BIO417 Midterm

Lectures 1-5

Genomics

• Focuses on the structure function, evolution, mapping, and editing of genomes

• Studies all its individual genes including interactions of those genes w/ each other, and also takes external signals (environment) into account

Genome biology

Studies quantification, packaging, storage, proof reading, and replication of genetic information

Genome size is measured by

Number of bases or C-value

C-value

Amount of DNA in a haploid nucleus (picograms)

The C-value paradox

Nuclear genome size varies strongly among species; there is no apparent relationship to the number of genes encoded in the genome that reflects the complexity of the organism

The phosphate groups and sugar backbone of the DNA are…

Polar and hydrophilic

The DNA bases are…

Hydrophobic; bases interact w/ each other to minimize interaction w/ the outside

Bases can’t mispair because…

It changes the space they take up (more or less) so they don’t fit into the backbone

Average twist angle for a single row

36º

Number of bp for a full twist

10

3 families of helicies

A-DNA, B-DNA, Z-DNA

Right handed helicies

A-DNA and B-DNA

Z-DNA

DNA-RNA hybrid structure; left handed

B-DNA

Average DNA conformation

Nuclosome

Basic unit of chromatin; made up of 147 bp of DNA, 2 H2A, 2 H2B, 2 H3, 2 H4, and a H1 linker histone

DNA-histone interaction

Small, basic, proteins attracted to negatively charged DNA; non-covalent

Histones

• Small basic proteins (100-220 residues; 11-15 kDa, H1 ~22 kDa)

• Conserved histone fold

• Structured motif (more so than DNA sequence) conserved

• N-terminal tail = variety of mods

Number of contact points between DNA and histones

12 overall (8 histones → 4 dimers x 3 contact points w/ DNA)

Histone dimers interact w/..

The minor grove

Total dimer interaction

147 bp / 10 bp for a full turn = 14.7 minor groves

Centromere function

Segment of chromosomal DNA that provides attachment sites for the kinetochore

Centromere structure

• CEN region → minimal region that supports chromosomal segregation

• Rich in repeats

• Humans: 𝛂-satelite DNA; 171-bp

• Heterochromatic

• Binds platform proteins (kinetochore) which binds microtubles

CENP-A

Centromere-specific H3 variant; not sequence specific

Kinetochore and microtubule interaction

Lose until there’s force on the microtubule, then it gets really tight

CENP-B

Protein in humans that recognizes centromere-specific repeats and turns sequences into centromere formation (accessory; not neccessarily required b/c the process isn’t sequence specific)

Telomere function

Region of specific repetitive DNA sequences at the end of a linear eukaryotic chromosome that protects chromosome ends and shortens slightly as cells divide

Telomere structure

• Short tandem repeat

• T_1-4A_0-1G_1-8

  • Human: TTAGGG

  • Tetrahymena: TTTGGGG

  • Arabidopsis: TTTAGGG

• Can form G-quadruplexes

• Length in humans: 5-20 kb

• Shelterin complex

• T-loops

Shelterin complex

Cover and compact repeats on the telomere; protect form repair

T-loops

Protruding single strand end, loops back into telomeric repeat

Hayflick limit

After a certain number of cell divisions, the telomere shortens until the cell stops dividing (apoptosis)

Telomerase

Attracted to telomeric repeats and synthesizes additional repeats; DNA pol unable to replicate the 3’ ends of DNA strand

Key features of the chromosome

• Centromere

• Telomeres

• Many origins of replication

• p and q arm (p = shorter; q = longer)

• Genes (interspaced)

• Kinetochore protein contact points

• Repetitive sequences

• Pericentromeric region

Metacentric chromosome

Centromere in middle (ex Chr1)

Submetacentric chromosome

Centromere towards the middle (ex Chr 4)

Acrocentric chromosome

Centromere towards the tip (ex Chr 14)

Telocentric chromosome

Centromere at the tip

Cytogenetic stains

• Allow to further characterize chromosomes

  • Giemsa → G-bands

  • Quinacrine → Q-bands

  • Reverse giemsa → R-bands

  • Telomere visualization → T-bands

  • Silver nitrate staining → NOR (nucleolar organization region; code for rRNA)

