SB 15: Ch 13.1-13.3 The Eukaryotic Chromosome Pt 1

What is a chromosome composed of?

Each chromosome (or chromatid, in terms of replication) contains one long linear molecule of double stranded DNA

• Shorter molecules recoil faster than longer ones

• Drosophila: longest DNA molecule = largest chromosome

How does DNA compact itself to fit in the cell nucleus?

• Interactions between two protein categories: histones and nonhistone chromosomal proteins

What are chromatin? What is a chromosome in terms of chromatin, what does chromatin have to do with RNA?

• Complexes of DNA, protein, and RNA found in a cell’s nucleus

• Chromosome = a piece of chromatin that contains a single DNA molecule

• Chromatin = 1/3 DNA, 1/3 histones, 1/3 nonhistone proteins + significant amounts of RNA

Histone proteins and core histones

• Small proteins with a preponderance of the basic, positively charged amino acids lysine and arginine

• Strong positive charge = bind to and neutralize negatively charged DNA

• Basic histone proteins are attracted to phosphate groups along DNA backbone

• 5 types of molecules: H1, H2A, H2B, H3, and H4

• H2A, H2B, H3, and H4 = core of nucleosome (DNA packaging unit), core histones

• All 5 appear in chromatin of all euk cells, very similar, changed little throughout evolution

Nonhistone (chromosomal) proteins

• Many different kinds

• Structural role, help package DNA > proteins that form scaffold of chromosome

• Active in replication, DNA Polymerase

• Chromosome segregation, motor proteins of kinetochores move cz along spindle and expedite transport of cz from parent to daughter

• Largest class = facilitate transcription during gene expression, influence when/where/how frequently genes are transcribed

Nucleosomes

• Winding of DNA around histones

• Compaction

• Basic unit of chromatin, exist in interphase cells and cells undergoing mitosis

Condensins

• Protein complexes that form rings around DNA, organize chromosomes into loops

• Condensin loops = greatest form of chromosome condensation in mitosis

• Compaction

Nucleosome structure

• DNA wraps twice around a core made of two copies each of histones H2A, H2B, H3, and H4 (8 total)

• Histone H1 associates with the DNA as it enters and leaves the nucleosome

• X-ray diffraction: DNA bends sharply as it wraps around core histone octamer

  • Only some DNA base sequences can bend

Linker DNA

• Connects one nucleosome with the next

• 40 or so base pairs

Histone chaperones?

What is the scaffold, how does chromosomal DNA appear relative to it?

The scaffold is the structural backbone of chromosomes. Chromosomal DNA appears as long loops anchored in the scaffold.

• Loops = chromosomal compaction

What are condensins, and what is their role in the scaffold?

Protein complexes that form one of the major components of the scaffold. They make the chromatin loops, which is the next step of chromosomal compaction.

• Ring-shaped

• Sit on chromatin by forming around nucleosome-studded DNA so the chromosome fiber passes twice through the ring's interior, making a small loop

How do condensins enlarge a loop?

Some subunits are molecular motors that use ATP hydrolysis to move the condensin relative to the chromatin. This extrudes the loop (makes it larger).

How fast is loop extrusion? How big?

• A single condensin complex can extrude about 1500 bp of chromatin per second.

• Roughly 30 to 400 kb of DNA.

• We don’t know when extrusion stops or what determines where condensin rings first form

Why does loop extrusion create a problem? What enzyme solves this and how?

• Condensin movement relative to chromatin twists the DNA and causes supercoiling. The supercoils would prevent further extrusion.

• DNA topoisomerase II relaxes the supercoils and prevents DNA from tangling in compacted chromosomes.

What are the two major components of chromosomal scaffolds?

Condensin complexes and DNA topoisomerase II.

How did biochemists study the requirements for metaphase chromosome assembly?

• They reconstituted chromosome structure in vitro using protein extracts from the large eggs of Xenopus (African frogs).

• Sperm chromatin incubated with the egg extract disassembles and then reassembles into metaphase chromosome structures.

Which four purified proteins were enough to mimic most steps of metaphase chromosome assembly?

Histones, histone chaperones, condensins, and topoisomerase II.

What do the reconstitution results suggest about mitotic chromosome condensation?

Researchers could mimic most steps of metaphase chromosome assembly

• The major features are nucleosome assembly (histones and histone chaperones) and chromatin loop extrusion (condensins, with help from topoisomerase II).

Why isn't loop formation alone the full story of condensation? What are two proposed explanations for additional compaction, and what is known about them?

• Large, loosely packed loops anchored by condensin don't reach the level of condensation seen in mitotic chromosomes.

1. Nucleosomes within loops interact to form a 300 Å superhelix. This has been seen under the electron microscope, but scientists disagree about whether much chromatin is actually organized this way in cells.

2. Condensin rings interact with each other in the scaffold, bringing loops closer together.

What is Giesma staining, what does it reveal?

• Chromosomes are gently heated, then exposed to Giemsa stain.

• The dye preferentially darkens certain regions, producing alternating dark and light

• The biochemical basis of G bands isn't understood.

  • Most think the bands reflect uneven packaging of loops, determined by the spacing and density of short repetitive DNA sequences along the chromosome.

Why do geneticists think banding patterns are an intrinsic property of each chromosome? What do the patterns reproduceability offer?

• They are highly reproducible from one generation to the next, so they must be determined by the base-pair sequence itself.

• Geneticist designate a gene’s location by its position relative to the bands on the p (short) or q (long) arm of a particular chromosome.

• Diagram of banding pattern = idiogram

What is FISH, what does it allow us to do?

• Fluorescence in situ hybridization

• Solves resolution gap. Karyotypes show the whole genome at low resolution, and whole-genome sequencing is ultra-high resolution

• Allows us to find the locations of specific DNA sequences with respect to the chromosomes in a karyotype.

Steps/basis of FISH

• It uses nucleotide complementarity to locate specific DNA sequences on chromosomes

• Spread and fix metaphase chromosomes on a slide, then gently denature the DNA. Add a heat-denatured fluorescent DNA probe. It hybridizes only to complementary regions, which are seen under a fluorescence microscope.

What does a FISH result with a single-gene probe look like?

Fluorescent spots marking where that gene sits. Sister chromatids are so close together that they show one spot per homolog.

What is SKY, and what are FISH and SKY used for?

SKY is a FISH variation with probes from DNAs scattered along each chromosome, labeled with a unique mix of fluors so all 24 human chromosomes show up as different colors. Both techniques detect chromosomal rearrangements (deletions, duplications, inversions, translocations).

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