theme 3 module 3/4

Why is gene expression regulated at multiple stages?

To ensure the correct proteins are produced in the appropriate amounts and at the appropriate times in response to cellular conditions.

At which stages can eukaryotic gene expression be regulated?

Transcription, RNA processing, RNA stability, translation, post-translational modification, protein transport, and protein degradation.

Why is measuring mRNA useful for studying gene expression?

The presence and abundance of mRNA indicate whether a gene is being transcribed and its relative level of expression.

What is in situ hybridization?

A technique that detects the location of specific mRNA molecules within intact cells, tissues, or organisms using complementary nucleic acid probes.

What type of probe is used in in situ hybridization?

A fluorescently labelled, single-stranded DNA or RNA probe complementary to the target mRNA.

How does a probe recognize its target mRNA during in situ hybridization?

The probe hybridizes with the target mRNA through complementary base-pairing between nucleotides.

What is spatial gene expression?

The location where a particular gene is expressed within different cells, tissues, or regions of an organism.

What is temporal gene expression?

The timing of gene expression during different stages of development or in response to changing conditions.

How can in situ hybridization reveal developmental changes in gene expression?

By detecting the location and timing of specific mRNA transcripts at different developmental stages.

What organism is used to demonstrate in situ hybridization in the lecture?

Drosophila embryos, in which different genes show distinct spatial expression patterns.

How many genes can typically be studied using in situ hybridization?

One or a few specific genes at a time.

What is the difference between in situ and in vitro?

In situ examines molecules in their original location within cells or tissues, whereas in vitro involves experiments performed outside the organism.

What is a DNA microarray?

An in vitro technique that measures the expression levels of thousands of genes simultaneously using complementary nucleic acid hybridization.

What is the main advantage of DNA microarrays over in situ hybridization?

Microarrays analyze thousands of genes simultaneously, whereas in situ hybridization generally examines one or a few genes.

What is the basic principle underlying DNA microarrays?

Complementary base-pairing between labelled cDNA molecules and known single-stranded DNA probes.

What is a DNA chip?

A glass slide containing thousands of spots with known DNA sequences attached to its surface.

What does each spot on a DNA microarray contain?

Many copies of a specific single-stranded DNA probe corresponding to a particular gene.

What are oligonucleotides?

Short sequences of nucleotides that can act as probes to detect complementary nucleic acid sequences.

How many oligonucleotide probes can DNA microarrays contain according to the lecture?

Up to approximately 100,000 probes.

What is the transcriptome?

The complete collection of RNA transcripts expressed in a cell or sample under particular conditions.

How do microarrays help identify genes involved in the same cellular process?

Genes showing similar expression patterns can be grouped into clusters that may represent coordinated functions or regulatory networks.

Why can different cell types have different gene expression patterns despite containing the same DNA?

Different genes are activated or repressed depending on cell type, developmental stage, and environmental signals.

Why are DNA microarrays useful for studying cancer?

They identify genes that are expressed differently between normal cells and cancerous cells.

Which two cell types are compared in the breast cancer microarray example?

Normal breast epithelial cells and breast carcinoma cells.

What is the first step in preparing samples for DNA microarray analysis?

Isolate mRNA separately from the two cell types being compared.

Why is mRNA converted into cDNA before microarray analysis?

To produce complementary DNA molecules that can be fluorescently labelled and hybridized to the DNA probes on the chip.

What enzyme converts mRNA into cDNA?

Reverse transcriptase.

What is complementary DNA (cDNA)?

DNA synthesized from an RNA template using reverse transcriptase.

How are fluorescent labels incorporated into cDNA?

Fluorescently labelled nucleotides are incorporated during reverse transcription.

Why are different fluorescent dyes used for normal and cancerous cell samples?

To distinguish the relative contribution of each sample to the fluorescence detected at each microarray spot.

What fluorescent colour represents normal epithelial cells in the lecture example?

Green.

What fluorescent colour represents breast carcinoma cells in the lecture example?

Red.

Why are equal amounts of labelled cDNA from both samples mixed?

