The cellular and molecular basis of inheritance

Dr. Mohammed T Tayeb

Emery's Elements of Medical Genetics

Mueller RF and Young ID11th edition

2001

This lecture :• Introduction:

– Definition of Genetics– Cell– Chromosome– DNA– Gene

•DNA Composition•Structure of DNA• The process of DNA replication• The types of DNA sequences• The genetic code• The processes of transcription and translation• The various types of mutations• Mutagenic agents and DNA repair.

What is genetics?

• Genetics is the study of heredity, or what makes organisms differ from one another…

• the transmission of characteristics from one generation to the next…

Genetics• Study of inheritance

– How traits are passed from one generation to the next

Cell• The fundamental unit of

life• Trillions in each human• Differentiated cells

– Cells that have specialized– Only use a small part of

genome• Stem cells

– Can differentiate in many possible kinds of cells

Nucleus

contains the genetic material

Chromosomes• Made of DNA and protein• 23 pair• Autosomes

– Pair 1-22• Sex chromosmes

– X and Y• Karyotype

– Picture of chromsomes

DNA

• Made of building blocks– Adenine (A)– Thymine (T)– Cytosine (C)– Guanine (G)

• Can copy itself• Is passed from one

generation to another• Stores genetic

information

Gene• Unit of inheritance• Made of DNA• Contain instructions

for making a specific protein

• The hereditary material is present in the nucleus of the cell.

• Protein synthesis takes place in the cytoplasm.

What is the chain of events which leads from the gene to the final products?

Chromosomes, DNA, and Genes

CellNucleus

Chromosomes

Gene

Protein

DNA: The hereditary material

A- Composition• Nucleic acid is composed of a long chain

of individual molecules called nucleotides.

• Each nucleotide is composed of nitrogenous base, a sugar molecule and a phosphate molecule.

G

A

U

P

5’ end

5’

3’

C

SSugar

phosphate

base

One nucleotide

• The nitrogenous bases fall into two types, purines and pyrimidines.

• The Purines include adenine (A) and guanine (G).

• The pyrimidines include cytosine (C), thymine (T), and uracil (U).

Structure of Bases

Pyrimidines Purines

cytosine thymine uracil adenine guanine

• There are two different types of nucleic acid:

1 -Ribonucleic acid (RNA) which contains five carbon sugar ribose.

2 -Deoxyribonucleic acid (DNA) in which the hydroxyl group at the 2‘ position of the ribose sugar is replaced by a hydrogen, i.e. an oxygen

molecule is lost.

Phosho-diester link between 3-OH of ribose and 5 phosphate of next nucleotide

SUGAR-PHOSPHATE BACKBONE

• DNA and RNA both contain A, G & C but T occurs only in DNA while U is only found in RNA.

• RNA is present in the cytoplasm and in particularly high concentrations in the nucleolus of the nucleus.

• DNA is found mainly in the chromosomes

(nucleus and mitochondria).

RNA DNA

Sugar Ribose Deoxyribose Bases AUCG ATCG Strand length Short Long No.strands One Two Helix Single Double

Comparison of DNA & RNA

B- Structure of DNA• The DNA molecule is composed of two

chains of nucleotides arranged in a double helix.

• The backbone of each chain is formed by phosphodiester bonds between the 3' and 5' carbons of adjacent sugars.

• The two chains being held together by hydrogen bonds between the nitrogenous bases which point in towards the centre of the helix.

• The DNA chain end terminated by the 5' carbon atom of the sugar molecule is referred to as the 5' end (5 prime), and the end terminated by the 3' carbon atom is called the 3' end (3 prime).

• In the DNA duplex the 5' end of one strand is opposite the 3' end of the other.

The arrangement of the bases in the DNA is not random:

– G in one chain always pairs with C in the other chain.

– A always pairs with T

i.e. this base pairing forming complementary strands.

