The artificial plasmid, pBR322, was constructed to act as a vector. It has often been used to insert human genes, such as the human insulin gene, into the bacterium, Escherichia coli.

The plasmid was constructed to include two genes, each giving resistance to a different antibiotic: an ampicillin resistance gene and a tetracycline resistance gene. The plasmid also has a target site for the restriction enzyme, BamHI, in the middle of the tetracycline resistance gene.

A pBR322 plasmid was cut using BamHI and the cDNA gene for human insulin inserted into it.

Fig. 2.1 shows pBR322 and the recombinant plasmid.
ampicillin resistance gene
pBR322
etracycline resistance gene
target site for BamHI

GlG A T C C
CCTA G^G

Fig. 2.1

One cause of the genetic disease severe combined immunodeficiency (SCID) is a mutation in the ADA gene. This mutation results in a deficiency of the enzyme adenosine deaminase (ADA).

Although ADA is found throughout the body, it is especially active in lymphocytes. The absence of functional ADA causes the build-up of toxic metabolites that kill lymphocytes and damage organs.

Babies are often diagnosed with SCID by six months old. Treatment can greatly improve the life expectancy of children with SCID.

Some treatment options are available.
- Enzyme replacement therapy with recombinant human ADA made by genetically modified (GM) Escherichia coli. Weekly intra-muscular injections are given.
- Bone marrow transplant if a well-matched donor, such as a close relative, can be found.
- Gene therapy.

The $\beta$-globin gene codes for the $\beta$-globin polypeptide of haemoglobin. It has two alleles, $\mathbf{H b}^{\mathbf{A}}$ (normal) and $\mathbf{H b}^{\mathbf{S}}$ (sickle cell). The sickle cell allele differs from the normal allele due to a base substitution mutation and this mutation results in a single amino acid change to the $\beta$-globin polypeptide.

There are three possible genotypes and phenotypes.
- $\quad \mathbf{H b}^{\mathbf{S}} \mathbf{H b}^{\mathbf{S}}$, sickle cell anaemia, a severe disease
- $\quad \mathbf{H b}{ }^{\mathbf{A}} \mathbf{H b}^{\mathbf{S}}$, sickle cell trait with mild or no symptoms of sickle cell anaemia
- $\quad \mathbf{H b}{ }^{\mathbf{A}} \mathbf{H b}^{\mathbf{A}}$, normal (healthy)

A man and woman who both have sickle cell trait may choose to have children by IVF. This allows the genotype of embryos to be determined by gene testing before the embryos are implanted. Embryos with the normal genotype can then be selected and implanted into the mother.

One technique that can be used in gene testing an embryo for the $\mathbf{H b}^{\mathbf{S}}$ allele is restriction fragment length polymorphism (RFLP) analysis. This involves digesting a DNA sample from an embryo with a restriction endonuclease and then separating the DNA fragments by gel electrophoresis. The position of the DNA fragments on the gel can show if the embryo has the $\mathbf{H b}^{\mathbf{S}}$ allele.

Mammals such as sheep, Ovis aries, and goats, Capra hircus, are important agricultural animals that are sometimes kept together in mixed flocks. Very occasionally, live offspring are born from a mating between a male sheep and a female goat.

In sheep 2 n=54 and in goats 2 n=60.

Sickle cell anaemia is a non-infectious chronic disease. If not treated, sickle cell anaemia can be painful and life-threatening.

Sickle cell anaemia is caused by a base substitution mutation in the gene coding for the $\beta$-globin polypeptide of haemoglobin. This leads to a change in the primary structure of the polypeptide, as valine is present instead of glutamine. This results in abnormal sickle-shaped red blood cells, which stick together in blood vessels.

Symptoms of sickle cell anaemia include painful attacks when red blood cells block capillaries in tissues and organs.

Lung epithelial cells have a thin layer of watery mucus on their surface.
The normal allele of the CFTR gene codes for a transport protein that transports chloride ions out of epithelial cells.

Fig. 4.1 is a diagram of part of the cell surface membrane and the mucus layer of an epithelial cell with normal CFTR proteins.

Cystic fibrosis (CF) is a genetic disorder caused by having two recessive alleles of CFTR. In severe cases of CF, the transport proteins are not added to the cell surface membrane. This causes the mucus layer to be thick and sticky.