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Cell division
Occurs in the nucleus of eukaryotic cells by mitosis and meiosis
o
Replacement of the entire lining of your small intestine
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Liver cells only divide for repairing
o
Nerve cells do not divide
Chromosomes
Long and thin for replication and decoding
Become short and fat prior mitosis → easier to separate due to compact form
Meiosis (reduction division) DIPLOID (2n →1n) HAPLOID
During the production of sex cells (gametes) in animals
In spore formation which precedes gamete production in plants
Haploid gametes (sperm ovum) - sexual reproduction
DNA in a cell replicates only once, but cell divides twice
The Cell Cycle
Interphase
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G1: Protein synthesis and growth (10 hours)
Preparation for DNA replication (e.g. growths of mitochondria)
Differentiation, only selected genes are used to perform different functions in
each cell
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S: DNA Replication (9 hours)
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G2: short gap before mitosis, organelles and proteins for mitosis are made (4 hours)
G0: Resting phase (nerve cells)
M-phase
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Mitotic division of the nucleus (Prophase, Metaphase, Anaphase, Telophase)
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Cytokinesis (division of the cytoplasm)
Interphase
Phase with highest metabolism (mitochondria have a high activity)
Muscles never complete the whole cycle
Mitosis
Process of producing 2 diploid daughter cells with the same DNA by copying their
chromosomes (clones)
Chromosomes can be grouped into homologous pairs
Mitosis occurs in
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Growth
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Repair
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Replacement of cells with limiting life span (red blood, skin cells)
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Asexual replacement
Controlled process, cancers result from uncontrolled mitosis of abnormal cells
Division of the nucleus (karyokinesis) and the cytoplasm (cytokinesis) are two processes of
mitosis
Division of cytoplasm after nucleus. Delayed if cells have more than one nucleus (muscle)
Active process that requires ATP
Prophase
Chromosomes become shorter and thicker by coiling themselves (condensation)
This prevents tangling with other chromosomes
Nuclear envelope disappears/breaks down
Protein fibres (spindle microtubules) form
Centrioles are moving toward opposite poles forming the spindle apparatus of microtubule
Metaphase
Centrioles at opposite poles
Chromosomes line up on the equator of the spindle
Centromeres (kinetochores) attach to spindle fibres
Kinetochores consist of microtubules and "motor" proteins which utilise ATP to pull on the
spindle
Anaphase
Spindle fibres pull copies of chromatids to spindle poles to separate them
Mitochondria around spindle provide energy for movement
Telophase
Chromatid at the pole
Sets of chromosomes form new nuclei
Chromosomes become long and thin, uncoil!
Nuclear envelopes form around the nucleus
Nucleic Acids - The Key to Life
Nucleic acids carry the genetic code that determines the order of amino acids in proteins
Genetic material stores information, can be replicated, and undergoes mutations
Differ from proteins as it has phosphorus and NO sulphur
Made up of several chains of nucleotides
DNA and RNA are types of nucleic acids
Nucleotide
Sugar-phosphate backbone (ensures stability of the molecule)
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Pentose sugar
Deoxyribose in DNA
Ribose in RNA
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Phosphate group
Organic bases
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Purines (double rings of C and N - bigger)
Adenine
Guanine
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Pyrimidines (single ring of C and N - smaller)
Thymine in DNA only
Uracil in RNA only
Cytosine
Deoxyribonucleic Acid (DNA)
Made up of 2 separate chains of nucleotides hold together by base pairing
o
Connected by weak hydrogen bonds
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Can easily be opened for replication
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Adenine-Thymine has 2 H-bonds
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Cytosine-Guanine has 3 H-bonds
DNA normally twist into a helix (coil) / forms a double helix
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Makes the molecule compact (store a lot in small space)
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Protects from damage as base pairs are facing inwards
Both chains of DNA are
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Directional → according to the attachment between sugars and phosphate group
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Antiparallel → essential for gene coding and replication
Semi-Conservative Replication of DNA
Semi-conservative replication: each DNA strand acts as a template for the formation of a new
strand
Happens during interphase S of the cell cycle
Unwinding
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Enzyme DNA helicase separates 2 strands of DNA by breaking hydrogen bonds
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Strands are separated a little at a time (not all at once!)