Base Pairing

ACCGTAA

ACCGTAA

DNA Base Pairs DNA-RNA base pairs

One strand of DNA RNA

Base Pairing

A TC GC GG CT AA TA T

A UC GC GG CT AA UA U

DNA Base Pairs DNA-RNA base pairs

One strand of DNA RNA

Structure of DNA

T ------- A

G ---------C

A --------- T

T ---------- A

PS

5’ end 3’ end

5’

3’

3’

5’

Structure of RNA

G

A

U

P

5’ end

5’

3’

C

SRibose

phosphate

base

One nucleotide

DNA Bases

Base Pairing

Hydrogen bonds

adenine = thymine (in DNA)adenine = uracil (in RNA)cytosine =guanine

C- DNA replication

How genetic information is transmitted from one generation to the next?

The process of DNA replication provides an answer to this question.

• During nuclear division the two strands of the DNA helix separate through the action of enzyme DNA helicase.

• Each DNA strand directing the synthesis of a complementary DNA.

• The process of DNA replication is termed semi-conservative, as only one strand of each resultant daughter molecule is newly synthesised.

• DNA replication, through the action of the enzyme DNA polymerase, takes place at multiple points known as origins of replication forming Y-shaped structure known as replication forks.

• The synthesis of both complementary DNA strands occurs in the 5` to 3` direction.

• The two strands joined together by the enzyme DNA ligase.

Types of DNA sequences

Nuclear genesIt is estimated that there are up to 80,000 genes in the genome. The distribution of theses genes varies greatly between different chromosomes.

– Unique single copy genes: most human genes are unique single copy genes coding for polypeptides which are involved in or carry out a variety of cellular functions. These include enzymes, hormones, receptor and structural and regulatory proteins.

– Multigene families:Many genes have similar functions making up what are known as multigene families.Multigene families can be split into two types :

1 -classical gene families which show a high degree of sequence homology.

2 -gene superfamilies which have limited sequence homology but are functionally related.

ORGANISATION OF GENES

1. Exons: are the functional portions of gene sequences that code for proteins.

2. Introns: are the noncoding sequences which separate the coding sequence (exons).

3. The open reading frame: a sequence with variable length that dose not contain stop codons and therefore can be translated. The sequence beginning with ATG which exist at the 5' end of genes.

4. TATA boxes: These regions are about 20-30 bases to the 5' end (left) of the open reading frame (ATG).

TATA boxes are thought to help direct important enzymes to the correct initiation site for transcription.

5. Termination codon: the end of translation is signified by a termination triplet at the 3' end of genes. The triplet could be TAA, TAG, or TGA.

Gene structure

Regulatory elements,promoter

Start codon

CAAT TATA AUG

Coding regionStop codon

AAAAAAAMature transcript

Introns discarded

AUGTATA

Extragenic DNA

• The reminder, 70-75%, of the human genome is made up of repetitive DNA sequences which are predominantly transcriptionally inactive.

Mitochondrial Chromosome• It is a circular, double-stranded DNA

molecule.

• All genes in mitochondrial DNA have been defined (37 genes).

• They are responsible for genes necessary for mitochondrial protein synthesis, and for proteins essential to oxidative phosphorylation.

Transcription• Transcription is the process whereby

genetic information is transmitted from DNA to RNA.

• The information stored in the genetic code is transmitted from the DNA of a gene to messenger RNA or mRNA.

• Every base in the mRNA molecule is complementary to a corresponding base in the DNA of the gene but with uracil replacing thymine in mRNA.

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1 .DNA Template

5’

3’ 3’-OH

5’PPP

sense strand

antisense strand

RNA DNA

Direction of elongation of RNA

Base Pairing

A TC GC GG CT AA TA T

A UC GC GG CT AA UA U

DNA Base Pairs DNA-RNA base pairs

One strand of DNA RNA

• mRNA is single stranded being synthesized by the enzyme RNA polymerase.

• In any particular gene only one DNA strand of the double helix acts as template strand.

