Bio230H1: Genes To Organisms

Front 1

Prokaryotes...

A) are bacteria, distinct from archae and eukaryotes

B) are very diverse, although none contain a nucleus and many have a cell wall

C) live in all sorts of ecological niches, ranging from sewage treatment plants, to hot acid springs, to the human mouth

D) B and C

E) all of the above

Back 1

D)

Front 2

Which of the following statements regarding DNA is NOT correct?

A) The sugar-phosphate bond between adjacent nucleotides in DNA is stronger than the hydrogen bonding between base-pairs

B) In all cells on earth, double-stranded DNA functions as the hereditary material

C) DNA functions as a template for the polymerization of RNA monomer

D) The rate of DNA replication and transcription is consistent in all species

E) Cells replicate DNA using one strand as a template for polymerization of nucleotide monomers

Back 2

D)

Front 3

In the absence of lactose and in the presence of glucose...

A) CAP binding prevents RNA polymerase activity

B) RNA is not transcribed from the LAC operon because of the bacteriophage lambda repressor

C) RNA is not transcribed from the LAC operon because the Lac repressor is bound

D) RNA is transcribed from the LAC operon because CAP is not bound

E) RNA is transcribed from the LAC operon because the Lac repressor is bound

Back 3

C) RNA is not transcribed from the LAC operon because the Lac repressor is bound

Front 4

Gene expression is...

A) Controlled only by the rate of transcription

B) Consistent in all the 25,000 genes of a given cell

C) High for the vast majority of the genes in all cells

D) Often affected by the extracellular environment, an example being the effect of glucocorticoids on liver cells

E) The same for all genes of a given species - this can be observed by transplanting a nucleus from a frog skin cell into an enucleated egg

Back 4

D) Often affected by the extracellular environment, an example being the effect of glucocorticoids on liver cells

Front 5

Which of the following statements about the E. coli tryptophan operon-repressor system is correct?

A) Five operators control each of the E. coli tryptophan-synthesizing enzymes

B) Low intracellular tryptophan leads to the transcription of genes that encode for tryptophan-synthesizing enzymes

C) There is no correct answer

D) The presence of tryptophan inhibits the expression of one gene

E) When two tryptophan molecules bind the tryptophan repressor, the repressor changes to an inactive conformation

Back 5

B)

Front 6

Which of the following is not true regarding eukaryotic gene regulatory proteins?

A) Most gene repressor proteins compete with RNA polymerase for access to DNA

B) Gene regulatory proteins may directly interact with general transcription factors

C) Gene activator proteins may bind histone modification enzymes to alter promoter chromatin structure

D) Gene activator proteins may increase the rate of transcription initiation

E) Gene activator and repressor proteins may compete for binding to the same regulatory DNA sequences

Back 6

A)

Front 7

If you isolated 2 nucleosomes in a test tube, which of the following would you typically have?

A) four molecules of H1

B) four molecules of H2A

C) eight molecules of H2B

D) sixteen molecules of H2A

E) two molecules of H2B

Back 7

B)

Front 8

Which of the following is not true regarding a gene control region?

A) It includes the promoter

B) It includes regulatory sequences

C) It is always located immediately next to the transcribed region

D) It may contain regulatory sequences within the gene's introns

E) C and D

Back 8

C)

Front 9

The regulatory system that determines the mode of growth in bacteriophage lambda (when infecting E. coli host cells) is a _______. The Cro protein turns on its own synthesis in a ___________, and turns off the repressor protein (cl) in a __________.

A) flip-flop device; positive feedback loop; negative feedback loop

B) memory device; feed-forward loop; negative feedback loop

C) feed-forward loop; positive feedback loop; flip-flop device

D) There is no correct answer

E) memory device; negative feedback loop; flip-flop device

Back 9

A)

Front 10

Histones are...

A) part of the histone code, which are a set of exceptions to the genetic code

B) primarily synethesized during mitosis

C) composed of H2A, H2B, H3, and H4 variants

D) continually being covalently modified

E) either completely methylated or completely acetylated

Back 10

D)

Front 11

Why does a cell need to regulate gene expression?

Back 11

External environmental conditions

Development cues

In response to hormone signals

Front 12

What is transcriptional regulation?

Back 12

Most common type of gene regulation in bacteria

Occurs in the first stage of gene expression before significant amounts of mRNA are synthesized

The lac operon is a well-characterized example of transcription-level gene regulation as well as the ara operon

Front 13

What is the ara operon?

Back 13

The ara operon contains genes that encode proteins required to metabolize a sugar like pentose sugar L-arabinose

When arabinose is present in bacterial growth medium, the genes in the ara operon may be transcribed at high levels, but when a more easily metabolized sugar such as glucose is present, there is little transcription of these genes

Proteins produced by lac and ara operons are not easily detectable but a plasmid called pGLO contains a reporter gene that produces an easily visualized gene product that inserts a marker so that the operon was switch on and an easily detectable protein was made

Front 14

What are the parts and functions on this plasmid map of pGLO?

