gene expression. have you ever wondered how a frog grows?

Gene expression

Have you ever wondered how a frog grows?

http://www.teara.govt.nz/en/frogs

Native male frogs sit on the eggs and the young froglets hatch almost fully

formed.

http://www.erodent.co.uk/GardenPond/PondPhotos/Tadpole3w.jpg

Why are their parts where they are?

The answers lie in their cells.

In particular – the answer can be found in their genetic material.

A right handed helix

Let’s revisit how we know that DNA is important.

Streptococcus pneumoniae

S: smooth

(virulent)

R: rough

(avirulent)

DNA is the genetic material - Griffith 1928

Avery, MacLeod & McCarty 1944 Chemically characterised the ‘transforming extract’ from virulent cells

observed by Griffith Removed the protein from ‘transforming extract’

and it still transformed Removed RNA

extract still transformed Removed DNA

prevented transformation Therefore transforming substance was DNA

Biochemically characterised the transforming extract, all its properties were consistent with DNA: High Mw (centrifugation) High charge (electrophoresis) Characteristic UV absorbance Chemical analysis, ratio of nitrogen to phosphorus

Hershey-Chase 1952 Left, T2 virus protein

radioactively labelled 35S Radioactivity outside

Right: T2 virus DNA radioactively labelled 32P Radioactivity inside

THEREFORE genetic material infecting E.coli was DNA and not protein

Genes are made from DNA (except some RNA viruses)

Genes are encoded within DNA

What is a gene?

Classical molecular gene: “A stretch of DNA sequence that codes for a particular protein that has a particular function”.(10,11) This can be an interrupted sequence within a chromosome.

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Evolutionary gene: “any portion of chromosomal material that potentially lasts for enough generations to serve as a unit of natural selection”.(39) As such a gene is an inherited unit which is somewhere between a nucleotide and a chromosome.

Systemic Concept: The gene is a combination of (one or more) nucleic acid (DNA or RNA) sequences, defined by the system (the whole cell, interacting with the environment, or the environment alone, in sub-cellular or pre-cellular systems), that gives origin to a product (RNA or polypeptide).(57)

Other definitions of a gene

The central dogma

RNA Precursors(ribonucleotides) RNA

Transcription

Protein Precursors(amino-acids) Protein

Translation

DNA Precursors(deoxyribonucleotides) Replication

DNA (Genotype)

(Phenotype)

http://vcell.ndsu.nodak.edu/animations/transcription/advanced.htm

Transcription is the first step in the process.

How many genes does it take to make a person?

20,000-25,000

26,000 19,000 13,000 6,000 4,000

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DSCAM: one gene 38,016 mRNAsAn immunoglobulin (Ig) superfamily member

an axon guidance receptor

Drosophila melanogaster Dscam gene contains 115 exons spanning ~60,000 bp20 exons are constitutively spliced (open boxes) and 95 exons are alternatively spliced

(shaded boxes).Alternatively spliced exons are orgnized into 4 clusters (exons 4,6, 9, 17) that contain

12, 48, 33 and 2 alternative exons each.

Exons in each cluster are spliced in a mutually exclusive manner.

Celotto & Graveley 2001 Genetics 159:599-608

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Immunoglobulin genes recombine within the DNA.

So what if nuclei are the wrong shape?

85 mm

16 mm

48129895 bp

249250621 bp

More or less matters!

DownEdwards

PatauKlinefelters

(XXY)

Turners(X)

22

Albert Einstein (1955)

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30-35% of cells are aneuploid

~4% aneusomy for chr 21

Affects survival, proliferation potential, and protein imbalances

24Falchi et al. (2014) Nat Genetics 46:492-498

Copy number affects your weight

How big is a vertebrate cell?

http://medicalpicturesinfo.com/human-cell/

~6 µm

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Progeria (Hutchinson-Gilford progeria)

Children with this disease typically have a stroke or heart attack caused by severe atherosclerosis at an average age of 13 years.

Nuclei in Progeria are the wrong shape.

Treating progeria cells with remodelin results in the nucleireverting to the ‘normal’ shape.

Shape directly affects the genes that are turned on.

31 of the altered genes affect vasculature and atherosclerosis

22 of the altered genes affect skeletal, limb and cartilage

Defects in transcription can cause problems. Who amongst us is lactose intolerant?

LCT gene 17 exons Chr 2q21

Lactase is encoded by a single gene

-24 Kb G-A

-14 Kb C-T

A single mutation (C changes to T) causes lactase persistence

Swallow; Annu Rev Genet 2003:37197-219

The frequency of lactase persistence varies dramatically in different

populations.