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This creates a replication fork which moves along the strand
Free DNA molecules join up to exposed bases by complementary base pairing
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Adenine with Thymine (A=T 2-H bonding)
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Cytosine with Guanine (CΞG 3-H bonding)
For the new 5' to 3' strand
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Enzyme DNA polymerase catalyses the joining of the separate nucleotides
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New strand is completed "all in one go"
For the 3' to 5' strand
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DNA polymerase produces short sections of strand
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These sections have to be joined by DNA ligase to make the completed new strand
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Specific base pairing ensures that two identical copies of the original DNA have been
formed
Ribonucleic Acid (RNA)
Ribose instead of deoxyribose
Single chain (shorter than DNA)
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Can pass through nucleus into cytoplasm
Base difference
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Uracil instead of thymine
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Adenine, guanine, and cytosine are the same
Messenger RNA (mRNA) carries the code from DNA that will be translated into an amino acid
sequence
Transfer RNA (tRNA) transfers amino acids to their correct position on the mRNA strand
Genetic Code
DNA codes for assembly of amino acids / forms a polypeptide chain (proteins - enzymes)
The code is read in a sequence of three bases called
o
Triplets on DNA (e.g. CAC TCA)
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Codons on mRNA (e.g. GUG AGU)
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Anticodons on tRNA (e.g. CAC UCA) - must be complementary to the codon of
mRNA
Each triplet codes for one amino acid
Single amino acid may have up to 6 different triplets for it due to the redundancy of the code
/ some amino acids are coded by more than one codon (degenerate code)
Same triplet code will give the same amino acid in all organisms (universal code)
We have 64 possible combinations of the 4 bases in triplets, 43
No base of one triplet contributes to part of the code next to it (non-overlapping)
Few triplets code for START and STOP sequences for polypeptide chain formation
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START: AUG
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STOP: UAA, UAG, UGA
DNA and Inheritance
Cell metabolism: reactions inside cells
Metabolic pathway: sequence of chemical reactions
Alleles: different forms of the same gene
Gene: length of DNA that carries the code for a protein (enzyme)
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Enzyme effect the cell's metabolism
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Visible changes are described with the phenotype
o
The phenotype is influenced by the metabolic pathway
Therefore
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DNA controls enzyme production
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Enzymes control metabolic pathways
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Metabolic pathways influence the phenotype of an organism
Alleles and Genes
Humans have 46 chromosomes
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22 of them are paired up as homologous chromosomes
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Females have an additional homologous pair of sex chromosomes (XX)
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Males have an X and Y sex chromosome
Pair of homologous chromosomes
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One of the pair is inherited from the mother, one from the father
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Gene is a small section of DNA that codes for a specific characteristic
Hair colour,
Eye colour, ...
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Found on both pairs of chromosomes at the same locus (position)
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A gene can have different alleles (forms)
Brown eyes, blue eyes, ...
Black hair, brown hair, ...
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This influences the phenotype
Multiple alleles
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Human ABO blood group is controlled by the gene called immunoglobulin I
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The immunoglobulin gene has 3 alleles IA, IB, I0
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These alleles code for antigen A, B, neither A/B, respectively
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Only 2 alleles can be present → IAIB is codominant, I0 is recessive
Genes Control Phenotype
Mutation
Change in one or more nucleotide bases in the DNA
Change in the genotype (may be inherited)
Deletion: reading frame shifts
Substitution: one base replaced by another
Duplication: repetition of part of the sequence
Addition: addition of extra base
Cystic Fibrosis
Revision from Unit 1 Section 3.1.3(c)
Autosomal recessive disorder
Mutation of the CFTR gene on chromosome 7
Deletion of 3 bases / allele is missing 3 nucleotides
Those nucleotides code for the amino acid phenylalanine
Phenylalanine is missing from the CFTR protein
Faulty CFTR protein cannot control the opening of chloride channels in the cell membrane
Phenylketonuria (PKU)
Autosomal recessive disorder
Gene mutation in DNA/gene coding for the enzyme phenylalanine hydroxylase
Phenylalanine hydroxylase is not produced
Amino acid phenylalanine cannot be converted to the amino acid tyrosine
Tyrosine
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Necessary to produce the pigment melanin
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Patients are fair-haired, fair skinned and blue eyed (phenotype)
Phenylalanine
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Accumulates in the blood and causes irreversible brain damage
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Found in most food that contains proteins
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Treated by avoiding food that contains phenylalanine (diet low in protein)
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Levels in blood are regularly measured by GP
All babies are screened shortly after birth to prevent learning difficulties
DNA Deoxyribonucleic Acid
Nucleotides are smaller units of long chains of nucleic acids. Each nucleotide has
