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SLE254 Lecture 1: SLE254 Genetics Lecture Notes-All Lectures
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Department
SLE
Course
SLE 254
Professor
Giorgio De Guzman
Semester
Winter

Description
SLE254 GENETICS LECTURE 2: CHROMOSOMES MITOSIS AND MEIOSIS INTERPHASE Before a cell enters mitosis (divides) it undergoes a period of growth called interphase. 90% of a cells time in the normal cellular cycle may be spent in interphase. The cell may appear to be dormant, however, biochemical activity is high during interphase. The genetic material exists as chromatin. The structure of chromatin during interphase is optimised to allow easy access of transcription and DNA repair factor to the DNA. Not tightly coiled. Different levels of packing on various regions of the same chromosome. • Euchromatin: loosely packed region on chromatin, active transcription. • Heterochromatin: densely packed region on chromatin, inactivated. Stages of interphase: • G 1hase (first gap): the period prior to the synthesis of DNA. For many cells, this phase is the major period of cell growth during its lifespan. New organelles are being synthesised, so the cell requires both structural proteins and enzymes, resulting in a great amount of protein synthesis and a high rate of metabolism in the cell. The DNA in a G 1iploid eukaryotic cell is 2n (2 sets of chromosomes present). • G 0hase: A cell may pause in the G ph1se before entering the S phase and enter a state of dormancy call the G 0hase. Cells exist in a quiescent (non-dividing) state but can re-enter the cycle at G1. Some cell types (liver and kidney) enter the G0phase semi-permanently and can be induced to begin dividing again only under very specific circumstances. Other types of cells, such as epithelial cells, continue to divide throughout an organism’s life and rarely enter G 0 • Restriction (R) point: present at the end of G 1hase. Depending on levels of nutrients, energy and external factors, cells must decide to enter the cell cycle or move into G phase. 0 • S phase: the period during which DNA is synthesised/replicated. The goal of this phase is to create exactly 2 identical semi-conserved chromosomes. Damage to DNA is detected and fixed during S-phase. Each chromosome now consists of 2 sister chromatids. The amount of DNA in the cell has doubled but the cell is still 2n, there are still 2 sets of chromosomes. • G 2hase: final suphase of interphase in the cell cycle preceding mitosis. Rapid cell growth and protein synthesis. Microtubules, proteins required for mitosis, are produced. Although chrmosomes have been replicated they cannot yet be dstinguished individually because they are still in the form of loosely packed chromatin. • G 2heckpoint: second checkpoint. Serve to preent the cell from entering mitosis with genomic DNA damage. If this checkpoint is passed, the cell initates the many molecular processes that signal the beginning of mitosis. MITOSIS One diploid cell divides to form 2 diploid cells that are identical to the parent cell. Mitosis is divided into 4 stages: • Prophase: nucleolus in the nucleus disappears. The chromatin condenses into double rod-shaped structures called chromosomes in which the chromatin becomes visible. The centrosomes move to opposite poles of the cell, forming a bridge of spindle fibres. • Prometaphase: nuclear membrane breaks apart. Microtubules emerging from the centrosomes at the poles of the spindle reach the chromosomes, now highly condensed. At the centromere region, each sister chromatid has a protein structure called a kinetochore. Other spindle microtubules make contact with microtubules coming from opposite pole. Proteins associated with spindle microtubules move the chromosomes toward the centre of the cell. • Metaphase: chromosomes align in the middle of the cell. The centromeres of the chromosomes convene themselves on the metaphase plate. Chromosomes are condensed and highly coiled in metaphase which makes them most suitable for visual analysis. Used to make karyotypes. • Spindle checkpoint: This checkpoint monitors the interaction between improperly connected kinetochores and spindle microtubules, and is maintained until kinetochores are properly attached to the spindle. Another regulator of checkpoint activation is kinetochore tension. When kinetochores are properly attached to opposite spindle poles, forces in the mitotic spindle generate tension. If activated, the spindle checkpoint blocks anaphase entry. Correct orientation of sister chromatids deactivates the checkpoint • Anaphase: Chromosomes move to opposite poles of the cell. Anaphase is initiated by a protease known as separase which cleaves cohesin, a protein responsible for holding sister chromatids together. While the chromosomes are drawn to each side of the cell, the non-kinetochore spindle fibre lengthen and elongate the cell. • Telophase: Two daughter nuclei form in the cell. The nuclear envelopes of the daughter cells are formed from the fragments of the nuclear envelope of the parent cell. The nucleoli reappear. The chromosomes also unwind back into loose chromatin • Cytokinesis: The cleavage furrow is the indentation of the cell's surface that begins the progression of cleavage. During cellular cleavage, the contractile ring (formed by actin and myosin filaments) tightens around the cytoplasm of the cell until the cytoplasm is pinched into two daughter cells Human cells are genetically programmed to divide about 50-80 times. This limit allows growth to adulthood, and repairs such as wound healing. Replicative exhausted cells undergo senescence, which is a cell cycle arrested state. They secrete proinflammatory factors to mediate their clearance from the body (macrophages). Alterations in this program can lead to genetic disorders (premature ageing) or to cancer. Cancer is a disease of the cell cycle. MEIOSIS A form of cell division that produces 4 haploid cells containing only 1 copy (paternal or maternal) of each chromosome. Meiosis produces 4 haploid cells (n). in humans the haploid number is 23. • Meiosis I: gametes receive random combinations of