Biology Topic 8

Mutations

Alteration in the DNA base sequence for a gene. Spontaneous and frequency of mutations increase with mutagenic agents.

Effect of mutations.

Mutations of DNA base sequences result in a different amino acid sequence, so hydrogen and ionic bonds are in different places (folds differently).

Different tertiary structure, so different 3D shape and non-functioning protein is produced.

Can cause cancer.

Types of mutations

Addition- extra base added and causes frameshift.

Deletion- Removal of a base and causes frameshift.

Substitution- base changed for another, so a different amino acid is coded for. (Can code for the same amino acid as the genetic code is degenerate)

Inversion- section of bases detach, invert then reattach, so different amino acid is coded for.

Duplication- particular base is duplicated, causing a frameshift.

Translocation- section of bases on one chromosome detach and reattach to another chromosome, which impacts gene expression.

Stem cells

Undifferentiated cells that continually divide to become specialised.

Types of stem cells- Totipotent

Occur for a limited time in early mammalian embryo and can divide into any type of body cell. Only translates part of their DNA, resulting in cell specialisation.

Types of stem cells- Pluripotent

Found in embryos and become almost any type of cell. Used to treat disorders and regrows damaged cells. Divides in unlimited numbers.

Issues: Treatment doesn’t always work and can be unethical.

Types of stem cells- Multipotent

Found in mature mammals and differentiates into a limited number of cells.

Types of stem cells- Unipotent

Found in mature mammals and differentiates into one type of cell. (E.g. Cardiomycocytes)

Types of stem cells- Induced pluripotent stem cells (iPS)

Created from adult unipotent/somatic cells and are altered in a lab to return to a state of pluripotency.

Genes thats make cell specialised are switched back on using transcriptional factors.

Self-renewal property, so used in medical treatment.

Control of transcription- Transcriptional factors

Transcriptional factors enter nucleus from cytoplasm and binds to promoter region on DNA and initiates transcription.

Once bound, it simulates RNA polymerase so transcription begins, creating mRNA for that gene.

Then translation in cytoplasm occurs to create protein.

No binding of transcriptional factors= inactive gene= No protein made.

Oestrogen

Lipid soluble hormone, so its diffuses through cell membrane.

Inside the cell, it binds to a receptor on a transcriptional factors enter which alters the shape and activates the TF.

Activated TF moves to nucleus and binds to specific DNA sequences in promoter region in target gene.

Stimulates RNA polymerase to bind, leading to transcription.

Epigenetics

Heritable change in gene function, without changing the DNA base sequence. Caused by changed in environment and can inhibit transcription.

Methylation of DNA

Increased methylation of DNA inhibits transcription. Methyl groups attach to cytosine base which prevents transcriptional factors from binding and attracts proteins that condense the DNA-histone complex.

Methylation prevents a section of DNA from being transcribed.

Acetylation of DNA

Decreased acetylation of DNA inhibits transcription. Acetyl groups are removed so histones become more positive and are more attracted to the phosphate group on DNA.

Makes DNA and histones more strongly associated and hard for transcriptional factors to bind.

RNA interference (RNAi)

RNAi inhibits translation of mRNA as the mRNA molecule that has already been transcribed is destroyed before it is translated to create polypeptide chain.

RNAi is important for regulating gene expression, controlling development and fighting viral infections. Potential therapeutic use also.

SiRNA Pathway

Double stranded RNA is split by a protein complex.

One strand of siRNA binds to complementary sequence on mRNA molecule. This binding guides and enzymes to cut the mRNA, breaking it down and preventing translation.

MiRNA Pathway

miRNA isn’t fully complementary to it’s target mRNA, so it binds to multiple mRNA, blocking ribosome attachments canor causing mRNA degradation.

Results in gene silencing.

Cancer

Results from mutations in genes that regulate mitosis.

Non-functioning proteins are made so mitosis isn’t regulated, so there is uncontrollable cell division and a tumour is formed.

Benign tumours

Grow large at a slow rate.

Non-cancerous as they are adhesive and stick to particular tissue.

Often surrounded by a capsule (easily removable) and impact is localised.

Malignant tumours

Cancerous as grow large rapidly.

