| Ames test | used to test mutagenic compounds. Uses sensitive bacteria to show mutations. Uses salmonella typhimurium as it cannot synthesise amino acid histidine is very susceptible to mutations and more permeable than wild type bacteria. |
| Asymmetric transcription | both DNA strands can be used as RNA templates the transcription direction is opposite to one another |
| RNA polymerase | dependant on DNA for template, multi-subunit protein of approx. 480KD. There are 3 types found in eukaryotes. Binds to promotor. |
| Promotors | directly bind to RNA polymerase. Are upstream of main part of gene and not transcribed. Have conserved sequences for example the TATA box as well as regulatory sequences that are recognised by transcription factors. |
| Transcription factors | Bind and recruit RNA polymerase. E.g. SPT, TFIID,TBP, regulate gene expression. Enhance, facilitate and stabilise and help create the transcription bubble. |
| Promoter clearance | after initiation RNA polymerase must clear the promoter. |
| Abortive initiation | the tendency for RNA polymerase to release RNA transcript and produce truncated transcripts. |
| The Ó factor | a protein needed for transcription in prokaryotes. Causes conformational changes to occur in core enzyme allowing for elongation. |
| Transcription | creation of RNA strand form template DNA. has 3 phases. Initiation Elongation And termination. |
| Initiation | promotor region is recognised by transcription factors and they bind to it to start transcription. |
| Elongation | RNA polymerase unwinds DNA through helicase activity the RNA polymerase incorporates nucleotides though complimentary base paring. RNA is synthesised as complex moves along the template DNA strand. The sequenced DNA re-joins, and the RNA disassociates. |
| Termination | Specific sequences in DNA signal the termination of transcription. They are encoded by polymerase. The RNA transcript is released from the DNA. |
| Termination In eukaryotes | synthesised mRNA must be processed before is exported to cytoplasm. 3 steps. 1- A-7 methylguanine (MG) cap added to 5 -end. 2- A poly-tail is added to 3’ end. 3- intron sequences removed. |
| Exons- the coding sequences in mature mRNA. | the coding sequences in mature mRNA. |
| Introns | non- coding regions of DNA. Are removed in intron splicing. Spliced out using RNA protein hybrids called spliceosomes. The intron segments are removed and remaining sequences are reattached. Remaining DNA will be exon only. |
| Translation | occurs in cytoplasm on ribosomes. 2 ribosomal subunits small subunit binds to mRNA and large to tRNA also has enzyme to form peptide bonds. 3 steps. Initiation- AUG start codon recognised my methionyl-tRNA. 2- Elongation, ribosome incorporates amino acids into polypeptide chains, RNA decoded by tRNA and transcribes specific amino acids into chain. Transfer RNA tRNA is used as adaptor between an amino acid and mRNA codon. Translates tRNA sequences to codons that signify amino acids. tRNA binds to specific amino acids by aminoacyl-tRNA synthase. 3- Termination, ends with stop codon, UAA, UAG or UGA. |
| Point mutations | substitution of a single base with another base nucleotide. Insertions or deletions. Cause frame shifts. |
| Silent mutations | base substitution that does not change amino acid encoded. |
| Missense mutation | base changes amino acid, can be harmless but some can be harmful like recessive lethal allele. |
| Nonsense | base substation that changes amino acids into stop codons. Catastrophic. Shortens proteins |
| Sense mutations | base change convers stops into sense codons lengthens proteins. |
| Trinucleotides repeat diseases | genetic diseases characterised by presence of unstable and abnormal expressions of DNA in triplets, can inactivate a gene or result in a toxic protein. |
| Loss of function mutations | impedes function. Most are recessive. Ones in trans or cis sites are always expressed. If found in lac 1 gene or in operator gene e can result in constitutive expression of lac Z.Y and A genes regrades of presence of lactose. |
| Null mutations | completly abrogates function. |
| Chemical mutagens | are base analogues and are incorporated into DNA. cause mispairing during DNA replication leading to mutations. Or can be base modifiers are alkylating agents transferring alkyl groups to nucleotide bases E.g. mustard gas. Intercalating agents fits in between bases caused DNA to unwind to fit can make good nucleotide acid stains. |
| Post replication repair | DNA replication may skip over a lesion such as a thymine dimer. Through recombination the correct sequence can be inserted into the gap. |
| Base exclusion repair | corrects DNA that is damaged. DNA glycosylase recognises broken base and binds between bases and sugar. Cuts sugar with missing base recognised by AP endonuclease that makes the cut. The gap is filled by DNA polymerase and signal ligase. |
| Nucleotide exclusion repair | repair bulky lesions that alter the double helix. Including UV induced thymine dimers. Usually a specific number of nucleotides are clipped on either side of lesion. |
