Some important points about Molecular Biology and Genetics in relation to DMD. K.A. Bettelheim (retired), London, U.K. Trustee; Action Duchenne formally

Some important points about Molecular Biology and Genetics in relation

to DMD.

K.A. Bettelheim (retired), London, U.K.

Trustee; Action Duchenne formally

Microbiological Diagnostic Unit

Public Health LaboratoryMelbourne, Australia

Genes, proteins and the Genetic

Code

All living organisms, whether bacteria, plants or animals including humans have the same way by which their characteristics are maintained and passed on through the generations.


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The basis of this is the genome of the organism in question. This material consists of Deoxyribonucleic acid (DNA) and in plants and animals is generally found within the nucleus of each cell. In bacteria it is free within the cell.


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The DNA in plants and animals is usually arranged as a series of Chromosomes, which are arranged in pairs, such that one half of each pair originates from each parent. When the cell divides the Chromosomes also divide thus exactly the same material goes into each daughter cell. This is known as Mitosis and is the basis how cells renew themselves and the body grows.


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Humans have 22 pairs of Chromosomes, as well as a pair known as the sex Chromosomes, known as the X and Y Chromosomes.

Females have two X Chromosomes.

Males have an X and a Y Chromosome.


Code On these 23 pairs of

Chromosomes are situated the genes which determine all the characteristics of the organism. As most of these genes are on the 22 pairs of Chromosomes, problems arising in genes as a result of mutations or other occasional damage can be compensated for by the other gene residing on the Chromosome, with which it is paired. This also applies to females, who have two X Chromosomes.


Code Parent Female Male

XX XY

Eggs/sperm X X X Y

Offspring XX XY XX XYGender F MF M


Code Parent Female Male

XX XY

Eggs/sperm X X X Y

Offspring XX XY XX XYGender F MF M

X has mutation on X chromosome.


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However, in males who have an X and a Y Chromosome, a deleterious mutation on the X Chromosome, is not compensated for, because the Y Chromosome only carries a few genes mainly associated with reproduction.

Note:The Dystrophin gene is on the X Chromosome.


Code


Code The Chromosomes and genes

within them are made up of DNA which consists of two strings consisting of a backbone of the sugar-like substance deoxyribose and phosphate to which are attached four bases in an apparently random order. These bases are listed in the next slide.


Code

Name of Base

Type of Base

Abbreviation

Adenine Purine A

Guanine Purine G

Thymine Pyrimidine

T

Cytosine Pyrimidine

C


Code These bases are arranged

such that each base will be linked with only one other on the opposite string, i.e. A and T form a pair and G and C form a pair.

-GGCTTAATCGT- ||||||||||||

-CCGAATTAGCA-


Code


Code


Code This forms the

spiral structurethat has becomeknown as theDoubleHelix.


Code

“I’m stunned that this one molecule is responsible for so much of what we are-for every cell in our body, and the way they all work. It’s even more incredible that we worked out what it lookslike!”Dr.Teresa TeixeiraAudience Researcher, Science Museum.


Code.Replication of the

DNA.


Code.Replication of the

DNA.


CodeRecombination involves the breakage

and rejoining of two chromosomes (M and F) to produce two re-arranged chromosomes (C1 and C2).


Code

Recombination is a process that sometimes occurs. It permits chromosomes to exchange genetic information thus producing new combinations of genes. This process increases the efficiency of natural selection and and is considered important in the rapid evolution of new proteins.


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Genetic recombination can also be involved in the repair of DNA. This is an especially important response of the cell to breaks in both strands.


Code


Code


Code


Code

The next stage in the process of forming a protein involves a second type of nucleic acid, namely RNA (Ribonucleic Acid).


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There are Three main differences between RNA and DNA.

1) RNA has Ribose as the sugar instead of Deoxyribose.


Code

2) RNA uses Uracil instead of Thymine to link with Adenine.

3) RNA is generally found single stranded in the cells.


CodeThere are three main types of RNA found in the cell.

1) Messenger RNA (mRNA) is the form that transports the message from the DNA to the protein making apparatus, (the Ribosome).


Code2) Transfer RNA (tRNA) is the form that transports the amino acids, which make up the proteins to the Ribosome.

3) Ribosomal RNA (rRNA) forms the backbone of the Ribosome, on which the enzymes synthesizing the protein reside.


CodeWhile in bacteria the genes on the DNA are continuous in plants and animals they consist of separate exons, which carry the message and introns which are not read and cut out of the message.


Code


CodeThus in the chromosomes the DNA acts as a template for the synthesis of RNA in a process called transcription. In most mammalian cells, only 1% of the DNA sequence is copied into a functional RNA (mRNA). Only one part of the DNA is transcribed to produce nuclear RNA , and only a minor portion of the nuclear RNA survives the RNA processing steps.


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One of the most important stages in RNA processing is RNA splicing.In many genes, the DNA sequence coding for proteins, or "exons", may be interrupted by stretches of non-coding DNA, called "introns".