Giemsa stain

• Typical stain

• Specific for the phosphate groups of DNA in AT-rich regions (heterochromatic regions = dark)

• Mix of methylene blue, eosin, and Azure B (readymixed powder)

• With cell arrest in metaphase or prometaphase

• Used to generate karyogram

Karyotype

• General appearance of the full set of chromosomes of an individual

• Can be obtained and represented in image (micrograph) of an individual’s metaphase chromosomes

Karyotype sorted by

• Size (assigned a number based on size, largest #1)

• Chromosome type (autosomes first, sex chr last)

• Centromere position

• Short (p) arm on top

• Further divided into groups based on size and centromere position

Ideogram for G-banding

• Standardized map of a chromosome set

• Centromeres = dark grey

  • Used as a reference point

• Can visualize missing/extra chromosomes or pieces of chromosomes

Holocentric chromosomes

• One size-restricted centromere per chromosome

• In diverse eukaryotic linages including algae, some nematodes, insects, spiders, plants (sedges)

• Convergent evolution

• Diffuse kinetochores/several distinct microtubule binding sites

Rules for karyotyping

• 22 autosomes by size

• Type (sex chromosomes at end)

• p arm oriented to the top

• Chr classified into 7 groups (A-G) by length and centromere position

Ideograms for G-banding of human metaphase chromosomes

• >400 bands (n)

• Centromeres = dark grey

Myrmecia pilosula

• Primitive ants

• 2n = 2-32

• Model for chromosome set evolution

Diploscapter pachys

• Single pair of chromosomes

• Relative of C. elegans

• Truncated meiosis (absence of functional meiosis I)

• Only asexual reproduction

• Lack of telomeric repeats and genes for telomere protection

Polyommatus atlantica

• 2N = 458

• 10x more chromosomes than most butterflies

• Highest number of chromosomes in non-polyploid organisms (smalles chr size possible)

• Result of rapid fragmentation of autosomes

• Centromeric region across whole chromosome

Chromosome aberrations

Substantial change to the structure of a chromosome; aka chromosome mutations

Types of chromosomal aberrations

Deletions, inversions, insertions, duplications, exchanges, fusions (translocations)

Deletions and duplications

Alter amount of genes

Inversions and translocations

Rearrangements of genes

Deletions

• Chromosomal breaks

• Recombination at incorrect places

• Often detrimental

• Phenotypic consequences often correlate with the size

Cri-du-chat syndrome characteristics

• High pitched cry

• Intellectual disability

• Delayed development

• Small head size

• Low birth weight and weak muscle tone

• 1:20,000 - 50,000 newborns

Cri-du-chat syndrome chromosomal defect

• Section of chromosome 5 missing

• 5p- / 5p15.2 / 46,XX,del(5)(p15.2)

Cri-du-chat syndrome molecular mechanism

• CTNND2 gene affected: delta catenin, neural migration

• Others (SEMA5A, hTERT)

Subfunctionalization

Domains are separated into different genes, allowing for more precise regulation

Neofunctionalization

Second copy takes on different function (evolution)

Duplications can result in…

Subfunctionalization, neofunctionalization, or degeneration/gene loss

Duplication and gene families example

Evolution of the globin gene family in humans

Hemoglobin β chain genes

• Has control region that regulates entire gene cluster

• Genes begin w/ embryonic globin chain and the order corresponds with development in the order of use

• Locus as chromatin hub

  • Shared locus control region

  • Folding and gene activation depending on developmental need

Inversion

• Chromosome segment is flipped in opposite orientation

• Classified according to location relative to centromere

• Often has no phenotypic consequence (genetic info still there)

• Variation in size; rather common

Pericentric inversion

Contains centromere

Paracentric inversion

Doesn’t contain centromere

Examples of inversions with a phenotype

Hemophilia (type A, X linked inversion) → factor VIII, blood clotting protein

Crossing over with paracentric inversions

• Creates a normal chr, an inverted viable chr, a dicentric chr, and an acentric chr

• Acentric fragment is lost and dicentric fragment randomly breaks, creating two non viable deletion products

