MOLECULAR GENETICS

RNA - a nucleic acid like DNA with 4 key differences

• ribose sugar instead of a deoxyribose sugar
• base URACIL instead of THYMINE
• single stranded (can still fold into a 3D structure)
• shorter than DNA

MESSENGER RNA (mRNA)

• carries the 'message' that codes for a paticular protein
• moves from the nucleus to the cytoplasm where the proteins are assembled
• single stranded, short lifetime and just long enough to contain 1 gene

TRANSFER RNA (tRNA)

• an 'adapter' that matches amino acids to their codon
• folds by complementary base pairing to form a clover leaf structure
• at the end of the sequence there is always the triplet code ACC where the amino acid binds
• on the middle loop there is a triplet code called an anticodon

RIBOSOMAL RNA (rRNA)

• rRNA + proteins forms ribosomes which are the site of mRNA translation and protein synthesis
• formed of 2 subunits - large and small
• assembled in the nucleolus and then exported to the cytoplasm
• rRNA is coded for by numerous genes
• ribosomes free in the cytoplasm make proteins for use in the cell, those attatched to the ER make proteins for export

• the sequence of bases on DNA codes for the sequence of amino acids in a protein
• sequence is read in groups of three called codons
• each codon codes for 1 amino acid
• the meaning of each of the 64 codons is called the genetic code

features of the genetic code:

• degenerate i.e. more than 1 codon for an amino acid
• degeneracy is often on the third base of each codon so it is less important
• one codon means start (AUG) it codes for methionine, thus every protein starts with this
• three codons mean stop, they do not code for amino acids
• the code is universal - it's the same in all organisms
• the code is non overlapping

• DNA never leaves the nucleus but proteins are synthesised in the cytoplasm
• a copy of each gene is made to carry the 'message' from the nucleus to the cytoplasm - mRNA
• the process of this copying is called TRANSCRIPTION

the process:

• the start of each gene is marked by a sequence of bases called a promoter
• DNA helicase acts at the region, seperating the two strands and exposing the nucleotide bases
• the RNA molecule is built up from nucleotides (A, U, C, G), these nucleotides attatch themselves via complementary base pairing to the exposed DNA strand (just like in DNA replication)
• the DNA strand that is being copied is called the template strand
• the new nucluotides are joined onto the DNA by RNA polymerase
• only a few bases remain attatched at any one time as the mRNA peels of from the DNA as its made
• at the end of the gene transcription stops, so the mRNA is only the length of the gene
• the mRNA diffuses out of the nucleus, through nuclear pores, to the cytoplasm
• here it attaches to ribosomes for translation

• eukayotic DNA contains non-coding sequences as well as coding sequences
• the non-coding sequences are called introns, the coding sequences are called exons
• introns need to be removed before translation
• this removal is done by SPLICING

process of splicing:

• initial mRNA that is transcribed is called pre-mRNA - it's an exact copy of the gene on DNA so contains introns and exons
• the introns in the pre-mRNA are cut out and the exons are joined together by enzymes in a process called splicing
• the result is a shorter mRNA strand containing only exons to which complementary tRNA can attatch
• the introns are broken down

prokaryotic DNA does not have introns, so splicing is not needed

• 1) a RIBOSOME attatches to mRNA at a start codon (AUG), the ribosome encloses two codons
• 2) the first tRNA molecule with an an amino acid attatched diffuses to the ribosome, its ANTI-CODON attatches to the first mRNA codon by complementary base pairing
• 3) next amino acid-tRNA attatched to the adjacent mRNA codon by complentary base pairing
• 4) the bond between the tRNA is cut and PEPTIDE BOND forms between the two amino acids
• 5) the ribosome moves along one codon so a new tRNA can attatch, the free tRNA leaves to collect another amino acid
• 6) the cycle repeats from step 3
• 7) the polypeptide chain lengthens 1 amino acid at a time and peels away from the ribosome, folding into a protein as it goes
• 8) the process continues until a stop codon is reached, when the ribosome falls apart and releases the finished protein

a single piece of mRNA can be translated by many ribosomes simultaneously, so many protein can be formed from 1 mRNA molecule  

