Biology 5: Section 2

There are four bases: Cytosine, Adenine, Thymine & Guanine but 20 different amino acids

Genetic code is described as:

• Universal - in all organisms the same codons code for the same amino acids
• Non-Overlapping - succesive codons are read in order, each nucleotide is part of only one triplet codon
• Degenerate Code - there are more codons than necessary to code for 20 amino acids, some amino acids are coded for by several different codons

mRNA molecules are effectively mobile copies of genes. They carry the code out of the nucleus to the site of translation - out of ribosomes in the cytoplasm

• mRNA is a single, long strand of nucleotides that is a copy of a gene
• tRNA is a small, cloverleaf shaped molecule that brings particular amino acids to the ribosome during translation

Most cells in the human body contain two complete sets of genes. However only a few genes are used. The steps of transcription:

• Step 1 - the two strands of DNA unwind along the length of the gene. This is catalysed by enzymes
• Step 2 - the enzyme RNA polymerase moves along one side of the DNA molecule - the sense strand that contains the genetic code. The enzyme catalyses the assembly of an mRNA molecule by the addition of matching nucleotides. When RNA is synthesised, the base thymine is replaced by uracil. So base pairing is A-U & C-G
• Step 3 - the mRNA molecule peels off the gene and passes out of the nucleus

Introns & Exons

In eukaryotic cells, the genes contain DNA sequences that are not used to make the final protein. These sequences are called introns and must be spliced (cut) out before translation. The DNA sequences that are going to be expressed are called Exons. Following transcription, a molecule of pre-mRNA exists for a short while, before the introns are spliced out the resulting mature mRNA leaves the nucleus and passes to the ribosomes for translation

Transfer RNA is the molecule that transfers amino acids to ribosomes during translation. It links the genetic code to the protein. At one end of the molecule is the anti-codon which binds to the codon on the mRNA. At the other end is the amino acid specified by the codon. A ribosome can be thought of as a giant enzyme that holds together all the components needed for translation

• Step 1 - the mRNA attatches itself to a ribosome
• Step 2 - the first codon is translated. The first codon is usually AUG, which codes for the amino acid methionine, so a tRNA molecule with the anti codon UAC will attatch, carrying a methionine molecule at the other end
• Step 3 - the second codon is translated in the same way. The second amino acid is held alongside the first, and a peptide bond is formed by condensation between them. The polypeptide chain has begun. ATP is split to provide energy to form the peptide bond
• Step 4 - the process is repeated - the mRNA moves along the ribosome until the polypeptide has been built. If a stop codon is encountered, translation ceases and the polypeptide is finished

Once the polypeptide has been assembled it folds and bends into its tertiary structure and accumulates on the inside of the RER. There it is packaged into vesicles and may pass into the Golgi apparatus where it is modified and/or activated.

• Subsitution
• Addition - causes frame shift
• Delection - causes frame shift

Can be lethal

• not perform function
• change shape of active site
• result in metabolic block
• addition & deletion more likely to be lethal

May have no effect

• may not alter the tertiary structure/shape

May be beneficial

• may confer a selective advantage, more likely to survive & reproduce

• Nomally occur by chance, random mistakes in the copying of DNA
• Rate of mutation can be greatly increased by mutagens
• Include: X-rays - Alpha and Beta radiation
• In cystic fibrosis mucus becomes thick & sticky, so the lungs are easily infected
• In haemophilia fault in blood clotting mechanism leads to major blood loss
• Phenylketonuria - phenylaline build up, interferes with brain development

Benign Tumours

• Enclosed in a capsule and grow in the centre, so they do not invade surrounding tissues
• They are not cancerous and are often easily removed by surgery

Malignent Tumours

• These tumours grow at the edges, invading surrounding tissues & organs
• They are cancerous and much more difficult to treat
• Often difficult to tell where the boudaries are, making surgery difficult
• Cells can break off and set up secondary tumours elsewhere in the body

Genetic Factors

• When protooncegenes mutate into oncegenes the cell loses its ability to control clee division
• Tumour supressor genes prevent cells from dividing too quickly
• Giving time for immune system to destroy rogue cells or for damaged DNA to be repaired
• If the tumour supressor genes mutate, safety mechanisms are lost, so development of cancer more likely

Cancer is more common in older people as their somatic (body) cells accumulate mutations. Sometimes these mutations occur in gametes, passed into offspring, people who inherit these have a genetic predisposition to cencer

Environmental Factors

• Smoking
• Diet
• Radiation
• Chemical Carcinogens - asbestos
• Microorganisms - viruses

Stem Cells

• 1. Self Renewal - the ability to go through mitosis remaining undifferentiated
• 2. Potency - capacity to differentiate into specialised cell types
• Totipotent - can differentiate into 216 different cell types - found in embryo
• Pluripotent - can differentiate into wide variety of cells - in adult tissues
• Multipotent - can only differentiate into closely related cell - majority of stem cells

