Genetics exam 3

Know the difference between chromatin and chromosomes

Chromatin is the generic term for any complex of DNA and protein found in a nucleus of a cell

Chromosomes are the separate pieces of chromatin that behave as a unit during cell division

Know the DNA:histone:non-histone ratio

Chromatin is ⅓ DNA, ⅓ histones and ⅓ non-histone proteins

What are histones?

Histones are small, positively charged proteins and highly conserved. They bind to and neutralize negatively charged DNA.

What are the different histone subunits, and how are they organized?

Five types, H1, H2A, H2B, H3 and H4. Core histones H2A, H2B, H3 and H4 make up the nucleosome.

What is the role of non-histone proteins?

Non-histone proteins act as structural scaffolding for chromosome organization, mediating transcriptional activation and repression and facilitating DNA damage responses.

Know what radial-loop scaffold is and the contribution of condesins.

Several non-histone proteins (NHPs) bind to chromatin, every 60-100kb and tether the 300 A fiber into structural loops. Other NHPs gather several loops together into daisy rosettes. Condensins may further condense chromosomes into a compact bundle for mitosis.

How does in situ hybridization work?

Chromosomes are spread on a glass slide and denatured to make them single stranded. A DNA sequence is labeled with a fluorescent tag to make a probe. The probe hybridizes to chromosomes at complementary regions.

What is the difference between FISH and SKY?

In FISH, it depends on hybridization between metaphase chromosomes and a labeled DNA sequence. In SKY, probes different fluorescent dye specific for each chromosome are labeled with a number of X,Y. SKY can be used to detect large chromosomal changes.

What is the difference between heterochromatin and euchromatin, and how do each contribute to gene expression?

Heterochromatin: highly condensed, usually inactive transcriptionally. Dark;y stained regions of chromosomes. Constitutive (condensed in all cells) and facultative (condensed in only some cells and relaxed in other cells).

Euchromatin: relaxed, usually active transcriptionally. Lightly stained regions of chromosomes

What are histone modifications, and why are they important in gene regulation?

Tails extend outward from nucleosome, enzymes can add chemical groups (ex: methyl groups) and modified tails can alter nucleosomes and bind chromatin modifier proteins.

What is the contribution of acetylation and methylation of histones on gene expression?

Acetylation causes histone acetyltransferases that add acetyl groups to histone tails. This prevents close packing of nucleosomes and favors expression of genes in euchromatin. Methylation causes histone methyltransferases that add methyl groups to histone tails. Effects depend on specific amino acids modified and adding methyl group to H3 lysine 9 favors heterochromatin formation. The process is reversed by methyltransferases.

What is a telomere?

Telomeres consist of specific repetitive sequences and don’t contain genes. Species specific sequences, TTAGGG in humans. 250-1500 repeats with variable numbers between different cell types. Prevent chromosome fusions and maintain integrity of chromosomal ends.

What is telomerase, and why is it essential to the maintenance of chromosomes?

Telomerase is a ribonucleoprotein that extends the end of chromatin. Telomerase RNA is complementary to telomere repeat sequences.

How does telomerase work?

 Serves as a template for addition of new DNA repeat sequences to telomere, additional rounds of telomere elongation occur after telomeres translocate to newly-synthesized end.

What is Shugoshin, and what does it do?

Shugoshin is a conserved family of proteins that protects sister chromatid cohesion at the centromere during cell division (meiosis and mitosis).

Know the four types of chromosomal rearrangements (Deletions, Duplications, Inversions, Translocations)

Deletion: region of the chromosome is removed

Duplication: region of chromosome is duplicated

Inversion: region of chromosome is flipped 180

Translocation: region of chromosome moves to a different chromosome

Know the two mechanisms by which chromosomal rearrangements arise (Double Stranded Breaks [DSB] followed by Non-homologous end joining [NHEJ] vs. Recombination at repetitive DNA)

Double Stranded Breaks [DSB] followed by Non-homologous end joining [NHEJ]: can occur through X-ray mutagenesis. NHEJ repairs DSB, KU70 and KU8- bind to the broken ends and recruit PKcs. PKcs recruit ligase, which repairs the break. Deletion can happen when two DSBs occur and NHEJ does not repair both breaks. Sometimes inversions can happen when the region of the middle is inverted. Duplications happen when DSBs occur on different sister chromosomes and the arms are swapped. Translocations happen when DSBs happen on different chromosomes and the arms are swapped. 

