BIOL2202 PART 2

How does prokaryotic gene regulation work?

A change in the environment (internal/external to cell) is detected by a protein sensitive to that specific condition. A signal molecule is produced which binds to a DNA reading protein. The protein changes its behaviour, up/down regulating specific genes by binding to nearby regulatory sequences.

What is an operon?

A cluster of genes, of related function, co-regulated by a single promoter (bacteria only).

What is a promoter region of a gene?

Region that integrates information, binding different complexes to control gene/operon expression

What are the lac operon genes for utilising lactose as an energy source?

LacZ → B-galactosidase → cuts lactose into glucose and galactose
LacY → Lactose permease → transports lactose into cell
LacA - Transacetylase → acetylates lactose

What DNA reading proteins regulate the lac operon?

CRP - cAMP receptor protein (homodimer)
RNAP - transcription (complex heteromer)
LacI - Lac operon repressor (homotetramer)

How is the lac operon switched on/off?

In the absence of lactose, LacI binds to the operator region and physically blocks RNAP. When lactose is present, allolactose binds to LacI and causes detachment so RNAP can proceed with transcription.

How does the presence of glucose regulate the lac operon?

The presence of glucose inhibits adenylate cyclase, which makes cyclic AMP → AMP binds to CRP which binds to the operon to enhance RNAP activity. This means when glucose is present, the operon is active but at a very low rate.

How do bacteria coordinate multiple operons across the whole genome?

By swapping the DNA-reading sigma subunit of its RNAP molecules, changing their programming, as different sigma factors recognise different operon promoters, due to small differences in their -10 and -35 binding sites. When a specific factor is bound, genes recognised by another factor are not off but suppressed.

What are the major bacterial factors and what genes do they activate?

Sigma D is the the housekeeping factor - matches most promoters, up regulating normal function genes in regular conditions. Sigma S is for the starvation response (recognises lac promoter).

What is the process of the starvation response in E. coli?

Starvation signals trigger enzymes to synthesise ppGpp ‘alarmone’/magic spot nucelotide which tells RNAP to swap sigma factors (RelA - uncharged tRNA molecules indicating a lack of amino acids, SpoT - low glucose). SpoT reverses activity to degrade alarmone when bacterium is no longer starving, without, RNAP swaps back to D factor.

How does gene regulation work in eukaryotes?

The nervous system, endocrine system or nearby cells release signal molecules which are detected by a receptor protein on or in the target cell. It activates several proteins in sequence which can activate/inhibit a transcription factor (signal transduction cascade). TF binds to enhancer sequence which typically increases mRNA production.

What is an enhancer sequence?

Sequences of DNA which TF binds to. Genes can be regulated together by a singular TF if they have the same enhancer sequence. Genes can have multiple enhancers for different TFs to be up or down regulated according to different signals.

Where are enhancers located relative to genes and why?

Can be far from the gene they regulate, upstream of promoter, downstream of stop or within an intron. Location can vary as DNA bends to bring TF into contact with transcription initiation complex.

How is transcription triggered from a TF?

DNA bends and brings TF into position. Basal transcription factors and other proteins recruit RNAP to the promoter to form the TIC. Contact between TF and TIC triggers transcription.

Explain the differing structures of TF molecule which allows activation.

All contain one or more signal molecule binding regions. Can be a small signal molecule or a protein that adds a molecular tag. All contain a DNA binding domain hard-coded to a specific enhancer sequence. Most contain dimerisation domains, operating as pairs or heterodimers = increases TF diversity.

Explain the TF pathway of the Androgen Receptor

Hormone testosterone passes directly from blood vessels into cell (small lipid) and binds to the androgen receptor (also a TF). Testosterone bound ARs form dimers which enters the nucleus and binds to the enhancers of androgen response genes.

Explain insulin signalling in C. Elegans (well fed)

When well fed, insulin is released into the blood. It cannot pass through the cell membrane and so interacts with a receptor on the surface (DAF-2). The signal is carried from the receptor to the DAF-16 TF through a cascade and deactivates the TF by preventing it from entering the nucleus.

