BIO 335 EXAM 4

What are the reagents used for the dideoxy method of DNA sequencing?

DNA template, 1 primer, DNA polymerase, dNTPs, labeled ddNTPs, and a buffer

What is the dideoxy method of DNA sequencing

a technique used to determine the exact order of nucleotide bases in a strand of DNA. AKA Sanger sequencing

What is the role of ddNTPs in DNA sequencing?

they cause synthesis arrest (chain termination) when incorporated in the growing DNA strand

Why does each ddNTP have its own color?

to let scientists know which nucleotide is present along the fragment sequenced

What is the general goal of In Situ Hybridization (ISH)?

to visualize the location or the presence of a DNA or RNA sequence

What information can be gathered from ISH done on chromosomes?

diagnosing a translocation, deletion and/or aneuploidy with the help of fluorescence

What information can be gathered from ISH done on embryos?

detecting gene expression, chromosome counts, and structure changes

What organism was the CRISPR-Cas system discovered in?

E. coli

What is the CRISPR-Cas system's role in E. coli?

It provides bacteria with a defense against bacteriophages

What does CRISPR-Cas do to its target sequence?

It uses a guide RNA (gRNA) to locate a matching DNA sequence, binds to the target sequence, and then makes a double stranded break

What are the 2 possible outcomes of a double-strand break?

Repair by nonhomologous end joining (NHEJ) or repair by homologous recombination repair (HRR)

What is nonhomologous end joining? Is it reliable?

a cellular pathway that brings together 2 ends of DNA fragments to repair double-stranded breaks. it is not reliable because it doesn't need a template

what is HRR (homologous recombination repair)?

a cellular pathway repair system using an undamaged DNA template sequence

How does the CRISPR-Cas system allow to create a mutation at a particular place in the genome?

1. gRNA guides Cas9 to sequence that is complementary to itself
2. Cas9 makes a double stranded cut
3. Cell repairs it by HRR or NHEJ but makes mistakes causing a mutation.

What is the favorite carbon source of E.coli?

glucose

What three enzymes allow E. coli to absorb and convert lactose into glucose

Permease (lacY), beta galactose (lacZ), and transacetylase (lacA)

What units is the lactose operon made of?

promoter, operator, and structural genes (lacZ,Y,A)

What is the promoter

DNA sequence where RNA polymerase binds to start transcription.

What is the operator

DNA sequence where repressor binds to block transcription.

What is the function of beta galactose (lacZ)

to break lactose into glucose and galactose

What is the function of permease (lacY)

allows lactose to enter the cell

What is the function of transacetylase

to help convert galactose into glucose

What is a polycistron

A single mRNA molecule that contains multiple genes called cistrons that are transcribed together (aka lacZYA) polycistron

What is the mechanism of lacZYA repression in the absence of lactose

the repressor protein (lacI) binds to the operator, blocking transcription (transcription OFF)

How is repression lifted in the presence of lactose

Allolactose binds to the repressor protein (lacI), changing its shape so it can't bind to the operator. (transcription ON)

What is the mechanism that stops LacZYA transcription when lactose becomes absent?

when lactose is depleted, allolactose is not available to bind to the repressor, so the repressor binds to the operator again. (Transcription OFF)

What makes lacZYA transcription regulation negative inducible?

It's negative in that a repressor blocks transcription, and inducible in that allolactose turns transcription on (Transcription ON)

What is negative repressible regulation

When a repressor is activated and blocks transcription (Transcription OFF)

What is positive inducible regulation?