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A pentose sugar (deoxyribose in DNA, ribose in RNA)
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A phosphate group
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An organic base which fall into 2 groups,
Purines (double rings of C and N - bigger)
Adenine or Guanine
Pyrimidines (single ring of C and N - smaller)
Thymine or Cytosine
Base pairing by weak hydrogen bonds
Adenine-Thymine 2 H- bonds
Cytosine-Guanine 3 H- bonds
Chains are directional according to the attachment between sugars and phosphate group
They are antiparallel which is essential for gene coding and replication
DNA molecule has 2 separate chains of nucleotides hold together by base pairing / DNA
normally twist into a helix (coil) / forms a double helix
Ribonucleic Acid (RNA)
Ribose instead of deoxyribose
Single chain (shorter than DNA - lower molecular mass)
Base difference: Uracil instead of Thymine. Adenine, Guanine and Cytosine are the same
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Ribosomal RNA (rRNA)
Located in the cytoplasm - ER
Reads mRNA code and assembles amino acids in their correct sequence to
make a functional protein (translation)
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Messenger RNA (mRNA)
Commutes between nucleus and cytoplasm
Copies the code for a single protein from DNA (transcription)
Carries the code to ribosomes in the cytoplasm
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Transfer RNA (tRNA)
In the cytoplasm
Transfer amino acids from the cytoplasm to the ribosomes
The Genetic Code
DNA codes for assembly of amino acids / forms a polypeptide chain (proteins - enzymes)
The code is read in a sequence of three bases called
o
Triplets on DNA
e.g. CAC TCA
o
Codons on mRNA
e.g. GUG AGU
o
Anticodons on tRNA
e.g. CAC UCA
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(must be complementary to the codon of mRNA)
Each triplet codes for one amino acid / single amino acid may have up to 6 different triplets
for it due to the redundancy of the code / code is degenerate. Some amino acids are coded
by more than one codon
Same triplet code will give the same amino acid in virtually all organisms, universal code
We have 64 possible combinations of the 4 bases in triplets, 43
No base of one triplet contributes to part of the code next to it, non-overlapping
Few triplets code for START and STOP sequences for polypeptide chain formation
eg START AUG
and STOP UAA UAG UGA
DNA Replication (Semi-Conservative Replication)
Happens during Interphase 'S'
Separate the strands, a little at a time to form a replication fork
Events:
o
Unwinding / Enzyme DNA helicase separates 2 strands of DNA by breaking hydrogen
bonds
o
Semi-conservative replication / each strand acts as a template for the formation of a
new strand
o
Free DNA molecules join up to exposed bases by complementary base pairing
Adenine with Thymine (A=T 2 -H bonding)
Cytosine with Guanine (CΞG 3 -H bonding)
o
For the new 5' to 3' strand the enzyme DNA polymerase catalyses the joining of the
separate nucleotides
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"All in one go" → completed new strand
o
For the 3' to 5' strand DNA polymerase produces short sections of strand but these
sections have to be joined by DNA ligase to make the completed new strand. Specific
base pairing ensures that two identical copies of the original DNA have been formed
Transcription: DNA to mRNA
DNA in nucleus unzips - bonds break
Single template strand of DNA used for mRNA (triplet on DNA = codon for amino acid on
mRNA)
Enzyme RNA polymerase joins nucleotides together
Free RNA nucleotides are assembled according to the DNA triplets (A-U / C-G / T-A)
mRNA bases are equivalent to the non-template DNA strand
Start and stop codons are included
Introns (Non-coding) and exons (coding) DNA sequences are present in the primary mRNA
transcript. Introns are removed before the mRNA is translated so that exons are only present
in the mature mRNA transcript
Total number of bases in the DNA sense strand and total number of bases in the mRNA are different
mRNA moves into cytoplasm and becomes associated with ribosomes
Translation: mRNA to Protein via tRNA
Translation is the synthesis of a polypeptide chain from amino acids by using codon
sequences on mRNA
tRNA with anticodon carries amino acid to mRNA associated with ribosome
"Anticodon - codon" complementary base pairing occurs
Peptide chain is transferred from resident tRNA to incoming tRNA
tRNA departs and will soon pick up another amino acid
Requirement for Translation
Pool of amino acids / building blocks from which the polypeptides are constructed
ATP and enzymes are needed
Complementary bases are hydrogen-bonded to one another
Structure involved in translation
Messenger RNA (mRNA)
Carries the code from the DNA that will be translated into an amino acid sequence
Transfer RNA (tRNA)
Transfer amino acids to their correct position on mRNA strand
Ribosomes
Provide the environment for tRNA attachment and amino acid linkage
DNA and Inheritance
Reactions in cells is referred to as cell metabolism
A sequence of chemical reactions is called a metabolic pathway
Different forms of the same gene are alleles
A gene is the length of DNA that carries the code for a protein (enzyme)
o
Enzyme effect the cell's metabolism
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Visible changes are described with the phenotype
The phenotype is influenced by the metabolic pathway
Therefore
o
DNA controls enzyme production
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Enzymes control metabolic pathways
o
Metabolic pathways influence the phenotype of an organism
Gene Mutations
Deletion, reading frame shifts
Substitution, one base replaced by another
Duplication, repetition of part of the sequence
Addition, Addition extra base
Change in one or more nucleotide bases in the DNA
Change in the genotype (may be inherited)
Cystic Fibrosis - Defective Gene
Mutation causes the deletion of 3 bases in DNA. One amino acid (phenylalanine) is not
coded for in the Cystic Fibrosis Transmembrane Regulator CFTR protein
Faulty CFTR protein cannot control the opening of chloride channels in the cell membrane
Results in production of thick sticky mucus, especially in lungs, pancreas and liver
Organs cannot function normally and infection rate increases
Phenylketonuria (PKU) - Defective Gene
Gene mutation in DNA coding for the enzyme phenylalanine hydroxylase
Phenylalanine hydroxylase not produced
Amino acid phenylalanine cannot be converted to the amino acid tyrosine
Tyrosine is necessary to produce the pigment melanin