maternal and paternal chromosomes (creates genetic diversity). Result of meiosis 1. Members of a pair of homologous chromosomes physically associate at this time each chromosome consists of 2 sister chromatids joined by a common centromere. • Crossing over: occurs which increases the genetic diversity in the gametes. Occurs during prophase. Matching regions on matching chromosomes (chromatids) break and then reconnect to the other chromosome. • Difference to mitosis: maternal and paternal homologous chromosomes pair during metaphase in meiosis 1. • Independent assortment: metaphase homologous chromosomes align in the middle and then anaphase chromosome pairs independently separate from each other. • Meiosis II: • Metaphase II: unpaired chromosomes align at the cell’s middle. There is no synthesis of chromosomes before meiosis II. Therefore, only the remaining on replicated chromosome. • Anaphase II: centromeres divide and daughter chromosomes move to opposite poles. STRUCTURE OF CHROMOSOMES Chromatid: one strand of a duplicated chromosome. Joined by a centromere to its sister chromatid. Sister chromatids: 2 chromatids joined by a common centromere. Each carries identical genetic information. Centromere: region of a chromosome to which microtubule fibres attach during cell division. Centromere location gives a chromosome its characteristic shape. Homologous chromosomes: maternal and paternal with identical gene loci (but often different alleles). They physically pair during meiosis. LECTURE 3: GENE INHERITANCE AND TRANSMISSION MENDELS BREEDING EXPERIMENTS 1856: performed hybridisation experiments of pea plants. Easy to grow and cross- breed experimentally. Reproduces well and grows to maturity in a season. Mendel created “pure breeding” strains for various traits. A Punnett square shows how the traits are inherited (A=dominant trait, a=recessive trait) Monohybrid cross: comparing 1 trait. 3:1 ratio between dominant and recessive. Dihybrid cross: comparing 2 traits. 9:3:3:1 FORKED LINE METHOD Relies on the simple application of the laws of probability established for the dihybrid cross. Each gene pair is assumed to behave independently during gamete formation. A male having genotype AaBBCc mates with a female having genotype aaBbCc. What are the expected genotype frequencies of their offspring? P = AaBBCc x aaBbCc The first step is to decompose the trihybrid cross into its constituent single-gene (monohybrid) crosses: Aa x aa BB x Bb Cc x Cc Determine the expected genotype frequencies for these monohybrid crosses. You can use the Punnett square. Pair every genotype from the first monohybrid cross with every genotype from the second cross with every genotype from the third. Now we traverse each path through the forked lines, one path at a time, in order to determine each trihybrid genotype and frequency PROBABILITY METHOD The most flexible and applicable method. A dihybrid cross is a situation in which two monohybrid crosses are involved and problems can sometimes be more easily solved by considering the two crosses independently (if the loci are unlinked) and then combining the results. The same principal applies; no matter how many gene loci are involved. There are two rules of probability you need to understand – Multiplication and Addition. CHI-SQUARED TEST In experiments, deviations from expected results can be obtained. The chi-square test converts deviations from expected values into a measure of the occurrence of the result by chance. A judgement can then be made as to whether we accept or reject the deviation as being significant. We therefore have a means of testing the validity of a hypothesis that formed the basis for an experiment. The point of the Chi- square test is to either reject or accept our null hypothesis. 1. State the hypothesis being tested and the predicted results 2. Determine the expected numbers for each observational class 3. Calculate chi-squared using the formula 4. Determine degrees of freedom 5. Use the chi-square distribution table to determine significance of the value. Locate the value closest to your calculated χ on the degrees of freedom df row. Move up the column to determine the p value. 6. State your conclusion in terms of your hypothesis. If p>0.05 accept null hypothesis, if p<005 reject null hypothesis. Degrees of freedom (df): number of possible outcomes minus 1. Eg. Flipping a coin df=2-1=1 Probability values (p): 95% sure of accepting or rejecting the null hypothesis. Probability value. NULL HYPOTHESIS When we assume data will fit a given ratio we establish a null hypothesis (H )0 The null hypothesis assumes there is no real difference between measured values and the predicted values. Any difference can be attributed purely to chance. WEEK TWO LECTURE 4: PEDIGREE ANALYSIS A pedigree analysis follows the inheritance of a trait through a family. Used to establish how a trait is inherited and to determine the risk of having an affected child. Pedigrees constructed using info from: medical records, interviews, photographs and family records. Pedigree: a diagram showing genetic information from a family which uses standardised symbols. Has 2 goals: • To determine whether the trait has a dominant or a recessive pattern of inheritance. • To discover whether the gene in question is located on a sex chromosome (X or Y) or on an autosome. Patterns of inheritance can be se to predict genetic risk for pregnancy outcomes, adult-onset disorders and recurrence risk of future offspring. PATTERNS OF INHERITANCE Genetic disorders can be inherited in a number of different ways. Males pass an X chromosome to all of their daughters but none of their sons. Females pass an X chromosome to all of their children. Most genes on the X chromosome are not on the Y chromosome. X-linked: pattern of inheritance that results from genes located on the X chromosome Y-linked: pattern of inheritance that results from genes located only on the Y chromosome. Hemizygous genes: A gene present on the X chromosome that is expressed in males in both the recessive and dominant condition. Or a gene present only in one copy in a diploid organism (most of the X