GENE223

CAULOBACTER: why study

*Caulobacter cresentus* - cell cycle control. - cellular asymmetry - Polar Morphogenesis (two growth forms, dependent on old/new pole) - applicable to eukaryotes due to differentiation.

ANIMAL DEVELOPMENT: Drosophila Sex Determination

*Cell Fate Example* - Every cell in Drosophila makes its own INEPENDENTLY decision to become male OR female - sex = CELL FATE decision *Gynandromorph* - half male, half female - indicate sex determination is cell autonomous *Aneuploid Drosophila* - XXY = Female - XO = Male - no relation to which sex chromosome is present - Determined by HOW MANY X chromosomes are present (X:A ratio) *X:A Ratio* - Female = X:A ratio of 1 - Male = X:A ratio of 1/2 - determines expression of Sex Lethal (Sxl) - High X:A ratio is more likely to from X chromosome dimers (rather than X chromosome/autosome heterodimers) - leads to the expression of Sxl at the Early promoter. - Early promoter creates an active Sxl protein. *Sxl Expression* - Late Sxl expressed in all cells - No early Sxl: forms truncated Sxl :. inactive - Yes early Sxl: active Sxl alternatively splices Sxl :. removes stop codon exon 3:. more active Sxl *Gender Cascade* - Active Late Sxl = alternatively spliced Transformer - Active Transformer = alternatively spliced double sex. (different DNA binding Domains) - Female double sex = no exon 4 - Male double sex = no exon 5 - Double sex is responsible for male/female specific RNAs *Behaviour* - Courtship determined by Sxl splicing - correct splicing of fruitless induces male courtship behaviour **also, rather than X inactivation in females, Male Drosophila double the transcription of X

CAULOBACTER: LIFE CYCLE

*1) G1 phase* *SWARMER* CtrA expression (silences Ori) GcrA inhibited - Mobile - requires oligotrophic environment - in search of new location to avoid nutrient competition *1.5) TRANSITION* CtrA proteolysis by ClpP/X protease - Ori liberated. PgcrA produced. - concomitant w/ DnaA expression DnaA expression - promotes GcrA alongside PgcrA *2) S PHASE* *STALK GcrA expression DnaA repression, degraded and inactivated (prep for cell division) - attached to substrate. INCREDIBLY STRONG - elongation of replication - chromosome segregation *2.5) TRANSITION* CtrA transcription activated by GcrA CtrA phosphorylated by CckA histidine kinase CtrA silences Ori CtrA represses GcrA transcription. *3) G2 PHASE* - Differentiation: stalk/swarmer CtrA proteolysis in stalked compartment GcrA proteolysis in swarmer

PLANTS: Mutant Analysis

*1. Is the Phenotype Heritable?* - does the phenotype show up in the next generation? *2. Is the Mutation Dominant/Co-Dom/Recessive* - cross w/ wild type and study offspring phenotype: i) wild type = recessive ii) mutant = dominant iii) mix between mutant + wild type = co-dom *3. One or More Nuclear genes?* - Examine segregation patterns of mutation phenotypes in F2 progeny (3:1 = one gene, 9:3:3:1 = two genes) OR - complementation! *4. Are the genes Functionally related?* - Double mutant analysis (phenotype of plant homozygous for both mutations)

CAULOBACTER: Cellular Asymmetry

*ANTERIOR/POSTERIOR* - PleC: new pole protein - DivJ: old pole protein - ZapA: middle/soon to be new pole *DORSAL/VENTRAL* - Crescentin: Creates Crescent Shape - Chemoreceptors: opposite side to crescentin. orientated towards new pole.

SACCHAROMYCES: Life Cycle

*ASEXUAL* - form colonies of genetically identical haploid yeast - G1: before replication - G0: Not dividing (often mutant) - S: DNA replication - G2: After replication, before division - M: Mitosis/ Division *SEXUAL* - can be induced by EXTRACELLULAR pheromones. STE gene signalling pathway. --- regulated by phosphorylation and protein interactions --- specific interaction of Ste12p (TF) in the nucleus triggers genes for reproduction (STE genes) - haploids/ mating types = alpha + A - form ascus with four hapoloid ascospores - haploid > shmoos > conjugate/fuse > zygote > diploid > bud > daughters

