BIOL1020 Module 2

What is the central dogma of biology?

- a summary of the information flow in the cell

- information is encoded in the DNA sequence of a gene

What is the process of DNA transcription

- transcribed into an intermediary called mRNA

1) RNA polymerase unzips the DNA helix

2) RNA nucleotides match up with one side of the DNA strand

3) a single-stranded mRNA is formed, and the DNA rezips

What is RNA splicing?

The process by which introns are removed from the pre-mRNA molecule.

How does RNA splicing occur?

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What is the process of RNA translation?

1) ribosomes build polypeptides

2) an initiation codon marks the start of an mRNA message

3) mRNA and the ribosome subunits assemble during initiation

4) elongation adds amino acids to the polypeptide chain until a stop codon terminates translation

What is a gene?

a unit of heredity that is transferred from a parent to offspring and is held to determine some characteristic of the offspring.

What is a trait?

a heritable physical characteristic

Beadle and tatum experiment

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Molecular location and anatomy of a gene

Anatomy

- has two parts

1) coding region

2) promoter (control region)

Location

- segment in the DNA

- genomic DNA is a very large molecule

How is DNA compacted into chromatin? and how does it unwind to be translated?

- wraps around histone proteins --> this forms nucleosomes (each consists of 8 histone proteins around which the DNA wraps 1.65 times)

- forms loops averaging 300 nm in length

- the 300 nm fibres are compressed and folded to produce a 250 nm wide fibre

- tight coiling of the 250 nm fibre produces the chromatid of a chromosome

Chromosome organisation

Problem: compacted chromatin makes DNA inaccessible

solution: make parts of chromosome accessible by relaxing the compaction level

- euchromatin (relaxed, transcribed)

- heterochromatin (compact, transcriptionally silent)

Karyotype

A display of the chromosome pairs of a cell arranged by size and shape.

23 pairs (22 pairs (autosomals) and 2 unpaired (sex chromsomes)

Steps of PCR

1) Denaturing (95-98C) - separates the DNA strands

2) Annealing (48-72) - allows the binding of primers to each DNA strand

3) Extension (68-72) - optimal temp for taq DNA polymerase to extend the new DNA strands

Enzymes not needed in PCR and why

Helicase: heat (95) breaks the H-bonds

SSBP, topoisomerase: heat keeps the H-bonds open

Primase: there are no RNA primers to be made. we add ready made DNA primers into our PCR reactions, these anneal due to temperature

ligase: there are no DNA fragments to be joined following primer repacement

DNA polymerase I: there are no RNA primers to be replaced

What are viruses

an obligatory (bc need hosts machinery for replication), intracellular infectious (infect host cells by breahing the membrane of cellular life) agent, whose genetic material (DNA or RNA) is surrounded by a protein capsid.

very small

Are viruses alive or not?

borderline between living and non-living

living in a sense that they:

- reproduce to produce progeny

- evolve thru mutation and natural selection

- DNA or RNA as genetic info

not like living:

- limited genetic material

- only replicate in living cells

- employ host's cell's replicative and metabolic pathways - "borrow" life

- don't have own ribosomes or ability to produce energy

- outside cells they are essentially inert macromolecules

Virus lifecycle (general)

1) attachment

2) penetration

- endocytosis - virus becomes internalised through being surrounded by an area of the plasma membrane

3) uncoating

4) transcription/translation

5) genome replication

6) assembly

7) release

lytic replication

1) attachment: bacteriophage binds to cell

2) penetration/uncoating: phage DNA enters the host cell

3) the host DNA is digested

4) new phage DNA forms using nucleotides from former host DNA

5)

lysogenic replication

Baltimore classification of viruses

RNA dependent RNA polymerase

A viral enzyme that makes a strand of RNA by reading a strand of RNa . All prokaryotic and eukaryotic RNa polymerases are DNa dependent; they make a strand of RNa by reading a strand of DNA.

Reverse transcriptase (retroviruses)

Gene expression in bacteria

What is an operon and what is its composition?

tryptophan synthesis (e.g. of bacteria genexpression)

Gene regulation in eukaryotes

chromatin regulation

transcriptional regulation

post-transcriptional regulation

translation gene regulation

Summary of gene regulation in eukaryotes

What is genetics?

the study of inheritance patterns

Basic principles of heretdity

1) inherited characteristics are determined by genes

2) all individuals of a species have the same base set of genes (variation between individuals for a given trait is due to gene mutation)

3) varients of the same gene (alleles) account for variant traits

What is a genotype?

the allelic constitution of an individual (at one or severeal genes - gene loci)

What is a phenotype?

the trait of an individual and is under control of its genotype.

Genetic variation comes from mutations of genomic DNA

types:

- point mutations

- insertions or deletions

- translocations, deletions or duplications (large scale)

can be:

- in the coding or non-coding region of DNA

- in somatic or germ cells

- spontaneous - result from imperfect cellular processes (e.g. errors in DNA replication)

- induced (e.g. by UV light)

loss of function vs gain of function mutations

loss of function - caused by a premature stop codon or if the regulatory region of a gene gets mutated so that the gene is no longer able to be activated

gain of function mutation - confers new or enhanced activity on a protein

Genetic variation in bacteria

How is DNA transfered in bacteria

1) conjugation - transfer by direct cell to cell contact via a pilus and a conjugative plasmid

2) tranformation - uptake and incorporation of naked DNA

3) transduction - transfer of chromosomal or plasmid DNA from cell to cell by bacterial virus (bacteriophage)

alternation of generations

life cycle that has two alternating phases—a haploid (N) phase and diploid (2N) phase

spore (n) --> gametophyte (n) --> gametes (archegonium - eggs; antheridium - sperm) --> fertilisation --> zygote (2n) --> sporophyte (2n) --> meiosis --> spore (n).

angiosperms

What is meiosis

a type of cell division that results in four daughter cells each with half the number of chromosomes of the parent cell, as in the production of gametes and plant spores.

two cell divisions

1. reducion division (diploid to haploid)

- reduces chromosome number from diploid to haploid by separating homologous chromosomes

2. separation of chromatids (haploid)

- 4 haploid gametes per starting cell

steps of meiosis

Mitosis 1

- prophase 1 - the chromsomes condsense and the nuclear envelope breaks down. crossing over occurs.