Phenylalanine collects in the blood and causes retardation in young children
Managed by controlling diet to eliminate proteins containing phenylalanine
Disease is tested by drops of blood taken from the baby
More on Cystic Fibrosis
Caused by a mutation of the Cystic Fibrosis Transmembrane Regulator (CFTR) gene
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Covered in Unit 2 Section 3.2.1
CFTR is a plasma membrane protein
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Normally, it transports chloride ions out of the cell by active transport
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In cystic fibrosis, a mutation alters the tertiary structure of CFTR
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The protein fails to reach plasma membrane
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Accumulation of Cl- and Na+ (attracted by negative Cl-) within the cell
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Secretions are thick as water stays inside the cell due to high internal Na+ (altered
water potential)
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NB: water always follows Na+
Lungs
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"Produce less mucus than normal "1
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Lung surface is dehydrated and mucus adheres to airways
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This favours the growth of bacteria causing chronic infection
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White cells engulf bacteria and die (phagocytosis)
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The DNA from dead inflammatory cells (pus) contributes to thick sputum
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Sputum has an increased viscosity and cannot be removed by the ciliary escalator
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Obstructs airways and causes further inflammation
Pancreas
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Thick digestive juice blocks passage from pancreas into small intestine (duodenum)
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Obstruction may cause chronic inflammation of the pancreas
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Pancreas fails to secrete digestive enzymes
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Food is not broken down and not absorbed
Sweat
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CFTR works differently in the skin
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Normally, chloride are transferred from the sweat into the cell
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Excessive NaCl remains on the skin - sweat taste saltier than normal
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Sweat can be collected and analysed to diagnose CF
Slow growth
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Less efficient energy/fooduptake due to malabsorption
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High energy consumption due to chronic inflammation of the lungs
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To compensate, children require high calorie diet (chocolate, crisps)
Treatment
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Pancreatic enzyme replacement therapy (PERT) to treat fat malabsorption
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Fat-soluble vitamin supplements (A, E, D, K) to prevent deficiencies
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Inhaled enzyme DNAse breaks down excessive DNA and thin mucus
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Antibiotics to treat lung infections (frequent use causes antibiotics resistance)
Chromosomes and inheritance
It is important to remember that all body cells (in situations that you are likely to come across) will be diploid.
In humans (except in red blood cells) there are 46 chromosomes in all body cells - 23 pairs. Each pair of
chromosomes is numbered and has its own particular genes.
In gametogenesis, (the production of sperm and eggs) this number is reduced to 23.
Only one chromosome of a pair can be inherited. Gametes are haploid. Which chromosome of the pair is
inherited is random (see Independent Assortment in Meiosis). When working out the chances of an offspring
inheriting a particular genotype, this fact must be remembered.
Monohybrid crosses - single gene inheritance
When studying genetics, the following conventions are used:
P is used as shorthand for the parent generation.
F1 is used for their offspring.
F2 is used if the offspring (F1) are crossed.
Capital letters are used to denote a dominant allele.
Lower case letters are used to denote a recessive allele.
For example:
Drosophila (fruit flies) can be either straight-winged or curved-winged, this characteristic is controlled by one
pair of genes. When straight- and curved-winged are bred together, all the offspring are straight-winged.
This means that straight-winged is dominant and curved-winged is recessive.
Therefore:
'straight' allele = S
'curved' allele = s
Since the allele for curved wings is recessive, if a fly has curved wings, it must have 2 alleles for curved wings
= ss
Since the allele for straight wings is dominant, a straight winged fly will have either SS alleles or Ss alleles.
Question 1:
What would be the result in the F1 generation of crossing a homozygous straight-winged fly with a curvedwing fly?
The easiest way to show what the offspring will look like, is to work through this sequence:
All F1 offspring are Ss.
This means that for each offspring there is a 100% probability that they will be Ss and therefore straightwinged.
Question 2:
What would be the result in the F2 generation of crossing 2 of the F1 flies?
This means that for each offspring there is a 75% probability that they will be straight-winged and 25%
probability that they will be curved-winged.
Another way of saying this is ratio of straight : curved is 3 : 1.
It does not necessarily mean that out of 4 offspring, 3 would definitely be straight-winged and 1 would be
curved-winged. It is possible, though unlikely, that all offspring could be curved or straight-winged.
Question 3:
How would you determine the genotype of any unknown straight-winged fly?
To find out whether a genotype is homozygous dominant (SS) or heterozygous (Ss), a test-cross needs to be
done since you cannot tell the genotype by looking at the fly.
The unknown is bred with a known. The only phenotype that gives a known genotype is homozygous
recessive (ss).
If the fly was SS:
All offspring, no matter how many, would be straight-winged.
If the fly was Ss:
Some offspring (it should be 50%), will have curved wings.