PLANTS: Agrobacterium tumefaciens Transgenics

*Agrobacterium Tumefaciens* - natural tool for plant transformation - intentional mutagenesis - reverse genetics - transgene insertion - usually forms tumour like calluses caused by gene insertion *Normal TDNA Plasmid* LB + RB: cis acting DNA elements Nos: opaline synthase = modifies amino acids in bacteria energy source Shi: shoot inducing = auxin synth Roi: Root inducing = cytokinin synth *Lab Plasmids* TDNA - Deleted auxin + cytokinin genes - Retain vir genes - reduce down to essential elements 2˚ PLASMID - LB+RB (transgene in between) - selectable marker Bacteria - selectable marker Plants (TDNA) - Cloning site for plant gene (TDNA) - Ori: e.coli - Ori: agrobacterium *INFECTION/INTRODUCTION* - Force on leaves: Transient gene expression - Flower Dipping: Stable gene integration. Enters developing flower + female gametophyte. - transfered into plant w/ viral proteins: VirD2 on 5' end - random insertion *METHOD* 1. Propogate and isolate binary vector from E.coli 2. Clone the gene/construct into vector 3. Reintroduce altered vector into E.coli to amplify 4. Isolate vector and insert into Agrobacterium w/ altered Ti Plasmid 5. Introduce agrobacterium (flower dipping = stable, on leaves = transient) 6. select for plant selectable marker in seeds (M1) 7. Self Fertilise M1 generation, pools of 1000 = M2 generation 8. Usually ¼ M2 gen are homozygous for t.gene

DEVELOPMENT: C. Elegans Vulva

*Anchor Cell* - inductive signalling/ paracrine signalling - secretes *lin 3*: determine cell fate in dose dependent manor (increasing dose changes cell fate) *Lin 39* - present in all precursor cells *Let 23* - Lin 3 receptor - all 3˚ without it *Let 60* - when expressed, produces 1˚ and 2˚ cells - gene expression that produces cell fate *Lin12* - juxtacrine signal from 1˚ cell to 2˚ cell - 2˚ cells still form even without Let 23/Lin3 *1˚ Cells*= highest lin 3 dosage *2˚ Cells*= juxtacrine signal of lin12 from 1˚ cells *3˚ Cells*= no lin3

PLANTS: Arabidopsis

*Arabidopsis thaliana* 30500 genes (~91% still have an unknown function) 140Mb of DNA (small and compact genome) 1 gene / 4.6kb - fast growing: 8 weeks from seed to seed - small ~20cm: space efficient, easily grown at high densities - diploid genome (many plants have a higher copy number) - can self fertilise - smal genome - efficient transformation via Agrobacterium tumefaciens - Forward genetics: identified many mutants (over 1500 freely available from stock centre) - reverse genetics: over 100000 insertions at precise locations

BACILLUS: why study

*Bacillus subtilis* - asymmetric cell division - altruism. - cell decision (initiation of sporulation) - intercompartmental communication between spore and parent (gene regulation during sporulation)

MODEL ORGANISMS: C. Elegans

*Caenorhabditis elegans* Phylum: nematoda - Free living (non-parasitic) nematode - easy to grow/culture on agar, will eat bacteria etc - transparent: can see internal organs and structures - EXACTLY 979 somatic cells, 2000 germ cells - mostly Hemaphrodites (XX) some males (XO) - 3.5 day life cycle: fast growing, fast development, get through multiple generations very quickly - 200-300 embryos *PROS* - All cells FATE MAPPED!: know from embryo what each cell lineage will become. including 131 cell suicides (Important in dev e.g. limb development) - Cell Ablation easy: laser targeted destruction of chosen cells. - mapped ALL connections between the 302 neurons and muscle connections - reverse + forward genetics possible (knock in + knock out) - homozygous mutation without crossing - easy to grow, maintain, can be frozen (put the experiment on hold) *CONS* - VERY different to humans. only 43% homologous genes - MOST diverged of the main 6 models + metazoan models - embryos hard to manipulate. not easy to study gene expression in embryos *What have we learnt?* - Genome sequencing - Incredible Survival abilities - apoptosis pathway - longevity research: can live for a LONG time, Found Daf16/FoxO TF linked to shortened life span. - micro-RNAs