• random changes to genes that are passed onto daughter cells
• happens during DNA replication

three kinds of mutations:

SUBSTITUTION

• MIS-SENSE - affects only one amino acid so tend to have less severe effects
• SILENT - if the substitution is on the third base of the codon it may have no effect at all, because the third base often doesnt effect the amino acid coded for e.g. all codons beginning CC code for proline
• NONSENSE - when a mutation leads to a premature stop codon, this leads to the protein being incomplete and certainly non functional

DELETION AND INSERTION

• have more serious effects because they cause a 'frame shift' which changes the codon reading frame

• mutations are normally very rare which is why memebers of the same species look alike
• however, the rate of mutations is increased by chemicals or radiation

these are called mutagenic agents or mutagens and include:

• high energy, ionising radiation e.g. x rays, UV rays, alpha rays and beta rays (these distrupt the DNA molecule so it doesnt form the correct base pairs)
• chemicals such as benzene, nitrous acid and tar in cigarette smoke (these react with DNA bases and interfere with transcription)
• some viruses also change the base sequence

mutations have positive and negative effects:

• they lead to increased genetic diversity, esential for speciation and selection
• however, they mean the organism is less suited to it's enviroment and they are often carcinogens since a common result of mutation is cancer

• mutations in some genes cause cancer - two of these genes are called proto-ongogenes and tumour supressor genes

PROTO-ONGOGENES -

• encode proteins that act as growth factors and therefore stimulate cell division by allowing the cell the participate in the cell cycle
• normally the PO are only expressed when needed for growth, a mutation causes them them to be expressed all the time - causes uncontrolled cell division and hence cancer
• the mutated gene is called a oncogene

TUMOUR-SUPRESSOR GENES -

• encode proteins that inhibit cell division by blocking the cell cycle
• some tumour supressor genes also initiate cell death
• thus, tumour-suppressor genes override the effect of oncogenes and so are known as anti-oncogenes
• a cancer occurs if a proto-oncogene and a tumour supressor gene mutate
• a tumour is a mass of identical cells formed by uncontrolled cell division

• a complementary growth factor attatches to a receptor protein on the cell surface membrane
• this releases relay proteins into the cytoplasm
• these relay proteins 'switch on' genes

proto-oncogenes work by either:

• coding for a growth factor which is then produced in excessive amounts or,
• the receptor protein is permenantly activated so the gene is 'switched on' even in the absense of the growth factor

• most body cells only express a few of their genes
• they do this in 2 ways:
• 1) preventing transcription and hence the production of mRNA for that gene
• 2) breaking down mRNA before its genetic code can be translated
• cells that do this are said to be SPECIALISED
• in animals specialised cells are irreversibly differentiated, so they and their daughter cells keep their specialisation and cannot become any other kind of cell
• there are few cells that remain undifferentiated and so become any type of cell
• these are called STEM CELLS
• stem cells have two main properties:
• 1) potency - potential to differentiate into specialised cell types
• 2) immortality - they can divide indefinitely
• potency can be TOTIPOTENT (where they can differentiate into any type of cell type and can even develop into a complete organism - they are found in embryos up to the 32 week stage), PLURIPOTENT (these can differentiate into nearly all cells and are found in 5 day old embryos, these are called embryo stem cells) or MULTIPOTENT (where they can differentiate into a related family of cells e.g. blood cells, muscle cells, these are called adult stem cells)

