Module 0 | Cell Division (ppt)

Rudolf Virchow- “Omnis cellula e cellula”, all cells come from pre-existing cells

Functions of cell division: Growth and development Repair of damaged tissues and organs Binary fission- asexual reproduction in unicellular organisms Formation of sex cells- gametogenesis

Mitotic cell division produces somatic (body) cells No recombination Produces 2 genetically identical diploid daughter cells

Meiotic cell division Produces sex cells (gametes) Genetic recombination occurs Produces 4 genetically different haploid daughter cells

DNA in Eukaryotic Cells stored in the nucleus Takes form of chromatin

Chromatin- DNA+histone proteins Chromosomes- condensed form of DNA Centromere- specialized sequence of DNA where sister chromatids are joined Kinetochore- protein complex where spindle fibers attach Ploidy- refers to number of sets of chromosomes in a cell Centrosomes- where spindle fibers are organized, come in pairs Centrioles- centrosome for animal cells

The cell cycle is 90% preparation and 10% execution

Interphase G1- growth phase, cells increase in volume by producing more cytoplasm and organelles S phase- DNA replication takes place G2- growth phase and preparation for mitosis, errors in DNA replication are addressed here G2- nucleolus and nuclear envelope are still intact

Mitosis- also known as Karyokinesis

Prophase Chromatin fibers condense into chromatids Nucleolus and nuclear membrane dissolve Formation of spindle fibers (microtubules) Centrosomes start to migrate to the poles

Prometaphase- period of movement of chromosomes toward the center of the cell

Metaphase Chromosomes align at the middle (metaphase plate) Spindle fibers attach to the kinetochore Centrosome are at the pole producing both kinetochore and nonkinetochore microtubules

Anaphase Sister chromatids separate and migrate toward the opposite poles Motor proteins- located in kinetochore complex, digest the spindle fibers, pulls on sister chromatids causing them to separate and move Spindle fibers facing away from the chromatids push on the cell, elongating it

Telophase Chromatid arrive at the opposite poles, becoming less condensed Spindle fibers dissolve Nuclear envelope and nucleus begin to reform Cleavage furrow- indentation in animal cells begin to form Cell plate- indentation in plant cells begin to form

Cytokinesis Cytoplasm is divided in half, forming 2 daughter cells At the end of this, chromatids are no longer condensed and nuclear envelope has reformed Nucleoli may be seen again

Meiotic Cell Division Reductional division One round of DNA replication (interphase) followed by two rounds of cell division (Meiosis I and Meiosis II) Gametogenesis- production of sperm and egg cells (n) from germ cells (2n) Primary basis for genetic variation in diploid organisms

Difference between Meiosis I and II? Meiosis I (2n→n) and Meiosis II (n→n) Meiosis I has interphase (IPMAT) and Meiosis II does not

Interphase (2n)- Genetic material is replicated Prophase I (2n) substages: Leptotene- homology search Zygotene- synapsis, pairing up Pachytene- recombination Diplotene- chiasmata formation Diakinesis- separation

Prophase I- chromatin condense intro chromatid, Nuclear envelope and nucleolus dissolve

Chiasmata- crossing over, Synaptonemal complex- holds synapsis together, chromosomes aligned Homologous chromosomes- have the same DNA length, same genes, centromeres are in the precise location Tetrads- homologous chromosomes align Recombination- exchange of genetic information

Metaphase I (2n)- spindle fibers attach to both kinetochores of the sister chromatids Anaphase I (2n)- chiasmata separate, homologous pair move toward opposite poles, sister chromatids are still attached Telophase I + Cytokinesis (n)- forms 2 haploid daughter cells with sister chromatids attached to each other

Prophase II (n)- no pairing of homologous chromosomes, no crossing over Metaphase II (n)- alignment, spindle fibers attach to the kinetochore Anaphase II (n)- sister chromatids separate and move toward opposite poles Telophase II + cytokinesis (n)- results to 4 haploid daughter cells