BIOTECH: E.g. Antibodies info

*Circulating antibodies* = protect from invading viruses, bacteria and toxins therapeutic = inflammatory diseases, cardiovascular diseases, infectious diseases, cancers *Secretory antibosied* = protects surfaces from pathogens + toxins, prevent entry + colonisation therapeutic = topical applications *Passive antibodies* = in colostrum + milk. passive immunity for neonates and infants *POLYCLONAL* - mixed population of antibodies targeting multiple epitopes on target antigen *MONOCLONAL* - single identity population of antibodies targeting one specific epitope on the target antigen - desired for therapeutic use (limits off target)

MODEL ORGANISMS: Zebrafish

*Danio Rerio* Phylum: vertebrata - embryo develops outside mother - egg transparent - 100-200 embyro *PROS* - genome editing - forward genetics - can be made transgenic - morpholine antisense gene knockdown - large embryos :. easy to manipulate - vertebrates! closer physiology to humans *CONS* - undergone whole genome duplication: more copies of each gene, inactive gene homologues. harder to create knockouts. - no limbs (not tetrapods) - large space required for aquaria - 4 month life cycle LONG *What have we learnt?* - developmental patterning genes - development and disease model (mainly fish) - toxicity screens

DEVELOPEMENT: Slime Moulds

*Dictyostelium discoideum* - social amoeba - 12,500 genes - single cell = myxamoebae - contains 3x cell adhesion molecules - Social aggragate/fruit body triggered by starvation - cells differentiate into different parts of the fruit body. *DIFFERENTIATION* LABILE COMMITMENT - cell mass forms pre stalk and prespore cells - if pre stalk was removed, cells would alter their expression and form new pre stalk cells STABLE COMMITMENT - 5 cell types: upper cup, spore sac (from prespore cells), lower cup, stalk, basal disc - can not change cell type at this point

AMPHIBIAN MODEL: Spemann's Organiser

*Discovery* - transplant of dorsal tissue into ventral side of the embryo - creates a new axis = siamese fish/ double headed fish - dorsal tissue fate map becomes notochord and spinal chord tissues... but must do something else if it caused dev of whole 2nd fish *Formation of the organiser* - Due to overlapping determinant signals. - endoderm signal + ß-catenin signal from nieuwkoop centre - Nieuwkoop centre pushed to dorsal side when sperm enters egg *Key Experiment in discovering function* - Explant from organiser + ventral explant put in close proximity - organiser develops into notochord (normal) - ventral explant develops into muscle tissue (not normal, usually develops into blood) -- shows proximity to organiser alters cell fate... dorsalised the ventral tissue! *Effect on Cells* - four cell types along organiser plane: notochord > muscle > kidney > blood - Mesoderm produced BMP proteins that instruct cells to become blood - Organiser releases diffusible BMP inhibitors - BMP gradient becomes four distinct categories *ALSO * - dorsalise ectoderm tissue = form neural tube (brain/CNS) - initiates movement of gastrulation - induces all three axis of symmetry (A/P, D/V, L/R)

MODEL ORGANISMS: Drosophila

*Drosophila melanogaster* Phylum: arthropoda - genetic model for over 100 years - lifecycle 2 weeks - 80-100 embryos *PROS* - fast breeding - small size - forward + reverse genetics - P-elements/Transposons (more controlled gene insertion possible) - Balancer chromosomes to maintain homozygous lethal mutations in offspring - 61% homologous to humans (w/ 75% of disease genes homologous) - great to study gene expression *CONS* - as they're insects, diverged from humans 600mya - VERY different physiology (circulatory system doesn't deliver oxygen to tissues, no internal skeletal system, totally different immune response) - THEY FLY ABOUT EVERYWHERE - limited micro manipulation *What have we learnt?* - Polytene Chromosomes - Base of genetics as a field of study - Heredity in animals - Hox genes - linkage and mapping in chromosomes - developmental toolkit/ Heidelberg screen

AMPHIBIAN MODEL: Embryo Patterning

*ECTODERM* = animal cap cells - skin, brain etc *MESODERM* = tissues next to endoderm - defined by proximity to endoderm (removal of mesoderm turns animal cap into mesoderm) - blood etc *ENDODERM* = vegetal cells - gut lining, accessory digestive organs, lungs etc