Multiple alleles
In the previous case, there were only 2 alleles for one gene. In the case of the ABO blood grouping, there are 3
alleles for one gene and in this situation they are written a little differently:
i : protein is produced but it is not antigenic - this allele is recessive
IA : protein with antigen A made - this allele is co-dominant
IB : protein with antigen B made - this allele is co-dominant
Blood group (phenotype): Possible genotype:
A
IA IA or IA i
B
IB IB or IB i
AB
IA IB
O
ii
Dihybrid crosses
This is where the inheritance of two characteristics is studied.
In this case we will look at a case where the genes are on separate chromosomes, the alleles are not linked
(they are not necessarily inherited together).
In a certain variety of rabbits, grey coat colour is dominant over white, and short hair is dominant over long.
Question:
A breeder has homozygous long-haired white rabbits and homozygous short-haired grey rabbits.
Short = S
Long = s
Grey = G
White = g
1. What would be the ratio of offspring in the F1 generation?
Offspring genotypes: all are SsGg.
Offspring pheotypes: all are short-haired grey rabbits.
2. If 2 offspring were bred together, what would the ratio of offspring be in the F2 generation?
F2 Punnett Square:
SG
Sg
sG
sg
SG SSGG SSGg SsGG SsGg
Sg SSGg SSgg SsGg SSgg
sG SsGG SsGg ssGG ssGg
sg SsGg Ssgg ssGg ssgg
Ratio of genotypes: Short grey Long grey Short white Long white
Ratio of phenotypes: 9
3
3
1
Incomplete dominance
This is when neither allele is dominant.
Both alleles are expressed and contribute equally to the phenotype.
A heterozygote has an intermediate phenotype as there is partial influence from both alleles.
Example:.
Snapdragons can be red (alleles = RR), white (alleles = WW) or pink (alleles = RW).
Codominance
In this case, both alleles are dominant.
They are independent, so there is no 'blending' as in the snapdragons, instead the phenotype is a result of the
full expression of both alleles.
Example:.
Blood group AB.
Sex determination
Gender is determined by sex chromosome s in many animals. The 3 most common systems are:
1. The XY System (e.g, in humans, Drosophila)
Female are XX, males are XY
Both sexes have 2 chromosomes but the females' chromosomes are the same, the males are different.
2.The XO System (e.g, grasshoppers, bugs)
Females are XX, males are XO
The male has only 1 sex chromosome whereas the female has 2.
3. The WZ System (e.g, birds, butterflies, some fish)
Females are ZW, males are ZZ
Both sexes have 2 chromosomes but the females' chromosomes are different, the males are the same.
Sex-linked genes
Some genes are part of the sex chromosomes and so are inherited with them. Usually it is the X chromosome
that is considered in which case the female will have two alleles, the male will only have one.
The genetic condition of haemophilia is carried on the X chromosome.
The normal allele is dominant (H), the allele for haemophilia is recessive (h).
XHXH = normal female
XHXh = carrier female
XHY = normal male
XhY = male sufferer
The ratio of males to females = 1 : 1
Of the males, there is a ratio of 1 : 1 normal : sufferer
Therefore there is a 25% probability that any offspring will be a sufferer. There is a 50% probability that a boy
is affected.
In cats (which are also XX if they are female and XY if they are male) the allele for coat colour is carried on
the X chromosome.
The alleles are black and orange but they are codominant.
Example.
XBXB = black female
XBY = black male
XOXO = orange female
XOY = orange male
XBXO = tortoiseshell female
All females are tortoiseshell.
All males are black.
Mother can provide XB or XO
Father can provide Y (accounting for orange & black males) plus 1 other allele.
Since 1 offspring is XO XO, the father must provide 1 of these allele.Therefore, the father's genotype is XOY
So far we have looked at situations where one pair of alleles controls one characteristic.
There are occasions where a number of genes interact together.
Pleiotropy
This is where one gene affects several characteristics. For example, a disease caused by one pair of alleles may
have several or many symptoms.
Polygeny
This is where one characteristic is affected by two or more genes (e.g, skin colour).
Several genes control skin colour, we will look at just two to make it a little simpler.
The alleles will be called A and B, and each of these has one alternative allele, a and b.
A and B cause the skin to be dark, a and b cause it to be light
A person who has the genotype AABB will have very dark skin;
A person who has the genotype aabb will have white (albino) skin.
A person with genotype AAbb, aaBB or AaBb will have medium colour skin.
Thus if two people of genotype AaBb have children, they may have any colour skin:
Epistasis
This is where one gene interferes with the expression of another gene.
Example
Mouse coat colour is controlled by two pairs of alleles: B and C
B = black coat colour, b = brown coat colour
C = pigment production, c = no pigment production
Therefore, if a mouse has cc, it will be an albino, if it has Cc or CC it will be black or brown.
Environment and phenotype
We have assumed in all the examples covered so far that the genotype always has exactly the same effect on
the phenotype.
For example: people with the alleles AaBb for skin colour will have medium dark coloured skin.
This is not always true. If this person is exposed to a lot of sunlight, it is very likely that their skin will be
darker as they tan.
In the case of height, the alleles determine the potential height that someone could achieve but with a poor diet,
they may be well short of their maximum height. Thus environment can have a large bearing on the extent to
which genes are expressed.