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In the cell nucleus, the DNA that includes all the exons and introns of the gene is first transcribed into a complementary RNA copy called "nuclear RNA," or nRNA. In a second step, introns are removed from nRNA by a process called RNA splicing. The edited sequence is called "messenger RNA," or mRNA.


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The mRNA leaves the nucleus and travels to the cytoplasm, where it encounters cellular bodies called ribosomes. The mRNA, which carries the gene's instructions, dictates the production of proteins by the ribosomes.


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Synthesis of RNA is usually catalyzed by an enzyme—RNA polymerase—using DNA as a template, a process known as transcription. Initiation of transcription begins with the binding of the enzyme to a promoter sequence in the DNA (usually found "upstream" of a gene).


CodeThe DNA double helix is unwound by the helicase activity of the enzyme. The enzyme then progresses along the template strand in the 3’ to 5’ direction, synthesizing a complementary RNA molecule with elongation occurring in the 5’ to 3’ direction. The DNA sequence also dictates where termination of RNA synthesis will occur.


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Summary of transcription.


Code


Code


CodeA transfer RNA (tRNA) molecule.


CodeSummary of activity of the Ribosome:Translation of mRNA (1) by a ribosome (2)(shown as small and large subunits) into a polypeptide chain (3). The ribosome begins at the start codon of mRNA (AUG) and ends at the stop codon (UAG).


Code


CodeHow many ribosomes

do you think are in an average human

cell?100

1,00010,000

100,0001,000,00

010,000,0

00


CodeHow many ribosomes

do you think are in an average human

cell?13,000,000

There are thirteen million ribosomes in a human liver

cell!


CodeA typical cell.

0.9 m = 0.00000090m


CodeZhu et al. 2013 Novel

mutation in exon 56 of the dystrophin gene in a child with Duchenne muscular

dystrophy.


CodeZhu et al. 2013 Novel mutation in exon 56

of the dystrophin gene in a child with Duchenne muscular dystrophy.

The Dystrophin Gene

The largest known gene is the human dystrophin gene. It has 79 exons spanning at least 2.4 million base pairs of genomic DNA on the X chromosome. Its 14 kb transcript encodes the full-length protein of dystrophin of 427 kiloDaltons.

The Dystrophin Gene

Detailed studies of transcription indicate that approximately 12 hours are required for transcription of 1,770 kb (at an average elongation rate of 2.4 kb per minute). Therefore this extrapolates to a transcription time of 16 hours for the complete dystrophin gene.

The Dystrophin Gene

Common Types of Mutation in the Dystrophin gene than can occur leading to DMD.1)A transcription triplet coding for an amino acid mutates to a Stop codon, causing transcrtiption to cease at this point.

2)An excision of part of the dystrophin gene.

A) If this happens without a frame-shift than a smaller Dystrophin molecule may formed.

B) If frame-shift occurs than nonsense is produced.

3)A duplication of part of the dystrophin gene.

A) If this happens without a frame-shift than a larger Dystrophin molecule may formed.

B) If frame-shift occurs than nonsense is produced.

The Dystrophin Gene

These can also be listed thus:

1) Large lesionsa) Large deletions (>= 1 exon)b) Large duplications (>= 1

exon)

2) Small lesionsa) Small deletions (< 1 exon)b) Small insertions (<1 exon)c) Splice sites (10 bp from exon)d) Point mutations

i) Nonsenseii) Missense

3) Mid-intronic lesions

The Dystrophin Gene

Taking the nonsense and missense mutations first, there a triplet that codes for an aminoacid has changed such that it now codes either for another amino acid or for a STOP codon. On some occasions the new codon codes for the same amino acid.

In a missense mutation the genetic change involves the substitution of one base in the DNA for another which results in the substitution of one amino acid in a polypeptide for another.

UUC (Phe) -> UUA (Leu)ACU (Thr) -> AAU (Asn)

The Dystrophin Gene

With a nonsense mutation, the new nucleotide changes a codon that specified an amino acid to one of the STOP codons

UGG (Try) ->UGA (STOP)CAA (Gln) ->UAA (STOP)

Due to the redundancy of the Genetic Code, there are some mutations, which do not cause changes.

UCU (Ser) -> UCC (Ser)CCU (Pro) -> CCA (Pro)

The Dystrophin Gene

With the other mutations there are two problems. 1)If the mutation maintains the reading frame, then the removal of one or more exons or the duplication of one or more exons may cause the Dystrophin molecule not to be able to function as well. 2)Far more important is if the mutation causes the reading frame to be shifted. This causes nonsense to be read and results in no Dystrophin being synthesized.

The Dystrophin Gene

Frame Shift Mutations.

In principle mutations can affect the reading frame in two ways.

1)1 or 2 bases are displaced so that a completely different message is produced.

2)3 or a multiple of 3 bases is removed which just causes a gap in the reading frame, which can then continue reading the message.

The Dystrophin Gene

Frame Shift Mutations.