Explain the starvation response in C. Elegans (DAF 16 TF pathway)

During starvation a lack of insulin means DAF-16 is not inhibited through the insulin receptor pathway, and enters the nucleus to bind the enhancers of starvation response genes.

What is the importance of gene regulation?

Genetic regulation is necessary as organism perceives the environment, detecting changes in resources, competition and optimal conditions. Regulation allows beneficial use of adaptive strategies e.g. reproduction vs survival trade-off.

What are common types of cell signalling?

Generating a small molecule that is detected by the next protein in the chain, adding or removing chemical groups directly to another protein (esp phosphate), physically moving or attracting a protein to another location. All are demonstrated in the DAF16 insulin signalling pathway.

Why do signalling pathways have multiple elements and signals? Why not just direct signalling?

Every step in a signalling pathway gives an opportunity for influence by other signals (“cross-talk”), such as from post-translational modifications. Cross-talk allows vast range of responses to be coordinated with limited components. Amount of cross-talk possible depends on a regulatory proteins role e.g. HOX TFs have only 2 modifiable site.

Explain how notch TF factors enable cell to cell communication

Notch TFs are transmembrane proteins with an extracellular signal receptor domain and an intracellular TF domain. When a neighbouring cell expresses a compatible signal on its cell surface its detected by the receptor domain. Contact causes Notch to cleave the amino acid chain holding domains together, releasing TF domain which enters the nucleus and binds to enhancers.

Explain HOX genes and their expression in terms of TFs

Highly conserved family of developmental genes where the product of each is a TF. They activate in early development based on a gradient of concentration of local signal molecules from the mother’s body. Once activated a HOX gene stays on permanently and suppresses others.

Explain the induction of genes in heat shock response

Heat stress results in the phosphorylation of heat shock TFs, which bind to gene enhancers for induction of response genes in 30 seconds and suppression of housekeeping genes in 300 seconds. Response is cell autonomous and is a general stress survival response including to starvation.

What cell roles are performed by heat shock proteins e.g. Hsp70?

Restoration of damaged proteins, stabilisation of membranes and changes to gene expression.

What is epigenetics?

Layer of gene regulation on top of the actual DNA sequence, created by your environment. Despite this, it is heritable between cell divisions and across whole generations.

What are the main forms of epigenetic influence?

Chromatin condensation, chromatin tagging and non-coding RNA

Explain histone tagging and its effect on gene expression

DNA is wrapped around histone protein complexes, histones have tails which can carry molecular signals. The patterns of signals regulates chromatin activation by changing chromatin condensation either directly or by indicating to other proteins what its condensation should be e.g. methylation, acetylation, phosphorylation. Condensed = inactive, relaxed = active.

Why are histone tags important?

DNA is unreadable when condensed, and is typically condensed and inaccessible for storage purposes. Cells use histone tags to identify whether a region should be active with chromatin modification providing access to DNA for essential genetic processes - replication, repair, recombination and transcription.

How is histone tagging heritable?

When cells divide, histones are shared randomly between new and old DNA, with new tagless histones made to fill the gaps. Protein complexes patrolling chromatin re-establish lost tags off what is exisiting - transferring tagging profiles.

How is DNA directly methylated and how does methylation persist through mitosis?

Methyl groups can be added to cytosines at CG dinucleotides or CNG trinucleotides, and tightly condenses chromatin. It persists through mitosis due to DNA methyltransferase enzyme which methylates the new strand with the same pattern of the old after replication.

Uses of DNA methylation

Imprinting, dynamic gene regulation, X chromosome inactivation, suppression of transposons (prevents gene disruption), methylation of repetitive DNA.

What is the purpose of methylation allowing dynamic gene regulation?

Most methylation patterns are erased in early embryogenesis and then re-established during one’s lifetime, reinforcing proper development and allowing the genome to learn from experience and respond more effectively to future conditions.

What are human imprinted genes?

Genes have one transcriptionally active copy, whilst the other is methylated (silent). Each particular gene is either methylated in sperm (parental pattern - female gene determines phenotype) or methylated in egg (maternal pattern - male gene determines phenotype). Methylated through imprinting control regions (either M or P).

What is the purpose of imprinted genes?