• The transcribed mRNA molecule is a copy of the complementary strand or what is called the sense strand of the DNA double helix.

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General Synthesis of RNA(transcription)

• DNA template (1 strand)• RNA polymerase• activated precursors

– ATP,GTP,UTP,CTP and Mg• RNA and DNA template antiparallel• No primer• Chain grows 5’ 3’• Primary transcript modified

m RNA

- long helix, high MW- base arrangement complementary to DNA

from which formed- COLINEAR: direct copy of opposite DNA

strand- nucleus cytoplasm - function: transcription

Post-transcriptional processing

Before the primary mRNA molecule leaves the nucleus, it undergoes a number of modifications or what is known as post-transcriptional processing.

mRNA splicing:

After transcription, the non-coding introns in the primary mRNA are removed, and the non-adjacent coding exons are spliced together to form a shorter mature mRNA.

5' capping:

• after transcription the mRNA is modified by the addition of a methylated guanine nucleotide to the 5' end of the molecule.

• The 5' cap is thought to facilitate transport of the mRNA to the cytoplasm and attachment to the ribosomes as well as protect the RNA transcript from degradation by endogenous cellular exonucleases.

Polyadenylation:

• The cleavage of the 3' end of the mRNA molecule from the DNA involves the addition of approximately 200 adenylate residues, the so-called poly(A) tail.

• The addition of the poly(A) tail is thought to facilitate transport of the mRNA to the cytoplasm and translation.

Translation

• Translation is the transmission of the genetic information from mRNA to protein.

• mRNA is transported from the nucleus to

the cytoplasm where it attached with the ribosomes which are the site of protein synthesis.

TRANSFER RNA:

• In the ribosomes the mRNA forms the template for producing the specific sequence of amino acids of a particular polypepetide.

• In the cytoplasm there is another form of RNA called transfer RNA or tRNA.

tRNA

• Small molecule,73 –93 nucleotides, lower MW than rRNA

• Single stranded: clover leaf shape– binds aa at one end– binds mRNA at the other end

• Different tRNAs• Function - adaptors to correctly order aa

on mRNA for protein synthesis.

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Charged tRNA

leucine

Anticodon

(3 Bases)

Acceptor end

Tψ LoopD loop

A A C

tRNA

t RNA

Diag.H Brezski

tRNAAmino acid

ACC

Anticodon

(3 Bases)

Acceptor end

Tψ LoopD loop

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Anticodons

• tRNA is an adaptor molecule.• No direct pairing between mRNA and

amino acids

mRNA

2.Binding1.No reaction

tRNA

amino acid

Sites which base pair with mRNA

Base pairing

mRNACodon AUG UGU UAU CAU UGG tRNA UAC ACA AUA GUA ACCanticodonamino acids met cys tyr his trp

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Function of Ribosomes

• Translation of information encoded in the mRNA

• holds mRNA and tRNA together• forms peptide bond between amino acids• ensures accuracy of protein synthesis

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Ribosome Structure

60S large subunit

40S small subunit

mRNA binding site

E P A

Peptidyl

sites for tRNAAminoacyl

Exit site

• The incorporation of amino acids into a polypeptide chain requires the amino acids to be covalently bound by reacting with ATP to the specific tRNA molecule by the activity of the enzyme aminoacyl tRNA synthetase.

r RNA

• Large molecule, double and single helix• Base sequence constant in all

organisms.• Constituent of ribosomes in association

with 55 different proteins• Several classes: S or Svedburg units

–e.g. 30S, and 50S in prokaryotes.• Function : translation - correct spatial

orientation of aa

rRNA

ribosomes

Electron micrograph of Endoplasmic Reticulum

• tRNA– adaptors for aa in protein synthesis

• r RNA– joins with various proteins to form

ribosome, vital in translation• mRNA

– sequence of bases which codes for the order of amino acids in polypeptide

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The Central Dogma

DNA

Transcription

rRNA mRNA tRNA

Ribosome

Translation

Protein

The process of the transfer of the genetic information from DNA to RNA to protein has been called the central

dogma.

