Cell Division & Human Genetic Diversity (Lecture Notes) PDF

Cell division & the human genetic diversity Zsolt Fábián M.D., Ph.D., Dr. Habil. 1  Cell division & the human genetic diversity Lesson 1 ‐ Cell division Zsolt Fábián M.D., Ph.D., Dr. Habil....

Cell division & the human genetic diversity Zsolt Fábián M.D., Ph.D., Dr. Habil. 1  Cell division & the human genetic diversity Lesson 1 ‐ Cell division Zsolt Fábián M.D., Ph.D., Dr. Habil. 2  The cell cycle Life stages of the cell t Cell division is the process by which one cell divides to produce two daughter cells Cells must replicate and properly distribute chromosomes The events that lead to formation of two daughter cells is called the cell cycle The principles of cell division are conserved in all kingdoms of life 3  The cell cycle Life stages of the cell S ‐ DNA and chromosomes replicate G2 ‐ Growth, metabolism, preparation for M G1 ‐ Growth and metabolism M ‐ The cell divides and chromosomes segregate into daughter cells Craig et al., Molecular Biology ‐ Principles of Genome Function, 2e, Oxford University Press, (2014) 4  The cell cycle Life stages of the cell 1. Reproduction (single‐cell organisms) 2. Growth and development (multi‐cellular organisms) 3. Renewal and repair Craig et al., Molecular Biology ‐ Principles of Genome Function, 2e, Oxford University Press, (2014) 5  The cell cycle DNA content during the cell cycle DNA content number (c) M G2 2 The number of copies of S DNA double helices of DNA content each chromosome in a cell G1 G1 1 The c number  2c during G1 phase  doubles during S phase  4c during G2 phase t  halved during M phase 6  The cell cycle DNA content during the cell cycle Ploidy number (n) The number of complete sets of chromosomes in a cell 2n throughout the cell cycle Homologous Chromosomes Pairs of nearly identical chromosomes One maternal one paternal In a single chromosome, sister chromatids are essentially identical to each other since the DNA in one chromatid was used as a template to synthesize the DNA in the other chromatid. See DNA replication later. The only exception are mutations in the new DNA molecule due to very rare DNA replication errors. Homologous chromosomes on the other hand are very similar in sequence to each other, but not identical. Since the human genome varies by about 0.1% between individuals, homologous chromosomes coming from different people (the mother and the father) would be expected to be about 0.1% different on average. In the eukaryotic cell cycle, DNA replication (synthesis) occurs during S phase. M phase is mitosis when cell division occurs. G1 and G2 are the first and second gap phases. In G1 phase cells are 2n, 2c. In other words, there are 2 copies of each chromosome (2n), and each chromosome has a single copy of the DNA double helix, so there are 2 total copies (2c). During S phase the DNA content doubles and in G2 phase each chromosome has 2 copies o the DNA double helix, so during G2 cells are 2n and 4c. During mitosis, the sister chromatids are pulled apart. The moment they separate 7 they are considered to be individual chromosomes. Each will segregate into 1 daughter cell during cell division, so each daughter cell will return to being 2n, 2c as the new cell enters the next G1 phase. In humans the vast majority of cells are diploid. The only haploid cells are the gametes (sperm and eggs) which are formed through Meiosis. 7  The cell cycle DNA content during the cell cycle Ploidy number (n) The number of complete sets of chromosomes in a cell 2n throughout the cell cycle Homologous Chromosomes Pairs of nearly identical chromosomes One maternal one paternal In a single chromosome, sister chromatids are essentially identical to each other since the DNA in one chromatid was used as a template to synthesize the DNA in the other chromatid. See DNA replication later. The only exception are mutations in the new DNA molecule due to very rare DNA replication errors. Homologous chromosomes on the other hand are very similar in sequence to each other, but not identical. Since the human genome varies by about 0.1% between individuals, homologous chromosomes coming from different people (the mother and the father) would be expected to be about 0.1% different on average. In the eukaryotic cell cycle, DNA replication (synthesis) occurs during S phase. M phase is mitosis when cell division occurs. G1 and G2 are the first and second gap phases. In G1 phase cells are 2n, 2c. In other words, there are 