Germ cells imprinting patterns are erased in gametogenesis and re-established based on sex of gamete producer - sperm imprinted paternally, eggs maternally by set pattern across all humans. Ensures sperm and egg cells match each others patterns to establish a functional set of genes in zygote when pairing, and prevents self-fertilisation.

Explain The Possible Imprinting Phenotypes

Traits controlled by imprinted genes have unusual/distinctive non-mendelian inheritance patterns. If a mutation is inherited from the parent whose imprinted gene copy is active, the child will express the mutation as well. But if it is inherited from the parent whose copy of that gene is methylated, it won’t be expressed (e.g. callipyge muscle overgrowth mutation can only be passed on in sheep if the mutation is inherited from the father, as the DLK1 gene is maternally imprinted (methylated in egg)).

What is the importance of mitotic epigenetic inheritance?

Chromatin code is passed onto daughter cells allowing a gene regulation pattern to be established once (e.g. for specialisation of skin cell) and maintained in all descendants for organisms life.

What is the importance of meiotic epigenetic inheritance?

Most epigenetic tags are eliminated during gametogenesis. However, exposure to environmental stimuli can directly affect 2 generations at the same time (3 for pregnant females), by effecting the germline cells of the next generation. Trans-generational = no direct exposure, have been observed (active area of study).

How was RNA interference discovered?

Scientists proposed a concept of antisense RNA as being able to block gene translation by binding entirely to complement, but had inconsistent results. However a research group using sense RNA as a control observed gene expression. It was realised that sense and antisense combined, and dsRNA was responsible for supression.

Why does dsRNA allow gene silencing?

Cells have built in system to destroy mRNA copies of any dsRNA that enters a cell, as a conserved defence mechanism against viruses.

What is the significance of miRNA?

dsRNA made by the cell for the purpose of regulating its own mRNA (natural existence of RNAi). After transcription, the sequence forms hairpin loops and is processed by dicer to release a small ssRNA fragment given to RISC. With ssRNA RISC can degrade mRNA, inhibit translation (more common purpose - mismatched with target deliberatley) or enter nucleus and remodel chromatin.

Further possibilities of miRNA and siRNA?

siRNA can be copied by RdRp, and siRNA and miRNA can be packaged into vesicles and shared between cells - systemic amplification and dissemination of a signal to coordinate regulation throughout an organism (or between parasite and host).

What are piRNA?

Small RNA molecules found to interact with germline PIWI proteins. Perform transcript degradation, chromatin remodelling and transposon suppression. Maintained across generations allowing transgenerational epigenetic inheritance and change after environmental traumas acting as memories of starvation, infection and near-lethal stress. Also found to be involved in neuronal development, function and repair.

What characteristics do good genetic model organisms possess?

Easy to grow and maintain in a lab - fast growth, short lifecycle, simple nutritional requirements. Small genome size, with considerable existing knowledge, easy to perform genetic crosses. Must have appropriate biology.

What are the benefits of using E. coli as a GMO?

Genetically simple model bacteria which is easy to culture and can demonstrate all simple molecular cell mechanisms. Extensive prior knowledge. Good usage for antibiotic/pathogen resistance studies, basic machinery studies

What are the benefits of using yeast (Saccharomyces Cerevisiae) as a GMO?

Model fungus, combines bacterium convenience with eukaryotic features. Can be cultured quickly and easily and screened for mutations (haploid). Good usage in cell cycle studies as an easily synchronised genetically identical population or for eukaryotic cell biochemistry and biology

What are the benefits of using C. elegans as a GMO?

Extensively studied nematodes. Transparent so cell processes can be directly observed, defined number of cells + neurons. Massive reproductive output for large samples, can be frozen and grown on plates. Useful models for development and RNAi.

What are the benefits of using drosophilia as a GMO?

Extensive previous knowledge. Easy to rear and maintain, easy to perform genetic crosses with a large collection of known mutants. Useful for fundamentals of development.

What are the benefits of using zebrafish (Danio Rerio) as a GMO?

Easiest vertebrate model organism to breed and maintain. External fertilisation and transparent embryos, with short generation time. Good models for vertebrate development, transgenics and large mapping populations.

What are the benefits of using mice as a GMO?

