Lecture 10: DNA Packaging and Meiosis

• The MFM II presentation covers DNA packaging and meiosis.

DNA Packaging and Key Players

• DNA packaging involves histones, nucleosomes, and chromatin.
• Histones are responsible for the first level of chromatin packing and nucleosome formation.
• Nucleosomes convert DNA molecules into chromatin fiber and are the organizing unit of chromatin.
• Nucleosomes are a complex of eight histone molecules (two copies each of H2A, H2B, H3, and H4).
• DNA wraps around the nucleosome, nucleosomes pack into solenoids, and solenoids pack to form chromatin.
• Chromatin is a complex of histone and nonhistone chromosomal proteins with nuclear DNA.
• Each human cell contains 23 pairs of chromosomes (46 total).
• Chromosomes package themselves into tightly wound, neatly arranged strands of DNA in preparation for cell division.
• DNA molecules are large; the total DNA from a single cell would cover over 6ft if unpacked.
• Histones are responsible for DNA packing and are key proteins
• Nucleosomes are the basic structural units of chromatin

Structural Hierarchy: DNA Wrapping of Histones

• Chromatin is a double-stranded helical structure of DNA at the simplest level.
• DNA complexes with histones to form nucleosomes.
• Each nucleosome has eight histone proteins around which DNA wraps about 1.65 times.
• Chromatosome includes a nucleosome plus the H1 histone.
• Nucleosomes fold to produce a 30-nm fiber.
• The 30-nm fibers compress and fold to produce a 250-nm-wide fiber.
• Tight coiling of the 250-nm fiber produces the chromatid of a chromosome.

Chromatin Regulation Mechanisms

• Histone proteins and DNA nucleotides can be modified.
• Highly methylated DNA regions with deacetylated histones are tightly coiled and transcriptionally inactive.
• Alterations can be inherited from parent to offspring.
• Epigenetic changes to the chromatin may result from development, environmental chemicals, drugs, aging and diet.
• Epigenetic changes may result in cancer, autoimmune disease, mental disorders and diabetes.
• DNA methylation and modification of histone tails alter the spacing of nucleosomes and change gene expression.
• DNA sequence is not altered; the pattern of gene expression is passed to the next generation.

Histone Modification Types

• DNA packaging around histone proteins, as well as chemical modifications to the DNA or proteins, can alter gene expression.
• Acetylation of lysine can reduce the affinity of the tails for adjacent nucleosomes, loosening the chromatin structure and allowing access to certain nuclear proteins.
• Histone acetyltransferases add an acetyl group to lysine residues to the tail of the histones.
• Histone deacetylases remove the acetyl groups from histones.
• Methylation results in tight packaging and gene silencing
• Acetylation results in looser packaging and gene activation
• Phosphorylation results in structural changes and cellular signalling

Mitosis

• Mitosis occurs in somatic cells and produces 2 diploid (2n) cells.
• A diploid cell with 2N becomes a diploid cell with 4N following replication in the S phase.
• Mitotic division of the 4N cell results in 2 diploid daughter cells that each contain 2N.
• Somatic cells are genetically identical to each other and to the original parent cell.
• Mitosis is a single cell division that is purposed for growth, repair, and asexual reproduction.

Meiosis

• Meiosis occurs in germ cells and produces 4 haploid (n) cells.
• Gametes are genetically unique from each other and the original parent cell.
• Meiosis involves two sequential cell divisions and is purposed for sexual reproduction and genetic diversity.

Comparative Analysis: Mitosis vs. Meiosis

• Mitosis occurs in somatic cells; products are genetically identical diploid cells; it involves a single cell division and is purposed for growth, repair, and asexual reproduction.
• Meiosis occurs in germ cells; products are genetically unique haploid gametes; it involves two sequential cell divisions and is purposed for sexual reproduction and genetic diversity.

Meiosis: Detailed Molecular Processes

• Key molecular events include the pairing of homologous chromosomes and recombination.
• Synapsis, via the synaptonemal complex, involves paired homologous chromosomes becoming intimately associated with one another.
• Alignment of genetic material occurs as DNA sequences on one chromosome locate corresponding sequences on the homologous chromosome.
• Recombination facilitates in crossing over, genetic material exchange, and the formation of chiasmata at sites where recombination has occurred.
• Meiosis is a mechanism of genetic diversity.