2 copies of each chromosome (2n), and each chromosome has a single copy of the DNA double helix, so there are 2 total copies (2c). During S phase the DNA content doubles and in G2 phase each chromosome has 2 copies o the DNA double helix, so during G2 cells are 2n and 4c. During mitosis, the sister chromatids are pulled apart. The moment they separate 8 they are considered to be individual chromosomes. Each will segregate into 1 daughter cell during cell division, so each daughter cell will return to being 2n, 2c as the new cell enters the next G1 phase. In humans the vast majority of cells are diploid. The only haploid cells are the gametes (sperm and eggs) which are formed through Meiosis. 8  The cell cycle Mitosis Entering mitosis from G2 phase n=2, c=4 each homologous chromosome pair: – 1 maternal, 1 paternal – NOT necessarily identical each chromosome – 2 identical sister chromatids During Mitosis each homolog behaves independently sister chromatids are pulled apart each daughter cell receives one identical chromatid from every chromosome Chromatid becomes the chromosome in the daughter cell Daughter cells are genetically identical n=2, c=2 See Read & Donnai pp. 34‐37 for a brief review of mitosis and meiosis Recall: Sister chromatids (within a single chromosome) are identical to each other (with the exception of very rare DNA replication errors). On the other hand, homologous chromosomes, while having very similar sequences, are not identical to each other – one copy is maternal, one copy is paternal – and different alleles may be inherited from each parent. So, the fact that the homolog's act independently of each other is important in generating genetically identical daughter cells. Each daughter cell will receive one identical chromatid from each of the homologs of every chromosome pair. 9  The cell cycle Mitosis ‐ prophase Craig et al., Molecular Biology ‐ Principles of Genome Function, 2e, Oxford University Press, (2014)  During prophase, separate condensed chromosomes are clearly apparent  Condensin is involved in chromosome compaction, but it is not clear how, although functional ATPase domains are required  It is thought that condensin associates with distant regions of chromatin and then brings them together  Loss of condensin does not lead to a complete loss of compaction, so other factors are probably involved too  The nuclear envelope breaks down at the end of prophase, so microtubules can access chromosomes – this is the start of prometaphase 10  The cell cycle Mitosis ‐ prometaphase Craig et al., Molecular Biology ‐ Principles of Genome Function, 2e, Oxford University Press, (2014)  Prometaphase ends when all chromosomes are properly attached to spindle microtubules  Microtubules interact with chromosomes via the kinetochore, a protein complex assembled on the centromere  Kinetochores also associate with motor proteins that move chromosomes on microtubules  Kinetochores ensure correct attachment and orientation of chromosomes on the spindle – each sister chromatid must attach to microtubules from opposite poles  Kinetochore proteins are highly conserved, but kinetochores vary in size – yeast kinetochores bind a single microtubule, animal kinetochores can bind more than 20  Kinetochores are very large (>5MDa for a simple yeast kinetochore).  More than 80 proteins have been so far identified, most of which have unknown functions  Kinetochore proteins are denoted as inner or outer kinetochore proteins.  Many kinetochore proteins are called CENP‐X (where the X can be A though U) for centromere protein  CENP‐A, a histone H3 variant found at the centromere, is an inner kinetochore protein 11  The cell cycle Mitosis ‐ prometaphase Craig et al., Molecular Biology ‐ Principles of Genome Function, 2e, Oxford University Press, (2014)  Kinetochores on sister chromatids must associate with spindles from opposite poles in order to segregate properly  This is amphitelic attachment, or bi‐orientation  Correct attachment creates tension, which stabilizes the kinetochore‐microtubule interaction and recruits more microtubules  Initial attachment to kinetochores by microtubules is random, so incorrect attachments can form  Incorrect attachments do not generate tension, and so are destabilized by aurora protein kinase  The kinetochore then releases the microtubule and waits for another random attachment – in this way, only correct attachments persist 12  The cell cycle Mitosis ‐ prometaphase Craig et al., Molecular