Recombination at repetitive DNA: simple sequence repeats or copy number variant. More rare than DSBs but products are similar. A recombination event at SSR in the same direction leads to a deletion, a different direction is an inversion. Duplications happen in different arms of sister/homologous chromosomes and translocation happens in different arms of unrelated chromosomes.

Deletions

Homozygosity for deletions is often lethal or harmful. Depends on size of deletions and affected genes. Heterozygotic deletions can have a mutant phenotype due to gene dosage effects (haploinsufficiency). Increases the risk of phenotype due to mutation in the other copy of the gene. May uncover existing recessive mutant alleles. Deletion lines can tell which gene is mutated. If the phenotype is mutant (fails to complement) then the mutant gene must lie inside the deleted region. If the phenotype is wild type (complement) then the mutant gene must lie outside the deleted region.

Duplications

Tandem means clustered together on the chromosome and dispersive means spread out on the chromosome. 

Dosage effects: increased copy number of a gene leads to an abnormal amount of gene production (protein or RNA) which disrupts normal cellular function

Enhancer trapping: positive effect, occurs when a chromosomal duplication relocates a gene or changes its surrounding regulatory landscape, causing inappropriate activation.

Inversions

Para-centric inversions: inversions do not include the centromere 

Paricentric: inversions include the centromere 

Usually do not have phenotypic effects, can have an effect if the rearrangement occurs in the middle of a gene. Inversion loops happen in meiosis, allowing for the tightest possible alignment of homologous regions. Crossing over within the inversion loop produces aberrant recombinant chromatids. The effect of an inversion loop is pericentric inversions (nonviable recombinant chromatids) and paracentric inversions (dicentric recombinant nonviable chromatids).

Translocations

Do not typically result in a phenotype since no genes are lost. When they do, this can happen because of novel gene products and loss of genes in heterozygous states. Homozygous translocation breakpoints of a reciprocal translocation do not affect gene function. In heterozygous translocations, the two haploid sets of chromosomes carry different DNA arrangements. 

Robertsonian translocations: two arcocentric chromosomes (13, 14, 15, 21 or 22) fuse at their centromeres, reducing the total chromosome count ot 45. Carriers are usually healthy but face infertility risks, recurrent miscarriages or having children with down syndrome.

Transposable elements

Transposable elements are any segment of DNA that evolves the ability to move from place to place within a genome. Have been found in all organisms, ranging from 50bp to 10kb. Can have retrotransposons (RNA intermediate) or DNA transposons (no RNA intermediate).

What are the different layers of transcriptional regulation in prokaryotes (direct, transcriptional control and post-transcriptional control).

Transcriptional control: binding of RNA polymerase to the promoter, the most critical step in regulation of prokaryotic genes. Shifts from initiation to elongation, release of mRNA at termination.

Posttranscriptional control: stability of mRNA, efficiency of translation initiation, stability of polypeptide.

Basic steps

Initiation: core RNA polymerase plus sigma factor 

Elongation: core RNA polymerase without sigma factor 

Termination: two kinds in bacteria, Rho-dependent and Rho-independent 

Know the difference between inducible and repressible regulation

Inducible regulation: genetic control mechanism where gene expression is normally turned off but is activated (induced) by a specific environmental molecule or stimulus.

Repressible regulation: gene control mechanism where a usually active operon (system of genes) is turned off in response to an accumulation of its end product.

• Negative regulation:

  • How is lactose utilized in bacteria? What are the required genes and what do they do

Using lactose and transcribing genes costs a lot of energy, so the genes are inactive if they do not to be on. Requires permease (transports lactose in cell), B-galactosidae (splits lactose into glucose and galactose) and Transacetylase (transfers acetyl group from acetyl CoA to galactosides, lactosides and glucosides. Assists in cellular detoxification). Lactose is the inducer of the genes encoding permease and B-gal. Induction is the stimulation of synthesis of a specific protein. Inducer is the molecule responsible for induction. Lac repressor is a negative regulatory element. 

Know how the lac operon works

Collective term for a group of genes all grouped together on DNA that are transcribed together and have a shared/common goal. Term for the set of genes that encodes permease, B-galactosidase and transacetylase. 

Lots of glucose and no lactose: cell wants to use glucose, operon OFF

Little glucose and lots of lactose: lac operon ON

Lots of glucose and lactose: lac operon OFF

What is the difference between cis- and trans-acting regulatory elements? (prokaryotes)

Cis acting elements: DNA sequences located on the same molecule as the gene they regulate, like promoters and enhancers, do not code for proteins.