Fertilization- fusion of genetically diverse gametes

Module 1.1 | Mendelian Pattern (ppt)

What accounts for the passing of traits from parent to offspring? Blending hypothesis- physical attribute from 2 parents blend to form the attributes of the offspring Particulate hypothesis- parent pass on discrete heritable units onto their offspring

Character- pertains to a physical aspect of the pea plant Traits- variation of each character

Mendel’s Main Findings Traits must be determine by discrete heritable units passed from parent to offspring One variation of these unit factors can mask the other variation Each member of the pairs units are segregated into separate gametes during meiosis Combination of male and female gametes during fertilization determine the genetic composition and the trait exhibited by the offspring

1857- mendel describes the units of heredity 1900s- sutton and boveri determine these units were carries in chromosome

Allele- bateson and saunders, variation in the part of the chromosome that codes for a specific traits Gene- johansenn

DNA- the biomolecule that contains the hereditary information of an organism in its sequence of nucleotide bases (A, T, C, G) Gene- a specific sequence in DNA that codes for a particular character Locus- where each gene is situated on a location in the chromosome Genotype- genetic composition Phenotype- physical appearance Reginald C. Punnett- creator of punnett square

Mendel’s Postulates: Unit Factors in Pairs- genetic characters are controlled by unit factors existing in pairs in individual organisms Dominance/Recessiveness- when two unlike unit factors responsible for a single character are present in a single individual, one unit factor is dominant to the other, which is said to be recessive Segregation- during the formation of gametes, the paired unit factors separate, or segregate, randomly so that each gamete receives one of the other with equal likelihood Independent Assortment- during gamete formation, segregating pairs of unit factors assort independently of each other

Independent assortment- the presence of one trait does not guarantee the presence of another, equal frequency

Module 1.2 | Non-Mendelian Pattern of Inheritance (ppt)

Incomplete Dominance- “Blending of traits”, traits are not permanently blended Incomplete Dominance- phenotype is a blend of contrasting homozygous parents Haploinsufficiency- when one copy of the functional allele is not enough for a standard/wild-type phenotype Snapdragons- examples of incomplete dominance Incomplete dominance follows paired unit factors and segregation

Codominance- patchwork, both alleles are evident Rabbits- examples codominance White-color coat and black color coat are dominant over albinism

Multiple Alleles- While an individual can only have at most 2 different alleles, the number of allele in a population can be more than 2

ABO Blood Groups Codominance Alleles A and B are dominant over O Phenotype is dependent on presence of antigens (glycolipids) on the surface of RBCs A and B alleles codes for functional glycosyltransferases Chromosome 9- ABO locus O allele- codes for nonfunctional glycosyltransferases O allele- cannot be detected by A or B antibodies, does not trigger agglutination Agglutination- clumping of blood cells when reacting with certain antigens

Epistasis- the expression of one gene masks the expression of another gene Epistasic- blocks Hypostatic- blocked Bombay Phenotype- FUT1 gene codes for fucosyltransferase that is responsible for synthesizing the H-antigen, recessive condition FUT 1 gene is mutated and codes for nonfunctional enzyme Masks the expression of A and B alleles, RBCs are type 0 phenotypically

Summary of Examples Incomplete dominance- snapdragons Codominance- rabbits Multiple alleles- ABO Blood Groups Epistasis- Bombay Phenotype

Module 1.3 | Karyotyping (ppt)

Gregor Mendel (1865)- discovered the basic principles of heredity through pea plants Brunn Society of Natural Science- where Mendel proposed his work but was largely appreciated

Why was his work unappreciated? It was too ahead of its time and and too controversial on popular beliefs about heredity

Walter Sutton and Theodor Boveri- independently determined that chromosome were carriers of Mendel’s units of heredity Sutton- observed meiosis in grasshopper Boveri- observed embryogenesis in sea urchins and parascaris nematode Walter Sutton and Theodor Boveri- Chromosome theory of inheritance Alfred Hershey and Martha Chase- Solidified DNA as genetic material, used T2 bacteriophage to show that DNA carries viral genetic information into the host cell