Biology ‐ Principles of Genome Function, 2e, Oxford University Press, (2014)  Persistence of incorrect microtubule‐kinetochore attachments leads to chromosome missegregation and can cause cell death  Incorrect attachments are destablized by aurora kinase, so if aurora kinase is missing or inactive (as in b), missegregation occurs  Incorrect attachments lead to activation of the spindle checkpoint pathway – this inhibits further progression through mitosis until the incorrect attachments are corrected  At the end of prometaphase, all chromosomes are correctly attached and held under tension at the spindle midzone – the cell is now at metaphase 13  The cell cycle Mitosis ‐ metaphase Craig et al., Molecular Biology ‐ Principles of Genome Function, 2e, Oxford University Press, (2014)  At metaphase, sisters are held together under tension as the sisters are attached to spindles from opposite poles.  Animal spindles have filaments called microtubules. Centrosomes are also present, at the poles of the cell  Microtubules nucleate on the centrosome and attach at the other end to different targets, such as a chromosome, another microtubule, or the cell membrane  Microtubules that associate with chromosomes are called kinetochore microtubules. Polar microtubules interact with microtubules from the other pole and astral microtubules extend to the cell periphery  Microtubules change in length throughout their lifespan, and movement involving microtubules is mediated by motor proteins 14  The cell cycle Mitosis ‐ metaphase Craig et al., Molecular Biology ‐ Principles of Genome Function, 2e, Oxford University Press, (2014)  Cells with aberrant numbers of centrosomes can arise: monopolar and multipolar spindles can’t separate chromosomes accurately (e.g., b)  Multipolar spindles are often seen in cancer cells 15  The cell cycle Mitosis ‐ anaphase Craig et al., Molecular Biology ‐ Principles of Genome Function, 2e, Oxford University Press, (2014)  At anaphase, cohesin is cleaved allowing sisters to be pulled apart 16  The cell cycle Mitosis – Spindle checkpoint Craig et al., Molecular Biology ‐ Principles of Genome Function, 2e, Oxford University Press, (2014)  Unattached kinetochores activate the spindle checkpoint  This activation causes proteins to bind and inhibit Cdc20, which inactivates APCCdc20  Thus, so long as there are unattached kinetochores, the cell cannot enter anaphase  The spindle checkpoint is essential – mutants in spindle checkpoint genes are lethal  A faulty spindle checkpoint is implicated in cancer ‐ cancer cells often have aneuploidy (the incorrect number of chromosomes) as a result of mis‐segregation 17  The cell cycle Mitosis ‐ anaphase Craig et al., Molecular Biology ‐ Principles of Genome Function, 2e, Oxford University Press, (2014)  In anaphase, sister chromatids separate and travel to opposite poles of the cell  Anaphase is a point of no return – any mistakes in chromosome‐microtubule attachments must be corrected before anaphase or daughter cells will inherit incorrect numbers of chromosomes  After cohesin has been removed, anaphase proceeds in two stages: In anaphase A, sister chromatid move towards their respective poles In anaphase B, spindle poles move apart from one another (mediated by motor proteins) 18  The cell cycle Mitosis ‐ telophase Credit: Michael Abbey (Science photo library) In telophase, the spindle disassembles Chromosomes become decondensed Nuclear envelopes form around the two sets of chromosomes 19  The cell cycle Cytokinesis Craig et al., Molecular Biology ‐ Principles of Genome Function, 2e, Oxford University Press, (2014)  Cells undergo cytokinesis after mitosis – this requires a new cell membrane to be formed between the daughter nuclei  In animal cells, a contractile ring (the actomyosin ring) binds to the membrane at the cleavage furrow  The contractile ring has actin filaments and motor proteins (myosin)  Myosin pulls the actin filaments together, contracting the ring until only a small hole is left, which is then filled with membrane 20  The cell cycle Cell cycle checkpoints Craig et al., Molecular Biology ‐ Principles of Genome Function, 2e, Oxford University Press, (2014) Checkpoints are quality control mechanisms that halt the cell cycle when things go wrong Checkpoints arrest the cell cycle and stimulate mechanisms to correct the problem In eukaryotes, arrest can occur at the G1‐S transition, S phase, the G2‐M transition, and mitosis 21  The cell cycle Tissue homeostasis ‐ atrophy Trayssac et al., J. Clin. Invest. 