BIOCHEM: Molecular Biology module lecture objective questions

Outline the “central dogma of molecular biology”.

DNA is replicated and transcribed into mRNA which is translated into amino acid sequence coded for by codons on the RNA, which together form a protein.

Describe the key elements of a gene.

• Promoter: region that controls gene expression.

• Transcribed region: a section of the gene that is copied into mRNA.

  Eukaryotic genes have:

• Exons are the translated regions of a gene.

• Introns are the untranslated regions.

Prokaryotic genes only have Exons.

Explain how transcription factors control gene expression.

Transcription factors are Proteins that bind DNA and control promoter binding of RNA polymerase. Two kinds: Activators allow RNA polymerase to bind to the promoter. Repressors block RNA polymerase from binding to a promoter region. Genes can have multiple transcription factors that control their expression.

Briefly outline the process of translation. (3 setps)

Initiation

• The ribosome, mRNA, and the first tRNA come together to form the translation initiation complex.

• The first tRNA always codes for a methionine (AUG codon/UAC anti-codon).

Elongation

• The ribosome moves along the mRNA, adding amino acids to the growing peptide chain. •

Termination

• A “stop codon” indicates the peptide chain has all the necessary amino acids and causes the translation complex to break apart.

• Releases the newly formed peptide chain (ready for protein folding and post-translational modification).

Outline the different types of genetic variants and their consequences on protein function. (2 in BIOC)

• SNP (said “snip”): single nucleotide polymorphism, a single base change in the DNA sequence (e.g. an A nucleotide changed to a G nucleotide). This will possibly change the amino acid in a protein in an exon or change expression if in the regulatory region.

• InDel: insertion-deletion, the addition or removal of one or more bases (e.g. AAT sequence changed to AACT sequence – C is added). This can cause a frame shift and change many amino acids in the protein, causing a complete loss of function. In a regulatory sequence, it may affect gene expression.

Explain how a genetic variant can change the function of a protein.

A change in a regulatory sequence could upregulate or downregulate how much of a protein is produced. A genetic variant in an exon can cause small structural changes in DNA that help or hinder it or can cause massive changes that render the protein usually useless.

Define monogenic and polygenic diseases.

Monogenic - one gene, polygenic - many genes

Describe how genetic variants can lead to monogenic diseases.

Small changes in a gene that codes for a certain function. This can lead to loss or gain of function for the genes protein which can have the phenotype causing a disease.

Outline how genetic testing can be used to determine an organism’s genotype.

• PCR primers are designed to amplify DNA surrounding genetic variant of interest.

• Restriction enzyme is chosen based on ability to cut one allele of the variant, but not the other allele.

• DNA fragments (after PCR and restriction digest) are separated by size on a DNA gel.

• Smaller fragments travel further through gel.

• Cut DNA has two fragments, uncut DNA has one fragment.

List the different techniques used to genotype organisms.

PCR is always used to amplify the DNA around the target sequence.

• Restriction enzymes and gel electrophoresis is used to cut out specific sections of DNA that could contain an alternate allele and then the DNA is placed in a gel and moved through it by an electric charge, we can determine exactly what allele is present by the mass of the fragment.

• Flourescence methods use probes to bind to certain allele strands in DNA. As the DNA is amplified the probe breaks down and releases dye. Only the probe that bound to an allele releases dye so a specialised PCR machine can pick up which allele is present in the DNA.

Define polygenic (or complex) disease.

A disease that is caused by a range of genes that often therefore has a continuous range of symptoms and severity.

Outline how genetic variants can lead to polygenic diseases.

Any type of protein can be changed by genetic variance, and proteins in any part of a pathway/process can be affected which means many different genetic variances can all affect the same bodily system in slightly different ways.

Describe how genes and environment can affect an organisms’ risk of developing a disease.

A genetic trait has a varying level of being affected by genetics or environment of an organism. Nearly all traits are a mixture of both.

Discuss how somatic mutations can lead to cancer.

Somatic mutations occur all over the body all the time. Usually, the bodies’ immune system removes these cells before they cause problems. If the immune system fails to eradicate these cells they can grow into tumors. Cannot be passed on to next generation. May cause cancer.

Define recombinant DNA technologies.

Joining bits of DNA together (sometimes from different species). These are then inserted into an organism to produce (express) a useful protein.

Describe the major tools used to manipulate and amplify DNA.

• PCR is used to amplify a section of DNA.