chromosome genes in a male human). X-linked recessive traits affect males more than females because males are hemizygous for genes on the X chromosome. Autosomal recessive inheritance: • All children of both affected parents are affected • Most affected individuals have unaffected parent • About ¼ children in large affected families show the trait • Both sexes affected in approximately equal numbers • The risk of an affected child with heterozygous parents is 25%. • For rare traits, most affected individuals have unaffected parents. Autosomal dominant inheritance • 2 affected individuals can have an unaffected child. • About ½ of children of an affected parent are affected. • Both sexes equally affected. • Every affected individual has at least one affected parent (except in traits with high mutation rates or incomplete penetrance). • If an affected individual is heterozygous and has an unaffected mate, each child has a 50% chance of being affected. X-linked dominant inheritance • Affected males produce all affected daughters and no affected sons. • A heterozygous affected female will transmit the trait to half of her children. (sons and daughters equally affects). • On average, twice as many daughters as sons are affected. X-linked recessive inheritance • Affected males receive the mutant allele from their mother and transmit it to all of their daughters, but not their sons. • Daughters of affected males are usually heterozygous. • Sons of heterozygous females have a 50% chance of being affected. • Hemizygous males are affected and transmit the mutant allele to all their daughters who become carriers. • Phenotypic expression much more common in males. Y-linked inheritance • Only males have Y chromosomes. Males are hemizygous for genes on the Y chromosome. • Genes on the Y chromosome are passed directly from father to son. All sons of affected males are affected. • All Y-linked genes are expressed. Non-Mendelian pattern of inheritance observed in traits controlled by mitochondrial genes Mitochondria: cytoplasmic organelles that convert energy from food into ATP. Carry DNA for 37 mitochondrial genes. Genetic disorders in mitochondrial DNA are associated with defects in energy conversion. The egg provides all the mitochondria to the zygote. The sperm does not pass on any mitochondria. Hence we have all our mother’s mitochondrial DNA. All siblings in a family will have the same mitochondrial DNA. VARIATIONS IN GENE EXPRESSION Variations in gene expression affect pedigree analysis and assignment of genotypes to members of the pedigree. Factors can affect gene expression: • Interactions with other genes in the genotype • Interaction between genes and the environment. Huntington disease: an autosomal dominant disorder associated with progressive neural degeneration and dementia. Adult onset is followed by death in 10 to 15 years. Porphyria: an autosomal dominant disorder that leads to intermittent attacks of pain and dementia. Symptoms first appear in adulthood. PHENOTYPIC VARIATION Expressivity: the range of phenotypic variation associated with a given phenotype. Penetrance: the probability that a disease phenotype will appear when a disease-related genotype is present. • Incomplete penetrance • Camptodactyly: a dominant trait (immobile, bent little fingers) with variable expression and incomplete penetrance. ONLINE MENDELIAN INHERITANCE IN MAN (OMIM) Genetic traits are described, catalogued and numbered in a database. LECTURE 5: EXTENSIONS OF MENDELIAN GENETICS LETHAL ALLELES Mutations occur giving rise to new forms of alleles that may become lethal mutations Dominant lethal: allele that causes death of the organism, whether homozygous or heterozygous for the allele. Rarely detected due to their rapid elimination from populations. Eg. Huntington’s disease but as onset is slow allele can be passed on. Recessive lethal: allele that is lethal when homozygous. In heterozygous, a lethal allele is masked by the presence of an allele for “wild type”. INCOMPLETE DOMINANCE (PARTIAL) Both alleles blend their effects. The phenotype of the heterozygote lies somewhere between those of the 2 kinds of homozygotes. The F generation shows only 1 pair of 2 alleles determines the phenotype. However, phenotype ratio is identical to genotype ratio and not 3:1 like complete dominance. Tay-Sachs disease: individuals who are homozygous for this recessive disorder are severely affected with a lipid storage disease that is fatal within the first 3 years of life. Neither allele is recessive so different symbols are used. CODOMINANCE Both alleles show their effects and do not blend. In codominance, neither allele is dominant and both are expressed. A cross between organism with 2 different phenotypes produces offspring which has both phenotypes of the parental traits shown. The MN blood group: in humans, 2 forms of a glycoprotein exist on the surface of RBCs (M and N). An individual may exhibit either one or both. MULTIPLE ALLELES Many genes have more than 2 alternative alleles. This increases the number of different genotypes and phenotypes that exist with respect to the particular gene. Multiple alleles can only be studied in populations. An individual diploid organism will only have at most 2 alternative forms of the same gene and a population will show all the alternatives. ABO blood groups: 3 alternative alleles of one gene, presence of antigens of the surface of RBCs. 4 phenotypes depending on the presence or absence of antigens. Bombay phenotype: Bombay cells can’t be converted to group A or B due to a mutation in the FUT1 gene which prevents synthesis of H substance, vital for producing functional A and B antigens. So individual may have I or I alleles but neither is added to the cell surface and they are functionally type O. binoSTASIS A form of gene interaction in which one gene masks the phenotypic expression of another. There are no new phenotypes produced by this type of gene interaction. The alleles that are masking the effect are called epistatic alleles. The alleles whose effect is being altered or suppressed are called the hypostatic alleles. PLEIOTROPY Occurs when 1 gene