When an inducer activates an activator protein, allowing transcription (Transcription ON)

What is positive repressible regulation

When an activator is turned off, preventing transcription (Transcription OFF)

What are the effects of mutations in the promoter vs. the operator in the lac operon

Mutations in promoter = lac operon permanently inactive Mutations in operator = lac operon is always ON

What is CRP activity in the lac operon

CRP is an activator protein that binds to the promoter, and helps RNA polymerase initiate transcription

What does CRP do

helps increase lac operon transcription when glucose levels are low by helping RNA polymerase to initiate transcription

Why is the mechanism of the trp operon control negative repressible

because trytophan activates the repressor which allows the repressor to bind to the operator (transcription OFF)

Why is the regulation of transcription important for the development of multicellular organisms

Because it provides the ability to control when, where, how long, etc. genes are expressed, allowing cell differentiation in complex organisms

Why is the regulation of transcription important for the evolution of new shapes

changes in gene regulation can lead to different morphologies (for example, bat wings and mouse limbs have the same genes, different regulation)

Why is the regulation of transcription important for regenerative potential?

gene expression regulation can explain why some organisms can regenerate limbs/organs while some cannot

What enzyme is responsible for gene transcription?

RNA polymerase II

What does RNA polymerase II do

transcribes mRNA from DNA

What is an enhancer

a noncoding DNA sequence that recruits transcription factors (proteins) to help stabilize RNA polymerase and increases gene expression

What is a silencer

A DNA sequence that inhibits/represses transcription

What is a transcription factor

A protein that binds to DNA/other proteins to regulate transcription

How is trasncription of a gene activated in a particular cell type but not another

It depends on which specific transcription factors are present to bind to specific enhancers

What is a reporter gene assay and its purpose?

A way for scientists to see where a gene is being turned on. A reporter gene (with glow dye) is attached to an enhancer and when that gene is turned on it glows

What is the purpose of a reporter gene assay

To show where the enhancer causes gene expression to occur

What are the three different gene types

housekeeping, luxury, and regulatory genes

What gene type drives development of complex organisms

regulatory genes

What is the function of housekeeping genes

Genes expressed in all cells providing basic function for survival (ex. dna repair, protein synthesis… etc.)

What is the function of luxury genes

they are expressed only in particular cell types for specialized functions

What is the function of regulatory genes

To drive differentiation by regulating transcription for cell specialization

What is the difference between a regulatory and effector gene

Regulator genes control expression of other genes (such as effector genes), and effector genes carry out specialized functions

What is a Gene Regulatory Network (GRN)

A series of interactions between several regulatory genes that work together to control/determine cell differentiation

What is chromatin remodeling

Process of changing chromatin structure to make DNA more or less accessible

How does chromatin remodeling influence transcription

open chromatin = transcription can occur closed chromatin = transcription can't occur

What are the two ways transcription factors influence chromatin state

1. preventing chromatin from reforming
2. recognizing closed chromatin

What common histone/DNA modifications modulate chromatin state

1. histone acetylation = activate chromatin
2. histone deacetylation = inactivate chromatin
3. DNA methylation = can inactivate chromatin

What proteins operate these histone or DNA modifications?

HATs = add acetyl groups to histones = activate chromatin HDACs = remove acetyl groups from histones = inactivate chromatin

What is cancer?

A disease characterized by uncontrolled cell division.

What are the characteristics of cancer?

Clonal: begins from a single cell, begins as a benign growth, multistep process that can be staged

What is malignant cancer

tumors that invade surrounding tissue

What is metastatic cancer

Cancer that has spread to other parts of the body

What are the percentages of inherited vs non-inherited forms of cancer?

5-10% inherited, 90-95% not inherited

What is a carcinogen?

environmental agent causing cancer by altering the structure/expression of genes

What evidence supports the idea that cell division is a regulated process

In human development, cells actively proliferate (multiply), but in adults only stem cells proliferate because most stem cells remain in he G0 phase to halt division.

How do growth factors regulate cell division?

Growth factors are signals released by cells that bind to specific receptors on other cells, triggering a pathway that changes gene expression and promotes cell division in that cell

Why might mutations affecting growth-factor pathways lead to unregulated cell growth

Mutations in growth factor signaling pathways can cause pathways to remain constantly active without a growth factor, leading to unregulated growth