Cell nucleus becomes larger and cell can become unspecialised again.

Metastasise- tumour breaks off and spreads to surrounding tissues and can develop its own blood supply.

Life threatening and recurrence is more likely.

Tumour development

Tumour development is due to a mutation in the tumour suppressor gene or proto-oncogene.

Abnormal methylation and increase oestrogen concentration.

Oncogenes

Mutated version of proto-oncogene and causes initiation of DNA replication/mitosis. Oncogenes are permanently activated, leading to continuous cell division.

Tumour suppressor genes

Produce proteins that slow down cell division and cause cell death when DNA copying errors are detected. Mutated TSG= continuous cell division and mutated cells aren’t identified and destroyed.

Abnormal methylation

1. Tumour suppressor genes are hypermethylated and gene is inactive.

2. Oncogenes are hypomethylated and gene is permanently switched off.

Oestrogen in breast cancer

Oestrogen produced by fat cells in breast issue linked to risk of breast cancer.

1.Oestrogen binds to receptor proteins in target cells, forming a complex that acts as a transcriptional factor.

2.Complex binds to specific DNA sites and can stimulate transcription of certain genes.

3.If oestrogen stimulates the transcription of a proto-oncogene, it will be over-expressed and there is an increase in proteins that stimulate cell division, forming a tumour.

Further breast tumour growth caused by?

Breast tumours that form in response to oestrogen may themselves stimulate further oestrogen production, creating a positive feedback loop that increases tumour growth.

Tumour environment can attract WBCs which release growth factors for further cell division and tumour progression.

Genome Sequencing

Determining the entire base sequence of an organism’s DNA. Used in a wide range of organisms, including humans.

Determining genome in simple organisms

In simple organisms (e.g. bacteria) genome sequencing allows the proteome of the organism to be determined.

This is possible due to the lack on non-coding DNA and regulatory genes, so genome and proteome relationship is straightforward.

Useful in identifying possible antigens for vaccine production as specific protein targets can be selected.

Determining the genome in complex organisms

More difficult to predict proteome due to large amounts of non-coding DNA and regulatory genes, so genome can’t be translated to proteome.

Advances in sequencing technology

Sequencing methods are more rapid, cost-effective and automated.

Enables large-scale genome projects and allows sequencing of more organisms faster and more accurately.

Recombinant DNA Technologies

Involves transferring fragments of DNA from one organism/species to another.

DNA recipient is called the transgenic organism.

Why recombinant DNA technologies work

1. Genetic code is universal, so same codons code for the same amino acids in all organisms.

2. Mechanisms for transcription and translation are universal, so genes from one species can be expressed in another. Inserted gene can be transcribed into mRNA and translated into functional proteins in the host cell.

Applications of recombinant gene technologies

1. Producing insulin in bacteria

2. Developing transgenic crops with beneficial traits (e.g. pest resistance)

3. Investigating gene function in research models.

DNA fragments and ways the create them

First step of recombinant DNA technologies is to isolate the fragments of DNA to combine with another piece of DNA.

3 methods to create fragments:

1. Reverse Transcriptase

2. Restriction endonucleases

3. Gene machine

Creating DNA fragments- Reverse transcriptase and one advantage

Reverse transcriptase naturally occurs in viruses and it makes DNA copies from mRNA.

1. A cell that produces the protein of interest is selected and these cells have a large amounts of mRNA for the protein

2. Reverse transcriptase joins the DNA nucleotides with complementary bases on the mRNA sequence

3. Single-stranded DNA is made (cDNA)

4. DNA polymerase is used to make this DNA fragment double stranded

Advantage: cDNA is intron free as it is bases on the mRNA template.

Creating DNA fragments- Restriction endonuclease

Restriction endonuclease are enzymes that cut a fragment containing the desired gene from DNA.

Normally occurs in viruses in bacteria as a defence mechanism.

Many restriction enzymes with active sites complementary to a range of DNA base sequences (recognition sequences), so each enzyme cuts the DNA at a specific location.

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

Exposed, staggered ends are called ‘sticky ends’ as the can join wuth DNA complementary base pairs.