influences multiple phenotypic traits. The gene codes for a product that is used by various cells or has a signalling function on various targets. A mutation in a pleiotropic gene may have an effect on some or all traits simultaneously. Phenylketonuria (PKU): symptoms include mental retardation, reduced hair and skin pigmentation. Caused by any of over 400 mutations in a single gene that codes for the enzyme phenylalanine hydroxylase, which converts the amino acid phenylalanine to tyrosine. Antagonistic pleiotropy: the expression of a gene resulting in multiple competing effects, some beneficial but others detrimental to the organism. Eg. Some genes responsible for increased fitness in younger years contribute to decreased fitness later in life or the p53 gene which suppresses cancer but also suppresses stem cells which replenish worn out tissue. GENETIC HETEROGENEITY Single phenotype or genetic disorder may be caused by any one of a multiple number of alleles or non-allele (locus) mutations. • Allelic heterogeneity: different mutations within a single gene locus (forming multiple alleles of that gene) cause the same phenotypic expression. Eg. 1000 known mutant alleles of the CFTR gene that cause cystic fibrosis. • Locus heterogeneity: variations in completely unrelated gene loci cause a single disorder. Ed. Retinitis pigmentosa has autosomal dominant, autosomal recessive and X-linked origins. PENETRANCE The probability of a gene or genetic trait being expressed. • Complete penetrance: the gene or genes for a trait are expressed in all the population who have the genes. • Incomplete penetrance: the genetic trait is expressed in only part of the population Penetrance can be difficult to determine reliably. Eg. In disease the onset of symptoms could be age related or affected by environmental codeterminants such as nutrition and smoking, as well as genetic cofactors. EXPRESSIVITY Refers to variations in a phenotype among individuals carrying a particular genotype. Eg. Drosophila flies homozygous for recessive mutant gene eyeless exhibit range of phenotypes from normal eyes to complete absence of one or both eyes. Other genes or environmental factors such as nutrition and temperature may be influencing or modifying the phenotype. ANTICIPATION A phenomenon where by the symptoms of a genetic disorder exhibit an earlier age of onset and are more severe as it is passed on to the next generation. Eg. Huntington’s disease – trinucleotide repeat disease. GERMLINE MOSAICISM Mosaicism: the presence of 2 or more populations of cells with different genotypes in one individual who has developed from a single fertilized egg. Germline mosaicism: a special form of mosaicism where some gametes (sperm or oocytes) carry a mutation but the rest are normal. Cause is usually a mutation that occurred in an early stem cell that gave rise to the gonadal tissue. This can cause only some offspring to be affected, even for a dominant disease. PHENOCOPIES An individual whose phenotype under a particular environmental condition, is identical to the one of another individual whose phenotype is determined by the genotype. The phenocopy environmental condition mimics the phenotype produced by a gene. UNIPARENTAL DISOMY Occurs when offspring receives 2 copies of a chromosome, or part of a chromosome, from one parent and no copies from the other parent. Most occurrences result in no phenotypical anomalies. If the event happens during meiosis II, the genotype may include identical copies of the uniparental chromosome (isodisomy), leading to the manifestation of rare recessive disorders. Eg. Prader-Willi syndrome and Angelman syndrome GENOMIC IMPRINTING Normally there is no difference of expression of the paternal and maternal alleles. Genomic imprinting causes selective expression of a gene or genes inherited from 1 parent. Not a mutation or permanent change. Plays a role in several genetic disorders. Eg. The same region of chromosome 15 mutated but cases a different disease if inherited from mother or father. • Prader-Willi syndrome: paternal copy • Angelman syndrome: maternal copy. LINKAGE 2 or more genes located on the same chromosome that do not show independent assortment and tend to be inherited together. In a given cross, the outcome depends on how the alleles are arranged on the chromosome. Example: a fly that is heterozygous for long wings (Ll) and heterozygous for long aristae (Aa) is crossed with another fly of the same type. AaLl x AaLl. In both cases the dominant alleles are located on the same chromosome. Example: a fly that is heterozygous for long wings (Ll) and heterozygous for long aristae (Aa) is crossed with another fly of the same type. AaLl x AaLl. In both cases the dominant alleles are located on separate chromosomes. The frequency of the genes exchanging during meiosis determines how far apart the alleles are on a chromosome. Alleles that are close together tend to stick together. Map units (MU): the distance between alleles. The long wing allele is 13MU from the aristae allele. CONTINUOUS VARIATION Some traits are controlled by 2 or more genes. Phenotypes can be discontinuous or continuous. Discontinuous variation: shows distinct (discrete) phenotypes. Eg. Pea plant colour, ABO blood group. Continuous variation: shows a series of overlapping phenotypic classes. Eg. Height, weight, hand span, milk yield. HOW ARE TRAITES DEFINED Polygenic traits: traits controlled by 2 or more genes. Patterns of inheritance that can be measured quantitatively. • Eg. Human eye colour. Multifactorial traits: polygenic traits resulting from interactions of 2 or more genes and 1 or more environmental factors. • Eg. Skin colour  controlled by 3 or 4 genes and environmental factors Complex traits: traits controlled by multiple genes and the interaction of environmental factors where the contributions of genes and environment are undefined. Many human diseases are controlled by the action of several genes. • Eg. Hypertension, obesity, cardiovascular disease, depression, autism. LECTURE 6: SEX DETERMINATION The mechanisms of sex determination vary from species to species. SEXUAL DIFFERNTIATION In