Swallow; Annu Rev Genet 2003:37197-219

Pastoralists and milk drinkers tend to have higher frequencies of lactase persistence

than nonpastoralists

Translation is important too.

tRNA: the adapter

What is the genetic code?

Redundant codons are all synonyms for the same protein building block.

Degenerate codons differ in their third positions; e.g. both GAA and GAG code for the amino acid glutamic acid.

SerCandida

Nons.Micrococcus Nons.

Micrococcus

Nons.Mycoplasma

TrpMycoplasmaSpiroplasma

CysEuplotes

Gln: Ciliates & Acetabularia

First position (5’ end)

Second Position

Third position (3’ end)

U C A G U UUU Phe UCU Ser UAU Tyr UGU Cys U

UUC Phe UCC Ser UAC Tyr UGC Cys C UUA Leu UCA Ser UAA Stop UGA Stop A UUG Leu UCG Ser UAG Stop UGG Trp G

C CUU Leu CCU Pro CAU His CGU Arg U CUC Leu CCC Pro CAC His CGC Arg C CUA Leu CCA Pro CAA Gln CGA Arg A CUG Leu CCG Pro CAG Gln CGG Arg G

A AUU Ile ACU Thr AAU Asn AGU Ser U AUC Ile ACC Thr AAC Asn AGC Ser C AUA Ile ACA Thr AAA Lys AGA Arg A AUG Met ACG Thr AAG Lys AGG Arg G

G GUU Val GCU Ala GAU Asp GGU Gly U GUC Val GCC Ala GAC Asp GGC Gly C GUA Val GCA Ala GAA Glu GGA Gly A GUG Val GCG Ala GAG Glu GGG Gly G

The genetic code is not always the same

Some people can’t use Glucose.Typically they have mutations in

GLUT1

37

These mutations occur because the codons are changed.

GLUT1 mutations are often in helix 4

38

R126H

R126L

R153L

Mitochondria in a mouse myoblast cell

39Image taken by James Markworth (Liggins Institute)

Mitochondria are red

Actin filaments are green

Nucleus is blue

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Differences in the human nuclear and mitochondrial genetic codesFirst

position (5’ end)

Second Position

Third position (3’ end)

U C A G U UUU Phe UCU Ser UAU Tyr UGU Cys U

UUC Phe UCC Ser UAC Tyr UGC Cys C UUA Leu UCA Ser UAA Stop UGA Stop A UUG Leu UCG Ser UAG Stop UGG Trp G

C CUU Leu CCU Pro CAU His CGU Arg U CUC Leu CCC Pro CAC His CGC Arg C CUA Leu CCA Pro CAA Gln CGA Arg A CUG Leu CCG Pro CAG Gln CGG Arg G

A AUU Ile ACU Thr AAU Asn AGU Ser U AUC Ile ACC Thr AAC Asn AGC Ser C AUA Ile ACA Thr AAA Lys AGA Arg A AUG Met ACG Thr AAG Lys AGG Arg G

G GUU Val GCU Ala GAU Asp GGU Gly U GUC Val GCC Ala GAC Asp GGC Gly C GUA Val GCA Ala GAA Glu GGA Gly A GUG Val GCG Ala GAG Glu GGG Gly G

Met

Trp

Stop

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Protein structure is important as well.

42

Prions and Alzheimer’s disease are examples of

pathological protein misfolding

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What’s different between these bears?

Polar bears and Brown BearsDivereged ~479-343 kya

Polar bears are adapted to high fat diets

Modified from Liu et al. Population genomics reveal recent speciation and rapid evolutionary adaptation in Polar bears. Cell (2014): 157, 785-794 and Ombostad, I. (2012) Relationships between persistent organic pollutants (POPs) and plasma clinical-chemical parameters in polar bears (Ursus maritimus) from Svalbard, Norway. Student thesis (Trondheim, Norway: Norwegian University of Science and Technology).

The environment is affecting the genes that Polar bears have.

Basic unit of muscle

Polar bears have seven missense substitutions in the LYST gene

These mutations occur in the same region as ones in

human disease

Chediak-Higashi syndrome Autosomal recessive

Mutation of lysosomal trafficking regulator (LYST)

What do Chimo and a Polar bear have in common?

Lyst affects melanosome maturation

The organelle that is involved in synthesis, storage and transport of

melanin

White fur is common in the arctic:

beluga whales, polar bears, arctic hare, arctic fox – but not all due to Lyst

mutations.

Confers a selective advantage?

www.katoa.ac.nz

Hands on experience for you and your students!

justin.osullivan@auckland.ac.nz.