Cytogenetics Finals Lesson 1 PDF

CYTOGENETICS FINALS LESSON 1 MODIFICATION OF ▪ Wild type allele is R1 = Red ▪ Mutant allele is R2 = White...

CYTOGENETICS FINALS LESSON 1 MODIFICATION OF ▪ Wild type allele is R1 = Red ▪ Mutant allele is R2 = White ▪ This type of mutation is loss of function MENDELIAN RATIOS ALLELES ALTER PHENOTYPES IN DIFFERENT WAYS Terminologies: Wild-type Allele – most frequently encountered allele in a population, arbitrarily considered as normal Null Allele – eventual loss of function of a gene due to mutation Mutation – modification of genetic sequence Gain of Function Mutation – increased activity of a gene due to mutation CODOMINANCE, THE INFLUENCE OF BOTH ALLELES IN HETEROZYGOTE Loss of Function Mutation – mutation that brought about diminished functionality of the gene CODOMINANCE Neutral Mutation – mutation in a gene that does In heterozygotes, both alleles are not necessarily alter its activity DOMINANT thereby expressing 2 distinct GENETICISTS USE A VARIETY OF SYMBOL FOR gene products. ALLELES Inheritance of both dominant genes (2-unit Recessive Allele – Lowercase and italicized factors) leads to expression of 2 dominant phenotypes Dominant Allele – Uppercase and italicized Both traits are equally visible, both alleles NEITHER ALLELE IS DOMINANT IN INCOMPLETE, OR are equally dominant PARTIAL DOMINANCE Ex. The MN Blood Group system INCOMPLETE/PARTIAL DOMINANCE Genes at certain locus in chromosome 4 regulate for Neither of the alleles are dominant nor the production of M and N recessive thereby expressing an substance (glycoprotein), intermediate phenotype (blended). inheritance of both genes lead to production of both glycoproteins. 1  MULTIPLE ALLELES OF A GENE MAY EXIST IN A POPULATION Multiple Alleles – in a population of organisms, the presence of three or more alleles of the same gene. THE ABO BLOOD GROUP MUTATIONS IN GENES THAT DO SUCH MAY BE LETHAL Simplest case of multiple alleles is that which three alternative alleles of one gene exist. Homozygous Recessive Lethal Allele - Lethal indeed. Discovered by Karl Landsteiner Heterozygous Recessive Lethal Allele Four Phenotypes of ABO Blood Group - The lethal mutation is present (mutation that 1. A antigen (A phenotype) led to total non-functionality of the gene) but there is another allele that may compensate. 2. B antigen (B phenotype) Dominant Lethal Allele 3. A and B antigen (AB phenotype) - Inheritance of even just one copy is lethal. COMBINATION OF TWO GENE PAIRS WITH TWO MODES 4. Neither antigen (O phenotype) OF INHERITANCE THE BOMBAY PHENOTYPE Modifies the 9:3:3:1 Mendel’s ratio A rare variant of the ABO antigen system in Genetic Linkage – it is defined as the which affected individuals do not have A or tendency for alleles close together on the B antigens and thus appear to have blood same chromosome to be transmitted type O, even though their genotype may together as an intact unit through meiosis. carry unexpressed alleles for the A and/or B antigens. PHENOTYPES ARE OFTEN AFFECTED BY MORE THAN ONE GENE Epistasis - The nonreciprocal interaction between nonallelic genes such that one gene influences or interferes with the expression of another gene, leading to a specific phenotype. LETHAL ALLELES Novel Phenotype - Other cases of gene interaction yield novel, Genetic mutation which might lead to death. or new, phenotypes in the F2 generation, in addition to producing modified dihybrid Genes function primarily for the ratios. expression/production of substances - Interaction between two genes produces essential to life. entirely new phenotypes. 2 CYTOGENETICS FINALS LESSON 2 KARYOTYPING Walter Flemming – earliest drawing of chromosomes in 1882. His depiction captures random distribution of INTRODUCTION chromosomes as they splash down on a slide. Karyotyping can be used in: III. SWELLING, SQUASHING, AND UNTANGLING 1. Prenatal monitoring (amniotic fluid) 1951 – the untangle of spaghetti-like mass 2. Cancer detection and monitoring (bone of chromosomes was solved. marrow aspirate and CBC) - Unknown etiology A technician mistakenly washed white blood cells being prepared for chromosome 3. Culture in enrichment media analysis in a salt solution that was less concentrated than the interiors of the cell. I. PROCEDURE IN KARYOTYPING Water rushed into the cells, swelling them and separating the chromosomes. Preparation of Metaphases 1. Culture (72 hours to 14 days) Albert Levan and Joe-Hin Tjio – found that 2. Synchronise drawing cell-rich fluid into a pipette and 3. Harvest dropping it onto a microscope slide prepared 4. Analyze Chromosomes with stain burst the cells and freed the mass 5. Stain Slides of chromosomes. Adding a glass coverslip 6. Prepare Slides spread the chromosome enough that they could be counted. II. PREPARING CELLS FOR CHROMOSOME OBSERVATION Karyotypes were once constructed using a Cytogenetics have tried to describe and microscope to locate a cell where the display human chromosomes since the 19th chromosomes were not