animals: • Primary: gonads, where gametes are produced. • Secondary: appearance of organism, including sexual characteristics. Unisexual, dioecious and gonochroic: individuals with male or female reproductive organs. Bisexual, monoecious and hermaphroditic: individuals with male and female reproductive organs. Produce eggs and sperm. HAPLODIPLOIDY Males develop from unfertilised eggs and are haploid. Females develop from fertilised eggs and are diploid. Determines the sex in all members of the insect order (Hymenoptera-bees, ants and wasps and the Thysanoptera) Honey bees: drones (males) are entirely derived from the queen, their mother. The diploid queen has 32 chromosomes and the haploid drones have 16 chromosomes. The queen stores sperm in an internal sac called the spermatheca. The queen controls the release of stored sperm from within the organ. If she releases sperm as an egg passes down the oviduct, the egg is fertilised. So the queen can modify sex ratios within colonies which increases relatedness. Relatedness: drones develop from unfertilised eggs and carry 1 copy of chromosomes (haploid) from their mother only. Females are fertilised and carry 2 copies of chromosomes (diploid). From a queen, all the daughters will share 50% genes from the father (since they are all the same), but 25% of their genes from the mother. The coefficient of relatedness among the offspring is therefore 0.75 (1 x 0.5 + 0.5 x 0.5). This is much higher than the 0.5 for sister-sister in a diploid organism (humans). The queen bee is the only fertile female in the hive of domesticated honey bees. If she dies the worker bees can start to lay eggs. Worker bees are unable to make and the unfertilised eggs produces only drones (males) which can mate only with a queen. Eventually all the worker bees die off and the new drones follow. XO SEX-DETEMINATION There is only 1 sex chromosome, referred to as X. • Males only have 1 X chromosome (X0) while females have 2 (XX). Maternal gametes always contain an X chromosome, so the sex of the offspring is decided by the male. Sperm contains either 1 X chromosome or no sex chromosome at all. In a variant of this system, some animals are hermaphroditic with 2 sex chromosomes (XX) and male with only 1 (X0) C. elegans: transparent nematode (roundworm) about 1mm in length that lives in soil. Has 2 sexes: hermaphrodites and males. Individuals are almost all hermaphrodite, with males comprising 0.05% of the total population. Hermaphrodites have a matched pair of sex chromosomes (XX) with the males only having 1 sex chromosome (X0). Males only have testes. Hermaphrodites have testes and ovaries. Eggs produced in adult stage are self-fertilised. When self-inseminated, the worm will lay approximately 300 eggs. A mating between an adult male and a hermaphrodite produces half male, half hermaphrodite offspring. When inseminated by a male, the number of offspring can exceed 1000. ZW SEX DETERMINATION In the ZW system it is the ovum that determines the sex of the offspring. • Males are the homogametic sex (ZZ) • Females are the heterogametic sex (ZW) The Z chromosome is larger and has more genes. Like the X chromosome in the XY system. No genes are shared between the avian ZW and mammal XY chromosomes. Comparing chicken and human, the Z chromosome appeared similar to the autosomal chromosome 9 in humans. XY SEX DETERMINATION Found in humans, most mammals, some insects and some plants. • Females are homogametic (XX) • Males are heterogametic (XY) The presence and absence of a Y chromosome is not the case in all XY organisms. Drosophila: unlike humans the Y chromosome does not confer maleness, rather it encodes genes necessary for making sperm. Sex is determined by the ratio of autosomes to X chromosomes. Each cell “decides” whether to be male or female independently of the rest of the organism. Can create gynadromorphs. • Normal (2X:2A) and triploid (3X:3A) females have a ratio of 1.0. If ratio >1 (more X to A) inviable metafemale produced. • Males have a ratio of 0.5 (X0:2A males are sterile). If ratio <0.5 (more A) then infertile metamale produced. • If ratio in between 0.5 and 1.0 then sterile flies expressing both sex morphologies produced. Chromosomal sex: established at fertilisation. Other aspects of sex depend on the interaction of genes and environmental factors, especially hormones. Humans: the formation of male and female reproductive structures depend on gene action and the interactions within the embryo, with other embryos in the uterus and the maternal environment. Sex of an individual is defined at 3 levels: • Chromosomal sex • Gonadal sex: for the first 7 or 8 weeks, the embryo is neither male nor female due to 2 undifferentiated gonads. Both male and female reproductive duct systems develop. Genes cause gonads to develop as testes or ovaries establishing gonadal sex. Alternative pathways can produce intermediates. • Phenotypic sex: mutations can uncouple chromosomal sex from phenotypic sex. A mutation in the X-linked androgen receptor gene (AR) causes XY males to become phenotypic females. Testosterone is produced, but not testosterone receptors therefore cells develop as females. The chromosomal sex of an individual (XX or XY) can differ from the phenotypic sex. Androgen insensitivity: X-linked genetic trait that causes XY individuals to develop into phenotypic females. Pseudohermaphroditism: an autosomal genetic condition that causes XY individuals to develop the phenotype of females. Caused by mutations in several different genes. Affected individuals have both male and female structures, but at different times of life. At puberty, females change into males. Y chromosome and testis development  SRY gene. • Sex-determining region of the Y chromosome. Located near the end of the short arm of the Y chromosome. Plays a major role in causing the undifferentiated gonad to develop into a testis. Before sexual differentiation, both male and female embryos have bipotential gonads. They possess both Wolffian and Mullerian ducts. These ducts can differentiate into male or female reproductive organs according to the hormonal status of the foetus. Female