some important points about molecular biology and genetics in relation to dmd. k.a. bettelheim...
TRANSCRIPT
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.
Genes, proteins and the Genetic
Code
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.
Genes, proteins and the Genetic
Code
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.
Genes, proteins and the Genetic
Code
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.
Genes, proteins and the Genetic
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.
Genes, proteins and the Genetic
Code Parent Female Male
XX XY
Eggs/sperm X X X Y
Offspring XX XY XX XYGender F MF M
Genes, proteins and the Genetic
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.
Genes, proteins and the Genetic
Code
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.
Genes, proteins and the Genetic
Code
Genes, proteins and the Genetic
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.
Genes, proteins and the Genetic
Code
Name of Base
Type of Base
Abbreviation
Adenine Purine A
Guanine Purine G
Thymine Pyrimidine
T
Cytosine Pyrimidine
C
Genes, proteins and the Genetic
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-
Genes, proteins and the Genetic
Code
Genes, proteins and the Genetic
Code
Genes, proteins and the Genetic
Code This forms the
spiral structurethat has becomeknown as theDoubleHelix.
Genes, proteins and the Genetic
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.
Genes, proteins and the Genetic
Code.Replication of the
DNA.
Genes, proteins and the Genetic
Code.Replication of the
DNA.
Genes, proteins and the Genetic
CodeRecombination involves the breakage
and rejoining of two chromosomes (M and F) to produce two re-arranged chromosomes (C1 and C2).
Genes, proteins and the Genetic
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.
Genes, proteins and the Genetic
Code
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.
Genes, proteins and the Genetic
Code
Genes, proteins and the Genetic
Code
Genes, proteins and the Genetic
Code
Genes, proteins and the Genetic
Code
The next stage in the process of forming a protein involves a second type of nucleic acid, namely RNA (Ribonucleic Acid).
Genes, proteins and the Genetic
Code
There are Three main differences between RNA and DNA.
1) RNA has Ribose as the sugar instead of Deoxyribose.
Genes, proteins and the Genetic
Code
2) RNA uses Uracil instead of Thymine to link with Adenine.
3) RNA is generally found single stranded in the cells.
Genes, proteins and the Genetic
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).
Genes, proteins and the Genetic
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.
Genes, proteins and the Genetic
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.
Genes, proteins and the Genetic
Code
Genes, proteins and the Genetic
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.
Genes, proteins and the Genetic
Code
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".
Genes, proteins and the Genetic
Code
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.
Genes, proteins and the Genetic
Code
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.
Genes, proteins and the Genetic
Code
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).
Genes, proteins and the Genetic
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.
Genes, proteins and the Genetic
Code
Summary of transcription.
Genes, proteins and the Genetic
Code
Genes, proteins and the Genetic
Code
Genes, proteins and the Genetic
CodeA transfer RNA (tRNA) molecule.
Genes, proteins and the Genetic
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).
Genes, proteins and the Genetic
Code
Genes, proteins and the Genetic
CodeHow many ribosomes
do you think are in an average human
cell?100
1,00010,000
100,0001,000,00
010,000,0
00
Genes, proteins and the Genetic
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!
Genes, proteins and the Genetic
CodeA typical cell.
0.9 m = 0.00000090m
Genes, proteins and the Genetic
CodeZhu et al. 2013 Novel
mutation in exon 56 of the dystrophin gene in a child with Duchenne muscular
dystrophy.
Genes, proteins and the Genetic
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!
Antioxidants:A Natural History.
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.
There was no Oxygen present!
Antioxidants:A Natural History.
With the absence of Oxygen all these reactions would be considered anaerobic.
Anaerobic reactions are far less energy efficient than Aerobic ones.
There was no Oxygen present!
Antioxidants:A Natural History.
Example:
Anaerobic Fermentation of sugars gives Alcohol.
Oxydation of sugars give CO2 and water and much more energy.
There was no Oxygen present!
Antioxidants:A Natural History.
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.
There was no Oxygen present!
Antioxidants:A Natural History.
Antioxidants:A Natural History. Antarctic studies uncovered hardy bacteria, which are
believed to be like the ones that survived when the earth
froze.
Antioxidants:A Natural History.
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!
Antioxidants:A Natural History.
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!
Antioxidants:A Natural History.
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!
Antioxidants:A Natural History.
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.
Antioxidants:A Natural History.
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.
Antioxidants:A Natural History.
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.
Antioxidants:A Natural History.
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