touching, century. photographing the cell, developing a print, cutting out the individual chromosomes, and Theophilus Painter (1923) – publish arranging them into a size-ordered chart. sketches of human chromosomes from 3 patients at Texas State Mental Hospital. Today: - Computer scans ruptured cells in a drop of stain and selects one in which the The difficulty in distinguishing between 46 or chromosomes are the most visible and 48 chromosomes was physical – it is well spread. challenging to prepare cell, a cell in which chromosomes do not overlap. - Image Analysis Software – recognizes the pattern of each stained chromosome Colchicine – cytogeneticist used to arrest pair, sorts the structures into a size- cells during division; an extract of the crocus ordered chart, and prints the karyotype. plant. 1  If the software recognizes an abnormal FISH (FLUORESCENCE IN SITU HYBRIDIZATION) band pattern, a database pulls out identical or similar karyotypes from More precise staining because it uses DNA records or either patient. probes that are complementary to specific DNA sequences. SkyView EXPO – the automatic spectral karyotyping system, lets you work with Probes are attached to molecules that banded images, multi-color SKY images and fluoresce when illuminated, producing a chromosome ideograms. flash color precisely when the probe binds to a chromosome in a patient’s sample. FISHView – is applied spectral imaging’s fully automated image acquisition and FISH can “paint” entire karyotypes by analysis system for FISH. This is helpful in probing each chromosome with several diagnosis of breast cancer. different fluorescent molecules. IV. STAINING A computer integrates the images and creates a unique false color with each In the earliest karyotypes, dyes were used chromosome. to chromosomes a uniform color. CHROMOSOMAL SHORTHAND Chromosomes were grouped into size, classes, designated A through G, in Geneticist abbreviate information in a karyotype by decreasing size order. listing: 1. Chromosome number 1959 – scientist described first chromosomal 2. Sex chromosome abnormalities: 3. Abnormal autosomes → Down Syndrome – an extra chromosome 21 Abbreviation What it Means → Turner Syndrome – also called XO 46, XY Normal Male syndrome, a female with only one X chromosome 46, XX Normal Female → Klinefelter Syndrome – also called XXY syndrome, a male with an extra X chromosome 45, X Turner Syndrome (Female) 1970 – Swedish scientist developed stains that create banding patterns unique to each chromosome. 47, XXY Klinefelter Syndrome (Male) Stains are specific for AT-rich and GC-rich, sketches DNA or for heterochromatin, which is dark staining. 47, XYY Jacobs Syndrome (Male) A band represents at least 5 to 10 million DNA bases. 2  A male missing part of For humans, white blood cells are used most 46, XY, del (7q) the long arm frequently because they are easily induced chromosome 7 to divide and grow in tissue culture. Sometimes, observations may be made on 47, XX, +21 A female with trisomy non-dividing (interphase) cells. The sex of 21 down syndrome an unborn fetus can be determined by observation of interphase cells (amnioti centesis and Barr body). 46, XY, t (7,9) A male with a (p21.1;q34.1) translocation between BARR BODY the short arm of chromosome 7 at band A normal human female has only one Barr 21.1 and the long arm body per somatic cell, while a normal of chromosome 9 at human male has none. band 34.1 A Barr body (named after discoverer Murray Barr) is the inactive X chromosome 48, XXYY A male with an extra in a female somatic cell. “X” and an extra “Y” IDEOGRAM An ideogram is a schematic chromosome map. It indicates chromosome arm (P or Q) and major regions delineated by banding patterns. STAINING Study of karyotypes is made possible by staining. Staining helps in the detection of the arrangement of chromosomes. A suitable dye such as Giemsa. It is applied after cells have been arrested during cell division by a solution of colchicine FUNDAMENTAL NUMBER (FN) usually in metaphase or prometaphase when most condensed. The fundamental number, FN, of a karyotype is the number of visible major In order for the Giemsa stain to adhere chromosomal arms per set of correctly, all chromosomal proteins must be chromosomes. digested and removed. 