Example

mRNA: UUU.CCU.GAG.UCA.GAU

Protein: Phe Pro Glu Ser Asp

Mutation

mRNA: UUU.CCU.AGU.CAG.AUC

Protein: Phe Pro Ser Gln Ile

Interventions

Missense Mutations

It has been shown that certain chemical compounds can cause the Ribosome to read past such an isolated STOP codon. A real STOP codon has other parts in the message which indicate STOPs. One of these chemicals is currently being trialled, with some success.

Interventions

Exon Skipping

The Dystrophin gene has 79 exons and it was considered some time ago that if the translational mechanism can be persuaded to skip over the damaged part a slightly smaller but still active Dystrophin molecule might be made.

Interventions

Exon Skipping

For this to be achieved two conditions have to be met:

1)The skip has to start at the right place.

2)It has to end at a point appropriate for the Dystrophin molecule to reform.

Interventions

Exon Skipping

For the skip to start at the right place an appropriate anti-sense RNA has to be constructed to fit just at the right place and nowhere else on the genome.

Interventions

Diagrammatic representation of the Dystrophin gene.

Interventions

Diagrammatic representation of the Dystrophin gene to show Exon-

skipping.

To skip exon 30 one can just skip from 29 to 31.

Interventions

Diagrammatic representation of the Dystrophin gene to show Exon-

skipping.

To skip exon 20 one must skip from 19 to 22.

Interventions

Exon-skipping of the Dystrophin gene.

Currently there are a number of trials on Exon-

skipping in progress.

Interventions

For the transcription of a gene a region of the chromosome in front

of the gene, known as the Promoter has to be activated.

These promoters can be up-regulated, i.e., transcription is

increased;

Or they can be down-regulated, i.e., transcription can be

decreased.

Interventions

For the transcription of a gene a region of the chromosome in front

of the gene, known as the Promoter has to be activated.

In foetuses a protein known as ‘Utrophin’ is made. It performs

a function similar to Dystrophin. After birth its

production is down-regulated and Dystrophin is made.

Studies are in progress to cause the up-regulation of the promoter of Utrophin in cases

of DMD.

Thank, you

for your

Attention.

Antioxidants:A Natural History.

The current view is that Life on earth began about 4 billion years ago, when the Earth’s atmosphere consisted predominantly of Hydrogen, Methane and related gasses.

There was no Oxygen present!


Life consisted of small bacteria-like organisms that obtained their energy from the degradation of minerals and primitive forms of photosynthesis, which used the sun’s energy to break down some minerals.



With the absence of Oxygen all these reactions would be considered anaerobic.

Anaerobic reactions are far less energy efficient than Aerobic ones.



Example:

Anaerobic Fermentation of sugars gives Alcohol.

Oxydation of sugars give CO2 and water and much more energy.



Two Billion years ago this anaerobic world suffered a severe reduction in temperature and ice covered most of the land and oceans.

Life held on only very tenuously as photosynthetic bacteria growing on the ice, like in Antarctica now.



Antioxidants:A Natural History. Antarctic studies uncovered hardy bacteria, which are

believed to be like the ones that survived when the earth

froze.


Then the earth warmed up, the melting glaciers brought a lot of nutrient rich soil into the oceans, and phosynthetic bacteria evolved which could use the sun’s energy to convert CO2 and water to sugars and Oxygen.

Oxygen began to appear in the atmosphere!


Bacteria evolved, which could use this Oxygen to split carbohydrates aerobically, gaining much much more energy and a great selective advantage, while photosynthetic bacteria continued to produce Oxygen.

Oxygen levels in the atmosphere increased to dangerous levels!


Predatory bacteria appeared that captured and digested these smaller bacteria, but some of these smaller bacteria were able to survive within the larger predators and form a symbiotic relationship.

Oxygen levels in the atmosphere increased to dangerous levels!


These symbionts became so efficient, they became the ancestors of all animals including humans and of course Elephants.

Oxygen levels in the atmosphere leveled off to the sustainable levels we now have.


The swallowed bacteria, which use up the oxygen to produce energy are now called ‘Mitochondria’.

Oxygen levels in the atmosphere leveled off to sustainable levels we now have.


Thus Mitochondria have converted a potentially lethal Oxygen poisoning situation into a valuable essential energy source.

However, too much Oxygen in the cell will overwhelm the Mitochondria, hence the importance of antioxidants.


An amoeba has swallowed bacteria, which continue to live inside it

Bacteria

Antioxidants:The relation to

DMD.As a result of the lack of

dystrophin, increased amounts of highly reactive oxygen is released. This is too much to be mopped up by the Mitochondria and damages the cells.

Taking Antioxidants reduces these ‘Reactive Oxygen Species’ and reduces the damage to the muscle cells.

Conclusion

Conclusion.

In order to understand the various treatments for DMD and how they

may work an understanding of the

genetics and molecular biology is necessary. I hope I have given what is a simplified summary

of the most salient points.

Thank you for your

attention.

K. A. Bettelheim

Conclusion

Documents

Some important points about Molecular Biology and Genetics in relation to DMD. K.A. Bettelheim (retired), London, U.K. Trustee; Action Duchenne formally