• Traditionally, we use Restriction enzymes to cut out a portion of DNA and use DNA ligase to rejoin recombinant DNA into the circular plasmid DNA strand.

• Modern Cloning is called Gibson Assembly. DNA fragments with overlapping ends are joined in a one-pot reaction using exonuclease, polymerase, and ligase enzymes. This allows multiple fragments to be spliced in.

Explain why a universal genetic code is critical for recombinant DNA technologies.

It means genes from other organisms will always be interpreted the same way and thus produce the same protein in any organism, allowing DNA from any organism to work in another.

Outline the steps required to make recombinant proteins.

1. Isolate or generate a construct for the gene of interest.

2. Clone into expression vector

3. Transform into bacteria for expression or isolation of more DNA for use in another expression system.

4. Grow cells expressing protein of interest in the appropriate expression system.

5. Isolate and purify the protein.

Explain why different expression systems are required to produce different types of protein.

Transforming prokaryotic cells with vectors is most cost-effective and pathogen free, however bacterial cells don’t read introns and lack some of the protein-folding machinery that eukaryotic cells have, which means they can make misfolded proteins, they also can’t do post-translational modifications on proteins to make them more target-specific.

A eukaryotic system must be used to do what prokaryotes can’t; many hormones produced by proteins require glycosylation before they can work in a certain organism.

Plant systems are eukararyotic and are often used because they are cheaper and more scaleable than mammalian culture and have less pathogen risk, however they cannot perform all PTMs so are not viable for some DNA recombinations.

Describe an example of the uses of recombinant proteins.

Human insulin was produced by recombinant bacteria, Insulin doesn’t require traditional PTMs but does have a long chain in the middle which is removed before the insulin is viable, to circumvent this scientists placed recombinant DNA to produce the A and B portions of insulin separately, where they can then be mixed, oxidise and bind together to make human insulin.

Describe how molecular mechanisms inform biotechnology methods.

Prokaryotic cells cannot perform post-translational modifications and cannot often properly fold proteins, so Plant or mammalian cell cultures are necassary.

Discuss how and why we might engineer proteins for biotechnology. (7)

Improved Therapeutics: engineered medicines can have better specificity, potency and less side effects.

Industrial Applications: Proteins are essential for food, biofuel production and bioremediation, increasing the efficacy, stability and specificity of proteins means more productive capacity.

Biotechnology and Biomedical Research: Changing certain proteins or enzymes can tell us more about their original function which helps research

Drug Discovery and Development: New pharmaceuticals, supplements and drugs can be created using biotechnology and recombinant DNA.

Agricultural and Environmental Applications: Increasing crop yield and decreasing crop growth damage on the environment.

Personalised Medicine: We can taylor medicines for individuals to minimise drug side effects and ensure efficacy

Biodefense and Biosecurity: Engineered proteins can be highly effective in better tracking and finding harmful pathogens and toxins in our immune systems.

Describe how Ozempic is created using biotechnology.

Ozempic is a modified version of the GLP-1 protein which increases insulin release, slows digestion and silences hunger. It was modified by making the protein shorter, taking out an amino acid that allowed for early degradation and made it able to bind to albumin in the blood. These changes make GLP-1 last much longer in the blood so hunger is massively decreased.

Describe how “impossible burgers” are made using biotechnology

To reproduce more closely the look, texture and taste of plant-based meats scientists added recombinant vectors to yeast that produce soy leghaemoglobin, which contains haem for the bloody flavour. These yeast produce soy leghaemoglobin in large quantities where it can then be added to plant-based meats.

Describe how a spectrophotometer measures the absorbance of a substance

Light of a single wavelength passes through a cuvette of solution. A deterctor detects the light that passes through the cuvette and compares the incident light (light in) with the transmitted light (light out). It takes the log of the ratio and that is the Absorbance.

Why are enzyme assays used to measure enzyme activity?

Enzyme assays measure the appearance of a product or disappearance of a reactant and as such give us a good graph of the different rates of enzyme activity.

outline the reaction catalysed by alcohol dehydrogenase

alcohol dehydrogenase breaks down primary alcohols into aldehydes. Alcohol dehydrogenase usually facilitate redox reactions so they require a co-enzyme to be reduced/oxidised. In humans dehydrogenase reduces NAD⁺ to NADH and H+, this is to turn ethanol to acetaldehyde. Ethanol is reduced to acetaldehyde.