Eukaryotic cells have more DNA than prokaryotic cells

DNA Organization (biggest to smallest) DNA strand (2 nm)- short region Nucleosome (11 nm)- 1st level of chromatin organization DNA + histones Solenoid Fiber (30 nm)- 2nd level of chromatin organization Nucleosome packed into helical coils Looped Domains (300 nm) Looping solenoid fiber attached to a protein scaffold Chromatid (700 nm)- 3rd level of organization Looped domains are coiled together Most condensed at metaphase Chromosome (1400nm) 2 sister chromatid joined together at the centromere DNA has replicated (700nm x 2)

Chromosome structure P-arm- short arm Q-arm- long arm Centromere- specialized sequence of DNA where sister chromatids are joined Kinetochore- protein complex where spindle fibers attach, generates movement of chromatids toward opposite poles

Centromere Location Metacentric: p = q 1, 3, 16, 19, 20 Submetacentric: p slightly shorter 2, 4 12, 17, 18, X Acrocentric: p very much shorter 13 15, 21, 22, Y Telocentric: p almost unobservable Not present in humans

Karyotype Preparation Cell division induced in cells During metaphase, a chemical substance is added to disrupt mitosis Cells are fixed onto a microscope slide and stained Chromosome are observed and photographed Image is edited to arrange into a karyotype

5ml venous blood Phytohemagglutinin and culture medium Culture at 37°C for 3 days Colchicine and hypotonic saline Cells fixed Spread cells by dropping Trypsin and stain with giemsa Photograph metaphase spread Karyotype

Colchicine- a mitotic inhibitor an alkaloid substance from autumn crocus anti-inflammatory Used to treat gout as a tubulin disruptor, downregulating multiple inflammatory pathways

The Metaphase Spread Highly condensed chromosomes Not aligned at the metaphase plate die to lack of mitotic spindles to arrange them Banding patterns are apparent

Euchromatin- loosely bound segments of DNA Possesses highly expressed genes, highly acetylated Stain as light bands

Heterochromatin- tightly bound segments of DNA Possesses deactivated or seldomly expressed genes, highly methylated Stain as dark bands

International System for HUman Cytogenetic Nomenclature (ISCN)- standardized karyotype notations

(chromosome#)(arm)

Aneuploidy- missing or extra chromosomes Polyploidy- more than two complete sets of chromosomes (3n/4n)

Structural variations Deletion Duplication Inversion Translocation

Nondisjunction- error in meiosis that lead to aneuploidy such as trisomies and monosomies

Trisomy 21 “Down’s Syndrome”, female = 47, XX, +21 Only one sex chromosome “Turner Syndome”= 45, XO

Aneuploidy- chromosome number is not equal to 46 Klinefelter syndrome= 47, XXY Turner syndrome= 45, XO Down syndrome (male)= 47, XY, +21 Patau syndrome (female)= 47, XX, +13

Polyploidy- more than two sets of chromosomes (3n, 4n, etc.) 69, XXX: due polyspermy, digyny (diploid ovum), or diandry (diploid sperm); offspring often miscarriages or dies shortly after birth 92, XXXX: much more rare than triploidy ; accounts for 1-2% of early miscarriages

Deletion- portion of a chromosome is lost Terminal deletion: loss of one end Intercalary deletion: loss of an interior portion Example: Cri du chat syndrome- 46,XX/Y, del (5p) DiGeorge syndrome- 46, XX, del(22q) Duplication- a single locus or a large piece is present more than once in a genome Cause: unequal crossing over, replication errors in meiosis Inversion- a segment is turned 180 degrees in a chromosome Double-strand breaks at 2 points and subsequent reinsertion Translocation- movement of a chromosomal segment to a new location in the genome Reciprocal translocation- exchange of segments between non-homologous chromosomes

Module 2.1 | Sex-Linked Traits (ppt)