2018;128(7):2702–2712. 22  The cell cycle Tissue homeostasis – hypertrophy, hyperplasia, dysplasia Trayssac et al., J. Clin. Invest. 2018;128(7):2702–2712. Loss of regulation of the cell cycle is accompanied by imbalance of tissue homeostasis. Loss of cell size and/or number result in atrophy Increased cell size results in hypertrophy. Hyperplasia occurs when there is an increase in the number of cells, but the morphology of the cells and their relationship to one another remains normal. Hyperplasia is a normal response to a specific stimulus, and the cells of a hyperplastic growth remain subject to normal regulatory control mechanisms. On the other hand, when cells have a distinctly abnormal and variable appearance; cells vary in shape and size, and there is evidence that cell to cell interaction has broken down resulting in a disorganized appearance of the tissue, we talk dysplasia. Dysplasia can revert back to hyperplasia, but it may also become malignant. 23  The cell cycle Anticancer drugs & the cell cycle Hertz et al., Molecular and Clinical Oncology, (2015) 3:37‐43 24  The cell cycle Key points Cell cycle is an ordered series of events of living eukaryotic cells Cell cycle consists of 2 major stages: interphase and cell division Intephase consists of the G1, S and G2 phases These phases have distinct functional characteritics Cell division consists of Mitosis and Cytokinesis Mitosis has substages with dedicated functions in the distribution of genetic material and the generation of daughter cells Cytokinesis is the finishing step in the formation of new cells Precisely controlled cell cycle is a key feature of tissue homeostasis 25  Cell division & the human genetic diversity Lesson 2 – Meiosis, Spermato‐ & Oogenesis Zsolt Fábián M.D., Ph.D., Dr. Habil. 26  The cell cycle Preserving the DNA content across generations 46,XX 46,XY 27  The cell cycle Meiosis I Generates haploid gametes Occurs only in the germline Entering Meiosis I from G2 n=2, c=4 During Meiosis I Homologous chromosomes associate forming bivalents – 2 joined chromosomes with 4 DNA double helices In Prophase I – Crossing‐Over or Homologous Recombination between homologues – In females, meiosis arrests for 10‐50 years Homologs are pulled into daughter cells Resulting cells are haploid n=1, c=2 Meiosis is a specialized form of cell division, differing from mitosis, that is used to produce spermatozoa and ova. It involves two different cell divisions, called Meiosis I and Meiosis II. While the behavior of chromosomes is largely identical in male and female meiosis there are other important differences we will look at soon. For example, male spermatogenesis begins at puberty and continues uninterrupted for the rest of the lifetime of the male. Female oogenesis begins during embryonic development and arrests in prophase I until the female begins ovulation at puberty. In each menstrual cycle only one cell continues through meiosis producing a single ovum. This continues until the female reaches menopause, usually in the 5th decade of life. So female meiotic arrest can last 50 years or more. Meiosis I is sometimes called the ‘reduction division’ since diploid cells divide to become haploid cells. 28  The cell cycle Meiosis II entering Meiosis II  n=1, c=2 during Meiosis II  sister chromatids are pulled apart into daughter cells resulting cells are gametes  n=1, c=1 gametes are not genetically identical paternal or maternal homologues crossing‐over/recombination Meiosis is a specialized form of cell division, differing from mitosis, that is used to produce spermatozoa and ova. It involves two different cell divisions, called Meiosis I and Meiosis II. While the behavior of chromosomes is largely identical in male and female meiosis there are other important differences we will look at soon. For example, male spermatogenesis begins at puberty and continues uninterrupted for the rest of the lifetime of the male. Female oogenesis begins during embryonic development and arrests in prophase I until the female begins ovulation at puberty. In each menstrual cycle only one cell continues through meiosis producing a single ovum. This continues until the female reaches menopause, usually in the 5th decade of life. So female meiotic arrest can last 50 years or more. Meiosis I is sometimes called the ‘reduction division’ since diploid cells divide to become haploid cells. 