development: requires the absence of the Y chromosome and the presence of 2 X chromosomes. Embryonic gonad develops as an ovary. In the absence of testosterone, the Wolffian duct system degenerates. In the absence of MIH, the Mullerian duct system forms female reproductive system. EXPRESSION OF THE X CHROMOSOMES Human females have 1 X chromosome inactivated in all somatic cells to balance the expression of X-linked genes in males and females. Females have 2 X chromosomes; males have 1 yet the amount of gene product is the same. Dosage compensation: is a mechanism that regulates the expression of sex-linked gene products. Barr bodies and X inactivation: • Lyon hypothesis: dosage compensation in mammalian females. Random inactivation of 1 X chromosome in females equalize the activity of X-linked genes in males and females. • Barr body: a densely staining mass in the somatic nuclei of mammalian females. An inactivated X chromosome, tightly coiled. The number of Barr bodies is 1 less than the number of X chromosomes. • Males (46,XY) have no Barr bodies. • Normal females (46,XX) have one Barr body • Female with 5 X chromosomes (49,XXXXX) have 4 Barr bodies In females, some cells express the mothers X chromosome and some cells express the father’s X chromosome. Inactivated chromosome can come from either mother or father, occur early in development and is permanent (all descendant of a particular cell have the same X inactivated). Mosaic expression occurs in female mammals: eg. Calico cats are always female. X inactivation centre: Inactivation begins and is regulated from the X inactivation centre (Xic) of the X chromosome. Inactivation begins here. Xic contains the gene XIST which encodes an RNA that coats the inactive X and somehow silences it. Effects of random X chromosome inactivation: random X inactivation can cause twins with identical genotype to have different phenotypes. The pedigree shows identical twins who are discordant for the phenotype of colour blindness. Almost all the active X’s in the colour bind twin carry the mutant allele, and in the non-colour blind twin, most of the active X’s carry the normal allele. WEEK THREE LECTURE 7: KARYOTYPES Karyology: the study of whole sets of chromosomes Karyotype: a complete set of chromosomes from a cell that has been photographed during cell division and arranged in a standard sequence. The human karyotype includes only 46 chromosomes. Used to study chromosomal aberrations, cellular function, taxonomic relationships and to gather info about past evolutionary events. Karyotypes reveal variations in chromosomal structure and number. Chromosome banding and other techniques can identify small changes in chromosomal structure. Any nucleus can be used to make karyotype  lymphocytes, skin cells, biopsies and tumour cells. Also sampling cells before birth: • Amniocentesis: foetal skin cells come off and float in the amniotic fluid which can be sampled, grown and analysed for chromosomes, enzymes or DNA. • Chorionic villus sampling (CVS): method of sampling foetal chorionic cells by inserting a catheter through the vagina or abdominal wall into the uterus. Used in diagnosing biochemical and cytogenetic defects in the embryo. Usually performed in the 8 or 9 week of pregnancy. The chorion is the outermost foetal membrane and the villi are projections from this membrane that can be sampled without harming the foetus. PERFORMING A KARYOTYPE FROM BLOOD • Blood is drawn from the body and treated to stop coagulation. • Mononuclear cells (lymphocytes and monocytes) are purified from the blood by centrifugation. The purified cells are cultured for 3-4 days in the presence of a mitogen, which stimulates the lymphocytes to proliferate. • The cells are treated with a drug such as colcemid which disrupts mitotic spindles and prevents completion of mitosis (and enriches the population of metaphase cells). • Cells are harvested and treated briefly with a hypotonic solution which makes the nuclei swell osmotically. • Swollen cells are fixed, dropped onto a microscope slide and dried. Slides are stained to induce a banding pattern. • The slides are scanned to identify a clear chromosome spread, and then photographed. • The image is cut up and rearranged in standard format. IDENTIFYING CHROMOSOMES • Size • Banding pattern: size and location of stained bands on chromosomes make each chromosome pair unique. • Centromere position: centromeres are regions in chromosomes that appear as a constriction • Metacentric: centrally places • Submetacentric: placed closer to one end than the other • Acrocentric: placed very close to, but not at one end • Telocentric: placed at one end of the chromatid and hence only one arm. Not seen in humans. KARYOGRAM (ideogram) Diagram of the chromosome banding patterns. Human metaphase chromosomes contain about 550 bands. More bands can be detected in later prophase chromosomes. System of naming chromosome bands: allows any region to be identified by a descriptive address (chromosome number, arm, region and band). • p: short arm • q: long arm • Each arm is divided into regions with bands within the numbered region. Eg. The arrow points to 1q2.4 (chromosome 1, long arm, region 2 and band 4). KARYOTYPE SYMBOLS An alpha-numerical karyotype is the designation of the chromosome constitution (46,XX), deletion (del), duplication (dup), translocation (t), inversion (inv), short arm of chromosome (p), long arm of chromosome (q), addition or loss of entire chromosome (+/-). CHROMOSOME PAINTING Chromosome-specific DNA probes are labelled with a fluorescent dye. Using combinations of probes and dyes can produce unique colours for each chromosome. INFORMATION FROM A KARYOTYPE 1. Differences in absolute sizes of chromosomes: chromosomes can vary in absolute size by as much as twenty-fold between genera of the same family. 2. Differences in the position of centromeres: this can be brought by unequal translocations, pericentric inversion and centric fusion and fission. 3. Differences in relative size of chromosomes: can be caused by translocations of unequal lengths. 