Best mammalian genetic model, with great genetic similarity to humans and extensive prior genome sequencing. Good models for studying human disease, testing carcinogens and genetic basis of traits.

What are the benefits of using Arabidopsis Thaliana as a GMO?

For study of gametophytic phenotypes. Small, easy to grow, short life cycle. Seeds can be collected and stored. Short + complete genome sequence.

What is CRISPR technology?

A DNA editing technique that works like a biological “find and replace” function utilising an enzyme complex with a guide molecule, a DNA copy and a cutting enzyme. Used to swap defective DNA with healthy but also to induce specific mutations in eukaryotes.

Forward vs Reverse Genetics

Determining the genetic cause of a known phenotype. Techniques - genetic crosses, complementation analysis, genetic mapping. VS determining the function/phenotype of a known genetic variant. Techniques - site directed mutagenesis, gene silencing, knock-ins or gene tagging.

What are the major methods of inducing mutations?

Using ENS (transitions, guanine alkylation) or ENU (transitions and transversions) on a parent generation and analysing phenotypes in the M2 generation. In M1, mutations will be generated by will be heterozygous → only dominant mutations can be detected. Chemical mutagenesis sometimes yield conditional mutants (e.g. temperature/media sensitive mutants).

What is insertional mutagenesis using t-DNA?

The inducing of a mutation in plants by inserting foreign DNA into the t-dna segment of a plant bacteria’s (agrobacterium tumefaciens) t-plasmid. Insert recombinant plasmid carrying a foreign gene into the chromosome of a plant cell and grow them in culture, then all cells in plant generated from cultured cell will contain.

What is the complementation test?

To determine if two recessive mutations causing the same observable phenotype are in the same or different genes. Cross different mutants for phenotype and observe F1 offspring.

How do you interpret the results of a complementation test?

If offspring have WT phenotype, they have inherited a functional copy of each mutated gene from the opposite parent, restoring the original phenotype. This indicates the mutations occur in different genes. If all progeny retain the mutated phenotype, it shows neither parent has a functional allele of the affected gene to be inherited - must be homozygous recessive for mutations in the same gene.

What is an epistatic genetic interaction?

Where the effect of one gene is dependent on the presence of one or more ‘modifier genes’. Determined by if you cross two different mutants and the phenotype of the double mutant represents the phenotype of only one mutant.

What is an additive genetic interaction?

If you cross two different mutants and the effects of both mutations are observable in the double mutant, genes effects are combining. Two genes are acting in mutually independent genetic pathways controlling the same phenotype.

What are enhancer and supressor genetic screens?

Where you mutate an already mutated individual to search for the effect of a secondary mutation - suppressors alleviate or revert the mutant phenotype and enhancers exacerbate.

Why do we use induce and observe mutants?

To understand how processes operate in biological systems. Even when genes are not molecularly characterised we can determine how they may act and suggest a testable genetic model.

What is the basis to producing genetic maps?

Crossing over produces recombinant chromosomes. Linked genes do not assort independently and crossing over is rarer depending on their separation distances. Ratio of recombinants observed for gene crossing is used to determine cM distance apart on map.

What is interference in genetic mapping?

In short chromosomal regions (<20cM), one crossover event may prevent another from occurring nearby. Interference estimate = 1 - c, where c is the ratio of observed frequency of double crossovers/expected frequency of double crossovers (single crossover frequency 1 x SCF 2).

What is the purpose of genetic markers?

To effectively reference points to illustrate relative positions in a genome. Molecular markers are DNA sequences which differ at specific locations in the genome across strains.

How to map a population’s genes

Create a mapping population by breeding two pure homozygous individuals with different alleles of many marker loci and getting an F1 population with mixed parental alleles. The gametes of the F1 population will be mosaics of F0 genotypes which you can calculate the RF between.

How can you map a mutant onto a genetic map?

Cross a mutagenised parent and a WT parent with alternative copies of select genetic markers. Select all mutant presenting progeny in the F2 generation and genotype them for which parents markers they carry. Determine which marker had the lowest recombination rate with the mutant phenotype to determine a relative position of the mutation.

How can you prove you’ve found a causative gene?