Central dogma

• The concept that genetic information is only transmitted from DNA to RNA to protein.

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Central dogmaDNA

transcription

translation

nucleus

cytoplasm

rRNA mRNA tRNA

Ribosome protein tRNA + aa

The Central Dogma

DNA RNA Proteins

Transcription Translation

Replication

The Central Dogma

Genetic Code

Groups of nucleic acid bases (codons) which code for the 20 amino acids

CRACKING THE CODE

1. How many bases make up a codon?

2. Do codons overlap?

3. Are there gaps in the code?

4. What are the code words?

1 .Number of bases in a Codon

4 bases code for 20 amino acids. How?

1base per aa 2 bases per aa

16 possible pairs

3 bases per aa

64 possible triplets

DNA is composed of four different nitrogenous bases, then a single base cannot specify one amino acid.

• If two bases were to specify one amino acid, there would only be 42 or 16 possible combinations.

• If three bases specified one amino acid then there would be would be 43 or 64.

• This is more than enough to account for all the 20 known amino acids and is known as the genetic code.

Genetic Code

Groups of nucleic acid bases which code for the 20 amino acids

• Unit of information is the codon - a sequence of 3 bases on mRNA

• 64 possible codons• each codon is assigned to one amino acid

or a punctuation signal

Genetic Code cont.

• Triplet code– Codon: trinucleotide in mRNA codes for a

specific amino acid or a stop-start signal• no overlap

– read sequentially in 5’ - 3’ direction

• 5’ - AGUCAGUCAAGUCAGUCAGUC- 3’

direction of reading

TRIPLET CODONS:• However, some amino acids are coded for

by more than one triplet.

• Termination of translation of the mRNA is signalled by the presence of one of the three stop or termination codons.

MUTATIONS

• Defn: Permanent chagne in nucleotide sequence.

• occur in somatic cells or germline cells.• only germline cells inherited.

• somatic mutations believed responsible for many medical problems

• many cancers?

• Heritable change in the nucleotide sequence of a chromosome.

• Mutations can arise through exposure to mutagenic agents but the vast majority occur spontaneously through errors in DNA replication and repair.

• Although mutations can occur either in non-coding or coding sequences, it is only when they occur in the latter that they are recognised as an inherited disorder or disease.

• A mutation arising in a somatic cell cannot be transmitted to offspring, whereas if it occurs in gonadal tissue or a gamete it can be transmitted to future generations.

TYPES OF MUTATIONS • mutations can be considered in two main

classes according to how they are transmitted.• mutations had been considered to be

transmitted unaltered as what are termed fixed or stable mutations.

• More recently a new class of mutations, known as dynamic or unstable mutations, have been demonstrated to undergo further alteration as they are transmitted in families.

A- Fixed/stable mutations

• Fixed/stable point mutations can be classified according to the specific molecular changes at the DNA level.

• These include single base pair substitutions, insertions, deletions or duplications of part of a gene or DNA sequence.

1- Substitutions:

• A substitution is the replacement of a single nucleotide by another.

• If the substitution involves replacement by the same type of nucleotide, i.e. a pyrimidine for a pyrimidine (C for T or vice versa) or a purine for a purine (A for G or vice versa), this is termed a transition.

• Substitution of a pyrimidine by a purine or vice versa is termed a transversion.

Nucleotide Misincorporation

-C-A-G-C-T--G-T-C-G-A-

-C-A-G-C-T-

-G-T-C-G-A-

-C-A-G-C-T--G-T-T-G-A-

-C-A-G-C-T--G-T-C-G-A-correctly copied

CT substitution

• A deletion involves the loss of one or more nucleotides.

• If it occurs in coding sequences and

involves one, two or more nucleotides which are not a multiple of three, it will disrupt the reading frame.