• the idea is to transplant tissue grown from stem cells into a patient where it would grow and replace damadged tissue
• some examples are: beta cells to treat diabetes, nerve cells to treat multiple sclerosis, parkinsons disease and strokes, muscle cells to treat muscular distrophy and cartilage cells to treat osteoarthritis
• tissues could be grown from the patients OWN cells, so there is no risk of rejection unlike traditional transplants
• stem cells from bone marrow are already being successfully used to treat leukaemia
• where do stem cells come from?
• EMBRYO STEM CELLS - human embryos created by IVF are made to help infertile couples reproduce, any embyro's not used can be used to create stem cells (with the consent of the donor couple) these are PLURIPOTENT so can create a wide range of different cells for clinical use, however, there are ethical concerns with creating stem cells in this way
• ADULT STEM CELLS - extracted from certain tissues in the body, most organs and tissues contain a small amount of undifferentiated cells which the body uses for repair, the use of these cells have no ethical concerns or problems with rejection but they are difficult to find and are only MULTIPOTENT

• in plants some cells remain totipotent throughout life, these cells have the ability to develop into whole plants
• totipotent plant cells are used natrually and artifically:
• ASEXUAL REPRODUCTION - natrual, bud grows from a non reproductive part of the plant e.g. stem, this develops into a completly new plant which eventually becomes detatched from the parent plant, since this relies on MITOSIS the offspring are all clones
• CUTTINGS - parts of the plant stem/leaves are cut off and replanted in wet soil, each cutting produces roots and grows into a new plant, this allows the orginal plant to be cloned numerous times, the division and differentiation are helped if a 'rooting hormone' is added e.g. IAA
• TISSUE CULTURES - modern and efficient way of cloning plants, small samples of plant tissue are grown on an agar plate, any plant tissue can be used for this, the tissue can be seperated into single cells which can grow into a mass of undifferentiated cells, if the correct plant growth regulators (PGRs) are added these cells can develop into whole plants and planted outside, important the conditions are sterile to prevent contamination, this method allows for thousands of clones to be produced quickly
• could be combined with genetic engineering to grow GMO from a single genetically-modified cell

• genes are expressed through transcription and tranlation into proteins which give cells their functions and properties
• cells dont express all their genes all the time
• gene expression can be switched on or off by: other genes, stimuli and hormones

there are 5 control points along the gene expression pathway:

• 1) transcription factors control which genes are transcribed
• 2) different splicing means different mRNA's are made
• 3) mRNA molecules can be destroyed quickly or slowly by enzymes
• 4) ribosome proteins control how much mRNA is transcribed
• 5) the activity of a protein can be altered by other enzymes

• for transcription to begin the gene need to be stimulated by specific molecules that move from the cytoplasm to the nucleus - TRANSCRIPTIONAL FACTORS (TF)
• each TF has a site that binds to a specific region on DNA in the nucleus
• when it binds it stimulates this region of DNA to begin the process of transcription
• mRNA is produced and the genetic code it carries is then translated into a polypeptide
• when a gene is not being expressed i.e. its switched of the site on the TF that binds to DNA is blocked by an inhibitor
• the inhibitor molecule prevents the TF binding to DNA and thus prevents transcription and polypeptide synthesis

hormones like oestrogen can switch on a gene by combining with the TF and causing it to release the inhibitor molecule:

• oestrogen is lipid soluble and so can diffuse across the cell surface membrane
• once in the cytoplasm oestrogen binds with the receptor site on the TF (complementary)
• this cause the shape of the TF to change and release the inhibitor molecule
• the transcriptional factor can now enter the nucleus through the nuclear pore and combine with DNA - this stimulates transcription as described above

• gene expression can be prevented by breaking down mRNA before its genetic code can be transated into a polypeptide
• this process uses small sections of RNA called small interfering RNA (siRNA)
• the process is as follows:
• an enzyme cuts large molecules of RNA into siRNA
• one of the two siRNA strands combines with an enzyme
• the siRNA guides the enzyme to the mRNA by pairing its bases with the complentary ones on mRNA
• once in position the enzyme cuts the mRNA into smaller sections
• the mRNA is no longer capable of being translated into a polypeptide
• this means the gene has NOT been expressed, its been blocked

siRNA has a number of potential scientific and medical uses:

• can be used to identify the role of a gene, some siRNA that blocks a paticular gene can be added to a cell, by observing the effects (or lack of them) we could determine what the role of the blocked gene is
• some diseases are caused by genes, we could use siRNA to block these genes