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Alex's Genetics > Lecture 15b > Flashcards

Lecture 15b Flashcards

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Biology 101 flashcards
1
Q

T/F: Bacteria have a lot of chromatin!

A

False! Bacteria don’t really have chromatin, although there are some proteins binding the DNA.

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2
Q

T/F: The 3D packing of chromatin is not important in gene expression.

A

False! The 3D packing of chromatin is an important parameter affecting gene expression.

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3
Q

T/F: Chromatin is a static structure.

A

False! Chromatin is a very dynamic structure that can alternate between two conformations.

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4
Q

What are the two conformations that chromatin can alternate between?

A

Heterochromatin and euchromatin

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5
Q

Describe the heterochromatin structure.

A

This is a closed conformation in which the chromatin is very tightly packed and transcription is difficult or impossible.

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6
Q

Describe the euchromatin structure.

A

This is an open conformation in which the chromatin is accessible to transcription factors and transcription can take place.

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7
Q

Is an open conformation present in heterochromatin or euchromatin?

A

Euchromatin

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8
Q

Is a closed conformation present in heterochromatin or euchromatin?

A

Heterochromatin

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9
Q

What types of chromatin have nucleosomes?

A

All types of chromatin have nucleosomes.

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10
Q

Using electron microscopy, what type of chromatin can we visualize and how does it appear?

A

Using electron microscopy, heterochromatin can be seen as darkly-staining regions in the interphase nucleus.

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11
Q

Where does heterochromatin localize itself in the nucleus?

A

Much of the heterochromatin is localized to the periphery of the nucleus and around the nucleolus.

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12
Q

Does heterochromatin in the nucleus stay in the same place at all times?

A

No, heterochromatin moves back and forth between the periphery of the nucleus and around the nucleolus.

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13
Q

What are the two types of heterochromatin?

A

Constitutive and Facultative

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14
Q

Describe constitutive heterochromatin.

A

It contains regions that are always heterochromatic or have a closed conformation.

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15
Q

Is a constitutive heterochromatin able to be transcribed?

A

No, it is permanently inactive with regard to transcription.

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16
Q

What is the purpose of a constitutive heterochromatin?

A

Genome stability

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17
Q

What always has the biggest block of heterochromatin?

A

Centromere

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18
Q

Describe a facultative heterochromatin.

A

This contains regions that can interconvert between euchromatin and heterochromatin.

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19
Q

What is the purpose of heterochromatin?

A

Allows for regulation/control of gene expression in multicellular organisms.

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20
Q

What causes a facultative heterochromatin to be assembled or disassembled?

A

Signals from gene expression cause a facultative heterochromatin to be assembled or disassembled.

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21
Q

Generally speaking, what from bacteria has been really helpful in the early study of chromatin?

A

Bacterial restriction enzymes

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22
Q

In regards to bacteria, what are bacterial restriction enzymes a part of?

A

Bacterial restriction enzymes are part of a bacterial immune system.

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23
Q

T/F: Bacterial restriction enzymes only cleave eukaryotic DNA that is packed in heterochromatin.

A

False! Bacterial restriction enzymes cleave eukaryotic DNA regardless of whether it is packaged in euchromatin or heterochromatin.

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24
Q

In regards to the chromatin, what does the restriction enzyme do?

A

It cleaves on both sides of a gene to release that gene and its flanking sequences. The cleaved DNA is now purified.

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25
Q

What is Eukaryotic DNase I?

A

It is an enzyme that cleaves DNA into many tiny pieces.

26
Q

Are we have our cleaved and purified gene, what should we expose it to?

A

We want to expose it to DNase I, which will cut the DNA into several tiny pieces.

27
Q

In regards to cleaving the chromatin, what is a difference between prokaryotic restriction enzymes and eukaryotic DNase I?

A

Prokaryotic restriction enzymes can readily cleave both heterochromatin and euchromatin.

With eukaryotic DNase I, it only cleaves the DNA if it is euchromatin, but not if it is heterochromatin.

28
Q

What happens when a gene in euchromatin is exposed to DNase I?

A

The euchromatin cannot stop the DNase I from cleaving the DNA into tiny pieces.

29
Q

What happens when a gene in heterochromatin is exposed to DNase I?

A

The heterochromatin is able to stop the DNase I from cleaving the DNA, because it repels the DNase I.

30
Q

In what cells does the B-globin locus express the hemoglobin subunit?

A

The B-globin locus expresses the hemoglobin subunit only in blood cells.

31
Q

How is the B-globin locus regulated?

A

It is regulated by chromatin.

32
Q

Where is hemoglobin produced?

A

Hemoglobin is produced in erythroid cells (RBCs) called reticulocytes.

33
Q

What cells produce the B-globin gene in euchromatin when expressed?