2- Deletions:

3- Insertions:

• An insertion involves the addition of one or more nucleotides into a gene.

• Again, if an insertion occurs in a coding sequence and involves one, two or more nucleotides which are not a multiple of three, it will disrupt the reading frame.

Added Nucleotides

-C-A-G-C-T--G-T-C-G-A-

-C-A-G-C-T-

-G-T-C-G-A-

-C-A-G-C-T--G-T-C G-A-

-C-A-G-C-T--G-T-C-G-A-

A

correctly copied

nucleotide added

B- Dynamic/unstable mutations

• unstable or dynamic mutations consist of triplet repeat sequences which, in affected persons, occur in increased copy number when compared to the general population.

• triplet amplification or expansion has been identified as the mutational basis for a number of different single gene disorders.

• The mechanism by which amplification or expansion of the triplet repeat sequence occurs is not clear at present.

Diseases arising from triplet repeat expansion

Repeat location Mutation number Repeat number Repeat sequence Disease

Coding 37-100 9-35 CAG Huntington's disease (HD)

3' UTR 50-4000 5-35 CTG Mvotonic dystrophy (DM)

5' UTR 200-2000 10-50 CGG Fragile X site A (FRAXA)

Coding 40-55 17-24 CAG Kennedy disease (SBMA)

Coding 43-81 19-36 CAG Spino-cerebellar ataxia I-(SCAT)

Coding 35-39 15-24 CAG Spino-cerebellar ataxia 2 (SCA2)

Coding 67->79 12-36 CAG Machado-Joseph disease (MJD, SCA3)

Coding 21-27 4-16 CAG Spino-oaebellar ataxia 6 (SCA6)

Coding 37-200 7-35 CAG Spuio-ce ebellar ataxia 7 (SCAT)

UTR 100->500 16-37 CTG Spmo-oaebellar ataxia 8 (SCA8)

Coding 49->75 7-23 CAG I tatorubral-pallidoluysian atrophy (DRPLA)

Intronic 200-900 17-22 GAA Friedreich's ataxia (FA)

Promoter >200 6-25 CCG Fragile X site E (FRAXE)

? >500 6-29 GCC Fragile X site F (FRAXF)

? 1000-2000 16-49 CCG Fragile 16 site A (FRA16A)

UTR = untranslated region.

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Triplet Expansion in Fragile X

(CCG)n

FMR-1

200

60

6

No.triplets

Affected

Carrier

Normal

5’ 3’

DNA

STRUCTURAL EFFECTS OF MUTATIONS ON THE PROTEIN

• Mutations can also be subdivided into two main groups according to the effect on the polypeptide sequence of the encoded protein, being either synonymous or non- synonymous.

A- Synonymous/silent mutations

• If a mutation does not alter the polypeptide product of the gene, this is termed a synonymous or silent mutation.

• A single base pair substitution, particularly if it occurs in the third position of a codon, will often result in another triplet which codes for the same amino acid with no alteration in the properties of the resulting protein.

B- Non-synonymous mutations

• If a mutation leads to an alteration in the encoded polypeptide, it is known as a non-synonymous mutation.

• Alteration of the amino acid sequence of the protein product of a gene is likely to result in abnormal function.

• Non-synonymous mutations can occur in one of three main ways.

1- Missense

• A single bp substitution can result in coding for a different amino acid and the synthesis of an altered protein, a so-called missense mutation.

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MIS-SENSE MUTATIONe.g. Sickle Cell Anaemia

Cause: defective haemoglobin due to mutation in β-globin geneSymptoms: severe anaemia and death in homozygote

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Normal β-globin - 146 amino acids val - his - leu - thr - pro - glu - glu - --------- 1 2 3 4 5 6 7

Normal gene (aa 6) Mutant geneDNA CTC CACmRNA GAG GUGProduct Glu ValineMutant β-globin val - his - leu - thr - pro - val - glu - ---------

2- Nonsense

• A substitution which leads to the generation of one of the stop codons will result in premature termination of translation of a peptide chain or what is termed a nonsense mutation.