3  Thus, FN ≤ 2 x 2n, the difference depending HAPLO-DIPLOIDY on the number of chromosomes considered single-armed (acrocentric or telocentric) Where one sex is diploid, and the other is present. haploid. It is a common arrangement in the Hymenoptera, and in some other groups. Humans have FN = 82, due to the presence of five acrocentric chromosome pairs: 13, 14, 15, 21, and 22. The fundamental autosomal number or autosomal fundamental number, FNa or AN of a karyotype is the number of visible major chromosomal arms per set of Autosomes (non-sex-linked chromosomes). ENDOPOLYPLOIDY CHROMOSOME ABNORMALITIES Endomitosis – a process by which PLOIDY chromosomes replicate without the division of the cell nucleus, resulting in a polyploid Is the number of complete sets of nucleus. chromosomes in a cell. Occurs when in adult differentiated tissues, Polyploidy, where there are more than two the cells have ceased to divide by mitosis, sets of homologous chromosomes in the but the nuclei contain more than the original cells, occurs mainly in plants. somatic number of chromosomes. It has been of major significance in plant In the endocycle (endomitosis or evolution according to Stebbins. endoreduplication), chromosomes in a ‘resting’ nucleus undergo reduplication, the The proportion of flowering plants which are daughter chromosomes separating from polyploid, was estimated by Stebbins to be each other inside an intact nuclear 30-35%, but in grasses the average is much membrane. higher, about 70%. In many instances, endopolyploidy nuclei Polyploidy in lower plants (ferns, horsetails, contain tens of thousands of psilotales) is also common, and some chromosomes (which cannot be exactly species of ferns have reached levels of counted). polyploidy far in excess of the highest levels known in flowering plants. The cells do not always contain exact multiples (powers of two), which is why the Polyploidy in animals is much less common, simple definition “an increase in the number but it has been significant in some groups. of chromosome sets caused by replication without cell division is not quite accurate. Polyploid series in related species which consist entirely of multiples of a single basic This process (especially studied in insects number, are known as euploid. and some high plants such as maize) may 4  be a developmental strategy for increasing Human chromosome 2 was formed by a the productivity of tissues which are highly merger of ancestral chromosomes, reducing active in biosynthesis. the number. The phenomenon occurs sporadically EUPLOIDY throughout the eukaryote kingdom from protozoa to man; it is diverse and complex, Some individuals possess one or more and serves differentiation and complete genomes in a cell, which may be morphogenesis in many ways. identical with or distinct from each other. ANEUPLOIDY Most common types are those in which two copies of the same genome are obtained. Is the condition in which the chromosome Since the basic chromosome number or number in the cells is not the typical genomic number is x, the above situation is number for the species. represented as 2x. This would give rise to a chromosome CHROMOSOMAL POLYMORPHISM abnormality, such as an extra chromosome or one or more chromosomes lost. Some species are polymorphic for different chromosome structural forms. Abnormalities in chromosome number usually cause a defect in development. The structural variation may be associated with different numbers of chromosomes in Down syndrome and Turner syndrome are different individuals, which occurs in the examples of this. ladybird beetle Chilocorus stigma, some mantids of the genus Ameles the European Aneuploidy may also occur within group of shrew Sorex araneus. closely related species. There is some evidence from the case of the Classic examples in plants are the genus mollusc Thais lapillus (the dog whelk) on the Crepis, where the gametic (= haploid) Brittany coast, that the two chromosome numbers form the series x = 3, 4, 5, 6, and morphs are adapted to different habitats. 7; DEPICTION OF KARYOTYPES Crocus, where every number from x = 3 to x = 15 is represented by at least one Cytogenetics employs several techniques to species. visualize different aspects of chromosomes. Evidence of various kinds shows that trends TYPES OF BANDING of evolution have gone in different directions in different groups. 1. G-banding 2. R-banding Closer to home, the great apes have 24x2 3. C-banding chromosomes whereas humans have 23x2. 4. Q-banding 5. T-banding 5  G-BANDING prior to staining leading to an almost complete depurination of the DNA. It is obtained with Giemsa stain following digestion of chromosomes with trypsin. After washing the probe, the remaining DNA is renatured again and stained with Giemsa It yields a series of lightly and darkly stained solution consisting of methylene azure, bands – the dark regions tend to be methylene violet, and eosin. heterochromatic, late-replicating and AT- Heterochromatin binds a lot of the dye, while rich. the rest of the chromosomes absorb only little of it. The light regions tend to be euchromatic, early-replicating, and GC-rich. This method The C-banding proved to be especially well- will normally produce 300-400 bands in a suited for the characterization of plant normal, human genome. chromosomes. Procedure: Q-BANDING 1. De-stain slides for 10 minutes in 95% ethanol. A fluorescent pattern obtained using 2. Place slide in PBS (phosphate buffer quinacrine for staining. solution) and incubate for 10 minutes at 56°C. The pattern of bands is very similar to that 3. Treat slide with 0.22 mL of stock Giemsa and seen in G-banding. 6.5 mL of PBS with a pH of 7.4. 