GENE TECHNOLOGY NOTES
Insulin and Genetic Engineering
Diabetes mellitus is the inability of beta cells of pancreas to produce insulin
Restriction enzymes/endonuclease cut DNA at specific recognition sites
o
This produces either "sticky ends" or "blunt ends"
o
DNA ligase can be used to re-join the ends
Recombinant DNA technology combines the DNA from two different organisms
Reverse transcriptase catalyses the formation of DNA from mRNA
Vector is a gene carrier. It will carry a human gene into the cell of a bacterium or yeast that
will be used to make human protein. Produces no benefit for viruses / carrier
Plasmid, circular strand of DNA, are useful vectors to make human protein from bacteria
Transgenic organisms contain another species DNA
Integration Link
Remove a particular gene from the DNA of an animal cell
Locate with the use of a gene probe
Use restriction enzymes
Use endonucleases to cut at specific base sequence by hydrolysing
Breaking sugar-phosphate bonds
Insert this gene into the genetic material of a bacterium
Same restriction enzymes
Cut at same base sequence in bacterial DNA
Leaving sticky ends/hydrogen bonds break
Join/splice with ligase
Use of plasmid
Task to find and insert the gene into bacterium for Insulin production
Isolate human gene, e.g. insulin, by using cytoplasmic mRNA (no introns)
Reverse transcriptase, taken from a retrovirus, makes DNA from mRNA
DNA is given "sticky ends" by using the enzyme restriction endonuclease
Insert into a plasmid from a bacterium
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Dissolve cell walls using enzymes
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Centrifuge to separate bacterial chromosome ring from plasmids
o
Cut open the plasmid
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Add sticky ends
Mix plasmid and DNA gene together and use DNA ligase to stick them together
Mix with bacteria //only ≈1% will take up the engineered plasmids
Identify by using antibiotic resistance. Add gene for antibiotic resistance next to insulin gene
in the plasmid. Add antibiotic to the culture / only bacteria surviving have insulin gene
Grow transformed cells using industrial fermenters
Isolate and purify human protein made by these cells
Moral and ethical issues associated with recombinant DNA technology
Transgenic bacteria or viruses may mutate and may become pathogenic
Genetically modified crops could "escape"
o
Forms a genetically modified population in the environment
o
Genetic modification may involve the resistance to herbicides
o
Escaped crops may become "superweeds" that are difficult to kill and control
Transgenic organisms could upset the balance of nature
o
Population of transgenic salmon have been produced in which individuals grow
rapidly
o
These transgenic fish could compete for food with other fish species
This controversial area of science raises many questions...
•
Crops that have been engineered to have resistance to a particular weeds, pests or diseases may
produce long term side effects that spread into wild species, making them difficult to eradicate.
•
The development of crops with additional genes poses potential risks. Genes may transfer to related
species by cross pollination, and affect the balance of natural communities.
•
Biodiversity of wildlife may be reduced by changes in the balance between food plants and wild stock.
•
Little is known as yet about the risks of movement of genes from crop to wild plants.
Raised ethical issues
There are also some ethical issues that have to be faced...
•
Traditional genetics makes use of natural selection processes, whereas the movement of genes across
species boundaries diminishes species uniqueness and has a degree of 'creating life'.
•
Questions are raised about whether foods should be labelled, so that consumer choice is retained.
•
Scientists are mistrusted and do not sell their ideas well to the general public, earning them little
confidence. The media is not well informed at times.
•
Patents have been taken out on genes, and this has added to the claims that humans are inventing life,
and laying claim to its ownership
What is gene therapy?
Gene therapy is the deliberate 'repair' or replacement of damaged genes. Success has been limited to somatic
(body) cells rather than sex cells (gametes). This means that any changes are not passed on to subsequent
generations (inherited). The modification of germ line cells (germ line therapy) is likely to be discouraged on
moral grounds.
Initial attempts at gene therapy
Using mice with artificially induced Cystic fibrosis (CF) copies of a gene CFTR enclosed in liposomes (tiny
lipid droplets) were squirted into their lungs.
The liposomes fused with the lung cell membranes, and this allowed the DNA in the CFTR genes to pass
through into the cells.
Not all cells were successfully changed, but those that had the gene added were then corrected for CF,
temporarily.
Later attempts were made using viruses as vectors (carriers) to introduce the gene as the virus enters (infects)
the cells.
Cystic fibrosis in humans.
Cause: The inheritance of two recessive alleles for cystic fibrosis.
Symptoms: The transport of chloride ions and water by the cells in the airways, lungs and gut is disrupted.
This causes thick mucus to line the lungs and ducts in the gut. Because the thick mucus is not shifted easily, it
is more likely that a sufferer will pick up a bacterial infection.
Gene therapy treatment: Copies of the DNA for the normal allele were inserted into other loops of DNA,
which were then attached to liposomes. The liposome complexes were then sprayed as an aerosol of fine
droplets into the patients' noses. The DNA is then taken up by some of the cells lining the airways.