Crossing over with pericentric inversion

Creates a normal chr, an inverted viable chr, and two duplication/deletion chrs that are nonviable

Translocations

• Cross over and non-homologous recombination

• Balanced/reciprocal are often without phenotype

• Can produce abnormal gametes (semisterility)

Philadelphia chromosomes

• Chronic myelogenous leukemia (CML)

• Translocation creates new fusion gene on chr 22

  • Kinase that becomes more active

• Cells proliferate without being regulated by cytokines

Ring chromosomes

• Fusion of broken ends

• Maintenance doesn’t work, so the cell tries to connect to something so its not completely unprotected

Usual ring chromosomes

14, 10, 13, 4, 20

Aneuploidy

Unbalanced chromosome set; number of chromosomes is not a multiple of a set (trisomy and monosomy)

Colchicine

• Inhibits microtubules polymerization (binds tubulin subunits)

  • Inhibits assembly of the mitotic spindle

• Can be used for generation of altered chromosome sets (polyploids, aneuploids)

  • Used in karyotyping

• Alkaloid from autumn crocus

Datura stramonium (jimsonweed)

• Early model for studies on chromosome sets

• Plant of nightshade family

• Produces tropane (nitrous, bicyclic) alkaloids, psychoactive compounds

• n=12 chromosomes; diploid 2n=24

Systematic study of trisomies in jimsonweed

Identified all 12 of the possible 12 forms (complete set)

Cocklebur

• 2n+Chr6

• Narrow leaves

• Weak

• Inclined to droop

Number of monosomies and tetrasomies in jimsonweed

Only 1 of 12 forms identified

Number of ploidies (1n, 2n, 3n, 4n) in jimsonweed

All 4 of 4 possible forms identified

Incidence of aneuploidy in humans

About 35% of spontaneous abortions but only 0.3% of live births

Most common aneuploidys in humans

Trisomy of chromosome 13, 18, or 21 (smaller chrs; only trisomies that can be tolerated)

Sex chromosome aneuploidies are…

Often nonlethal

How to detect aneuplodies?

Karyogram or FISH

Monoploid individuals

Drone bees

Polyploidy is common in…

Plants

Polyploidy is common in plants and crops b/c…

• Plants larger in size

• Better stress resistance

• Larger flowers

• Higher yields

• Better adaptability

Polyploidy in wheat

Hexaploid; result of a hybridization between a domesticated tetraploid

Autopolyploids

Multiple genomes of a single species

Allopolyploids

Multiple genomes derived from 2 or more species

Polytene chromosomes

• Model for genetic experiments and analyses

• Found in Drosophila and other dipteran flies; salivary gland (larvae); interphase nuclei

Endomitosis

Specialized cells undergo repeated round of DNA replication without cell division; found in interphase nuclei

Mutations

• Any change in base-pair sequence of DNA

• Source of genetic variation

• Raw material for natural selection

• Occur spontaneously

• Can be induced by external factors (e.g. chemicals, radiation)

• Can be detected and repaired: proofreading, correction of replication errors, BER (base excision repair), homologous recombination repair

Mutation classification is based on

• Molecular change

• Phenotypic effects

• Location

• Mode of generation

Frameshift

Deletion or insertion of any number of bases, except multiples of 3

Substitution

Missense (difference amino acid), nonsense (stop), silent (same amino acid), transition, or transversion

Transition

Pyrimidine replaces pyrimidine, or purine replaces purine

Transversion

Purine replaces pyrimidine or v.v.

Sickle cell anemia cause

Missense mutation

Causes of duplications and deletions

• Abnormal events during recombination (non-allelic homologous recombination)

• Cross over at misaligned sites

Depurination

• Loss of a base in intact double helix (mostly A or G)

• Most common type of naturally occurring chemical change

• Release of purine base, leaving apurinic site

• 1:10,000 purines

• Recognized by repair, but if missed → mutation

Deamination

• Amino group (NH2) → keto group (O)

• Changes cytosine to uracil and 5-methylcytosine to thymine (keto)

• U can be recognized by repair, it not, mutation during replication

Guanine damination

Changes to xanthine (still pairs with C)