- metaphase 1 - pairs of homologous chromosomes move to the equator of the cell

- anaphase 1 - homologous chromsomes move to the opposite poles of the cell

- telokinesis 1 & cytokinesis - chromsomes gather at the poles of the cells. the cytoplasm divides.

Meiosis 2

- prophase 2 - a new spindle forms around the chromsomes

- metaphase 2 - chromsomes line up at the equator

- anaphase 2 - centromeres divide. chromatids move to the opposite poles of the cell.

- telokinesis 2 & cytokinesis - a nuclear envelope forms around each set of chromosomes. the cytoplasm divides.

genetic variation from meiosis

independent assortment, crossing over, errors during meiosis

- homologous chromsomes segregate and the non-homologous chromsomes assort independently during gamete formation creating different combinations.

alleles segregate because the two homologus chrmosomes that carry them segregate in meiosis 1

genetic variation by crossing over

recombination events (crossovers) occur at pachytene (a stage in prophase 1) at different sites along chromosomes

chiasma - site of crossing over

linkage is broken by crossing over as it is a recombination event (2-3 crossover ovents/chromosome pair)

Genetic variation comes from

1) mutations

2) independent assortment of non-homologous chromsomes

3) recombination/crossing over

4) fusion of gametes (fertilisation)

What happens if meiosis goes wrong?

if chromosomes fail to seperate properly (nondisjunction) in anaphase 1 or 2, gametes may have +/- 1 chromsome

therefore zygote will be aneuploid (e.g. down syndrome)

Mendel's method

1) remove stamens from purple flower

2) transferred pollen from stamen of white flower to carpel of purple flower

3) pollinated carpel matured into pod

4) planted seeds from pods

5) examined offspring: all purple

Mendel's Terminology

P = parents from 2 'pure breeding' lines crossed

F1 - first filial generation (pheotype scored then self-fertilised)

F2 - second generation (phenotype scored)

What is a test cross and what is it used for?

used to determine the genotype conferring a dominant phenotype

cross the individual in question to a known homozygous recessive

Monohybrid cross

one trait lost in F1

reappeared in F2

3:1 phenotype ratio in F2

reciprocal cross gave similar results

other characters gave similar results

Findings and conclusions of momohybrid cross

inheritance is particulate

2 copies of heritable factor

one trait is dominant

Mendel's conclusions

1. a) inheritance is particulate (heritable factors = genes)

b) alternative versions of genes account for variations in inherited characters

--> different alleles confer different traits of the same character

2. a) organisms inherit 2 alleles for each character, one from each parent (peas are diploid)

b) only one allele is passed on to gamete (gemetes are haploid)

3. if the 2 alleles differ one may be dominant (other recessive)

4. some of mendels genes were actually far apart but on the same chromosome (due to recombination)

Dihybrid cross (heterozygous at 2 loci) - observations and conclusions

hypothesis: of independent assortment

F1 dihybrid cross --> F2 phenotype ratio = 9:3:3:1

Conclusions:

- seed colour is controlled by one factor (gene)

- seed shape is controlled by a different factor (gene)

- each gene is inherited by progeny independently of the other = Law of independent assortment

- all genes were inherited independently of each other --> TOO BROAD

Mendel's 3 principles

1) Principle of Dominance: phenotypes depend on inheritance of dominant and recessive alleles

2) Segregation (first principle): the two alleles sepearate from each other into the gametes (due to seg. of homologous chromosomes that carry them)

3) Independent Assortment (second): genes segregate independently when gametes are formed (due to independent assortment of non-homologous chromosomes)

What are the 3 regions of DNA?

Regulatory region

- the binding site for RNA polymerase that regulates the transcription of a downstream segment of DNA

Transcribed region (inbetween region)

- the template for mRNA synthesis

Termination region

- the transcription stop signal causes the RNA polymerase to fall off the DNA that it is transcribing, once the appropriate region has be transcribed.

Lac operon

Restriction enzymes

- sequence specific. they recognise and bind to specific DNA sequences

- can be used to diagnose mutations

- once they bind to their recognition sequence, they cut the sugar-phosphate backbones

--> stick ends and straight

- they can be reattached by ligase

Reconnecting DNA fragments using DNA ligase

Key components of gel electrophoresis

1) smaller fragments move faster and further - less frictional drag

2) to tell size - use DNA ladder - can compare unknown to known to determine size

3) if you increase the concentration of ager, you get higher resolution of smaller fragments, but lower resolution of larger fragments (and vice versa)

Example of using synthetic biology

can self-fertilisation still give rise to genetic variation?

self fertilisation can still give genetic variation (crossing over and independent assortment). Cutting of the plant will result in genetically identical offspring.

Increasing genetic variation:

1. independent assortment of chromosomes in metaphase 1

2. crossing over in prophase 1

3. fusion of gametes (random)

*double fertilisation

what is a locus?

Location of a gene on a chromosome