To allow comparison of their relative hybridization to the same DNA probes.

What happens when labelled cDNA is applied to the DNA microarray?

cDNA molecules hybridize with complementary single-stranded DNA probes attached to the chip.

Why is the microarray washed after hybridization?

To remove unbound or weakly bound cDNA before measuring fluorescence.

How is gene expression measured after microarray hybridization?

A scanner detects the fluorescence colour and intensity at each spot.

What does a green spot indicate on the microarray?

The gene is expressed more strongly in normal epithelial cells than in breast carcinoma cells.

What does a red spot indicate on the microarray?

The gene is expressed more strongly in breast carcinoma cells than in normal epithelial cells.

What does a yellow spot indicate on the microarray?

The gene is expressed at approximately equal levels in both cell types.

What does a spot with no fluorescence suggest?

The corresponding gene is not detectably expressed in either sample.

Why does a highly expressed gene produce greater fluorescence intensity?

More mRNA produces more fluorescently labelled cDNA, resulting in stronger fluorescence after hybridization.

What does a brighter fluorescence signal indicate?

Greater relative abundance of the corresponding labelled cDNA and therefore higher mRNA expression.

What does it mean if a gene is upregulated in cancer cells?

Its expression level is higher in cancer cells compared with normal cells.

What does it mean if a gene is downregulated in cancer cells?

Its expression level is lower in cancer cells compared with normal cells.

How are microarray results analyzed to identify related genes?

Computational software compares expression patterns and groups genes with similar expression profiles.

Why must gene expression be regulated after transcription?

Because existing mRNA molecules can continue to be translated into proteins even after transcription has stopped.

How can removing mRNA alter gene expression?

Degradation of mRNA prevents further translation, reducing production of the encoded protein.

What is mRNA stability?

The length of time an mRNA molecule remains intact and available for translation before degradation.

How does mRNA stability affect protein production?

More stable mRNA can remain available for translation longer, whereas rapidly degraded mRNA generally produces less protein.

How does the poly-A tail affect mRNA stability?

The poly-A tail helps protect mRNA from degradation, and shortening of the tail can promote mRNA decay.

What is the role of the 5′ cap in mRNA?

It is important for translation initiation.

What is the role of the 5′ untranslated region (5′ UTR)?

It contains regulatory sequences that can influence translation initiation and mRNA transport.

What is the role of the 3′ untranslated region (3′ UTR)?

It contains regulatory sequences that can influence translation and mRNA stability.

What is RNA interference (RNAi)?

A post-transcriptional regulatory mechanism in which small noncoding RNAs direct the inhibition or degradation of specific mRNA molecules.

What are noncoding regulatory RNAs?

RNA molecules that are not translated into proteins but instead regulate processes such as gene expression.

What is microRNA (miRNA)?

A small noncoding RNA that regulates gene expression by binding to target mRNA and inhibiting translation.

How is a miRNA precursor initially formed?

It is transcribed as an RNA molecule containing complementary regions that fold into a hairpin structure.

Why does the miRNA precursor form a hairpin loop?

Complementary nucleotide sequences within the same RNA molecule base-pair with one another.

What happens to the miRNA hairpin before it becomes functional?

It is processed into a smaller regulatory RNA that can associate with the RNA-induced silencing complex.

What does RISC stand for?

RNA-induced silencing complex.

What is the function of RISC?

It uses a small regulatory RNA to recognize complementary target mRNA and inhibit its expression.

How does miRNA recognize its target mRNA?

Through imperfect or non-exact complementary base-pairing.

What happens when miRNA-guided RISC binds to target mRNA?

Translation of the target mRNA is inhibited, reducing protein production.

Does miRNA regulation require perfect complementarity with target mRNA?

No. miRNA typically binds through imperfect complementarity.

What is the final result of miRNA-mediated translation inhibition?

Reduced production of the protein encoded by the target mRNA.

What is small interfering RNA (siRNA)?

A short regulatory RNA that guides RISC to complementary target mRNA and promotes its cleavage and degradation.