Trans acting elements: diffusible molecules, usually proteins, produced by distant genes that bind to cis-elements to regulate expression across the genome.

What is the biochemical evidence that the Lac repressor binds to the lac operon?

PajaMo experiments, These were a seminal set of

experiments done by Jacob, Manod

and Arthur Pardee. They started with Bacteria that had

mutations in LacI and LacZ and then

introduced the genes transiently lacI+ lacZ+ DNA transferred into lacI−

lacZ− cells. At first, no β-gal was present, but

shortly after the cell began to make

β-gal from the LacZ+ gene

What does the protein structure of the Lac repressor show?

Different ‘domains’, one of them is a DNA binding domain, one is a inducer binding domain, and another is a heterodimer domain. Also, know that the DNA binding domain shows a helix-turn-helix motif, which is a conserved motif for DNA binding domains.

• Positive regulation:

  • How does CRP-cAMP work as a positive regulatory of the lac operon?

  • How does the presence of glucose influence the levels of cAMP?

The CRP-cAMP complex acts as a positive regulator of the lac operon by binding to the promoter region when glucose is absent, directly facilitating RNA polymerase binding to initiate high-level transcription. Conversely, the presence of glucose lowers cAMP levels by inhibiting its synthesis, preventing the formation of the complex and ensuring the operon remains largely inactive

Know the basics of regulation at RNA – Trp operon

The trp operon is a repressible system in E. coli that synthesizes the amino acid tryptophan. It is primarily regulated by negative feedback: when tryptophan levels are high, it binds to a repressor, activating it to block RNA polymerase, thus turning transcription "off." Conversely, low levels allow for transcription "on”

• Be able to name a few similarities and differences between gene regulation in eukaryotes and prokaryotes

Similarities

Promoters are essential

to gene regulation in both

life forms

Many aspects of gene

regulation in eukaryotes

relies on DNA binding

proteins

Regulation is often at the

level of transcription

initiation/permission

Differences

Chromatin dynamics are

often involved

Additional RNA

processing is critical to

regulation

Gene regulation is much

more complex, and

millions of cells need to

transcribe different sets

of genes

Eukaryotes use

enhancers and

promoters, whereas

prokaryotes only use

Understand the process by which a gene goes from DNA to a functional protein, and know how expression is regulated at each step

Gene expression is the multi-step process, known as the central dogma (DNA to RNA to protein), by which information in DNA is used to synthesize functional proteins. It is regulated at every stage—chromatin, transcription, RNA processing, translation, and post-translation—to control which proteins are made, when, and in what amounts

Understand what are cis-regulatory and trans-regulatory elements in eukaryotes (and in prokaryotes in chapter 16)

cis-acting element: an element that is continuous or on the

same area and the system it’s modulating. A promoter, because

it is on the same DNA strand and is adjacent to the gene it’s

regulating, is considered a cis-acting element

trans-acting element: an element that is away from and must

migrate to the area it’s regulating. The inducer such as one in

the Lac Operon is a trans-acting element

What are promoters and enhancers?

Promoters: areas where polymerase binds, cis-acting regulatory elements. DNA sequence that is directly adjacent to the gene. In eukaryotes

Enhancer: cis acting element, increases likelihood that a specific gene will be transcribed. In eukaryotes, DNA sequence that can be far from gene

What is a TATA box and why is it called a TATA box

Conserved sequences which help recruit RNA polymerase to initiate transcription. Have a sequence of T’s and A’s (TATA). Allow a basal level of transcription, helps to recruit to RNA pol II.

How does a basal promoter work in eukaryotes? What’s the sequence of basal activation (TBP -> TAF -> RNA pol II)

the DNA sequence immediately surrounding the transcription start site (TSS) that acts as the docking site for the preinitiation complex (PIC), enabling basal-level transcription. It typically contains conserved motifs, like the TATA box, which are recognized by general transcription factors (GTFs) and RNA polymerase II to initiate gene expression. [1, 2, 3, 4, 5]

How are reporters useful in examining the function of promoters and enhancers

They fuse cis-acting regulatory elements to a reporter coding sequence, researchers can determine exactly where and when a gene is expressed and how transcription factors control its expression.

Know things like what happens in a enhancer fusion?

An enhancer fusion puts a specific regulatory sequence in control of the reporter gene.

What happens to an enhancer fusion if the promoter is deleted or mutated

If the promoter is deleted or mutated then the enhancer cannot function effectively.

What is a transcription factor, and why are they essential to gene-regulation?