4. Differences in basic number of chromosomes: number can change due to a number of reasons. • Chromosome elimination: during development • Chromatin diminution: found in some roundworms, portions of the chromosomes are cast away in particular cells. • Aneuploidy: where the chromosome number in the cells is not the typical number for the species. • Polyploidy: more than 2 sets of chromosomes, this can be normal for some species. The number of chromosomes in the karyotype between (relatively) unrelated species is highly variable. Eg. Pilosula ant males are haploid n=1 or the Ophioglossum fern has an average of 1262 chromosomes. 5. Differences in number and position of satellites: small bodies attached to a chromosome by a thin thread of chromatin. The secondary constrictions are always constant in their positions and can be used as markers. 6. Differences in degree and distribution of heterochromatic regions: heterochromatin stains darker than euchromatin, indicating tighter packing and mainly consists of genetically inactive repetitive DNA sequences. LECTURE 8: CHROMOSOMAL ABNORMALITIES CHANGES IN CHROMOSOMAL NUMBER Polyploidy and aneuploidy are major causes of reproductive failure in humans. • Polyploidy: chromosomal number that is a multiple of the normal haploid chromosomal set. Seen only rarely in live births • Triploidy: chromosomal number that is 3 times the haploid number, having 3 copies of all autosomes and 3 sex chromosomes. • Tetraploidy: chromosomal number that is 4 times the haploid number, having 4 copies of all autosomes and 4 sex chromosomes. • Aneuploidy: chromosomal number that is not an exact multiple of the haploid set. The rate of aneuploidy in humans is much higher than in other primates and mammals. Cause of aneuploidy is nondisjunction which is the failure of homologous chromosomes to separate properly during meiosis • Monosomy: condition in which one member of a chromosomal pair is missing; one less than the diploid number (2n-1). ▪ Autosomal monosomy: lethal condition which causes spontaneous abortion. • Trisomy: condition in which one chromosome is present in 3 copies, and all others are diploid; one more than the diploid number (2n+1). Maternal age is the leading risk factor for trisomy, by age 42 about 1/3 identified pregnancies are trisomic. ▪ Autosomal trisomy: relatively common, most result in spontaneous abortion but four types can result in live births (8, 13, 18, 21) • Trisomy 13 (Patau syndrome): lethal condition (1/15,000 live births) where half die within 1 month, average survival is 6 months. Facial malformations, eye defects, extra digits, malformation of brain and nervous system and congenital heart defects. • Trisomy 18 (Edwards syndrome): lethal condition (1/11,000 live births) where survival is 2-4 months and 80% are female. Small size, slow growth, mental retardation, clenched fists with malformed feet and heart abnormalities. • Trisomy 21 (down syndrome): first chromosomal abnormality discovered in humans and the only autosomal trisomy that allows survival into adulthood. 1/800 live births. Growth and development is retarted, high rate of congenital heart defects, leukaemia and respiratory infections. 94% of nondisjunctions occur in the mother, 6% in the father. The majority of nondisjunction events occur in meiosis I in oocytes. ▪ Most autosomal trisomy’s are lethal and up to 50% of chromosomal abnormalities in miscarriages. • Sex chromosomes: involves both X and Y chromosomes. A balance is needed for normal development with at least one copy of the X chromosome. Increasing numbers of X or Y causes progressively greater disturbances in phenotype and behaviour. Changes in the number of sex chromosomes have less impact than changes in the number of autosomes. ▪ Turner syndrome (monosomy of X) resulting in female sterility. Females are short and wide- chested with rudimentary ovaries. ▪ Klinefelter syndrome: aneuploidy (XXY) 1/1000 live births. Males with some female features that become apparent after puberty. Some have learning difficulties. Can have more severe forms such as XXXY and XXXXY ▪ XYY syndrome: males are relatively normal, tend to be taller than average and some have personality disorders and subnormal intelligence. Maternal age: Meiosis I is not completed until ovulation, so eggs produced at 40 have been in meiosis I for more than 40 years. Intracellular events of environmental agents may have damaged the cell so aneuploidy can result when meiosis resumes at ovulation. Maternal selection: embryo-uterine interactions that normally abort abnormal embryos become less effective. CHANGES IN CHROMOSOMAL ARRANGEMENT • Deletions: loss of chromosomal material associated with genetic disorders. • Cri du chat syndrome (5p-): associated with an array of congenital malformations, with most characteristic of which is an infant cry that sounds like a cat. • Prader-Willi syndrome • Translocations: exchange of chromosome parts with often produces no overt phenotypic effects and can result in genetically imbalanced and aneuploidy gametes. • Robertsonian translocation: 2 acrocentric chromosomes are joined at their centromere. Eg. Chromosome 14 and 21 resulting in Down syndrome. Someone carrying the translocation is phenotypically normal but produces 6 types of gametes (3-lethal, 1-DS, 1-translocation carrier and 1 normal). • Duplications • Inversions Uniparental disomy (UPD): condition in which both copies of a chromosome are inherited from a single parent. Associated with several genetic disorders such as X-linked disorders and autosomal recessive disorders (Prader-Willi syndrome and Angelman syndrome). Fragile sites: appear as gaps or breaks in chromsomespecific locations. One fragile site on the X chromosome is associated with a common form of mental retardation in males. • Fragile X is an X chromosome that carries a nonstaining gap at band q27. LECTURE 9: DNA AND CHROMOSOME PACKAGING STRUCTURE OF NUCLEIC ACIDS Nucleic acids are composed of strings and nucleotides linked together. Consists of a phosphate group, pentose sugar (DNA or RNA) and a nitrogen-containing base (A, G, C, T or U). Nucleoside: base plus a sugar. Eg. Cytosine + deoxyribose = deoxycytidine. Nucleotide: