Chapter 10: Molecular Structure of Chromosomes

What are Chromosomes?

Structures that contain the genetic material

What complexes are Chromosomes?

Of DNA and Proteins

What is Genome?

Comprises all the genetic material that an organism possesses

The genome of bacteria is?

Typically a single circular chromosome

The genome of eukaryotes is?

Refers to one complete set of nuclear chromosomes

What other genome characteristics do Eukaryotes possess?

Possess a mitochondrial genome

What genome characteristic do plants have?

Have a chloroplast genome

What is the main function of genetic material?

To store the information required to produce the traits of an organism

How does genetic material store information required to produce the traits of an organism?

Accomplished via protein-encoding genes

What are four reasons DNA sequences are necessary?

1. Synthesis of RNA and cellular proteins

2. Replication of chromosomes

3. Proper segregation of chromosomes

4. Compaction of chromosomes (So they can fit within living cells)

Eukaryotic species contain?

One or more sets of chromosomes

What are the sets of chromosomes in Eukaryotic species composed of?

1. Each set is composed of several different linear chromosomes

2. Humans have 2 sets of 23 chromosomes

Each chromosomes contains?

A single, linear molecule of DNA

What are 2 characteristics of each chromosome?

1. Typically tens to hundreds of millions of base pairs

2. Typically a few hundred to several thousand genes

What does a Chromosome look like?

What is an example of simpler eukaryotes?

Yeast

What is the gene structure of yeast?

•Relatively short (e.g., several hundred bp long)

What are 2 examples of more complex eukaryotes?

1. Mammals

2. Flowering plants

What is the gene structure of more complex eukaryotes?

1. Genes are long

2. They tend to have many introns (noncoding intervening sequences)

3. Intron lengths from less than 100 to more than 10,000 bp

What are 3 types of DNA sequences are required for chromosomal replication and segregation?

1. Origins of Replication

2. Centromeres

3. Telomeres

What are Origins of Replication?

1. Chromosomal sites necessary to initiate DNA replication

2. Eukaryotic chromosomes contain many origins

What are Centromeres?

Regions that play a role in segregation of chromosomes

What are Telomeres?

1. Specialized regions at the ends of chromosomes

2. Important in replication and for stability

What are four key features of the Organization of Eukaryotic Chromosomes?

1. Eukaryotic chromosomes are usually linear.

2. Eukaryotic chromosomes occur in sets. Many species are diploid, which means that somatic cells contain 2 sets of chromosomes.

3. A typical chromosome is tens of millions to hundreds of millions of base pairs in length.

4. Genes are interspersed throughout the chromosomes. A typical chromosome contains between a few hundred and several thousand different genes.

What are the four chromosomal features of Organization of Eukaryotic Chromosomes?

1. Each chromosome contains many origins of replication that are interspersed about every 100,000 base pairs.

2. Each chromosome contains a centromere that forms a recognition site for the kinetochore proteins; required for chromosome sorting during mitosis and meiosis.

3. Telomeres contain specialized sequences located at both ends of the linear chromosome.

4. Repetitive sequences are commonly found near centromeric and telomeric regions, but they may also be interspersed throughout the chromosome.

The total amount of DNA in eukaryotic species is typically?

Much greater than that in bacterial cells

The total amount of DNA in eukaryotic species contains?

Many more genes

Eukaryotic genomes vary substantially in?

Size

The sizes for Eukaryotic Genomes in many cases?

This variation is not related to the complexity of the species

What are 2 examples of the variation in sizes for Eukaryotic Genomes that is not related to the complexity of the species?

1. For example, there is a two-fold difference in the size of the genome in two closely related salamander species

2. The difference in the size of the genome is not because of extra genes

3. Rather, the accumulation of repetitive DNA sequences

4. These do not encode proteins

What do Genome sizes for nucleotide base pairs per haploid genome, Plethodon richmondi and Plethodon Iarselli look like?

What is Sequency complexity?

Refers to the number of times a particular base sequence appears in the genome

What are the 3 types of repetitive sequences?

1. Unique or non-repetitive

2. Moderately repetitive

3. Highly repetitive

What are Unique or non-repetitive sequences?