Transcription factor: trans-acting proteins, sequence specific DNA binding proteins, bind to promoters and enhancers, recruit other proteins to influence transcription, basal factors, activators and repressors.

What’s the structure of a transcription factor?

a DNA-binding domain (DBD), which attaches to specific promoter or enhancer sequences, and an activation/repression domain (AD) that interacts with other proteins to regulate RNA polymerase activity

How do enhancers work?

They modify the genome so that TFs can more easily bind to the promoter. Stimulate recruitment of basal factors and RNA pol II to promoters. They also recruit coactivators to open chromatin structure.

What’s the difference between an enhancer being an activator and a repressor?

when an activator protein binds, it boosts transcription, turning a gene "on" or increasing its rate. Conversely, when a repressor protein acts upon regulatory DNA (or binds to a silencer), it decreases or stops transcription, turning a gene "off" or lowering its rate.

How does CREB modulated transcription?

by acting as a molecular switch, binding to DNA as a homodimer and recruiting the co-activator CBP (CREB-binding protein) upon phosphorylation at Ser133

How can an enhancer be both an activator and a repressor?

An enhancer acts as both an activator and repressor based on the specific transcription factors (TFs) and co-regulators bound to it, which dictate whether it recruits machinery to initiate transcription or complex with co-repressors to silence it.

How does dorsal work?

Dorsal, a Drosophila transcription factor, acts as a bifunctional regulator (activator/repressor) depending on the binding site context, typically functioning as an activator alone, but converting to a repressor when recruiting the co-repressor Groucho.

What is competition in enhancers?

Competition in enhancers: multiple promoters vie for the limited activity of a single enhancer determining which gene is expressed 

• Quenching: enhancer interference, protein binds to near the enhancer through an insulator and inhibits its activity or a promoter acts as a sink for the enhancer

• Cytoplasmic sequestering: a transcriptional regulator or transcriptional factor is held in the cytoplasm, preventing it from entering the nucleus and activating an enhancer

• Heterodimerization: two different transcription factors bind together (heterodimerize) to form a functional complex that binds to the enhancer to regulate gene expression

How can enhancer regulate expression in different tissues?

binding unique combinations of transcription factors (TFs) present in specific cell types, acting as molecular switches to activate or repress gene transcription 

How is the even-skipped locus expressed in stripes?

The even-skipped (\(eve\)) locus in Drosophila melanogaster is expressed in seven distinct stripes along the anterior-posterior axis of the early embryo, regulated by five specific modular enhancers. These enhancers function independently to drive precise, localized expression patterns by integrating spatial information from maternal morphogens and gap gene repressors

What are the following, how are they used, and what are the pros and cons of each?

• Enhancer bashing: identifies which part of a DNA fragment is responsible for gene expression, for discovery specific transcription factor binding sites within an enhancer. Very precise and direct by time consuming. 

• Enhancer deletion: removal or silencing of an endogenous enhancer element from its natural location in the genome to determine its necessity. CRISPR/CAS9 stuff. Tests the requirement of an enhancer in its environment but enhancers are oftentimes redundant, so removing one may not show a result because another enhancer compensates. 

• Enhancer trapping: identifies previously unknown enhancers across the genome. Enables the discovery of new enhancers without prior knowledge of their location but requires screening many lines to find the desired enhancer (random process) 

What is DNA methylation? How does is DNA methylated, and what function does DNA methylation serve?

DNA methylation is a fundamental epigenetic mechanism where a methyl group (\(CH_{3}\)) is added to DNA, typically at cytosine bases, altering gene expression without changing the underlying genetic sequence. It acts as a "silencer" that turns genes off, playing a crucial role in development, cell differentiation, and silencing harmful genetic elements

• What is Gal4/UAS, and how is it used in model organisms research?

• What is the Q-system? What are the similarities and differences between Q-system and Gal4?

The Gal4/UAS system is a powerful, two-part genetic tool used to control gene expression in model organisms, primarily Drosophila melanogaster (fruit flies) and zebrafish. It acts as a binary system where a Gal4 transcription factor activates the transcription of a specific gene located downstream of a UAS (Upstream Activation Sequence) enhance

How does alternative splicing modulate gene function?

Alternative splicing modulates gene function by enabling a single gene to produce multiple, distinct mRNA transcripts—and consequently, diverse protein isoforms—from one primary RNA transcript. It increases proteomic diversity by selecting different coding segments (exons) to include or exclude, affecting protein structure, function, interaction, localization, and enzymatic activity