Fortunately only about 10% of these cells need to take up the DNA for symptoms to be relieved.
Rennet (rennin or rennilase) and cheese making:
Back in the 1960's, the world faced a severe shortage of calf rennet.
Rennet is a protease enzyme added to milk, along with certain bacteria, which coagulate milk proteins,
producing the curds. This then separates from the liquid, whey.
The semi-solid curds are then treated by adding salt and then matured in containers to make the cheddar-style
cheeses.
(Rennet is an enzyme found in the stomachs of young mammals, like calves, and is important in the digestion
of milk proteins.)
Over the past 30 years, alternatives to calf rennet have been sought.
Alternative sources of Protease for coagulating milk:
There are a number of alternatives available, which are not derived from animal sources directly.
One form is comes from fungi, such as Rhizomucor miehei. Fungi produce natural proteolytic enzymes as part
of their extracellular digestion process.
The other are from genetically modified microbes, such as E.coli bacteria, a fungus Aspergillus niger and
food yeasts.
Obtaining Chymosin from yeast cells:
About 90% of hard cheeses like Cheddar are made now using chymosin from GM microbes.
Advantages of using GM Chymosin:
1.
Chymosin was the first enzyme to be approved for used in the food industry, so its effects are well
known.
2.
Chymosin behaves exactly the same way as the animal equivalent, but due to its structure, it is much
more predictable and there are fewer impurities.
3.
As it is made in yeast, it is fully accepted by vegetarians and some religious authorities.
Chymosin and GMO's:
Cheese produced using genetically engineered Chymosin is not regarded as a problem as it does not contain
the organism, but the product (enzyme) of that GMO.
The enzyme does not remain in the cheese either, but soon breaks down, as it is a relatively unstable protein.
GENETIC FINGERPRINTTING
Devised by Alex
Jeffreys at Leicester University and now widely used in forensic science and other uses of identification.
Different bands, resembling a supermarket bar code, are produced, which are unique to any one individual,
except identical twins.
DNA is obtained from the white blood cells, mixed with restriction endonuclease, which 'cuts' the DNA into
millions of fragments, but not the repeated regions, which retain their original length.
The DNA fragments are then loaded into a slot at the end of an agarose gel.
An electric current is passed across the gel, with the positive electrode at the furthest end from the fragments.
Since DNA molecules have a negative charge, they will migrate through the gel towards the positive electrode.
Larger fragments move more slowly than the smaller ones. The result is a series of bands down the gel, but
these are invisible at this stage.
Since the gel is difficult to keep, the DNA bands are then transferred to a nylon membrane, which is incubated
overnight with radioactive probes.
The radioactive probes bind with the DNA that has repeating regions.
A sheet of X-Ray photographic film is laid over the membrane in total darkness. The radiation will affect the
film, which is later developed, as a photograph.
The developed film reveals the series of DNA bands, which are unique to an individual.
Genetic counselling
Many illnesses are now being traced back to particular defective genes. Some, like Huntington's disease, only
appear later in life - between 30 and 50 years of age.
While pinpointing the genes responsible for these terrible diseases is a breakthrough, it also poses a dilemma...
In the past, children who have a 50:50 chance of inheriting the disease faced a long wait to find out if they
have been affected. Now they can choose to be tested, and then cope with the devastating news.
Screening tests can reveal whether an unborn child has Down's syndrome or cystic fibrosis for example.
Termination of pregnancy may be a decision that parents have to face, and this is where counselling is
required, for such a difficult decision.
With high-risk parents, embryos may be produced outside the body (so called ''test-tube babies'') and after
screening for defective genes, only normal embryos are implanted.
Transplant surgery
Transplanting foreign tissue carried great risk of rejection by the body's immune system. The patient faces the
rest of their life with a cocktail of anti-suppressant drugs.
There is a great shortage in organs suitable for transplantation, resulting in may patients suffering or even
dying before they get a chance to have the transplant operation.
Xenotransplantation.
Organs from other animals can be used in human transplantation, but they pose a potentially greater risk of
rejection.
One way to avoid this is to change the chemical signature of the surface of the organ. This is achieved by
adding the human gene that produces the correct chemical signals onto the surface of the organ so that the
recipient's antibodies recognise it as 'self'.
Causes of genetic variation
There are several causes for variation being present within a population:
1.
Each gene has different alleles. Therefore, different individuals may have different alleles.
2.
During prophase I of meiosis, chiasmata (crossing over) occurs whereby sections of DNA are
swapped between sister chromatids.
3.
During metaphase I of meiosis, there is independent assortment of the homologous pairs of
chromatids.
4.
Mutation - gene and chromosome.
5.
Random fertilisation.
This shows that there is variation of genotype and phenotype between individuals. The environment also exerts
an effect and can cause variation.
Types of variation
For each characteristic, the population may show either continuous or discontinuous variation.
The genetic basis for discontinuous variation
This is where different alleles for one gene have a large effect on the phenotype.
Example:
ABO blood groups, there are no intermediates; you are either A, B, AB or O.
