Gene Regulation and Genomes

All the cells in your body possess the same DNA, yet a neuron and a muscle cell are very different. Explain what causes this difference with reference to gene expression.

What is a constitutive gene and what is a regulated gene? Give an example of each type and explain why it is advantageous for the cell to regulate some of its genes.

What does it mean that a gene is silenced in a cell? Explain one way in which the cell can prevent a given gene from being transcribed.

What is an operon? State in what type of organisms they are found and name the basic components of an inducible operon such as the lac operon.

The diagram represents the lac operon of Escherichia coli in two situations (A and B). State in which of them lactose is present in the medium, name the elements labelled (P, O, Z-Y-A and ?), and explain what happens to the expression of the structural genes in each situation.

A strain of E. coli grows in a medium with glucose and without lactose. Explain what the state of the lac operon will be under those conditions and describe what will happen to the expression of its structural genes when the glucose runs out and lactose is added to the medium.

In a laboratory, a mutant strain of Escherichia coli displaying a complete deletion in the regulatory gene lacI (I⁻ phenotype) is studied. A student predicts that this bacterium will maximally transcribe the structural genes of the lac operon under any circumstance, since the repressor will never bind to the operator.

To test their hypothesis, the strain is cultured in three media with equivalent concentrations of basic nutrients:

• Medium A: glucose as the sole carbon source.

• Medium B: simultaneous presence of glucose and lactose.

• Medium C: lactose as the sole carbon source.

1. a) Evaluate whether the student's prediction is correct. Justify what level of operon transcription (null, very low basal, or high) will be observed in each of the three media, detailing the molecular mechanism involved (role of the CAP-cAMP complex and adenylyl cyclase).
2. b) Predict with reasoning what would happen to transcription in Medium C if the strain were to acquire a second mutation that inactivated the CAP protein gene.

In haploid bacteria, both a null mutation in the repressor gene (I⁻) and a point mutation in the operator sequence (Oᶜ) prevent repression of the lac operon, leading to a constitutive phenotype in the absence of lactose.

To discriminate between the two mechanisms, François Jacob and Jacques Monod generated partial diploid strains (merozygotes) using F' plasmids. Analyze the following two strains:

• Strain 1: I⁺ Oᶜ Z⁺ / I⁺ O⁺ Z⁻

• Strain 2: I⁺ O⁺ Z⁻ / I⁻ O⁺ Z⁺

(where Z⁺ encodes functional β-galactosidase and Z⁻ a non-functional enzyme).

1. a) Predict with reasoning whether active β-galactosidase will be produced in Strain 1 and Strain 2 when cultured in a medium lacking lactose.
2. b) Based on your prediction, classify the repressor and the operator as cis-acting or trans-acting regulators, justifying the molecular difference between both types of control.

What is a transcription factor? Explain briefly what role it plays in the regulation of gene expression and to which regions of the DNA it binds.

What is a promoter and what role does it play in the regulation of transcription? Explain what may happen if a mutation disables the promoter of a gene.

In eukaryotic cells, DNA is packaged bound to histones forming chromatin. Explain how the degree of chromatin compaction influences gene expression.

In eukaryotic cells there are regulatory sequences called enhancers that can be located thousands of base pairs away from the gene they regulate. Explain how they can act over such a distance, referring to the folding of chromatin and to transcription factors.

MicroRNAs are small RNAs that do not encode proteins but regulate gene expression. Explain how they act on the target mRNAs, whether their effect is activating or inhibitory, and why they are considered a post-transcriptional level of regulation.

Compare the regulation of gene expression in prokaryotes and in eukaryotes (number of control levels, speed of response to the medium, organisation of the genes) and reason why more possible points of regulation exist in eukaryotic cells.

In an experiment with cultured hepatocytes, the mechanism of action of two hormones is compared: adrenaline (a water-soluble hormone that acts through a G protein-coupled membrane receptor) and cortisol (a lipid-soluble steroid hormone that binds to an intracellular receptor).

To investigate the signaling pathway, actinomycin D (a potent transcription inhibitor that blocks RNA polymerase II) is added to half of the cultures. Next, the cellular response (enzyme activation and glucose synthesis) is measured over time.

1. a) Interpret and justify why the cellular response triggered by adrenaline takes only seconds or minutes to manifest, whereas that of cortisol requires several hours.
2. b) Predict how the presence of actinomycin D will affect the response generated by each of the two hormones. Justify your answer at the molecular level.
3. c) Explain why, after washing the medium and removing both hormones, the metabolic effects induced by cortisol persist for a much longer time than those of adrenaline.