Micropropagation - clones large numbers in short time

• small piece of plant -explant- grown in culture medium with hormones
• can be divided again and again, complete plants grown from explant
• have to be 'hardened' to survive in greenhouses and outside

Advantages:

• rapidly produces high numbers, takes little space, produce specialised plants (orchids)

Disadvantages

• doesn't work for all plants, infection passed on, labour intensive

• Making new skin for burns victims, or people with skin ulcers
• Making pancreatic cells for diabetes
• Repairing spinal cord injuries and other nerve damage
• Repairing muscle damage e.g. after heart attack
• Making certain brain cells for sufferers of Alzheimers & Parkinsons disease

The Regulation of Transcription & Translation

• Transcription factors, selective activation of genes
• RNA polymerase and proteins bind to area adjacent to gene (Promotor Region)
• Assemble into a transcription initiation complex (TIC)
• Transcription factors activated by signal proteins e.g. hormones, growth factors
• Can be prevented by repressor molecules which attach to promotor region

Role of Oestrogen in Gene Transmission

• Oestrogen passes through cell membrane & cytoplasm into nucleus
• Binds with and activates nuclear oestrogen receptors (ERs)
• Regulate gene expression & stimulate cell to make specific proteins

siRNA

• Can act as repressor molecules
• 20-25 nucleotides long and double stranded
• Can be made artificially and introduced into cells to bring about the specific knockdown of a particular gene

Restriction Enzymes & Ligases

• Restriction endonucleases protect against viral attack cutting viral DNA
• A particular restriction enzyme will cut DNA at a specific recognition site
• Typically between 4&8 nucleotides long and many palindromic sequences
• Sequence on one strand needs the same as the corresponding sequence on the complimetary strand when read in the reverse direction
• The cuts are staggered, strands longer than others, known as sticky ends
• DNA ligase joins pieces of DNA cut at restriction sites, have complementary sticky ends

• Isolate the gene and cut it out using a restriction enzyme
• Isolate the plasmid from a bacterial cell and cut it with the same type of restriction enzyme, producing complementary sticky ends
• Splice the human DNA into the plasmid using DNA ligase to join the sticky ends
• Treat the bacterium so that it takes up the recombinant plasmid; once successful, the bacterium will multiply so that either the human gene or product of the gene can be used

This process is very unreliable, need to add genetic marker to see which has accepted gene. In replica plating, bacteria are grown in medium that contains antibiotic, only those bacteria that took up the plasmid with the new gene and the gene for antibiotic resistance will survive and grow

Advantages

• Gene can be expressed (used to make protein)
• Proofreading enzymes correct copying mistakes

Disadvantages

• Relatively slow
• More complex purification

Required is: DNA sample, nucleotides, DNA polymerase & primers

• Heat sample of DNA to 95 degrees so that strands seperate
• Add primers and cool to 40 degrees so that they bind to DNA
• Increase temperature to 70 degrees; DNA polymerase copies each strand, starting at primers, adding complimentary nucleotides

One cycle lasts a few minutes, so you can get millions of copies within an hour

Advantages

• Very quick
• Little purification of final sample required

Disadvantages

• Mistakes copying base sequence common 

Gene Therapy

• Isolate allele that is causing the disease
• Work out base sequence of the healty allele
• Make lots of copies of the healthy allele (clone it)
• Deliver it into the cells/tissues in which defective allele expressed

Genetic Probes

• Used to test individual for a particular gene
• Small piece of DNA, probe complementary to base sequence on gene
• Has a flourescent/radioactive label; if bind, will show presence

Restriction Mapping

• Restriction endonucleases used to cut DNA at restriction sites
• Restriction map shows length of fragments cut
• Shorter the fragment, the further the fragment moves

Ingredients: strand of DNA, primers, DNA polymerase, normal & special nucleotides

Process: 4 test tubes, labelled nucleotides, primers, DNA polymerase, 1 special nucleotide

• Occurs until enzymes try to add one of the special nucleotides
• It then stops and we can see the sequence of bases
• Newly synthesised and labelled DNA fragments are double stranded
• Denatured by heat and seperated by size by gel electrophoresis
• The smallest fragments will travel the furthest

• Within the non-coding DNA there are hypervariable regions - vary in length
• These regions consist of particluar base sequences which are repeated
• Different people have a different number of repeats

Processing the DNA

• Cut molecule using restriction enzymes
• Electrophoresis - place fragment mixture in wells with gel
• Apply a current and DNA fragments move to postive terminal
• Fragments seperate into bands according to size

Process:

• DNA sample
• DNA extracted & purified
• Enzyme digestion cuts DNA into fragments
• Electrophoresis sorts DNA fragments
• DNA transfer from gel to nylon membrane (southern blotting - capillary action)
• Hybridisation probes bind to certain DNA fragments
• Results: pattern of DNA bands (stained to see hypervariable regions)