Medical Genetics Wk 6

Question 1

DNA Is Organized
into Chromatin in Eukaryotes

Answer 1

After chromosome separation and cell division, cells enter the interphase stage of the cell cycle, at which time the components of the chromosome uncoil and decondense into a form referred to as chromatin. While in interphase, the chromatin is dispersed throughout the nucleus.

As the cell cycle progresses, cells may replicate their DNA and reenter mitosis, whereupon chromatin coils and condenses back into visible chromosomes once again. This condensation represents a length contraction of some 10,000 times for each chromatin fiber.

Question 2

Chromatin Structure and Nucleosomes

Answer 2

Eukaryotic chromatin has a substantial amount of protein associated with the chromosomal DNA in all phases of the cell cycle. The associated proteins can be categorized as either positively charged histones or less positively charged nonhistone proteins. Histones contain large amounts of the positively charged amino acids lysine and arginine, making it possible for them to bond electrostatically to the negatively charged phosphate groups of nucleotides.

Question 3

The five main types of histones

Answer 3

H1 - Lysine rich 23,000
H2A- slightly lysine rich 14,000
H2B- slightly lysine rich 13,800
H3- Arginine rich 15,300
H4- arginine rich 11,300

Question 4

Chromatin Structure and Nucleosomes /cont./

Answer 4

1. Chromatin consists of some type of repeating unit, each of which protects the DNA from enzymatic cleavage except where any two units are joined. It is the area between units that is attacked and cleaved by the endonuclease.
2. Electron microscopic observations of chromatin have revealed that chromatin fibers are composed of linear arrays of spherical particles (Figure 12.8). The particles are called nucleosomes. These findings conform to the above observation that suggests the existence of repeating units.

Question 5

Chromatin Structure and Nucleosomes /cont./

Answer 5

3. Studies of the chemical association between histone molecules and DNA in the nucleosomes of chromatin show that histones H2A, H2B, H3, and H4 occur as two types of tetramers, (H2A)2 # (H2B)2 and (H3)2 # (H4)2. Roger Kornberg predicted that each repeating nucleosome unit consists of one of each tetramer (creating an octomer) in association with about 200 base pairs of DNA. Such a structure is consistent with previous observations and provides the basis for a model that explains the interaction of histones and DNA in chromatin.

Question 6

Chromatin Structure and Nucleosomes /cont./

Answer 6

When nuclease digestion time is extended, some of the 200 base pairs of DNA are removed from the nucleosome, creating what is called a nucleosome core particle consisting of 147 base pairs. The DNA lost in the prolonged digestion is responsible for linking nucleosomes together. This linker DNA is associated with the fifth histone, H1

● At moderate salt concentrations, H1 is removed - the result is the classic beads-on-
a-string morphology.
● At low salt concentrations, H1 remains
bound - beads associate together into a more compact morphology.

Question 7

Chromatin Structure and Nucleosomes /cont./

Answer 7

Predicting chromatin structure and its condensation into chromosomes. In this model, illustrated in Figure (a) 147-bp length of the 2- nm-diameter DNA molecule coils around an octamer of histones in a left-handed superhelix that completes about 1.7 turns per nucleosome. Each nucleosome, ellipsoidal in shape, measures about 11 nm at its longest point [Figure (a)]. The formation of the nucleosome represents the first level of packing, whereby the DNA helix is reduced to about one-third of its original length by winding around the histones.

Question 8

Chromatin Structure and Nucleosomes /cont./

Answer 8

In the nucleus, the chromatin fiber seldom, if ever, exists in the extended form (that is, as an extended chain of nucleosomes). Instead, the 11-nm-diameter fiber is further packed into a thicker structure, initially called a solenoid, but now referred to as a 30-nm fiber. [Figure (b)]. This thicker structure, which is dependent on the presence of histone H1, consists of numerous nucleosomes coiled around and stacked upon one another, creating a second level of packing. This provides a sixfold increase in compaction of the DNA. It is this structure that is characteristic of an uncoiled chromatin fiber in
interphase of the cell cycle.

Question 9

Hierarchical levels of chromatin packaging in a human chromosome

Answer 9

Interphase nucleus
Solenoid
Nucleosome fiber
Double helix

Question 10

Chromatin Structure and Nucleosomes /cont./

Answer 10

In the transition to the mitotic chromosome, still further compaction must occur. The 30-nm structures are folded into a series of looped domains, which further condense the chromatin fiber into a structure that is 300 nm in diameter [Figure (c)]. These coiled chromatin fibers are then compacted into the chromosome arms that constitute a chromatid, one of the longitudinal subunits of the metaphase chromosome [Figure (d)].

While Figure shows the chromatid arms to be 700 nm in diameter, this value undoubtedly varies among different organisms. At a value of 700 nm, a pair of sister chromatids comprising a chromosome measures about 1400 nm.

Question 11

Chromatin Structure and Nucleosomes /cont./

Answer 11

In the overall transition from a fully extended DNA helix to the extremely condensed status of the mitotic chromosome, a packing ratio (the ratio of DNA length to the length of the structure containing it) of about 500 to 1 must be achieved. In fact, our model accounts for a ratio of only about 50 to 1. Obviously, the larger fiber can be further bent, coiled, and packed to achieve even greater condensation during the formation of a mitotic chromosome.

General model of the association of histones and DNA to form nucleosomes, illustrating the way in which each thickness of fiber may be coiled into a more condensed structure, ultimately producing a metaphase chromosome.