1. Found once or a few times in the genome

2. Includes protein-encoding genes as well as intergenic regions

3. In humans, make up roughly 41% of the genome

What are Moderately Repetitive Sequences?

Found a few hundred to several thousand times

What are examples of moderately repetitive sequences?

1. Genes for rRNA and histones

2. Sequences that regulate gene expression and translation

3. Transposable elements

What are Highly repetitive sequences?

1. Found tens of thousands to millions of times

2. Each copy is relatively short (a few nucleotides to several hundred in length)

What are characteristics of Highly Repetitive Sequences?

1. Some sequences are interspersed throughout the genome

2. Other sequences are clustered together in tandem arrays

What is an example of Some sequences are interspersed throughout the genome?

Alu family in humans

1. Approximately 300 bp long

2. Represents 10% of human genome

3. Found every 5000-6000 bp

What is an example of Other sequences are clustered together in tandem arrays?

AATAT and AATATAT sequences in Drosophila

1. These are commonly found in the centromeric regions

What do Classes of DNA sequences look like?

What is a nucleosome?

The repeating structural unit within eukaryotic chromatin

What are characteristics of Nucleosome?

1. Composed of a double-stranded segment of DNA wrapped around an octamer of histone proteins

2. A histone octamer is composed of two copies each of four different histone proteins

3. 146 bp of DNA make 1.65 negative superhelical turns around the octamer

What does Nucleosomes showing core histone proteins look like?

What are characteristics of Histone Proteins?

1. Basic

2. Contain many positively-charged amino acids (Lysine and Arginine)

3. These bind to the negatively-charged phosphates along the DNA backbone

4. Have a globular domain and a flexible, charged amino terminus or ‘tail’

What are the five types of Histones?

1. H2A

2. H2B

3. H3

4. H4

5. H1

Which histones are core histones?

1. H2A

2. H2B

3. H3

4. H4

How many of each core histone make up the octamer?

Two of each

Which histone is called the linker histone?

H1

Why is H1 a linker histone?

1. Binds to DNA in the linker region

2. Less tightly bound to DNA than core histones

3. Helps to organize adjacent nucleosomes

What does the Molecular model for nucleosome structure look like?

What do nucleosomes showing linker histones and nonhistone proteins look like?

Who proposed in 1745 the model of nucleosome structure?

Roger Kornberg

What did Kornberg base his proposal on?

Various observations about chromatin

What were the 3 observations about chromatin that Kornberg made?

1. Biochemical experiments

2. X-ray diffraction studies

3. Electron microscopy images

Who tested Kornberg’s Model?

Markus Noll

How did Markus Noll test Kornberg’s model?

1. Digest DNA with the enzyme DNase I

2. Accurately measure the molecular mass of the resulting DNA fragments using gel electrophoresis

3. The rationale is that the linker DNA is more accessible to DNase I than the DNA bound to core histones (Thus, the cuts made by DNase I should occur in the linker DNA)

What is Noll’s experiment test?

The beads-on-a-string model of chromatin structure

If Noll’s model was correct, DNase 1 should?

1. Preferentially cut DNA in the linker region

2. Thereby producing DNA pieces that are about 200 bp long

What was the process of Noll’s Experiment?

1. Incubate the nuclei with low, medium, and high concentrations of DNase I. The conceptual level illustrates a low DNase I concentration.

2. Isolate the DNA. This involves dissolving the nuclear membrane with detergent and treating the sample with organic solvent phenol.

3. Load the DNA into a well of an agarose gel and run the gel to separate the DNA pieces according to size. On this gel, also load DNA fragments of known molecular mass (marker lane).

4. Visualize the DNA fragments by staining the DNA with ethidium bromide, a dye that binds to DNA and is fluorescent when excited by UV light.

What did Noll’s Experiment look like?

What was the data of Noll’s Experiment

Note: The marker lane is omitted from this drawing.

In Noll’s Experiment, At low or medium concentrations?

DNase I did not cut at every linker region

In Noll’s Experiment, At low or medium DNase I concentration?

Longer pieces were observed in multiples of 200 bp

In Noll’s Experiment, At high concentration?