Meiosis I

• Prophase 1 involves homologous chromosome pairing.
• Metaphase I involves chromosome alignment.
• Anaphase I involves chromosome separation.
• Telophase 1 is cell division.
• Cells undergo DNA replication and form duplicate chromosomes during interphase 1.
• Chromatin strands coil and condense during prophase 1.
• Synapsis is where homologous chromosomes pair up and exchange fragments (crossing over), forming a tetrad with 4 chromatids and chiasmata formation.
• Attachment occurs between chromosomes, which move toward the equatorial plane, and the spindle begins to form in the cytoplasm as the nuclear envelope disappears.
• Completion of the spindle formation occurs during metaphase 1.
• Bivalents (still attached at the chiasmata) align at the equatorial plane.
• Tetrads line up on the equatorial plate and kinetochores of each sister chromatid pair are associated with microtubules from the same pole.
• Anaphase 1 allows each sister chromatid pair to migrate to an opposite pole
• Only half of the original number of chromosomes migrate toward each pole.
• One member of each pair of autosomes and one of the sex chromosome.
• Chromosomes uncoil slightly and a new nuclear membrane begins to form
• 2 haploid daughter cells duplicated chromosomes
• Cells are genetically different

Meiosis II

• Meiosis II is similar to mitotic division and separates sister chromatids.
• In mitosis, chromosomes make an exact replica of themselves and split evenly, but during meiosis, homologous chromosomes line up in groups of four in bundles called tetrads, then swap places and cross over to the other chromosome.
• Chromosomes thicken as they coil, the nuclear membrane disappears, and new spindle fibers form during prophase II.
• Spindle fibers pull the chromosomes into alignment at the equatorial plane during metaphase II.
• Two sister chromatids of each chromosome are not genetically identical because of crossing over in Meiosis I, and kinetochores of sister chromatids are attached to microtubules.
• During anaphase II, centromeres split and each carries a single chromatid toward the pole of the cell, separating chromatids.
• Chromosomes uncoil and new nuclear membranes form around each group of chromosomes, and the 4 daughter cells are genetically distinct from one another and the parent cell.

Gametogenesis: Spermatogenesis

• Spermatogenesis is a continuous process in mature males involving seminiferous tubules of testes that contain spermatogonia (diploid cells) and produces millions of sperm cells.
• Spermatogonia undergo several mitotic divisions and produce 1º spermatocytes (23 ds chromosomes).
• 1º spermatocytes undergo meiosis I and produce 2º spermatocytes.
• 2º spermatocytes undergo Meiosis II and produce a pair of spermatids (23 ss chromosomes).
• Spermatids lose most of their cytoplasm and develop tails for swimming, becoming mature sperm cells.
• Meiosis and cytodifferentiation leads to mature sperm

Gametogenesis: Oogenesis

• Oogenesis involves a limited number of potential gametes that are arrested at different developmental stages and involves asymmetric cell division and polar body formation.
• Females undergo meiosis and cytodifferentiation to mature ovum
• Spermatogenesis is constantly recurring; much of oogenesis is completed before birth
• The Diploid oogonia divide mitotically to produce 1º oocytes by the 3rd month of fetal development.
• 1º oocytes are arrested in prophase I.
• At puberty, 1º oocyte undergoes meiosis I to produce a secondary oocyte and a polar body.
• The 2º oocyte is arrested in metaphase II until fertilization
• Once fertilized it completes meiosis II and the second polar body is released.

Genetic Diversity and Variation

• Genetic diversity is influenced by homologus recombination, corssing over, genetic material exchange and increased genetic combinations.
• Independent assortment is also a factor because of the random chromosome orientation and the exponential increase in genetic possibilities.
• 2^n possible combinations

Homologous Recombination and Genetic Diversity

• Homologous chromosomes line up in groups of four in bundles called tetrads
• Homologous chromosomes have the same types of genes, genetically diverse offspring are produced.
• Pieces of the chromosomes swap places and cross over to the other chromosome; the chromosomes must break apart for this recombination to happen and genetic diversity.
• DNA-repair proteins fix these chromosomal breaks that occur during cell division by rejoining (or ligating) the chromosomes.
• Chiasmata are points where homologous chromosomes exchange genetic material.
• Synaptonemal Complex helps align duplicated homolog pairs.

