Cell division

Occurs in the nucleus of eukaryotic cells by mitosis and meiosis

o Replacement of the entire lining of your small intestine

o 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

o 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

o S: DNA Replication (9 hours)

o G2: short gap before mitosis, organelles and proteins for mitosis are made (4 hours)

G0: Resting phase (nerve cells)

M-phase

o Mitotic division of the nucleus (Prophase, Metaphase, Anaphase, Telophase)

o 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

o Growth

o Repair

o Replacement of cells with limiting life span (red blood, skin cells)

o 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)

o Pentose sugar

� Deoxyribose in DNA

� Ribose in RNA

o Phosphate group

Organic bases

o Purines (double rings of C and N - bigger)

� Adenine

� Guanine

o 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

o Can easily be opened for replication

o Adenine-Thymine has 2 H-bonds

o Cytosine-Guanine has 3 H-bonds

DNA normally twist into a helix (coil) / forms a double helix

o Makes the molecule compact (store a lot in small space)

o Protects from damage as base pairs are facing inwards

Both chains of DNA are

o Directional → according to the attachment between sugars and phosphate group

o 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

o Enzyme DNA helicase separates 2 strands of DNA by breaking hydrogen bonds

o Strands are separated a little at a time (not all at once!)

o This creates a replication fork which moves along the strand

Free DNA molecules join up to exposed bases by complementary base pairing

o Adenine with Thymine (A=T 2-H bonding)

o Cytosine with Guanine (CΞG 3-H bonding)

For the new 5' to 3' strand

o Enzyme DNA polymerase catalyses the joining of the separate nucleotides

o New strand is completed "all in one go"

For the 3' to 5' strand

o DNA polymerase produces short sections of strand

o These sections have to be joined by DNA ligase to make the completed new strand

o 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)

o Can pass through nucleus into cytoplasm

Base difference

o Uracil instead of thymine

o 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)

o Codons on mRNA (e.g. GUG AGU)

o 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

o START: AUG

o 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)

o Enzyme effect the cell's metabolism

o Visible changes are described with the phenotype

o The phenotype is influenced by the metabolic pathway

Therefore

o DNA controls enzyme production

o Enzymes control metabolic pathways

o Metabolic pathways influence the phenotype of an organism

Alleles and Genes

Humans have 46 chromosomes

o 22 of them are paired up as homologous chromosomes

o Females have an additional homologous pair of sex chromosomes (XX)

o Males have an X and Y sex chromosome

Pair of homologous chromosomes

o One of the pair is inherited from the mother, one from the father

o Gene is a small section of DNA that codes for a specific characteristic

� Hair colour,

� Eye colour, ...

o Found on both pairs of chromosomes at the same locus (position)

o A gene can have different alleles (forms)

� Brown eyes, blue eyes, ...

� Black hair, brown hair, ...

o This influences the phenotype

Multiple alleles

o Human ABO blood group is controlled by the gene called immunoglobulin I

o The immunoglobulin gene has 3 alleles IA, IB, I0

o These alleles code for antigen A, B, neither A/B, respectively

o 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

o Necessary to produce the pigment melanin

o Patients are fair-haired, fair skinned and blue eyed (phenotype)

Phenylalanine

o Accumulates in the blood and causes irreversible brain damage

o Found in most food that contains proteins

o Treated by avoiding food that contains phenylalanine (diet low in protein)

o 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

o A pentose sugar (deoxyribose in DNA, ribose in RNA)

o A phosphate group

o 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

o 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)

o 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

o 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

o (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

o "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

o Visible changes are described with the phenotype

The phenotype is influenced by the metabolic pathway

Therefore

o DNA controls enzyme production

o 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

o Covered in Unit 2 Section 3.2.1

CFTR is a plasma membrane protein

o Normally, it transports chloride ions out of the cell by active transport

o In cystic fibrosis, a mutation alters the tertiary structure of CFTR

o The protein fails to reach plasma membrane

o Accumulation of Cl- and Na+ (attracted by negative Cl-) within the cell

o Secretions are thick as water stays inside the cell due to high internal Na+ (altered

water potential)

o NB: water always follows Na+

Lungs

o "Produce less mucus than normal "1

o Lung surface is dehydrated and mucus adheres to airways

o This favours the growth of bacteria causing chronic infection

o White cells engulf bacteria and die (phagocytosis)

o The DNA from dead inflammatory cells (pus) contributes to thick sputum

o Sputum has an increased viscosity and cannot be removed by the ciliary escalator

o Obstructs airways and causes further inflammation

Pancreas

o Thick digestive juice blocks passage from pancreas into small intestine (duodenum)

o Obstruction may cause chronic inflammation of the pancreas

o Pancreas fails to secrete digestive enzymes

o Food is not broken down and not absorbed

Sweat

o CFTR works differently in the skin

o Normally, chloride are transferred from the sweat into the cell

o Excessive NaCl remains on the skin - sweat taste saltier than normal

o Sweat can be collected and analysed to diagnose CF

Slow growth

o Less efficient energy/fooduptake due to malabsorption

o High energy consumption due to chronic inflammation of the lungs

o To compensate, children require high calorie diet (chocolate, crisps)

Treatment

o Pancreatic enzyme replacement therapy (PERT) to treat fat malabsorption

o Fat-soluble vitamin supplements (A, E, D, K) to prevent deficiencies

o Inhaled enzyme DNAse breaks down excessive DNA and thin mucus

o 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 curved-

wing 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 straight-

winged.

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 i i

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

o Dissolve cell walls using enzymes

o Centrifuge to separate bacterial chromosome ring from plasmids

o Cut open the plasmid

o 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

F1: all EeFf

Parental genotypes: EeFf x EeFf

Gametes: 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

Geographical:

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.

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.

Behavioural:

A population becomes separated because two groups behave differently. For example; one

group of birds may sing one song, another group sings a different song and neither group

recognises the other.