All chromosomal DNA digested into fragments that are ~ 200 bp in length

Nucleosomes join to form?

A 30 nm Fiber

Nucleosomes associate with each other to form?

A more compact structure termed the 30 nm fiber

What plays a role in the compaction of Nucleosomes?

Histone H1

How does histone H1 play a role in the compaction of nucleosome structure?

1. At moderate salt concentrations, H1 is removed (The result is the classic beads-on-a-string morphology)

2. At low salt concentrations, H1 remains bound (Beads associate together into a more compact morphology)

What does H1 histone not bound-beads on a string look like?

What does H1 histone bound to linker region-nucleosomes more compact look like?

What does the 30 nm fiber shorten?

The total length of DNA another seven-fold

The 30 nm fiber structure has proven difficult to determine?

1. The DNA conformation may be substantially altered when extracted from living cells

2. Zigzag model has been proposed as a model for the structure of the 30 nm fiber

What does the micrograph of a 30 nm fiber and the zigzag model look like?

What is Third level of compaction?

30nm fiber folds into loop domains

What is CCCTC binding factor (CTCF)?

Binds to 3 regularly spaced repeats of the sequence CCCTC

How is a loop formed?

1. Two different CTCFs bind to the DNA and then bind to each other to form a loop

2. A second mechanism of loop formation is carried out by SMC proteins

3. An SMC protein forms a dimer that can wrap itself around two DNA segments and form a loop

4. In addition, SMCs may wrap around sites that are bound by CTCF dimers

What does the formation of a loop look like?

Each chromosome in the cell nucleus can be found in?

A discrete chromosome territory

How is each chromosome in the cell nucleus known to be location in a discrete chromosome territory?

In the studies by Thomas and Christoph Cremer and others through fluorescent staining in which each chromosome is shown in a different color

What do Metaphase Chromosomes look like?

What do Chromosomes in the cell nucleus during interphase look like?

The compaction level of interphase chromosomes is not?

Completely Uniform

What is Heterochromatin?

1. Tightly compacted regions of chromosomes

2. Transcriptionally inactive (in general)

3. Loop domains compacted even further

What is Euchromatin?

1. Less condensed regions of chromosomes

2. Transcriptionally active

3. The 30 nm fiber forms loop domains

What are the 2 types of Heterochromatin?

1. Constitutive heterochromatin

2. Facultative heterochromatin

What is Constitutive Heterochromatin?

1. Regions that are always heterochromatic

2. Permanently inactive with regard to transcription

3. Usually contain highly repetitive sequences

What is Facilitative Heterochromatin?

Regions that can interconvert between euchromatin and heterochromatin

What does Constitutive/Facultative heterochromatin look like?

How does the structure of eukaryotic chromosomes change during cell division?

1. As cells enter M phase, the level of compaction changes dramatically

2. By the end of prophase, sister chromatids are entirely condensed

3. Two parallel chromatids have an overall diameter of 1,400 nm and are much shorter than in interphase

4. These highly condensed metaphase chromosomes undergo little gene transcription

What does Figure 10.23 (a. Nucleosomes (“beads on a string” and b. 30 nm fiber) look like?

What does Figure 10.23 (c. loop domains and d. metaphase chromosome) look like?

What are four characteristics of Metaphase Chromosomes?

1. Controversy regarding whether or not nonhistone proteins form a scaffold to organize metaphase chromosomes

2. When a metaphase chromosome is treated with salt to remove histones, loops are still attached to a darkly staining scaffold

3. Scaffold appears to contain SMC proteins

4. However some researchers are not convinced a protein scaffold plays a major role in metaphase chromosome organization

What does a Metaphase chromosome look like?

What does Metaphase chromosome treated with high salt to remove histone proteins look like?

Condensin and Cohesin promote?

The Formation of Metaphase Chromosomes

Two multiprotein complexes help to form and organize?

Metaphase chromosomes

What are 2 examples of two multiprotein complexes?

1. Condensin

2. Cohesin

What is Condensin?

Plays a critical role in chromosome condensation

What is Cohesin?

Plays a critical role in sister chromatid alignment