The genetic basis for continuous variation
Different alleles for one gene have small effects.
Different loci have the same or additive effects. (When a large number of loci produce a combined effect it is
called polygeny.)
Example:
Imagine height is controlled just by two genes (though in reality, many genes will contribute to height). Each
has two alleles; E and e, F and f.
E and F contribute 2cm to height whereas e and f contribute just 1cm to height.
Therefore:
EEFF = 8cm
eeff = 4cm
If EeFf is crossed with EeFf, the outcome will be...
Parental genotypes: EEFF x eeff
Gametes:
All EF x All ef
F 1:
all EeFf
Parental genotypes: EeFf
Gametes:
x EeFf
EF, Ef, eF, ef x EF, Ef, eF, ef
F2 punnett square: EF
Ef
eF
ef
EF
EEFF EEFf EeFF EeFf
Ef
EEFf EEff EeFf Eeff
eF
EeFF EeFf eeFF eeFf
ef
EeFf Eeff eeFf eeff
Phenotypes: 8 cm tall 7 cm tall 6 cm tall 5 cm tall 4 cm tall
Ratio:
1
4
6
4
1
This crudely shows the continuous variation in a population with regard to height.
Don't forget that environment also causes variation of the phenotype. This will not be passed on to offspring.
The gene pool and allele frequencies
In any population, the total variety of genes and alleles present is called the gene pool.
This gene pool can change in content (new alleles arriving, existing alleles being lost) or the ratio of
alleles altering due to the following:
1.
Mutation
2.
Natural selection
3.
Emigration
4.
Immigration
5.
Mate selection
The factors favouring stability of the gene pool are:
1.
No mutation
2.
No natural selection
3.
The population being large
4.
No gene flow (due to individuals emigrating or immigrating)
5.
Random mating
If these factors favouring stability are fulfilled, the ratio of the alleles for a gene can be established using the
Hardy-Weinberg Equilibrium.
For a species to survive, it must reproduce. However, the population is limited by environmental factors and so
remains more or less constant over time.
There is competition between individuals of the same species (intraspecific competition) or between members
of different species (interspecific competition) for resources.
Since there is variation within a population, some individuals are less well adapted to a particular environment.
The less well adapted are 'weeded out' as the selection acts on the phenotype of the individual.
These individuals fail to reproduce or die, the more successful ones reproduce and pass on their genes to the
next generation.
Note: Adaptations are environment-specific; an advantageous characteristic can become disadvantageous if the
environment changes in a particular way.
The change in adaptation that occurs is called evolution.
There are three types of selection that occur in nature:
•
Stabilizing selection.
•
Directional selection.
•
Disruptive selection.
In each case we will use the illustration of a population of mammals and the characteristic being selected for or
against is fur length.
In each situation, the population is normally distributed - there are a few individuals that have very short or
very long fur length but most have an intermediate fur length.
Stabilising selection
Initially there is a wide range of fur length about the mean of 1.5cm.
Due to rapid breeding in either very cold or very warm weather, animals with extreme fur lengths survive.
When the temperature remains constant with little variation, the individuals with very short or very long hair
become less numerous and are eventually eliminated from the population.
Directional selection
If the temperature falls, the individuals with longer fur length are at an advantage as they have better
insulation against the cold.
There is a selection pressure favouring the animals with longer fur so these animals are more likely to survive
and thus reproduce.
Over several generations, the average fur length increases as more young have inherited the genes for long fur.
When the mean fur length has reached the most advantageous length, the selection pressure ceases.
Disruptive selection
If the temperature difference between summer and winter increases, long hair for animals being active during
the winter or short hair for animals being active during the summer is advantageous.
Intermediate fur length is disadvantageous. Therefore, two sub populations are formed over time.
It is thought that natural selection, as well as altering allele frequencies according to the advantage they give, is
the force behind the production of all the different species that have ever lived on Earth.
Definition of a species
A group of organisms with similar morphological, physiological and behavioural features, which can
interbreed to produce fertile offspring, and are reproductively isolated from other species.
Therefore, donkeys, which look and behave like horses, can breed with horses, but their offspring (mules) are
infertile. Donkeys and horses belong to different species.
Speciation
For one species to form 2 species, they must therefore be reproductively isolated. This may happen because of
several reasons.
Isolating mechanisms
A population becomes physically separated by a barrier that prevents them from mixing.
For example; a stretch of water (as has happened in the Galapagos Islands) or a road being
cut into a forest.
Geographical:
In the two areas there could be very different selection pressures, resulting in
different alleles being advantageous and thus increasing in frequency.
Over time, the morphological, physiological and behavioural differences are so
great that they can no longer interbreed.
Habitat:
A population becomes separated because two groups may live on the same mountain but
at different altitudes, or in the same area but in differing types of soil.
Seasonal:
A population becomes separated because two groups breed at different times.
A population becomes separated because two groups behave differently. For example; one
Behavioural: group of birds may sing one song, another group sings a different song and neither group
recognises the other.