29  The cell cycle Meiosis versus Mitosis 30  The cell cycle Summary of the n & c numbers Stage n (ploidy) c (DNA Content) Cell Cycle During G1 Phase 2 2 During G2 Phase 2 4 Meiosis After Meiosis I 1 2 After Meiosis II (gametes) 1 1 31  The cell cycle Spermatogenesis versus Oogenesis 32  The cell cycle Spermatogenesis versus Oogenesis Stage Females Males Begins Early embryonic life Puberty Duration 10‐50 years 60 days Number of mitoses before meiosis 20‐30 30‐500 Gametes produced/meiosis 1 ovum+3 polar bodies 4 spermatozoa Gametes produced 100‐200 million/ejaculation 1 ovum / period Chromosomes behave identically in male and female meiosis, however there are important differences between spermatogenesis and oogenesis Males produce far more spermatozoa than females produce ova Many more rounds of mitosis are required in males before meiosis begins to produce the larger number of gametes Each round of mitosis requires 1 round of DNA replication Each round of DNA replication potentially introduces mutations so more mutations are inherited from fathers than from mothers 33  Cell division & the human genetic diversity Lesson 3 ‐ Human genetic diversity Zsolt Fábián M.D., Ph.D., Dr. Habil. 34  Lesson 3 ‐ Human genetic diversity Generation of Genetic Diversity In Sexual Reproduction 1. Independent Assortment of Chromosomes – Individuals have 23 pairs of maternal and paternal chromosomes – For each pair, they pass only one, either their maternal or paternal, onto their offspring. – Selection is random and independent for each chromosome pair. – 223 or 8.4 million possible combinations Independent assortment of maternal and paternal homologs at meiosis I produces the first level of genetic diversity. There are 223 or 8.4 million different ways of picking one chromosome from each of the 23 pairs in a diploid cell. Gametes A‐E show just five of the possible combinations of maternal and paternal chromosomes. The independent assortment of chromosomes is the basis of Mendels 3rd law – see Inheritance Patterns. Chromosomes segregate randomly and independently. Eg. For chromosome 1, either the maternal or paternal homolog will be randomly passed onto the offspring. Which chromosome is passed has no bearing on whether or not the maternal or paternal copy of any other chromosome will be passed on. 35  Lesson 3 ‐ Human genetic diversity Generation of Genetic Diversity In Sexual Reproduction 2. Crossing‐Over / Homologous Recombination – Occurs at least once in every bivalent during Prophase I An average of 2 times in humans – Resulting chromatids have a combination of maternally and paternally inherited DNA – The number of possible combinations effectively becomes infinite 36  Lesson 3 ‐ Human genetic diversity Human genetic variation Human genomic sequences average ~0.1% variation – ~3 million base‐pairs differ out of 3 billion base‐pairs – Individuals carry ~10 unique ‘sequence variants’ – variations in sequence arise by mutation & then spread throughout population during evolution causing genetic “polymorphism” – Alleles are different versions of an identified gene normally differ by only a small number of nucleotides – “Wild‐type allele” = the most common allele for a gene within a population – “a polymorphism” = an allele that is less common than the wild‐type allele, but occurs more than 1% of the time – “Variant” – generally just refers to a change in DNA sequence (that may or may not produce changes in observed features) – ”Rare variant:” found at a frequency of < 1% The haploid human genome is ~3.1 billion base pairs, so 0.1% of that is ~3.1 million base‐pairs. With ongoing human genome sequencing allowing the comparison of thousands of individuals, it is becoming apparent that this estimate is likely to be conservative. “Poymorphism” simply means having things that exist in different forms. Polymorphism, as used here, describes the condition of having variation in sequence for known genes = having different versions of a known gene. Most polymorphisms represent small changes in sequence. 37  Lesson 3 ‐ Human genetic diversity Diversity in Human Populations Out of Africa Hypothesis Anatomically modern humans first appeared ~250,000 years ago in Africa – Most human genetic diversity arose during human evolution in Africa – Today there is more genetic diversity in Africa than anywhere else 2 waves of migration i. 130,000‐115,00 years ago Died out but may have established in China ii. 77,000‐69,000 years ago A small group (1% of the population, rare variants are found at