MUTANTS: Heidelberg Screen

*Example Of a Genetic Screen* - Screened for all genes involved in development - Scorable phenotype = larvae cuticle: dentricals (can be made flourescent),pattern development, Visible Segments. Dorsal/Ventral. Anterior/Posterior. Thorax + Abdomen. (swallow on head) *METHOD* - flies treated w/ EMS - saturation screen: continued to mutate until no more mutations could be found - mutagenised 5800 flies - use balancer chromosomes to create homozygous mutants w/ lethal mutations. - Virgin females with Balancer Chromosomes. Males with EMS mutations. - complementation test to asses whether the mutations were different genes *RESULTS* - identified 15 individual genes that affected the cuticle pattern. - All developmental drosophila genes

PLANTS: World Issues

*Food Crisis* - major objective in plant science is to increase food production: food production unable to keep up with increasing population - need to increase production by 70% in the next 40 years to support population - Food shortage and high food prices linked to unrest and conflict - ea year >1billion people are chronically hungry - hunger kills 1000 people every hour - ea year >2billion people are diagnosed as chronically anemic (iron deficiency) *Solution* - previous solutions were using traditional breeding methods (crossing for hybrid vigor etc) but these techniques are unlikely to keep up - GM seems to be very promising method. (but not the only answer) - e.g. reducing loss w/ herbicide tolernance and insect resistance *Goals* - increase nutrition: fortification - increase drought + stress tolerance: can be done by a single gene change - require less fertiliser or water - resistant to pathogens: biggest threat to our main food supplies (potato and wheat)

MODEL ORGANISMS: Chicken

*Gallus gallus* Phylum: vertebrata - 1 embryo *PROS* - amniote: closer to humans than fish or frogs (segmented spine during embryo dev etc) - majority of development outside the mother - cheap + easy to manipulate - Large limb bunds (great for studying limb dev) - retroviral insertion of transgender - gene expression studies easy w/ flat embryo *CONS* - No genetics - Hard to breed in labs - earliest stages not accessible (inside mother) *What have we learnt?* - limb development - somitogenesis (formation of segmented vertebrae body parts which go on to form vertebrae and muscles) - cell migrations in the nervous system (neural crest)

ANIMAL DEVELOPMENT: Mammalian Sex Determination

*General Case* - Usually determined by Y chromosome - presence of Y = male - Y chromosome encodes SRY gene *SRY Gene* - Dominant. - Recombination: SRY onto X chromosome = male - SRY gene = Male gonad - no SRY = Female gonad = Non Cell Autonomous sex determination

MUTANTS: Tübingen Screen

*Genetic Screen* zebrafish *Method* - ENU mutagen (similar to EMS) - Scorable Phenotype: ANY altered development - sperm from mutagenised lines stored for mutant stock. (no need for balancer chromosomes) - sperm can be expressed from fish by rubbing - 4264 mutants identified.

SACCHAROMYCES: Genome Character

*Genome size: 12mb* *Chromosomes: 16* - can study chromosome organisation and segregation *Number of Genes: 6000* - 30% with unknown function. (50% of those are similar to other species tho) - smaller than human. - has ncRNA genes *Human Homologs: 25%* - pretty good *Average Gene Size: 1.5kb 0.03intron/gene* - small. - mostly intronless. *Transposons: only a small proportion of the DNA*

BIOTECH: e.g. Antioxidant rich tomato

*Goals* - increase nutrition and quality of widely used crop: tomato *METHOD* Anthocyanin production - intro DEL and ROS1 genes for anthocyanin production (from snapdragon flower) - E8 fruit specific promoter - Kanamycin resistance for *ANTI-CANCER* used as mouse feed in Trp53-/- mice - lack p53 :. cancer prone. - antioxidants = reduced tumour incidence = increased mouse lifespan - Consumer survey = tastier! *OTHER BENEFITS* - decreased ripening after 2x months - slower softening - increased skin firmness (decreased cell-wall-modifying proteins) - decreased infection - decreased botrytis on skin --- EXPERIMENTAL EVIDENCE --- - used VIGS to reduce DEL + ROS1 = increased botrytis susceptibility = decreased storage time = decreased antioxidant quality *NEXT...* - production in canada - produce juice from tomatoes (no viable seeds, easier export)