In a colorectal carcinoma biopsy, it is detected that the tumor suppressor gene MLH1 is completely silenced, which prevents DNA mismatch repair. When sequencing the gene in the tumor, its nucleotide sequence is identical to that of healthy tissue; however, molecular analysis reveals dense hypermethylation of cytosines in CpG islands of its promoter region. When the tumor cells are cultured with 5-azacytidine (a DNA methyltransferase inhibitor), MLH1 expression resumes.

1. a) Based on these data, justify why the silencing of MLH1 in this tumor constitutes an epigenetic event and not a genetic mutation.
2. b) Explain the physical-molecular mechanism by which the presence of methyl groups on promoter cytosines prevents gene transcription, referring to the binding of transcription factors and chromatin remodeling.
3. c) When these 5-azacytidine-treated tumor cells divide in a drug-free medium, new cell generations can inherit the methylation pattern and silence the gene again. Justify how DNA methylation is maintained and transmitted during cell replication by maintenance methyltransferases.

At the end of the 19th century, August Weismann proposed that cell differentiation during embryonic development occurred through a selective and progressive loss of genes: each cell lineage would retain only the DNA necessary to perform its specialized function.

1. a) Describe the basis of a historic experiment (such as Gurdon's nuclear transfer in amphibians or the cloning of Dolly the sheep) and explain how its results definitively refuted Weismann's hypothesis.
2. b) If a neuron and a hepatocyte from the same adult human individual possess exactly the same genome, justify which molecular mechanisms (specific transcription factors and epigenetic modifications such as DNA methylation and histone acetylation) allow each to express a largely different set of proteins and stably maintain its identity.

In Drosophila melanogaster, the dominant gain-of-function mutation Antennapedia (Antp) causes the Antp gene, whose normal expression is restricted to the second thoracic segment to induce leg development, to be erroneously transcribed in the imaginal discs of the head. As a consequence, the flies develop fully formed legs instead of antennae.

1. a) Based on this case, deduce what molecular function the proteins encoded by homeotic (Hox) genes perform, and explain how the activation of a single gene is capable of triggering the coordinated morphogenesis of an entire appendage.
2. b) A student puts forward the hypothesis that, if a fly with a homozygous null deletion mutation of Antp (total loss of function) is isolated, the embryo will be born without a head. Refute this hypothesis and predict, with reasoning, what anatomical alteration you would expect to observe in the thorax of said individual.

Name two general differences between the genome of a prokaryotic cell and that of a eukaryotic cell (location, DNA shape, presence of introns).

Define intron and exon, and state what happens to each of them during the maturation of the mRNA in eukaryotic cells.

The human genome contains about $20000$ protein-coding genes, but these occupy only a small part of the total DNA. State what other types of sequences the genome contains and what functions are attributed to some of the non-coding regions.

Only about $1.5\ \%$ of the human genome corresponds to protein-coding sequences. Calculate how many base pairs that percentage is equivalent to in a genome of $3 \times 10^9$ base pairs and explain what is known today about the rest of the genome (regulatory sequences, introns, mobile elements, repetitions).

Analyze the following table with genomic data from three representative organisms:

1. a) A student claims that, since Paris japonica has a genome nearly 50 times larger than the human genome, it must possess a proportionally higher biological and anatomical complexity. Refute their claim using the data and explain what the C-value paradox consists of.
2. b) In light of the data in the table, explain which types of sequences make up the vast majority of non-coding DNA in eukaryotes and why a bacterium cannot tolerate such a high proportion of non-coding DNA.

A biotechnology team designs a plasmid so that Escherichia coli produces recombinant human insulin. In their first trial, they directly cloned the human gene extracted from nuclear DNA (with its introns and exons) alongside a strong constitutive bacterial promoter. The result was a failure: the cell culture died prematurely due to the toxic accumulation of an anomalous, non-functional protein.

1. a) Identify the two fundamental mistakes made in the design and explain the biological and molecular causes of said failure.
2. b) Redesign the strategy using the lac operon regulatory system: state what type of nucleic acid they must use as a template for the coding sequence, what indispensable bacterial regulatory elements they must include in the expression vector, and how they will temporally control induction in the fermenter so that the bacteria first reach a high cell density before starting protein synthesis.