4. Flood slide with stain solution for 15 They can be recognized by a yellow minutes. Rinse with water and air dry. fluorescence of differing intensity. Most part 5. View slide under bright field, oil immersion of the stained DNA is heterochromatin. microscope. Quinacrine (atebrin) binds both regions rich in AT and GC, but only the AT quinacrine- R-BANDING complex fluoresces. Since regions rich in AT are more common in heterochromatin It is the reverse of G-banding (the R stands than in euchromatin, these regions are for “reverse”). labelled preferentially. The dark regions are euchromatic The different intensities of the single bands (guanine-cytosine rich regions) and the mirror the different contents of AT. bright regions are heterochromatic (thymine-adenine rich regions). Other fluorochromes like DAPI or Hoechst 33258 lead also to characteristic, C-BANDING reproducible patterns. Each of them produces its specific pattern. In other words: Giemsa binds to constitutive the properties of the bonds and the heterochromatin so it stains centromeres. distribution of AT and the association of AT with other molecules like histones, for The name is derived from centromeric or example, has an impact on the binding constitutive heterochromatin. The properties of the fluorochromes. preparations undergo alkaline denaturation 6  T-BANDING Karyotypes are arranged with the short arm of the chromosome on top, and the long arm Visualize telomeres on the bottom. Some karyotypes call the short and long arms p and q respectively. In Silver staining: Silver nitrate stains the addition, the differently stained regions and nucleolar organization region-associated sub-regions are given numerical protein. This yields a dark region where the designations from proximal to distal on the silver is deposited, denoting the activity of chromosome arms. rRNA genes within the NOR. For example, Cri du chat syndrome T-Banding by Thermal Denaturation: Method 1 involves a deletion on the short arm of chromosome 5. It is written as 46, XX, 5p-. 1. Bring 94 mL of distilled water and 3 mL of The critical region for this syndrome is phosphate buffer (pH 6.7) to 87°C in a deletion of p15.2 (the locus on the Coplin jar. chromosome), which is written as 46, XX, 2. Add 3 mL of Giemsa stain. del (5) (p15.2). 3. Add slides to jar; stain for 5 to 30 minutes. 4. Rinse in distilled water, air dry, and examine. SPECTRAL KARYOTYPE (SKY TECHNIQUE) For Fluorescent Observation Spectral karyotyping is a molecular cytogenetic technique used to 5. Destain, rehydrate through a series of simultaneously visualize all the pairs of alcohols, rinse in distilled water. chromosomes in an organism in different 6. Stain in acridine orange (5mg/100mL) for 20 colors. minutes. 7. Rinse in phosphate buffer, mount, and Fluorescently labeled probes for each examine with a fluorescence microscope chromosome are made by labeling (excitation: 450-490 nm; suppression: 515 chromosome-specific DNA with different nm) fluorophores because there are a limited number of spectrally distinct fluorophores, a CLASSIC KARYOTYPE CYTOGENETICS combinatorial labeling method is used to generate many different colors. Karyogram from a human female lymphocyte probed for the Alu sequence Spectral differences generated by using FISH. combinatorial labeling are captured and analyzed by using an interferometer In the “classic” (depicted) karyotype, a dye attached to a fluorescence microscope. often Giemsa (G-banding), less frequently Image processing software then assigns a Quinacrine is used to stain bands on the pseudo color to each spectrally different chromosomes. Giemsa is specific for the combination, allowing the visualization of the phosphate groups of DNAs. individually colored chromosomes. Quinacrine binds to the adenine-thymine DIGITAL KARYOTYPING rich regions. Each chromosome has a characteristic banding pattern that helps to Digital karyotyping is a technique used to identify them; both chromosomes in a pair quantify the DNA copy number on a genomic will have the same banding pattern. scale. Short sequences of DNA from specific 7  loci all over the genome are isolated and Group F: chromosomes 19-20 are short with enumerated. median centromere This method is also known as virtual Group G: chromosomes 21-22 are very short with karyotyping. acrocentric centromere CLASSIFICATION OF CHROMOSOMES FOR Chromosome X is similar to group C KARYOTYPING Chromosome Y is similar to group G Chromosomes are arranged into seven groups based on size and centromere location. The centromeres can be found in the middle of the chromosome (median), near one end (acrocentric), or in between these first two (submedian). Group A: chromosomes 1-3 are largest with median centromere Group B: chromosomes 4-5 are large with submedian centromere Group C: chromosomes 6-12 are medium sized with submedian centromere Group D: chromosomes 13-15 are medium sized with acrocentric centromere Group E: chromosomes 16-18 are short with median or submedian centromere 8