DEVELOPMENT: Hox Genes + Homeosis

*HOX GENES* - All have Homeobox DNA binding Domain - Transcription factors - regulated by segmentation cascade - Guide Cell decisions *Homeosis* - where one body part of an organisms is replaced by another - usually a heritable change *Homeotic Genes* - induce Homeosis when mutated *Homeotic Mutants* - Recessive mutants = next most anterior segment - dominant mutants = next most posterior degment *Lewis Model* - though there were 10 BX-C genes - each with limited expression pattern - segment identity determined by overlap - no genes in T2 (no hox genes = all T2) *Current Model* - 3 BX-C genes. - overlap is key - extra genes identified by lewis actually just regulatory elements. *E.g. Drosophila* - BX-C: posterior segment identity. 3 Genes. - ANT-C: anterior segment identity. 5 genes. - conserved over almost all animals tho - Mice have genes all homologous to drosophila HOX genes (4 complexes tho)

DEVELOPEMENT: Fate Determination

*INDUCTIVE SIGNALLING* - components of cell localised so that a specific daughter cell inherits that cell determinant - cell determinant induces signalling pathways in the cell its contained in, - can influence cell types around it too. e.g. neurons. have an extra Nun protein OR no extra protein. ?????? *DIFFERENT ENVIRONMENTS* - environmental exposures influence cell type e.g. inner cells vs outer cells of the embyro = trophectoderm or blastocyst *PARACRINE SIGNALLING* - signals into extracellular space - cell fate determined by signals from cells all around it - can alter cell fates to different extents based on amount of signal molecule received *JUTACRINE SIGNALLING* - signal molecules sent to cells in immediate proximity - only alters cell fate of neighbouring cells. - signals NOT diffused

: Techniques for looking at Gene Expression

*Immunohistochemistry* - tagged antibody *In situ hybridisation* - utilise Antisense RNA - binds to target mRNA, produces colour *Reporter Gene* - genome modification so that reporter gene is transcribed alongside target gene - can visualise expression of target by monitoring reporter gene *Gene Fishing* - extract target tissue (organiser) - extract mRNA, transcribe into cDNA - construct expression library.

SACCHAROMYCES: Homologous Recombination

*Knock Out* - Reverse Genetics GENE DISRUPTION - target a known sequence to recombine. - disrupt gene with marker gene e.g. Ura3 - positive selection for that marker gene (uracil free media) GENE DELETION - target a known sequence to recombine. - disrupt gene with marker gene. - marker gene has identical sequences on either side. - positive selection for marker - recolonise - hope for recomb b/w identical marker sequences to remove marker gene - NEGATIVE selection for marker gene (FOA kills URA3 species) *SELECTION* E.G. Ura3 system - Encodes orotodine phosphate decarboxylase - Ura3-52 mutant strain: requires uracil in media POSITIVE SELECTION - Strains with URA3 killed by 5-fluoro-orotic acid (FOA) in media. NEGATIVE SELECTION

TRANSGENICS: why?

*LAB* - Reverse genetics - find what parts of the gene do what - see if a known gene rescues a phenotype - knock in /knock out - consequences of a misregulated gene (ectopic or HIGH expression) - examine gene expression (fusion proteins) *MEDICINE* - bulk production of therapeutic protein e.g. Atryn (antithrombin in ruminant milk) *COMMERCIAL* - increased nutrition of food e.g. increased caseins in milk e.g. omega 3 in pig meat - increasing plant resilience e.g. herbicide resistance e.g. insecticide resistance

BIOTECH: GE why?

*LAB* - controlled mutagenesis - reverse/forward genetics *MEDICINAL* - gene therapy for genetic disorders - prod of therapeutic proteins - ex vivo applications, out side of the body and reintroduced (in vivo would require high accuracy + efficiency, tissue type targeting and control of repair pathways NHEJ/HR) *COMMERCIAL* - GE for desired ag traits (accelerated trad breeding) e.g. accelerate breeding, quantitative trait loci is known - improving resilience for ag e.g. virus resilience in pigs: 5 base changes = african swine fever resistance *ENVIRONMENTAL* - maintain genetic diversity in extinct species - gene drives (reduce retrod fitness of invasive/pests)

MUTANTS: types of mutation

*Loss Of Function* - can be rescued by extra copy of gene NULL - same as phenotype deletion. - gene completely deffective HYPOMORPHIC - similar, but not as severe, as deletion *Gain Of Function* - can not be rescued by extra copy of the gene HYPERMORPHIC - similar result as over expression NEOMORPHIC - new function entirely. - Not rescued by deletion either.

