BIO 130.02 Long Test #1

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