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1.Types of Mutationa) Substitution

Codon amino acidMissense GAG glu

AAG lysNonsense GAG gluChange to termination UAGstopSilent GAG glu

GAA glu

3- Frameshift

If a mutation involves the insertion or deletion of nucleotides which are not a multiple of three, it will disrupt the reading frame and constitute what is known as a frameshift mutation.

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Frameshift

RNA AUG UGU UAU CAU UGG

Insertion AUG UUG UUA UCA UUG G

Deletion AUG UGU UUC AUU GGANote amino acids translated

Mutations in non-coding DNA

• Mutations in non-coding DNA, in general, would not be expected to have a phenotypic effect unless they occur in the DNA sequences involved in gene regulation, e.g. the TATA box, or in the splicing of introns, e.g. the highly conserved GT and AG dinucleotides at the end of introns.

• Mutations in regulatory elements can affect the level of gene expression while mutations in splice junctions can result in coding sequences being lost or intronic sequences being added to the mRNA molecule.

FUNCTIONAL EFFECTS OF MUTATIONS ON THE PROTEIN

• The mutations effect can appear either through loss- or gain-of-function.

1- Loss-of-function:

• These mutations can result in either reduced activity or complete loss of the gene product.

• Loss-of-function mutations in the heterozygous state would be associated with half normal levels of the protein product.

2- Gain-of-function mutations

• Gain-of-function mutations, as the name suggests, result in either increased levels of gene expression or the development of a new function(s) of the gene product.

MUTAGENS AND MUTAGENESIS

• Naturally occurring mutations are referred to as spontaneous mutations and are thought to arise through chance errors in chromosomal division or DNA replication.

• Environmental agents which cause mutations are known as mutagens.

MUTAGENS

• These include natural or artificial ionizing radiation and chemical or physical mutagens.

• Ionizing radiation includes electromagnetic waves of very short wavelength (X-rays and gamma rays), and high energy particles (α particles, ß particles and neutrons).

Causes of Mutations• radiation (nuclear accident

or x-ray)• chemicals

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Mutagenesis

“Process of producing a mutant”

spontaneous induced

Mutagen: physical agent or a chemical which causes or increases the rate of mutation

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Causes of Spontaneous mutations

• error in DNA replication• faulty DNA repair e.g. mis-match • bases form tautomers which pair

differently• natural mutagens

DNA Mismatch Repair

A G C T G

T C T A C

T C G A C

A G C T G

A G C T G

T C T A C

Base pair mismatch

Normal DNA repair

Mutation introduced by

unrepaired DNA

T C T A C

A G A T G

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Mutagens

PHYSICAL CHEMICAL BIOLOGICAL

USE ResearchDiseaseWarfare

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Physical Mutagens

Radiation• Non-ionising

– Ultraviolet (UV)

• Ionising– X-Rays

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Mutagenic chemicals in food contribute to 35% of cancers!

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Biological Mutagens

Insertion of a transposon within a gene• disrupts the reading frame • loss of functionJumping genes- transposable elements

move to different positions in the chromosome. Transposon carries other genes with it.

• (Antibiotic resistance)

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1. UV (260nm)

Absorbed by bases of DNA and RNA• forms pyrimidine dimers• 2 adjacent bases (CC or TT) are covalently

joined• insertion of incorrect nucleotide at this point

likely• during replication Skin cancer, sterilisation of

equipmentPoor penetration

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2. Ionising radiation

X-rays, cosmic rays, gamma raysMore powerful than UVPenetrates glassWater etc ionises: mutagenic effect• free radicals e.g. OH-• inactivate DNA• cell death

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Chemical Mutagens

1.Base analoguesResemble DNA basesFaulty pairing propertiesExample 5-bromouracil• incorporated like T• faulty pairing with G

THE END