BACILLUS: Intercompartmental Communication *SIGMA E*

*MOTHER CELL* Spo0A-PO4 greatest activity in mother cell after division *1. SIGMA E* - After Sigma F activation in prespore - Synthesised inactive proprotein. *Activated by proteolysis* - Sigma F dependent messenger SpoIIR secreted into mother cell. - SpoIIR activates SpoIIGA: protease cleaves inhibitory amino terminal domain. *FUNCTION* - Prevents Asymmetric division at 2˚ FtsZ ring. - Triggers englulfment of prespore - initiates spore coat assembly - Directs transcription of Sigma K

PLANTS: Evidence for ABC model

*MUTANTS* - can see changes in RNA ISH Ap2/Aclass - no sepal or petal dev - A usually inhibits C. - With no A, C in all tissues Ap3/Bclass - no petal or stamen dev - only A/C axis Agamous/Cclass - no stamen or carpel dev - With no C, A in all tissues *RNA IN SITU HYBRIDISATION* - shows each gene expressed in the location of the associated phenotype - (not always the case for all genes) *DOUBLE MUTANTS* No A or B - Only Carpels No B or C - Only sepals No A or C - whorls 1 + 4 = weird modified leaves - Whorls 2 + 3 = weird modified petals/ stamens (halfway between each growth form) *TRIPLE MUTANT* No A B or C - Whorl pattern still present - no Flower structures *TRANSGENIC ARABIDOPSIS* Pairing each floral dev gene with whole plant promoters 35s:AG (C everywhere) - Like an A class mutant - inhibits A 35s:AP2 (A everywhere) - like a C class mutant - inhibits C

BIOTECH: Platforms for GE protein production

*Microbes: bacteria + yeast* - understood + well characterised - not the best for mammalian proteins - often over glycosylated proteins in yeast *Insect Cell Lines* - better for mammalian proteins - very simple glycosylation *Mammalian Cell line and Mammals* - protein/protein interactions - difficult to maintain expression levels and the cell line - huge range of glycosylation patterns *Plant Cell Lines and Plants* - easy to culture - complex GLYC, not as large a range as mammals

PLANTS: Mapping

*Molecular Markers* A. thaliana (Columbia) A. thaliana (Landsberg) - 50000 polymorphisms between the species. - their location is PRECISELY known *Insertions/Deletions* - different sized PCR fragments *Restricion Site Polymorphisms* - different sized PCR fragments *Mapping* - compare locations of markers of mutagen offspring of the same parents - strong cosegregation w/ marker in all offspring = good marker for that gene - to isolate gene location more, add more markers.

MODEL ORGANISMS: Mouse

*Mus muculus* Phylum: vertebrata *PROS* - Mammalian - inbred strains to reduce variation readily available - short lifecycle (only 3 months) - forward + reverse genetics (mostly reverse tho) - sperm can be frozen *CONS* - expensive! - hard to access post implantation embryo (have to kill mother) - small litters (only 8-9) - no micromanipulation possible *What have we learnt?* - Disease models - embryonic stem cells - comparative genomics - drug testing - obese mouse - Nude mouse (no T cells) - SCID mice (immune deficient) - iPS cells

BIOTECH: Bulk Protein in Animals e.g. Milk

*NUTRITION* *Caseins* - 80% of milk proteins - bind CaCO3 :. increase calcium content - alpha s1-, alpha s2-, Beta- and kappa- casein - casein composition + ratios alter milk casein properties *Transgenics* - Introducing additional copies of Beta and Kappa Casein (engineered fibroblasts introduced into cow embryos) *ALLERGENS* - Beta- Lactoglbulin (BLG): in cow milk but not human milk. - RNAi targets two regions of the BLG gene - downregulate BLG production

BACTERIA MODELS: old vs new concepts

*OLD SCHOOL THOUGHTS* - homogenous static structures - good model for eukaryotic molecular biology. poor for cell biology - small size. - lack organelles - no organised internal structure - single celled *NEW IDEAS* - NOT SMALL: can be around 0.6mm. e.g. Epuliscium fishelsoni: 600um, 80-120000 copies of its genome. daughter cells develope within mother. - NOT SINGLE CELLED: capable of multicellular behaviour! can signal each other to coordinate. e.g. myxobacteria: social prokaryotes. social gliding/swarming. fruiting body development (for endospore formation) - Highly ordered + dynamic - capable of polarising and differentiation - Intracellular organisation: cytoskeleton w/ homologues of tubulin + actin. protein localisation, proteins targeted to specific locations with precision AND rapid directed changes in localisation.