1) Sequence candidate genes to find mutation - molecular lesion evidence
2) Cross mutant to other existing mutant alleles in the candidate gene to prove via no complementation that the mutation is from that gene - genetic evidence
3) Transform the mutant with the complementary WT allele of the gene and demonstrate that the mutant can be rescued - functional complementation evidence

What do you do when you want to map a gene in a population with no mapping population e.g. humans?

Creating mapping populations requires self and/or back crossing. In absence of this ability association mapping can be used. Uses a naturally occurring, genetically diverse population, with a population history of genetic variation and LD between genetic markers and causal variants, to identify statistical associations between markers and phenotypic variation.

What are quantitative/complex traits?

Traits affected by multiple genes and the environment. Have complex inheritance patterns with a continuous distribution of phenotypes.

How do we analyse complex traits?

Continuous distribution of phenotypes means complex traits can be described in terms of a sample mean and dispersion of trait values (standard deviation) - quantification & use of statistics. When organisms are genetically identical, variation = environment. When they are grown under identical conditions, variation = genetic.

Explain the inheritance pattern of complex traits via East’s experiment.

Start with two pure (homozygous) lines. Variation between individuals is environmental, as they are genetically identical. When crossed, so is F1 generation, variation = environment. At the F2 generation variance increases since genetic component of trait is now segregating into different allelic combinations at multiple loci. Final generation demonstrates both environmental and genetic variation between individuals.

How many genes control a quantitative trait?

If you cross two pure lines, homozygous for all loci controlling a trait then upon two generations of selfing you would expect a small number to be identical to parents - 1/4^n genes.

What is a QTL and why is it harder to map then the locus of a simple trait?

The locus for a gene that effects a quantitative trait. Simple trait = co-segregation analysis of mutant phenotype with genotype. Continuous variation prevents this → investigate whether allelic variation is significantly associated with phenotypic variation instead.

QTL mapping

Identifying genomic regions associated with phenotypic variation in a quantitative trait. Using a marker gene, different genotype groups can be established and the average phenotypic mean for each determined, testing for association of allelic variation with phenotypic (significant difference?)

What are the considerations of running a QTL map?

1) If there is no genetic variation in chosen parents then QTL impact on trait will not be observable
2) Environmental factors must be controlled for and minimised by replication
3) If epistatic interactions are occurring, identifying all QTLs will not be possible

Example of QTL mapping with tomato fruit weight

1) Cross varieties that differ in weight and carry different molecular markers at many loci throughout the genome
2) Self-fertilise F1
3) Measure fruit weight in F2
4) Determine which markers are present in each F2 plant (PCR/SNP analysis)
5) Analyse relationship between marker gene locus and fruit weight across groups, testing for significance

How can you demonstrate the role of a suspected QTL locus on a trait?

Use the associated genetic marker to hone into the region suspected to cause the trait, clone it and use recombinant plasmids to introduce one parent’s copy of the QTL to the other parent (transgenic complementation) and determine whether it produces the predicted phenotypic change e.g. if the QTL is associated with increased fruit weight, introducing the large-fruit parent's QTL allele into the small-fruit parent should increase fruit weight by approximately the estimated QTL effect size.

Problems with Identifying QTLs

Time consuming, requires phenotyping and genotyping a large number of individuals. Environment has to be precisely controlled and replicated to differentiate variation. Genetic effects such as epistasis, dominance and multiple QTLs affecting the same trait make true detection difficult. Cloning the QTL to prove an observation requires recreated variation.

Formula for Variance of A Complex Trait

VT = VG + VE. VT from F2 generation standard deviation. VE can be estimated from parental populations which are genetically uniform and inbred - VE = (VA + VB + VF1)/3. Then determine VG.

Broad Sense Hertiability

The proportion of total phenotypic variation due to genetic differences, H2 = VG/VT. Close to 0 = little to no genetic influence, close to 1 = most influence. Summarises relative contributions of factors to observed variability and is population-specific, even for the same trait.

What factors make up genetic variance?

Additive effects of individual alleles (VA) + dominance relationships (VD) + epistatic interactions (VI) = VG. Only additive variance is useful in predicting phenotypes of offspring from parents, as it is the only one that can be mapped to segregation of alleles at multiple loci.