How does siRNA recognize its target mRNA?

Through exact or near-perfect complementary base-pairing.

What happens when siRNA-guided RISC binds to target mRNA?

The target mRNA is cleaved and subsequently degraded.

Why does siRNA-mediated cleavage prevent protein synthesis?

The mRNA is destroyed and can no longer serve as an intact template for translation.

What is the key difference between miRNA and siRNA in the lecture?

miRNA binds imperfectly and inhibits translation, whereas siRNA binds with near-perfect complementarity and promotes mRNA cleavage.

What do miRNA and siRNA have in common?

Both are small regulatory RNAs that associate with RISC and regulate gene expression after transcription.

Why is RNA interference considered post-transcriptional regulation?

It acts on mRNA after transcription rather than directly controlling DNA transcription.

Is RNA interference a natural cellular mechanism?

Yes. RNA interference occurs naturally and helps regulate gene expression.

How can scientists use RNA interference to study gene function?

By silencing a specific gene and observing the resulting changes in cellular activity or phenotype.

Why is RNA interference useful in disease research?

It allows researchers to investigate how reducing expression of specific genes affects disease-related processes.

What is post-translational regulation?

Regulation of protein activity, function, or abundance after a protein has been synthesized.

Why is post-translational regulation important?

It allows cells to rapidly alter protein activity or remove proteins without waiting for changes in transcription or translation.

What are post-translational modifications?

Chemical or structural changes made to proteins after translation that can affect their maturation, activity, or stability.

What post-translational modifications are discussed in the lecture?

Protein cleavage, disulphide bond formation, acetylation, phosphorylation, and methylation.

How can post-translational modifications regulate protein function?

They can activate, inactivate, stabilize, or otherwise alter a protein's function.

What is selective protein degradation?

The targeted breakdown of specific proteins that are damaged, misfolded, or no longer needed.

Why must protein degradation be selective?

Cells need to remove particular proteins without destroying proteins that are still required for normal function.

What is a proteasome?

A large protein complex that degrades selected proteins by breaking peptide bonds.

What types of proteins can proteasomes degrade?

Unneeded, damaged, misfolded, or specifically targeted regulatory proteins.

How do proteasomes break down proteins?

They unfold targeted proteins and cleave their polypeptide chains into smaller peptide fragments.

What happens to the peptide fragments produced by proteasomes?

They can be further broken down into amino acids that may be reused to synthesize new proteins.

What is ubiquitin?

A small protein that can be attached to other proteins to mark them for degradation.

What is ubiquitination?

The enzymatic attachment of ubiquitin to a target protein.

What is polyubiquitination?

The attachment of multiple ubiquitin molecules in a chain to a target protein.

What is the function of a polyubiquitin chain?

It serves as a signal that allows the proteasome to recognize a protein targeted for degradation.

What are the three major stages of ubiquitin tagging?

Ubiquitin activation, ubiquitin conjugation, and ubiquitin ligation.

What happens during ubiquitin activation?

Ubiquitin is activated through an ATP-dependent enzymatic reaction.

What happens during ubiquitin conjugation?

Activated ubiquitin is transferred through the ubiquitin-conjugating enzyme system.

What happens during ubiquitin ligation?

Ubiquitin is attached to the target protein, allowing formation of a ubiquitin chain.

Why is ATP required during ubiquitin tagging?

Energy is needed to activate ubiquitin for its subsequent transfer and attachment to target proteins.

What happens after a protein has been tagged with a polyubiquitin chain?

The tagged protein is recognized by the proteasome and directed toward degradation.

What happens to a tagged protein before it enters the proteasome?

It is unfolded so that its polypeptide chain can enter the proteasome.

Why is protein unfolding necessary for proteasomal degradation?

The protein must be unfolded to pass into the proteasome's internal degradation chamber.

What happens inside the proteasome?

The polypeptide chain is cleaved into smaller peptide fragments.

What happens to ubiquitin during proteasomal degradation?

Ubiquitin molecules are removed from the target protein and can be recycled.