Independent Assortment

• Homologous chromosomes line up independently during Metaphase I
• Paternal and Maternal homolog can line up in many different combinations in Metaphase I
• Maternal and paternal chromosomes are not always oriented on the same side
• Homologs segrate and each daughter cell has a different combination
• Independent Assortment can lead to many possible gametes.

Independent Assortment and Increased Diversity

• Children that share the same parents are genetically different.
• Exceptions exist with identical twins
• Genetic combonations in gametes relies on number of chromosomes
• Human (23 chromosomes) can have 8,388,608 possible combonations of gametes

Meiosis Is Not Flawless

• Nondisjunction occurs
• Homologs can fail to seperate (trisomy 21) or (monosomy X)
• Some haploid cells lack a chromosome and other may have more than one copy
• Gametes result in abnormal embryos
• Germline abnormality (translocated, deleted, or flipped [inverted]), results in every cell in the zygote being affected.

Clinical Implications

• Abnormal meiosis results in non-disjunction which causes chromosomal abnormalities
• Chromosomal abnormalities are responsible for: Down Syndrome, Turner Syndrome and Klinefelter Syndrome.
• There are genetic counseling considerations, risk assessment, inhertiance analysis and prenatal screening available.

Diagnostic Techniques

• Can detect genetic abnormalities
• Techniques include: Karyotyping, FISH, Chromosomal Microarray, Next Generation Sequencing
• Some techniques have limitations
• A translocation of trisomy 21 is an example.

Cytogenetics and Abnormalities

• Cytogenetics is the study of chromosomes and their abnormalities
• Aberration to the number and structure of chromosomes
• Can be result of spontaneous abortions (50% in 1st trimester and 20% in 2nd trimester)
• Can cause of for intellectual disability and pregnancy loss.
• Molecular Techniques can identify deletions of small regions of chromosomes, genes that contribute to phenotypes of cytogenetic syndromes as well as DNA polymorphism in parent and off-spring.

Classification of Chromosomes

• Use size and shape
• Shape is location of centromere (Acrocentric, Metacentric, Submetacentric)
• Short arm is p
• Long arm is q
• Bands are numbered and are used for classification

Chromatin forms

• Heterochromatin is highly condensed with 10% of an interphase chromosome with locations at the centromere/telomeres with lacking expressed genes
• Euchromatin is less condenses occupying 90% of an interphase chromsome as well as have higher levels of gene expression
• Heterochromatin contains inappropriate packing and can cause disease

Karyotype Characteristics

• Chromosomes are easily visualized during metaphase of mitosis.
• Two chromosomes are sex chromsomes; the 1-22 are termed autosomes.
• Acrocentric Chromosomes feature tiny p-arms (13-15; 21-22)

Tissues for Studying Chromosomes

• Study using tissue that can be stimulated to undergo cell division in vitro.
• Types of tissues include: Amniotic fluid, Peripheral blood (lymphocytes), Skin (fibroblasts) and Bone marrow

Chromosomal Banding and Identification

• Treatment of chromosomes for identify individual chromosomes/ structural abnormalities.
• Technique includes: Quinacrine, Giemsa, Reverse, C and NOR banding
• Chromosomal location of a gene.
• Increases number of observable bands to the chromosomes

Fluorescence In Situ Hybridization and Labeling

• Labeled for a segment of chromosomes
• Can have probes hybridized with microscope for testing.
• Prenatal/ Deletion test for abnormalities.
• A fluorescent DNA probe to bind specific gene sites is used

Other Techniques for Genomes

• Comparative/ spectral genome used for identification by colors and chromosomes.
• Detectes: Loss or duplication, location, cells types, rearrangement.
• Can be limited to CGH and not balanced abnormalities.
• Not used as much in the clinic