by coding for proteins that stimulate cell division

What are oncogees?

Mutated form of proto-oncogene

Explain how proto-oncogenes can be involved in developing cancer

• A mutation in a proto-oncogene could turn it into a permanently activated oncogene

• The oncogene could be permanently activated for 2 reasons:

• The receptor protein on cell surface membrane can be permanently activated so cell division is switched on even in absence of growth factors

• The oncogene may code for a growth factor that is then produced in excessive amounts, stimulating excessive cell division

• They may also lead to decreased DNA methylation or increased histone acetylation which cause excess transcription

• Results in uncontrolled cell division and formation of a tumour

Describe the function of tumour suppressor genes

Code for proteins that:

• Inhibit/slow cell division

• Causes self-destruction (apoptosis) of potential tumour cells

Explain the role of tumour suppressor genes in the development of tumours

• If there is a mutation in DNA base sequence - amino acid sequence altered

• Hypermethylation can cause tumour suppressor gene to become inactivated by inhibiting transcription so lack of tumour suppressor protein leads to tumour formation

• Or it produces a non-functional protein - stops inhibiting cell division

Explain the relevance of epigenetics in cancer treatment

Drugs could reverse epigenetic changes that cause cancer -

• Increasing DNA methylation or decreasing histone acetylation of oncogene to inhibit transcription

• Decreasing DNA methylation or increasing histone acetylation of tumour suppressor gene to stimulate transcription

Explain the role of increased oestrogen concentrations in the development of some breast cancers

• Some breast cancers have oestrogen receptors, which are inactive transcription factors

• If oestrogen concentration is increased, more oestrogen binds to oestrogen receptors, forming more oestrogen-receptor complexes which are active transcription factors

• These bind to promoter regions of genes that code for proteins stimulating cell division

• This increases transcription, increasing the rate of cell division

Suggest how drugs that have a similar structure to oestrogen help treat some breast cancers

• Drugs bind to oestrogen receptors (inactive transcription factors), preventing binding of oestrogen

• So no/fewer transcription factors bind to promoter regions of genes that stimulate cell division

What is the genome?

The complete set of genetic information contained in the cells of an organism

What is genome sequencing?

Identifying the DNA base sequence for all the DNA in a cell so amino acid sequences of proteins that derive from an organisms genetic code can be determined

What is the proteome?

The complete set of proteins that can be produced by a cell

Do prokaryotic cells contain introns?

No

Can the genome be used to sequence to proteome?

In simpler organisms - yes. As they do not contain introns in their DNA

In complex organisms - no. As they have introns and regulatory genes

Give an application of sequencing the proteome in simple organisms

Identifying potential antigens for use in vaccine production

Suggest some other potential applications of genome sequencing projects

• Identification of genes/alleles associated with genetic diseases - new targeted drugs/gene therapy can be developed or can screen patients, allowing early prevention/personalised medicine

• Identification of species and evolutionary relationships

Describe how sequencing methods are changing

• They have become automated (so are faster, more cost-effective and can be done on a larger scale)

• They are continuously updated

What is recombinant DNA technology?

The combining of different organisms DNA, which could enable scientists to manipulate and alter genes to improve industrial processes and medical treatments

Why does recombinant DNA technology work?

Because the genetic code is universal, so transcription and translation result in the same amino acid sequence across organisms

Give 3 methods to create fragments of DNA for recombinant DNA technology

1. Reverse transcription

2. Restriction endonucleases

3. Gene machine

Explain the process of using reverse transcriptase to produce DNA fragments

1. A cell that naturally produces the protein of interest is selected

2. These cells should have large amounts of mRNA for the protein

3. mRNA is isolated and mixed with DNA nucleotides and reverse transcriptase

4. Reverse transcriptase enzyme joins the DNA nucleotides with complementary bases to the mRNA sequence

5. Single stranded DNA is made (cDNA)

6. To make this DNA fragment double stranded, DNA polymerase is used

Suggest 2 advantages of obtaining genes from mRNA rather than directly from the DNA removed from cells

• Much more mRNA in cells making the protein than DNA - easily extracted

• cDNA is intron free because it is based on the mRNA template. In mRNA, introns have been removed by splicing. So it can be transcribed and translated by prokaryotes who cant remove introns by splicing

Explain the process of using restriction endonucleases to produce DNA fragments

1. Restriction endonucleases are enzymes that cut up DNA at specific base ‘recognition sequences’ either side of the desired gene

2. Different REs have different active sites complementary to a range of different DNA base sequences

3. Some enzymes cut in the same location in double strand to create a blunt end, others cut to create staggered ends and exposed DNA bases

4. The exposed staggered ends are referred to as sticky ends as they have the ability to join DNA with complementary base pairs

Explain the process of using gene machine to produce DNA fragments

• DNA fragments can be created in a lab using a computerised machine

• Scientists first examine the protein of interest to identify the amino acid sequence, and work out what the mRNA and DNA sequence would be

• The DNA sequence is entered into he computer, which checks for biosafety and biosecurity that the DNA being created is safe and ethical to produce

• The computer can create small sections of overlapping single strands of nucleotides that make up the gene, called oligonucleotides

• The oligonucleotides can then be joined to created the DNA for the entire gene

• PCR can be used to amplify the quantity and to make the double strand

In which 2 ways can we amplify DNA fragments? Give examples of each

• In vitro (outside a living organism) - polymerase chain reaction

• In vivo (inside a living organism) - transferring the fragments to host cell using vector

Explain the importance of sticky ends

• If same restriction endonuclease is used to cut DNA, then all fragments produced will have ends that are complementary to each other

• This allows us to combine strands of DNA from different organisms

Describe the reaction mixture required for PCR

• DNA fragments to be amplified

• DNA polymerase

• Primers - short, single stranded sequence of nucleotides that are complementary to base sequences at the ends of the DNA fragments

• Nucleotides to match up to exposed bases

• Thermocycler - machine that varies the temperature

Explain how DNA fragments can be amplified by PCR

• Separate of DNA strand - mixture heated to 95C which separates DNA strands by breaking hydrogen bonds between bases

• Addition of primers - mixture cooled to 55C, causing primers to join (anneal) to their complementary bases at the ends of DNA fragment strands by forming hydrogen bonds between complementary bases. This is because DNA polymerase can only attach nucleotides to the end of an existing chain

• Synthesis of DNA - mixture heated to 72C, optimum temperature for DNA polymerase to join complementary nucleotides to exposed bases along each of the separated DNA strands, forming phosphodiester bonds

Explain the role of primers in PCR

• Primers are short, single stranded DNA fragments

• Complementary to DNA base sequences at edges of region to be copied

• Allows DNA polymerase to bind to start synthesis, as it can only add nucleotides onto an existing chain

• 2 different primers are requires as base sequences at ends are different

• They also prevent the 2 separate strands from rejoining

Suggest one reason why DNA replication eventually stops in PCR

There are a limited number of primers and nucleotides which are eventually used up

Explain why promoter and terminator regions are added to DNA fragments that are used to genetically modify organisms

• Promoter regions allow transcription to start by allowing RNA polymerase to bind to DNA near the gene to be transcribed

• Terminator regions ensure transcription stops at the ends of a gene by stopping RNA polymerase

What are the roles of vectors in in vivo gene cloning?

Used to transport the DNA into the host cell

Explain the process of inserting a DNA fragments into a vector

• Restriction endonucleases cut vector DNA - the same type that was used to cut the gene out so vector DNA and DNA fragments have sticky ends that can join by complementary base pairing

• DNA ligase joins DNA fragment to vector DNA by forming phosphodiester bonds between adjacent nucleotides