Chapter 5 Chromosomes

Chromosomes In order to compact DNA molecules to fit inside a cell, specific proteins interact with DNA Forms a condensed nucleoprotein complex called Chromatin Chromatin structure varies greatly between and within the 3 domains of living organisms

Bacterial Chromatin E. coli DNA is a closed circle Length ≈ 1,600 μm Fits into a cell 0.5 μm in diameter x 1μm long DNA forms a condensed nucleoprotein complex called the nucleoid

Electron micrograph of released E. coli DNA reveals chromatin loops Mostly supercoiled DNA loops with some relaxed loops (caused by nicks) Each loop appears to be insulated from the others Current model proposes supercoiled loops attached to a protein core E. coli estimated to have 400 loops Each loop is topologically independent

Macromolecular crowding and DNA binding proteins are critical contributors to DNA compaction MukB protein Organizes and compacts DNA H-NS (histone-like nucleoid structuring) protein Forms bridges across DNA segments DNA bending proteins Three nucleoid associated proteins bend DNA

Eukaryotic Chromatin Highly condensed structures called chromosomes Germ cells (reproductive cells) have a characteristic number of chromosomes (n) also called the haploid number Somatic cells are diploid (2n)

Mitosis Mitosis – a type of nuclear division that produces two daughter cells with the same number of chromosomes as the parent cell Eukaryotic cells spend only a small fraction of their life cycle in mitosis Remainder is spent in interphase

Interphase chromatin exists in two forms Euchromatin – less condensed Actively transcribed Heterochromatin – more condensed Tends to be located near the nuclear membrane Not actively transcribed

Mitosis a liver cell nucleus

Interphase is broken into 3 phases S phase: DNA replication and histone synthesis Bracketed by 2 gap phases G1 and G2 G1→S→G2→M(mitosis) S, G2 and M tend to be relatively uniform in length for a given cell type G1 is quite variable

The cell cycle of a typical mammalian cell growing in tissue culture with a generation time of 24 hours

Four stages of mitosis Prophase Metaphase Anaphase Telophase

Sister chromatids held together by cohesin Nucleolus disappears Prophase Chromatin condenses Sister chromatids held together by cohesin Nucleolus disappears A mitotic spindle made of microtubules begins to form Chromosome behavior during mitosis in an organism with two pairs of chromosomes

Metaphase Spindle fibers attach to the kinetochore (a region of the centromere) Chromosomes move toward the cell center line

Anaphase Cohesin is cleaved Sister chromatids (now chromosomes) move toward opposite poles

Telophase Spindles disappear Nuclear membrane forms Nucleoli re-form Chromosomes de-condense The cell divides - cytokinesis

Meiosis Used to reduce the chromosome number from 2n to n Meiosis produces gametes with a haploid chromosome number Requires 2 successive nuclear divisions

Meiosis: First Meiotic Division Prophase I Chromatin begins to condense Homologous chromosomes pair up (synapsis) Fully paired chromosomes: bivalents or tetrads Crossing over occurs and generates chiasmata: cross connections between homologous chromosomes

Metaphase I Spindle fibers from one pole connect with one chromosome in a homologous pair Each chromosome moves into the metaphase plate

Anaphase I Homologous chromosomes are pulled apart Each pole has a haploid number of chromosomes

Telophase I The single Spindle dissasembles and two new spindles form Seamless transition between Telophase I and Prophase II No chromosome replication occurs between the 1st and 2nd meiotic divisions

Meiosis: Second Meiotic Division Prophase II Chromosomes begin moving to the midpoint Metaphase II Chromosomes align Anaphase II Centromeres split, chromatids (now considered chromosomes) move to opposite poles Telophase II Chromosomes decondense, nuclear membrane re-forms

Karyotype Cytogenetics: study of the physical appearance of chromosomes Examined during metaphase when chromosomes are most condensed Centromere position determines the chromatid arm lengths Shorter: p arm (petite) Longer: q arm

Chromosomes can be stained to reveal reproducible band patterns A karyotype of a normal human male. (a) The chromosomes as seen in the cell by microscopy

Each chromosome is assigned a number Largest assigned 1 Short arm - p Human chromosome 1 Each chromosome is assigned a number Largest assigned 1 Short arm - p Long arm - q Regions and bands are numbered from the centromere outward

Chromosome pairs are arranged in decreasing order of size Produces an arrangement called a karyotype Human karyotype: 23 pairs of chromosomes 22 pairs of autosomes 1 pair of sex chromosomes

Fluorescence in situ hybridization (FISH) Uses fluorescent dyes bound to specific DNA probes Probes are hybridized to the chromosomes Chromosome painting combines multiple dyes and specific DNA probes to uniquely label each chromosome

The fluorescent in situ hybridization (FISH) technique Spectral karyotyping of human chromosomes. (b) The same chromosomes rearranged so that homologous chromosomes are shown as pairs.

The Nucleosome Multiple levels of chromatin packing First level of organization: interactions between DNA and histones Chromatin contains 5 major classes of histones Each histone has a high percentage of the basic amino acids lysine and arginine

Histones are electrostatically attracted to the negatively charged phosphates of DNA Attraction can be broken by high salt concentration Purified DNA and purified histones mixed together will reconstitute chromatin Extremely well conserved proteins

Uncondensed chromatin from interphase nuclei resemble beads on a string Bead is a nucleoprotein complex called a nucleosome DNA wound around 8 histone protein core Treatment with micrococcal nuclease cleaves DNA in the linker region to release free nucleosomes

”Beads on a string” structure of chromatin Electron micrograph of chromatin. The beadlike nucleosome particles have diameters of approximately 11 nm ”Beads on a string” structure of chromatin

Free nucleosomes can be further digested to the nucleosome core particle made up of 164bp DNA Octameric protein complex: 2 copies each of H2A, H2B, H3 and H4

X-ray crystallography reveals the atomic structure of nucleosome core particles Histone octamer has two-fold symmetry DNA is negatively supercoiled in the nucleosome A-T rich minor grooves tend to contact the histone octamer

Nature of H1 interaction and the core particle is not known When the fifth histone (H1) is present the particle is called the chromatosome 166bp DNA wrapped around octameric histone core held in place by H1

The next level of chromatin organization is not yet resolved 30nm fiber

Condensins and topoisomerase II help stabilize condensed chromosomes Topo II - disentangles DNA from different chromosomes Condensin I - shows DNA dependent ATPase activity that can in vitro introduce positive supercoils into closed circular DNA Condensin II - activity not established All localized along the chromatid long axis

Scaffold model Predicts that non-histone proteins form a central scaffold along the long axis Histone depleted chromosomes have DNA loops attached to a protein scaffold

The Centromere The Centromere Chromosomal site of microtubule attachment Last point of attachment between sister chromatids Centromere DNA consists of repetitive DNA sequence Proteins assemble to form the microtubule attachment site (Kinetochore)

A chromatin fiber in the centromere may fold into a cylindrical coil

The Telomere Telomeres: the chromosome ends X-ray induced deletion experiments generate stable chromosomes with internal deletions, but never end deletions

Chromosome end has 3’ overhang Single strand ranging from 100’s bp in yeast to 1,000’s bp in vertebrates Folds back to form a loop Telomeres are located at the ends of the sister chromatids. Each telomere has a G-rich 3′-overhang

Structure of telomere t- and D-loops

Werner syndrome helicase Protein involved in telomerase formation Defective in Werner syndrome A genetic disease associated with premature aging

Eukaryotic chromosomes lose DNA from their ends during replication Require the enzyme telomerase Failure to replace telomeres is potentially responsible for finite number of divisions of cells in culture Most cancer cells have telomerase