nucleoside + phosphate. Eg. Cytidine 3’-phosphate. Polynucleotides: are directional. Nucleotides are joined together to form a polynucleotide chain. Polynucleotide chains have slightly different structures at either end (phosphate group at 5’ end and OH group at 3’ end). DNA is a double helix of 2 polynucleotides held together by hydrogen bonds. The 2 strands run in different directions. Ratios of bases: C=G and T=A, therefore pyrimidines=purines. DNA DOUBLE HELIX Right handed double helix structure. Two polynucleotide chains held together by hydrogen bonding between the bases and hydrophobic interactions. Repeating substructure every 0.34 nanometers (base stack). The 2 strands are held together by complementary base pairing. The stability of the helix is due to: • Large number of hydrogen bonds • GC rich DNA is more stable than AT rich DNA because the GC pair has 3 hydrogen bonds compared to 2. • Hydrophobic bonding (stacking forces) between bases. There are 3 biologically active double helical structures   DNA can be negatively supercoiled. The relaxed form of DNA is twisted into the “biologically active” negatively coiled structure. This state of supercoiled DNA is maintained within the cell by a series of enzymes called topoisomerases. If the DNA of a human cell was stretched out it would span 2 meters. At interphase the DNA is dispersed so that it can be replicated and expressed but must be condensed into a chromosome during cell division. CHROMATIN The combination of DNA and proteins that make up the contents of the nucleus of a cell. Primary functions: • Package DNA into a smaller volume to fit in the cell • Strengthen the DNA to allow mitosis and meiosis and prevent DNA damage • Control gene expression and DNA replication. The primary protein components of chromatin are histones that compact the DNA. Histones: provide the 1 level of packaging for DNA as they compact the DNA by a factor of approximately 7. Histone protein sequence is highly conserved among eukaryotes. Rich in lysine and arginine. 5 main types: • H1: attached to the nucleosome and involved in further compaction of the DNA (conversion of 10nm chromatin to 30nm chromatin). • H2A 2 copies in each nucleosome • H2B “histone octomer”. DNA wraps • H3 • H4 around this structure 1.75 times. Non-histone proteins: other proteins that are associated with the chromosomes. Highly variable in cell types, organisms and at different times in the same cell type. Amount of non-histone protein varies. May have role in compaction or be involved in other functions requiring interaction with the DNA. Many are acidic and negatively charged, bind to the histones, binding may be transient. FUTHER COMPACTION DNA is further compacted when the DNA nucleosomes associate with one antoher to produce 30nm chromatin. If H1 is absent then chromatin cathot be converted from 10 to 30nm. DNA is condensed to 1/6 its unfolded size. 30nm  300nm: compaction continues by forming looped domains from the 30nm chromatin which compacts the DNA to 300nm chromatin. Human chromosomes contain about 2000 looped domains. 30nm chromain is looped and attached to a nonhistone protein scaffolding. DNA in looped domains are attached to the nuclear matrix via DNA sequences called MARs (matrix attachment regions). Level of DNA compaction changes throughout the cell cycle, most compact during mitosis and least compact during synthesis. 2 types of chromatin related to level of gene expression: • Euchromatin: areas that stained lightly. Chromsomes or regions that exhibit normal patterns of condensation and relaxation during the cell cycle. Usually areas where gene expression is occurring. • Heterochromatin: areas that stain darkly. Chromosomes or regions that are condensed throughout the cell cycle. CENTROMERE Part of a chromosome that links sister chromatids and acts as the site of assembly of the kinetochore. Two types: • Point centromeres: very small regions of DNA capable of genetically conferring centromeric function on any DNA segment into which they are transferred. • Regional centromeres: more complex. Human and mammalian regional centromeres contain alpha satellite DNA. 171bp sequence arranged in tandem repeats which binds to centromere-associated proteins. Large centromere bind 30-40 microtubules. TELOMERE Sequences at the ends of eukaryotic chromosomes which play critical roles in chromosome replication and maintenance. Telomeres keep chromosomes protected and prevent them from fusing into rings or binding with other DNA. The telomere DNA sequences of a variety of eukaryotes are similar, consisting of repeats of a simple sequence DNA containing clusters of G residues on one strand. E.g. the sequence of telomere repeats in humans and other mammals is AGGGTT, in Tetrahymena (a protozoan) it is GGGGTT, in Arabidopsis (a plant) it is AGGGTTT. These sequences are repeated hundreds or thousands of times, Spanning up to several kilobases and terminate with an overhang of single stranded DNA. Telomere DNA loops back on itself to form a circular structure that protects the ends of chromosomes. Maintenance of telomeres appears to be an important factor in determining the lifespan and reproductive capacity of cells. Progerias: accelerated ageing diseases in humans. Somatic cells from patients with progerias have shorter telomeres. Cancer cells up-regulate telomerase which can prevent the telomeres from getting shorter and even elongate them. Telomerase is activated in approximately 90% of tumours. WEEK FOUR LECTURE 10: DNA REPLICATION To carry the genomic information to daughter cells, the DNA molecule must replicate using itself as a template. TRITIATED THYMIDINE EXPERIMENT Evidence for semiconservative replication of eukaryotic chromosomes. Labelling of DNA in bean shoots with tritiated thymidine. The labelled DNA strands in the chromosomes is detected by autoradiography. The second round of DNA replication was carried out without the radioactive isotope so the new strands are not labelled. Note that only one of each chromatid is labelled, as predicted from the semiconservative replication model.
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