A

Erythroid cells called reticulocytes.

34
Q

What cells produce the B-globin gene in heterochromatin?

A

Non-erythroid cells

35
Q

Once we have exposed the euchromatin and heterochromatin to DNase I, how do we determine which chromatin actually got chopped up?

A

We get rid of the protein portion on the DNA. To visualize the presence or absence of this one restriction fragment, people then used a Southern Blot.

36
Q

What is the Southern Blot?

A

This is a technique used to visualize the presence or absence of restriction fragment in chromatin from DNase I.

37
Q

On an agarose gel, how do we determine the size of the DNA fragments?

A

The gel is usually run with a few size markers in one lane, which acts as a “ruler” to show the precise sizes of DNA fragments.

38
Q

How does DNA migrate on the agarose gel?

A

Since DNA has a negative charge, it migrates to the positive electrode.

39
Q

If we saw super long bands on the agarose gel, what could we assume it is?

A

These are the huge number of restriction fragments that had been protected from DNase I by heterochromatin.

40
Q

After the DNA has been run on the gel, what do we do with it?

A

The DNA is then transferred from the agarose gel to a nylon membrane that resides on top of the gel.

41
Q

How does the DNA from the gel reach the nylon membrane?

A

There is a buffer that will sit under the agarose gel. The buffer will soak upwards, taking the DNA off of the agarose gel and putting it up onto the Nylon membrane.

42
Q

How does the DNA from the gel get STUCK to the nylon membrane?

A

The DNA is negatively charged and the Nylon Membrane is positively charged so it gets stuck. Then, the Nylon Membrane is exposed to UV light to covalently attach the DNA to the Nylon Membrane.

43
Q

What do we have to add in order to visualize the DNA on the nylon membrane?

A

We add in a probe DNA, heat the fluid, then cool it.

44
Q

What will the single-stranded DNA probe anneal to? What won’t it anneal to?

A

It will anneal to the intact gene DNA from the heterochromatin.

It will NOT anneal to the fragmented euchromatin because there is nothing to bind to, its just a bunch of pieces.

45
Q

How long is the DNA strand we are typically looking at before it is cut up by DNase I?

A

About 4.6 KB.

46
Q

What size fragments run further on the agarose gel?

A

Smaller DNA fragments run further on the gel

47
Q

Explain this gel.

A

We are looking at the 4.6 KB fragment cuz that is our gene of interest.

On the left side, there has not been that much DNase added, so these could be euchromatin that did not get chopped up. Thus, the 4.6 KB fragment is present

The wells up to 1.5 with no 4.6 KB are likely euchromatins that had their DNA chopped up as DNase concentration increased. Thus, the 4.6 KB is not present because the probe DNA could not anneal.

The well at 1.5 with a 4.6 KB is likely a heterochromatin that did not allow DNase I to chop it up. Thus, the probe could anneal and be visualized.

48
Q

How many genes does the B-globin locus have?

A

It has 5 genes. The one at the top is a pseudo-gene so it is non-functional.

49
Q

Is this a heterochromatin or euchromatin?

If a heterochromatin, what type?

A

This is a facultative heterochromatin. We know it is facultative because of the black binding site at the bottom.

50
Q

What is the black site at the bottom?

A

This is the Locus Control Region (LCR), which will bind proteins to become a euchromatin.

51
Q

What happens when the LCR binds proteins?

A

A facultative heterochromatin can be turned into a euchromatin.

52
Q

What type of chromatin is this?

A

A euchromatin

53
Q

What is Beta-Thalassemia?

A

A type of anemia caused by a deletion in one copy of the B-globin locus.

54
Q

What is anemia?

A

When less oxygen is delivered to the body.

55
Q

Name 3 B-Thalassemia mutations.

A

Dutch deletion, English deletion, and Hispanic deletion.

56
Q

Memorize this

A

Dumb idiot

57
Q

What did the Hispanic deletion lead to the discovery of?

A

The Locus Control Region (LCR)

58
Q

What does the Hispanic deletion cause the deletion of? What impact does this have?

A

The LCR. This means that the chromatin is seen only in heterochromatin.

59
Q

What did Southern blots of the Hispanic Thalassemic deletion show?

A

The Hispanic deletion leads to DNase I insensitivity at the b-globin gene.

Basically, no matter how much DNase I is added, the DNA will never be chopped up or degraded,

60
Q

What does the hispanic deletion remove but still keep?

A

It removes the LCR but not the genes.

61
Q

Which one shows the hispanic deletion? Explain.

A

As the DNase I concentration is increased, the middle chromatin still does not degrade. This is indicative of the hispanic deletion.

62
Q

What is always the shape of the chromatin with a Hispanic deletion?

A

Always in heterochromatin.

Alex's Genetics (38 decks)

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