What is narrow sense heritability?

The proportion of total phenotype variance that is due to the additive effects of alleles - h2 = VA. Tells us how closely the average trait value of offspring will resemble the average of their parents for that trait. If h2 = 1, it is the midparent value.

How to calculate the h2 value for a selected trait?

h2 = R/S. S = selection differential = difference between mean of parents and mean of population. R = response to selection = difference between offspring mean and population mean.

How is h2 estimated from linear regression?

The slope of offspring trait values plotted against mid parent traits values.

How is narrow sense heritability used to predict the outcome of a selective breeding program?

TO = u + h2[Ts - u]. If a breeding process is repeated, the effect of selection will be cumulative, moving the population mean towards the selected parental mean over time.

What assumptions under the HW principle keeps a population in equilibrium?

No mutations, no natural selection, no gene drift or flow, random mating = no differential survival or reproduction among members, genotype frequencies should persist through generations

How do you determine if a popualtion is in equilibirum?

Perform a chi-squared test with the expected and observed counts of genotypes

How does non random mating disrupt equilibrium?

May lead to an excess of homozygous individuals. Inbreeding = across whole genome, positive assortative mating (between alike individuals) = for genes associated. Negative assortative = increase heterozygosity.

How do mutation and natural selection disrupt equilibrium?

If the rate of mutation from one allele to another changes, frequencies shift. The elimination of individuals carrying deleterious phenotypes shifts frequencies towards non deleterious genotypes e.g. sickle cell anaemia.

What effect does population subdivision have on allelic frequencies?

Genetic differences accumulate in isolated populations with genetic drift as a driving force. The introduction of genotypes via migration generally increases heterozygosity, however when two previously segregated populations merge it generally results in a deficit of heterozygotes (Wahlund Effect) due to the weighted averages of their specialised allelic frequencies.

What is natural selection and its requirements?

The systematic change in allele frequencies in a population due to differential survival and reproduction among genotypes. Requires individual variation, fitness differences and heritability. Occurs by laws of probability not biology.

Fitness

The ability of an organism to survive and reproduce including ability to utilise resources, avoid predators, find mates etc. The fitness of a genotype is demonstrated by its phenotype, and can differ in different selective environments.

How do we quantify fitness?

Fitness = w, each population member has own value. 0 = dies/no offspring, 1+ = based on number of viable offspring. Growing population mean w (average fitness) > 1, stable w = 1, declining w < 1.

What is relative fitness and how is it quantified?

Between 0 and 1. Relative fitness of superior genotype = 1, inferior is a deviation from 1. Fitness deviation gives s, the selection coefficient which measures the intensity of natural selection acting on population genotypes e.g. if deviation from 1 is 1, genotype is lethal. Relative fitness does not inform on absolute reproductive abilities of genotypes but competitive ability.

Steps for calculating next generation genotype frequencies with selection coefficient.

1) Take given frequencies of p an q and determine starting genotype frequencies
2) Determine genotype contribution to next generation by starting frequency x relative fitness value
3) Determine proportional contribution by dividing contribution/sum of contributions

What are the three forms of selection for quantitative traits?

Directional - favours trait values at one extreme
Disruptive - favours trait value at either extreme, at the expense of intermediate values
Stabilising - favours intermediate values

What is genetic drift and its contributing factors?

Unpredictable changes in allele frequencies due to reproductive uncertainty. Mechanisms - alleles are randomly incorporated and segregated into gametes, random variation in number of offspring. The more parental alleles there is in a gene pool, the lower the chances are of two randomly selected gene copies in the next generation originating from the same parental gene copy.

Relationship Between Population Size and Genetic Drift

Drift causes heterozygosity to decline at a factor of 1/2N per generation, so drift is weaker in larger populations. H’ (heterozygotes in t generations) = (1 - 1/2N)^t x H (current).

What is the ultimate outcome of drift?

The fixation of one allele at a locus in a population at the loss of all others. Probability of fixation is equal to allele current frequency in population.

What is dynamic genetic equilibrium?

When there is no net change in allele frequencies due to evolutionary forces acting in opposing ways.