Lecture Cycle 2

• Why is genetic complementation so important?

  proving that is the gene that caused the mutation and presentation of phenotype
• What would you do with the identified mutated chlamys?

  determine the sequence of the surrounding DNA, then insert non mutated DNA into mutant, if flagella is present once again, we know we found the gene that causes the no flagella mutant
• What do each of the green dots on the agar plate represent?

  each dot represents a colony from a single mutated cell, each dot is a different mutant
• How do we generate a mutagenized population?

  insert mutagen and then electrocute the cell so that the nuclear membrane opens so that the DNA can integrate
• What are the 4 steps of reverse genetics? What happens at each stage?

  Step 1: generate mutagenized population of cells (insert mutagen, electrocute cells to open membrane, grow cells, each colony is growth from a single mutated cell), step 2: screen the mutant population(screen for particular phenotype on a well plate with a row of WT and a row of previously characterised genes), step 3: identify mutated gene (determine the sequence of the neighbouring DNA), step 4: rescue the mutagen (take non mutated DNA, insert into mutant, rescue mutant phenotype)
• What are two foundational ideas of Insertional Mutagenesis (forward genetics)?

  where mutagen goes in random, 1 mutation per cell
• What is the basic idea of Insertional Mutagenesis? Why do we do it?

  insert foreign DNA into genome, allowing us to identify mutated genes
• What is reverse genetics? What is an example?

  intentionally mutate/knock out the gene of interest to analyse the phenotype, an example is modifying viral genomes
• What is forward genetics? What is an example?

  start with a mutant phenotype to figure out the genotype, an example is cystic fibrosis
• What is post-translational regulation?

  apoprotein + cofactor
• How do genes affect biosynthetic pathways?

  create the enzymes that catalyse the pathway
• How do proteins affect mRNA stability, subsequently gene expression?

  proteins can either increase or decrease stability after transcription, effects gene expression respectively
• How do transcription factors affect expression and transcript abundance?

  transcription factors enhance or decrease gene expression, increase or decrease transcript abundance respectively
• What is the role of the promoter? What is the transcription unit?


  promoter: region of gene that doesn't get transcribed, regulates transcription, proteins bind to it, transcription unit: part of the gene that gets transcribed
• What are three types of regulation?

  transcription, RNA stability regulation, post-translational regulation
• How would you determine if two sequences are evolutionarily related or homologous?

  the DNA sequences would have significant nucleotide sequence similarity
• Complementary base-pairing is due to "hydrogen bonding", what is this type of bond?

  noncovalent bond formed by unequal sharing between hydrogen atoms and oxygen, sulfur and nitrogen
• Concept of "complementary base pairing" occurs between the single stranded DNA probe and the RNA molecules (the GAL transcript) stuck on the membrane".


  The probe has been designed to have a sequence complementary to the target RNA, the probe then binds to the RNA molecule, allowing for detection
• Why is it important the probe is labelled and single stranded?

  probe must be labelled with fluorescent dye or radioactive atom so the RNA molecule to be detected, it must be single stranded so that it can bind to the target RNA
• What are the basic steps of RNA blot analysis or Northern Blotting?

  RNA isolation, electrophoresis separates the molecules, the separated contents are blotted onto membrane, membrane is treated with probe that will complementary base pair(hybridise) with the target RNA, detection
• What is the purpose of an RNA blot analysis?

  to detect specific RNA molecules amongst a mixture of RNA
• Why might gene expression change in a transcriptome profile during a growth curve?

  rate of transcription of genes varies along the curve
• Why do proteomics and metabolomics provide more meaningful data?

  can screen out key proteins and metabolic pathways
• What are genomics, transcriptomics, proteomics, metabolomics? What is the usefulness of each (in broad terms)?

  genomics: DNA, do they carry?

  transcriptomics: mRNA, is gene expressed?

  proteomics: is protein made corresponding to 'cancer' gene?metabolomics: is protein changing metabolic products, growth factors hormones
• What are omics?

  characterization of groups of molecules (provides a better understanding of biological processes)
• What are house-keeping genes?

  genes that are transcribed in all cells of the body that encode products that are required for cells maintenance and metabolism
• What is constitutive gene/protein expression? What is induced gene/protein expression? What is repressed gene/protein expression?

  constitutive: doesn't change, induced: induced by stress, repressed: gene expression is lower than some wildtypes
• What are the key takeaways from a transcript abundance and protein abundance blots?

  how the expression changes, what causes the change, and the type of expression
• What is heat shock? What does HSP do?

  Heat shock is a cell's protective mechanism that becomes activated when an organism is exposed to high temperatures. HSP helps other proteins stay functional at high temperatures
• What distinguishes intact versus degraded RNA? How would you understand a gel picture?

  intact has bright band of rRNA shown, degraded has no visible bands
• Why is it a good thing that mRNA breaks down?

  temporal regulation of gene expression (quality and quantity)
• What makes RNA more reactive?

  the reactive oxygen in the hydroxyl group that wants to bond with phosphate
• What are the two reasons that RNA is less stable than DNA?

  more reactive, ribonuclease chew up mRNA
• What are the key structural differences between DNA and RNA?

  DNA: double stranded, deoxyribose, thymine RNA:single stranded, ribose, uracil
• What determines the amount of a particular mRNA or a protein that is in a cell?

  rate of transcription/translation, and rate of breakdown i.e. transcript/protein abundance
• What is the pathway required for gene expression?

  -transcription, transcript abundance, (mRNA breakdown), translation, protein abundance, protein breakdown
• What is a ribozyme? What are two examples of a ribozyme?

  a ribozyme is a class of RNA molecules that are catalytic, the two types are ribosomes and spliceosome
• Why is RNA the first to evolve?

  RNA can drive chemical reactions, it also carries genetic information
• How does hydrogen bonding work in DNA vs RNA?

  DNA(hydrogen bonds are used to form complimentary base pairs) RNA(can hydrogen bond to itself, i.e. bonding)
• How abundant are the different forms of mRNA, tRNA, and rRNA?

  mRNA:5% tRNA:10% rRNA:85%