TRANSGENICS: Drosophila

*P- ELEMENTS* special drosophila transposons - require transposase for insertion - encode own transposase w/ developmental regulation: active in germ line, inactive in somatic cells (alternative splicing) *LAB P-ELEMENTS* - Lab strain: Transposase removed. marker gene added. - transposase ∆2-3 (produced in ALL cells) added on another plasmid w/ defective P element *TRANSGENICS W/ P-ELEMENTS* - mount desired gene in a P-element - inject into EARLY embryo (<1hour) - :. more likely to reach genome before cellularisation. - aim is to alter germline cells. - find offspring with marker gene e.g. white gene to produced Red eyes, in white eye Drosophila strain. - around 1/100 genes transformed

BACILLUS: Intercompartmental Communication *SIGMA F*

*PRESPORE* High SpoIIAB, High SpoIIE (enriched after or during division), Low SpoIIAA SpoIIAB = anti sigma factor SpoIIAA = anti- anti sigma factor. Regulated by AB phosph + E dephosph SpoIIE = dephosphs spoIIAA (high abundance in prespore as it determined prespore) *1. SIGMA F* - Co-synthesised with SpoIIAB :. inactive when first transcribed. - Low C of SpoIIAB as septum transiently excludes gene (near oriC) + protein is unstable. (15min before chromosome is imported) - less phosphorylation of SpoIIAA by SpoIIAB - more dephosphorylates SpoIIAA by SpoIIE - release of sigma F from SpoIIAA - SpoIIAA + SpoIIAB bind instead - Less repression of sigma F *FUNCTION* - protease from sig F degrades Sigma E (hypothetical) - secretes SpoIIR int0 Mother compartment

SACCHAROMYCES: Advantages + Limitations as Human Model

*PROS* - nucleus - chromosomes - mitotic checkpoints mostly homologous - Key amino acids conserved - good fro fundamental processes - introns/splicing *CONS* - Cancer: no p53 equiv, mutated in 50% of human cancer - no cell specialisation or larger cell organisation into organs

BACILLUS: Intercompartmental Communication *SIGMA G + SIGMA K*

*SIGMA G* PRESPORE - kept inactive until engulfment (unsure how) - activated by mother cell *Function* - Couple late prespore + mother cell gene expression - protect spore from hazardous conditions - prepare spore for germination *SIGMA K* MOTHER - pro-protein requires activation by prespore signals *function* - activation of genes involved in the formation of spore coat and spore coat maturation

SACCHAROMYCES: Plasmids

*Shuttle vectors* e.coli + s.cerevisiae elements for ease of use in both organisms - no yeast ori: requires integration into yeast genome - selectable yeast marker e.g. Leu 2 - Selectable bacterial marker e.g. ampicillin resistance - bacterial ori - cloned region of interest *e.g. YIp: integrative plasmid* - stable integration *e.g. YEp: episomal plasmid* - stable without integration - often high copy number

PATTERNING: universal tool kit

*Signalling Molecules* - TGF betas (e.g. BMP, Nodal) - FGF + EGF family - Insulin Family - Hedgehog Family - WNT family - Notch Family *Transcription Factors* - Homeodomain (hox) - Paired Box (pax) - Zinc finger - bHLH - T-box - Fox - Nuclear receptors - HMG- box

CAULOBACTER: Developmental regulation processes

*Temporal changes in gene expression* - ~586 Proteins expressed in response to biological clock/ cell cycle - "Just in time Transcription": peak expression of proteins before or coincide with timing of their event. *Spatial positioning of proteins and other signal molecules* *Protein phosphorylation* *Proteolysis*

BIOTECH: History

*Traditional* 5000 BC/3000BC - Selection for certain traits - development of modern Corn! - Utilising Microbes for Beer brewing, cheese making, wine fermentation *Conventional* 1800s-1960 - Mendelian Genetics - hybrid crop development - Chemical agriculture - Mutagenesis, Tissue culture, plant regeneration - GREEEN REVOLUTION!!!!! *Modern* 1970-Now! -Gene transfer - Recombinant DNA - Embryo rescue + protoplast fusion in plant breeding - Insulin commercial product of transgenics - Genetic fingerprinting - GE vaccines + Hormones - GE plants - Bio informatics - genomics/proteomics/metabolomics/systems biology - synthetic biology, genome editing.... AND MORE!

