BIO ON STEROIDS

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What experimental technique identifies lac operon mutants?

Replica plating.

In replica plating, what indicates a colony cannot metabolize lactose?

Colony grows on glucose master plate but not on lactose replica plate.

What is a constitutive lac operon mutant?

Mutant expressing lac genes even without lactose.

How is constitutive expression measured in lac mutants?

ONPG assay (detects β-galactosidase activity).

What does a partial diploid experiment show about the lac operon?

Repressor is trans-acting; operator is cis-acting.

Define “operon.”

Cluster of genes with a common function under shared regulatory sequences.

When is the lac operon fully ON?

Lactose present, glucose absent → repressor inactive, cAMP-CAP bound.

What happens to lac operon transcription if lactose is absent?

Repressor binds → transcription blocked.

What is positive control?

Increases gene expression (activator proteins).

What is negative control?

Decreases gene expression (repressor proteins).

Why combine positive and negative control in lac operon?

Ensures energy-efficient regulation; avoids wasteful simultaneous metabolism.

In lac operon, what is the effect of glucose on cAMP-CAP?

Glucose lowers cAMP → prevents CAP binding → lowers transcription.

Default state of lac operon?

OFF (inducible).

Default state of trp operon?

ON (repressible).

Lac operon: what turns transcription ON?

Allolactose inactivates repressor.

Trp operon: what turns transcription OFF?

Tryptophan activates repressor.

What is the additional regulation mechanism in lac operon?

cAMP-CAP positive control (glucose dependent).

What is the additional regulation in trp operon?

Attenuation (ribosome speed affects transcription termination).

Both lac and trp operons rely on what type of control?

Negative control (repressor blocks transcription).

What is genomic imprinting?

Expression of only one allele based on parent of origin.

How does imprinting affect gene expression?

Silences one allele → organism expresses only one working copy.

Name one molecular mechanism of imprinting.

DNA methylation, histone modification, or noncoding RNAs.

How do DNAse-susceptibility experiments indicate gene expression?

Open chromatin → DNA degraded → gene expressed. Condensed chromatin → DNA protected → gene not expressed.

Define distal and proximal regulatory elements.

Distal = far from promoter; proximal = near promoter.

Function of enhancers?

Increase transcription; provide tissue-specific control.

Function of promoter-proximal elements?

Help recruit RNA polymerase and fine-tune transcription.

How do enhancers and proximal elements work together?

Enhancer loops to promoter → TFs recruit coactivators → proximal elements stabilize transcription complex → efficient, tissue-specific expression.

What are iron response elements (IREs)?

mRNA sequences regulating ferritin and transferrin receptor based on iron levels.

How do IREs regulate ferritin?

5’ UTR binding → blocks translation when iron low; releases when iron high.

How do IREs regulate transferrin receptor?

3’ UTR binding → stabilizes mRNA when iron low; mRNA degraded when iron high.

What is RNA interference (RNAi)?

Post-transcriptional gene silencing by miRNA or siRNA.

Name one way miRNA silences genes.

Translational repression, mRNA destabilization, or mRNA cleavage.

Why regulate gene expression early vs. late?

Early: energy efficient, strong control. Late: fast response, reversible, but energy costly.

What is a transgenic organism?

Organism containing genes not normally present.

Why breed transgenic founders?

To produce true-breeding homozygous lines.

How is a gene knockout organism made?

Remove or inactivate a gene using embryonic stem cells and selection strategies.

What are chimeric animals?

Animals with tissues from both host and genetically modified cells.

What is a conditional knockout?

Gene is removed in specific tissues or times using Cre-Lox.

What is “floxed”?

Gene flanked by loxP sites for Cre-mediated excision.

Difference between transgenic organisms and gene therapy recipients?

Transgenic → heritable, affects germline; gene therapy → affects somatic cells only.

What is CRISPR?

DNA loci in prokaryotes storing memory of past infections.

What is Cas9?

Endonuclease that cuts DNA guided by CRISPR RNA.

How does CRISPR guide Cas9 to its target?

crRNA or sgRNA matches target sequence; Cas9 binds and cuts DNA.

Difference between NHEJ and HDR?

NHEJ → error-prone repair → knockouts. HDR → template-based repair → precise edits.

Why add nuclear localization signals to Cas9?

To allow Cas9 to enter the eukaryotic nucleus.

What is prime editing?

Cas9 nicking + reverse transcription → precise single-strand DNA edits.

How is heterozygosity detected in DNA sequencing?

Two peaks at the same genomic position (roughly 50/50 ratio).

What does a single clean peak indicate?

Homozygous allele.