| Double stranded break repair | uses homologues recombination. Uses sequence complimentary on sister chromatin. Double stranded breaks caused by ionising radiation. |
| Ames test | used to test mutagenic compounds. Uses sensitive bacteria to show mutations. Uses salmonella typhimurium as it cannot synthesise amino acid histidine is very susceptible to mutations and more permeable than wild type bacteria. |
| Constitutive- genes that are always on, usually housekeeping genes. | genes that are always on, usually housekeeping genes. |
| Inducible | only turned on as needed. Structural genes and enzymes. |
| Operons | are sections of DNA that contain a cluster of genes under control of a single promoter. They contain the sequence that control transcription (in eukaryotes). |
| Polycistronic mRNA | one mRNA mol translated into separate proteins |
| Lac operon | proof of principal in E.coli. negative control of inducible gene expression a repressor operator interaction. Positive control uses CAP that depends of the sugar glucose. RNA binding not efficient under lack control without CAP. Binds to promoter, binding of CAP requires CAMP. If glucose if low CAMP is high. Glucose inhibits adenyl cyclase decreasing CAMP. |
| Lactose | RNA polymerase is inhibited in the presence of lactose. Sugar binds to repressor causing a conformational change to the molecule. Repressor operator interaction readies structural gene to be translated. In absence of lactose the repressor gene encodes a molecule that blocks transcription of structural genes. |
| Lac Z gene | encodes the enzyme b-galactosidase that cleaves the disaccharide lactose into a monosaccharide |
| Lac y gene | encodes for the enzyme permease which allows lactose to enter bacterial cells. |
| Lac A | removal of waste by-products from the digestion of lactose. |
| Self-limiting repressible operon | tryptophan (trp) operon. E.coli. can produce enzymes needed for biosynthesis of amino acids. 5 genes code for trp operon. In its presence all enzymes are repressed, the repression molecule cannot bind to operator but binds to trp. Is a co-repressor and has negative regulation of its own production. |
| Attenuation | leader sequence contains attenuator sequence which can cause transcription of translation to stop and an attenuated mRNA sequence is formed. |
| Riboswitches | leader sequence contains binding site which if bound by ligand can cause transcription to terminate. Are regulatory segments of leader sequence mRNA that binds to small molecules. Responds to intracellular concentration of specific small molecules. Can bind to target molecules by regulating gene expression . |
| Indictable operons | controlled by a regulator protein with both negative and positive controls. The ara operon metabolises the sugar arabinose by the enzymatic products of ara B,A and D. the operons is regulated by regulator promotors enclosed by ara C that interacts with aral and ara o2. Like the lac operon induction includes CAP and CAMP. |
| Chromatin remodelling | opens up regions of chromatin for association with transcription machinery. Uses chromatin remodelling complexes. Complexes use ATP to loosen attachment between histones. Loose DNA strand detach and disrupt nucleosome core. |
| Histone remodelling | H2A is core histone along with H2B, H3 and H4. H2A variant that exchanges with conventional H2A core protein. Is important for gene activation and silence. Histone modification can include addition of acetyl, methylene or phosphate groups. Or acetylation with allows proteins to interact with exposed DNA that allows transcription to occur. |
| Tumour development | hyperplasia- benign and does not spread. Dysplasia- abnormal cell changes in size, shape, and organisation. Often early step in cancer. Malignant- rapid growth invades tissue. metastasises, cell differentiation usually poor. |
| Chromosomal change and cancer | changes in number and structure of chromosomes can lead to cancer. E.g. chronic myeloid leukemia is a translocation between chromosome 9 and 22. The bcr is on the breakpoint of chromosome 22 translocation of this chromosome fuses genes together, the oncogenic fusion gene activates signals for cell growth. |
| Proto-oncogenes | genes that increase the rate of cell division. Are usually switched on when an increase in cell division is recognised. |
| Oncogene | non normal version of proto-oncogene. The mutant version. Has gained function permanently switching on gene. |
| Tumour suppressor genes | genes that decrease the rate of cell division. |
| The RAS gene | a proto-oncogene, normal function is to switch an cell division gene cycles between an active and inactive state. |
| Restriction enzymes | exist naturally in bacteria to attack and chop viruses into smaller pieces. Recognise specific sequences to chop. Are specific bacteria endonucleases that evolved as a defence mechanism. |
| Vector | a DNA molecule that can be used artificially to incorporate and replicate the target sequence of interest into another cell. Must be capable of replicating themselves. |