Question 12

Metaphase chromosome

Answer 12

The radial loops are highly compacted and stay anchored to scaffold
The scaffold is formed from the nuclear matrix

Question 13

Two multiprotein complexes help to form and organize metaphase chromosomes

Answer 13

(A) Cohesins tie together the two adjacent sister chromatids in each duplicated chromosome.
(B) Condensins help coil each sister chromatid (in other words, each DNA double helix) into a smaller, more compact structure that can be more easily segregated during mitosis.

Question 14

The alignment of sister chromatids via cohesin

Answer 14

End of s phase to g2 phase (decondensed sister chromatids, arms are cohered)
Beginning of prophase (condensed sister chromatids, arms are cohered)
Middle of prophase (condensed sister chromatids, arms are free)
Anaphase (condensed sister chromatids have separated)

Question 15

Chromatin Remodeling

Answer 15

When present in several levels of compaction within the chromatin fiber, DNA is inaccessible to interaction with other important DNA- binding proteins. For example, the various proteins that function in enzymatic and regulatory roles during the processes of replication and transcription must interact directly with DNA. To accommodate these protein–DNA interactions, chromatin must be induced to change its structure, a process now referred to as chromatin remodeling.

To allow replication and gene expression, chromatin must relax its compact structure and expose regions of DNA to these proteins, and there must also be a mechanism for reversing the process during periods of inactivity. There are unstructured histone tails that are not packed into the folded histone domains within the core of the nucleosomes but instead protrude from it. The importance of histone tails is that they provide potential targets along the chromatin fiber for a variety of chemical modifications that may be linked to genetic functions, including chromatin remodeling and the possible regulation of gene expression.

Question 16

Chromatin Remodeling /cont./

Answer 16

Several chemical modifications are recognized as important to genetic function.

Question 17

Chromatin Remodeling /cont./

Answer 17

One of the most well-studied histone modifications involves acetylation by the action of the enzyme histone acetyltransferase (HAT). The addition of an acetyl group to the positively charged amino group present on the
side chain of the amino acid lysine effectively changes the net charge of the protein by neutralizing the positive charge.

Lysine is in abundance in histones, and geneticists have known for some time that acetylation is linked
to gene activation. It appears that high levels of acetylation open up, or remodel, the chromatin fiber, an effect
that increases in regions of active genes and decreases in inactive regions. In the well-studied example of the inactivation of the X chromosome in mammals, forming a Barr body, histone H4 is known to be greatly underacetylated.

Question 18

Chromatin Remodeling /cont./

Answer 18

During methylation methyl groups can be added to both arginine and lysine residues in histones, and this change
has been correlated with gene activity. Interestingly, while methylation of histones within nucleosomes is often positively correlated with gene activity in eukaryotes, methylation of the nitrogenous base cytosine within polynucleotide chains of DNA, forming 5-methyl cytosine, is usually negatively correlated with gene activity.

Methylation of cytosine occurs most often when the nucleotide cytidylic acid is next to the nucleotide guanylic acid, forming what is called a CpG island. We must conclude, then, that methylation can have a positive or a negative impact on gene activity.

Question 19

Chromatin Remodeling /cont./

Answer 19

Phosphate groups can be added to the hydroxyl groups of the amino acids serine and histidine, introducing a negative charge on the protein. During the cell cycle, increased phosphorylation, particularly of histone H3, is known to occur at characteristic times. Such chemical modification is believed to be related to the cycle of chromatin unfolding and condensation that occurs during and after DNA replication.

It is important to note that the above chemical modifications (acetylation, methylation, and phosphorylation) are all reversible, under the direction of specific enzymes. It is clear that the dynamic forms in which chromatin exists are vitally important to the way that all genetic processes directly involving DNA are performed. We will return to a discussion of the role of chromatin remodeling when we consider the regulation of eukaryotic gene
expression later.

Question 20

Heterochromatin

Answer 20

The whole chromosome is not structurally uniform from end to end. In the early part of the twentieth century, it was observed that some parts of the chromosome remain condensed and stain deeply during interphase, while most parts are partially uncoiled and do not stain. In 1928, the terms euchromatin and heterochromatin were coined to describe the parts of chromosomes that are uncoiled and those that remain condensed, respectively.

Two types of heterochromatin…
Constitutive - always contain heterochromatin
Facultative- regions of euchromatin and heterochromatin

Question 21

Heterochromatin

Answer 21

Subsequent investigation revealed a number of characteristics that distinguish heterochromatin from euchromatin.
1.Heterochromatic areas are genetically inactive because they either lack genes or contain genes that are repressed.

2.Heterochromatin replicates later during the S phase of the cell cycle than does euchromatin.

3.The discovery of heterochromatin provided the first clues that parts of eukaryotic chromosomes do not always encode proteins. For example, one particular heterochromatic region of the chromosome, the telomere, is thought to be involved in maintenance of the chromosome’s structural integrity, and another region, the centromere, is involved in chromosome movement during cell division.

4.The presence of heterochromatin is unique to and characteristic of the genetic material of eukaryotes. In some cases, whole chromosomes are heterochromatic. Ex. mammalian Y chromosome, much of which is genetically inert, Barr body.

5.When certain heterochromatic areas from one chromosome are translocated to a new site on the same or another nonhomologous chromosome, genetically active areas sometimes become genetically inert if they now lie adjacent to the translocated heterochromatin. This influence on existing euchromatin is one example of what is more generally referred to as a position effect. That is, the position of a gene or group of genes relative to all other genetic material may affect their expression.