Back 14

Arrows indicate open reading frames or genes

araC gene:

• Origin of replication for plasmid (ori), a bla gene that codes for a protein that makes the bacterial cell resistant to the antibiotic ampicillin

GFP:

• Gene that codes for green fluorescent protein, a fluorescent marker

• Made in large amounts in bacterial cells and these cells are exposed to long-wave ultraviolet light that makes GFP bright green fluorescence

PBAD:

• The ara operon promoter

• Normally regulates the transcription of genes in the ara operon (allow uptake and metabolism of arabinose) but in pGLO these genes have been replaced by the GFP gene

• GFP would normally have its own eukaryotic promoter (from jellyfish Aequoria victoria) but instead is controlled by bacterial promoter PBAD

Whenever environmental conditions of the bacterium would have caused an increase in transcriptional activity from the PBAD promoter, GFP will be produced instead

Only one gene from native ara operon present in the pGLO plasmid is the araC gene and that is has its own promoter

Front 15

Describe the differential regulation of the ara operon in the pGLO system

Back 15

AraC is an allosteric activator protein with two binding sites

When arabinose is present in the environment, AraCi (inactive) binds to sugar

This binding causes a conformational change in AraCi to AraCa (active) that allows it to recognize and bind to aral, an activator sequence located just upstream of the PBAD promoter

It is hypothesized that AraCa helps RNA polymerase (RNAP) bind to the PBAD promotor. Therefore, in absence of arabinose:

• AraC is not activated (remains as AraCi) and thus cannot bind to aral sequence

• RNAP will not bind efficiently to the PBAD sequence

• Very little transcription will occur (little GFP will be made)

Second mechanism to regulate transcription involves cyclic adenosine monophosphate (cAMP) and the catabolite activator protein (CAP)

Front 16

How would you get the most efficient binding of RNAP to PBAD?

Back 16

In order to get most efficient binding of RNAP to PBAD, not only must two molecules of AraCa bind to the aral sequence, but CAP must bind to the cAMP-CAP binding site (CBS) located just upstream of the aral sequence

Front 17

What are the two forms found of CAP and what happens with CAP in bacterial cells?

Back 17

2 forms:

I) When cAMP is bound to CAP, CAP is in its active form and can bind to the CBS sequence

II) But when cAMP is not bound, CAP is inactive and cannot bind to CBS

In bacterial cells:

High concentration of glucose = reduce the synthesis of cAMP = CAP is less likely to be activated and bind to CSB sequence

• Consequently even in in presence of arabinose, high concentrations of glucose will prevent the formation of active cAMP-CAP complex and the binding of RNAP to PBAD will not be efficient

• Little transcription of GFP will occur

In presence of arabinose and low concentrations of glucose = two molecules of AraCa will bind to aral sequence, activated cAMP-CAP will bind to CBS = binding of RNAP to PBAD will be efficient

• Transcription of GFP will be relatively high and it would be expected that bacterial colonies will glow bright green when exposed to long-wave UV light

Front 18

What is the heat shock method?

Back 18

Laboratory procedure

Treatment produces bacteria transiently permeable to DNA by introducing brief heat shock

Mechanism Not well understood, believed to involve the neutralization of electrostatic repulsion between DNA and the cell surface induced by interaction with divalent cations and by increased fluidity and permeability of the membrane induced by an increase in temperature

Front 19

How can we differentiate between bacterial cells containing our plasmid of interest and those that do not?

Back 19

We cannot differentiate these using a regulate light microscope

Key is in the bla gene found on the pGLO plasmid

This gene codes for beta-lactamase, an enzyme that makes the bacterial cell resistant to the antibiotic ampicillin

This gene is called a "selectable marker" because if we include ampicillin in the E. coli growth medium, only those E. coli cells that have been successfully transformed with the pGLO plasmid and therefore expressed the enzyme beta-lactamase will be able to grown on the medium

Front 20

What is the Aseptic technique?

Back 20

Refers to the procedures that are used to establish and maintain a sterile environment or prevent contamination of a pure environment

Procedure goes:

I) Wiping down (decontaminating) your workspace with a bleach or ethanol solution before and after working with bacterial cultures

II) Pipetting bacterial cultures carefully to prevent the formation of aerosols containing bacteria

III) Wearing lab coats, safety glasses and gloves at all times when dealing with bacteria

IV) Minimizing the amount of time that your petri dishes are uncovered

V) Making sure that you do not contaminate materials that may come in contact with your bacterial cultures

Front 21

What is 3C-Seq technology?

Back 21

Couples chromosome conformation capture to high throughput sequencing

Measures interaction frequencies between a viewpoint (DNA fragment of choice) and (distal) regulatory elements on a genome wide scale

Recently used for unbiased analysis of spatial organization of the Myb proto-oncogene locus in erythroid cells

Since 3C-based technologies only provide topological information, their functional relevance should be interpreted with caution and needs to be supported by additional experiments

Front 22

What is Myb?

Back 22

Myb is a critical hematopoietic regulator required for the proliferation and expansion of all blood progenitors and is dramatically down regulated during terminal differentiation

Failure to silence Myb expression is linked to impaired differentiation and may play a role in leukemogenesis

Myb transcription is regulated by an array of distal intergenic enhancers localizing up to 109 kb upstream of the gene, which are occupied by the essential hematopoietic