Creating DNA fragments- Gene machine

Protein of interest is examined to identify the amino acid sequence, and from that the mRNA and DNA base sequence is determined.

DNA base sequence is entered in the computer, checking for biosafety and biosecurity to ensure DNA being created is safe and ethical.

Computer created oligonucleotides- small sections of overlapping single strands of nucleotides that make up the gene.

Oligonucleotides are joined to create the DNA for the entire gene.

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

Process is quick and accurate and makes intro-free DNA so it can be transcribed in prokaryotic cells.

In Vivo Cloning- Promoter region

A DNA base sequence is placed before the gene and acts as a binding site for RNA polymerase and transcription factors, which initiates transcription.

Correct promoter region must be used for host organism so gene is expressed properly.

In Vivo Cloning- Terminator region

A DNA base sequence is placed after the gene to signal the end of transcription and ensure RNA polymerase stops transcribing at the correct point.

Why Promoter and Terminator regions are important

No promoter region= gene isn’t transcribed

No terminator region= transcription continues to unintended regions (wasted resources or disrupts other genes)

These sequences are essential for producing functional mRNA, which can transcribed into the correct protein.

Inserting DNA into a vector

Vector- carries isolated DNA fragments into host cell (e.g. plasmids)

1. Plasmid is cut open with the same endonuclease which creates sticky ends.

2. DNA fragment sticky ends (exposed nucleotides) are complementary to sticky ends on plasmid.

What is the enzyme used to anneal DNA fragments and cut plasmid?

DNA ligase catalyses the condensation reaction to form phosphodiester bonds between nucleotides. (Sticks DNA fragment sticky ends to cut plasmid)

How does the vector enter the host cell (Transformed cell)?

The cell membrane increases permeability when hosts cells are mixed with Ca2+ and heat shocked (sudden increase in temp).

Enables vector to enter host cell.

Why don’t all host cells successfully take up recombinant plasmids?

3 issues can occur:

1. Recombinant plasmid doesn’t enter the cell.

2. Plasmid rejoins before DNA fragments enter.

3. DNA fragments stick to itself rather than inserting into the plasmid.

Marker genes

Marker genes on the plasmid can be used to identify which bacteria successfully took up the recombinant plasmid.

3 types:

1. Antibiotic resistant genes

2. Genes coding for fluorescent proteins

3. Genes coding for enzymes

In Vitro Cloning- PCR (polymerase chain reaction)

1. Temperature is increased to 95 degrees to break the hydrogen bonds and splits DNA into single strands.

2. Temperature is decreased to 55 degrees so primers can attach (annealing).

3. DNA polymerase attaches complementary free nucleotides and makes a new strand to align next to each template (synthesis).

4. -Temperature is increased to 72 degrees for this stage (optimum for taq DNA polymerase).

Advantages of PCR

1. Automated- more efficient

2. Rapid- 100 billion copies of DNA made in a few hours

3. Doesn’t require living cells- quicker and less complex techniques needed

DNA Probes

A short single-stranded piece of DNA that is complementary to a specific allele/gene. Labelled either with a radioactive isotope or a fluorescent marker to allow detection.

Presence of the probe indicates the presence of the target allele and can be detected using X-Ray film or UV light depending on the label used.

DNA Hybridisation

Process when a probe binds to it’s complementary base sequence within a DNA sample.

To prepare the sample, DNA is first extracted and heated to separate it’s two strands and the probe is then added. If the target sequence is present, it will hybridise with it.

DNA Probes in screening/ medical genetics

DNA Probes used in medical genetics to screen individuals for specific alleles linked to heritable conditions (e.g. cystic fibrosis, Hunington’s disease and BRCA1-linked breastt cancer)

Used to identify variations in genes that affect drug responses and allow doctors to predict how patients will react to certain medication.

This screening can detect individuals at increased risk of disease, even before sympromes, allowng early intervention and preventative treatment.

Personalised medicine and Genetic counselling

Screening for the presence of particular alleles allows doctors to predict select medicines and give health advice based on your genotype.

Some drugs are more/less effective depending on alleles, so enables more effective and cost-effective treatment.

Genetic Counselling- social work where advice and information following the screening of disease causing alleles is given.