Drosophila melanogaster, aka the Fruit Fly One of the most prized organism in biological research as a model organism Widely studied and used as a representative for other organisms Easy to cultivate and maintain in a laboratory setting Ideally their whole genomes have been characterized and sequenced

Why is the fruit fly a good model organism? Logistical advantages- short life cycle, easy of culture and maintenance Advantages for genetic research- low number of chromosomes, small genome size in terms of number of base pairs, homologous gene in humans

Short Life Cycle- 4 life stages in 12 days Embryo- 1 day Larval- 5 days Pupal- 4 days Adult-2 days

Ease of Culture and Maintenance High fecundity: a female can lay 750-1500 eggs in her lifetime Easy containment because of minute size, e.g. Gatorade bottles Easy food source, e.g. oatmeal-agar mixture

Low Number of Chromosomes Fewer chromosomes make it easier to map out and locate genes Polytene chromosome- found in salivary glands of larvae Caused by endomitosis- 10 rounds of repeated DNA replication without cell division Easily visualized with dark and light bands which can serve as a road map for gene location Polytene puffing- can be used to study transcription activities 4 pairs of chromosomes Chromosome 1: sex chromosomes Chromosome 2-4 autosomes

Small Genome Size The whole genome sequence was published in Science in March 2000 Comparison to the human genome: Only 5% the size of human genome at 132 million base pairs Gene density per chromosome is higher (15,500 genes on four chromosomes vs. 22,000 genes on 23 chromosomes) 60% similar in terms of gene sequence

Homologous Genes in Humans Developmental Genetics Fruit flies and humans have similarities in the genes that are responsible for the development of specific body segments The hedgehog gene (hh) Partially responsible for the control of segmentation and future head-tail body patterning in fruit fly embryos If disrupted, mutant tend to have tiny projections covering their entire surface

3 Homologues of the hh gene Desert- dhh Indian- ihh Sonic- shh, development of the notochord, neutral tube, gut, and limbs Birth defect of dhh- cleft palate, no separation of brain hemisphere, abnormal eyes

Fruit-Fly Mutant Phenotypes: White- eye trait Eye color: Wild type (W+): red eyes, phenotype of the typical form of a species as it occurs in nature Mutant (w): sex-linked mutation, linked to x-chromosome, recessive trait

Sex-linked inhertitance Most sex-loinked condition are coded in x chromosome Red-green color blindness Male pattern baldness Hemophilia Duchenne muscular dystrophy X-linked recessive Females- both X chromosomes must have the allele, if not, carrier only Male- only 1 X chromosome needed

How to know if a trait is sex linked?

In hybridization experiments, perform reciprocal crosses Crossing a pair of parents with contrasting traits with the sexes reversed. Sex linked inheritance should lead to different F1 phenotypes. E.g. reciprocal crosses for the white eye trait in fruit flies. X linked recessive Trait skips generation. Males are more often affected. Affected fathers usually don’t have affected sons. Affected mothers always have affected sons. X linked dominant Trait is more common. Males and females are equally likely to be affected. Affected fathers have all their daughters affected.

Module 2.2 | Polytene Chromosome (ppt)

Polytene Chromosomes Found in the salivary glands of dipteran flies Drosophila Chironomus Rhynocosciara Multiple copies of gene allow for high expression rate of important proteins; e.g. adhesive proteins needed for pupation Produced through endometriosis Repeated DNA replication without cell division Strands of DNA still attached to each other Leads to cell expansion and massive chromosome

Euchromatin vs. Heterochromatin When stained (e.g. with aceto-orcein), banding patterns are very visible

Heterochromatin Dark staining bands More condensed Often with inactive genes More DNA, less RNA

Euchromatin Light-staining bands Less condensed Often with active genes More RNA, less DNA

Polytene Puffing Chromosomes “puff” up at certain points Site of active transcription of genes in the area DNA, RNA, transcription proteins Different points in the chromosome may puff up depending on the development stage May indicate which gene are more active per stage of the larva → function

Dissection Protocol