BACILLUS: Sporulation Decision

*Triggers* cell crowding, starvation for N/C/P. Depends on Phosphorylation state of Spo0A. Requires high density and starvation to actually cause response. No singular effect. *Pre-Sporulation/Final Go* - release killing factor + signalling protein. - act together to lyse sister cells - cannibalism = nutrients :. delays sporulation. *1. Quorum Sensing* - Cells release small signalling peptides - increased mass of cell culture = increase peptide - sensed. activates Spo0A. *2. Starvation* - Cell exhausts all nutrients. - Spo0A phosporelay initiated (to phosphorylate Spo0A) *3. Checkpoint* - Cell monitors intracellular conditions: chromosome integrity + replication, krebs cycle. - asses whether or not sporulation can be completed if its started *4. Spo0A-P Action* - represses abrB - abrB usually represses stationary phase + sporulation genes

BIOTECH: E.g. Antibodies GE prod in animals

*Xenomouse* - Cross Antibody knockout mouse + Human Antibody transgene mouse - mouse producing human antibodies! *PROS* - 7 current antibody drugs available from xenomouse platform - Xenomouse can produce FULL human anitbodies while retaining its own antibody responses *CONS* - antibody knockout mouse very vulnerable

MODEL ORGANISMS: Xenopus

*Xenopus laevis* Phylum: vertebrata - embryo develops outside mother - 500-3000 embryo *PROS* - genome editing - egg transparent - limbs - can be made transgenic - morpholine antisense gene knockdown - large embryos :. easy to manipulate (esp frogs) - vertebrates! closer physiology to humans *CONS* - allotetraploid: 4 copies of each gene, required double knockouts - 9 month life cycle LONG - large space required for aquaria *What have we learnt?* - pregnancy test - spemans organiser - nuclear transfer for cloning and genomic equivalence - developmental patterning genes - development and disease model (mainly fish) - toxicity screens

PATTERNING: Other organisers (besides froggo)

*ZebraFish* = Sheild - dino homologous to chordin - 3 noggin genes, noggin 1 = organiser specific USING MORPHOLINOS/ knockdowns - suggests redundancy between Nog1, Chd and Follistatin like -2. *mouse* = node - Noggin + chordin = redundant *Overall* - organiser structure present in Echinoderms, Cephalochordates and vertebrates (no single evolutionary event) - same genes present in protostomes and invertebrates but, do different roles

BACTERIA MODELS: Cell Polarity

- Cell that inherits old pole exhibits signs of aging - old pole considered an again parent repeatedly producing offspring. - old pole cells can die of old age.

PLANTS: ABC floral Development

- Determining the fate of the Apical Meristem: leaves, shoot, flowers etc *floral cues > signal detection > transduction pathways > switch on LEAFY gene (flowers not leaves)* Arabidopsis flowers - organs arranged in whorls *Homeotic Selector Genes* - Ap2 =Aclass - Ap3 = Bclass - Agamous = Cclass A alone = Sepal A + B = Petal B + C = stamen C alone = Carpel

SACCHAROMYCES: Wine!

- Different Strains used for different wines - single polymorphisms + copy number variations affect the phenotype - Can trace strains to the use in different wines e.g. French Wine vs NZ wine *Identifying genes for Desired Traits* 1. Crossing + Linkage Analysis - strain with high 4MMP thiol cross with lab strain with no 4MMP thiol - identify haploid offspring w/ High 4MMP 2. PCR + Genome Analysis - sequencing genomes to find genes in common with all high 4MMP strains (IRC7) 3. Knock In/ Knock out - identify IRC7 function: phenotype with, phenotype without

DROSOPHILA PATTERNING: Maternal Genes

- Early acting. - Establish posterior/anterior - RNA produced in maternal tissue, transfered into oocyte (unfertilised egg) tissue via cytoskeleton. *Discovery* - mutations in maternal phenotype cause mutated embryo, independent of embryo genotype. *Bicoid* - moprhogen - maternal effect gene - encodes a TF, activator + repressor - master regulator of anterior dev - mRNA translated after fertilisation - gradient formed: greatest @ anterior, lowest @ posterior - where ever gradient is highest, anterior structures form, even if its the middle of the oocyte - higher concentrations change location of cephalic furrow

SACCHAROMYCES: Conditional Mutants

- Only express mutated phenotype at certain conditions. *e.g. Temperature Sensitive Mutants for Essential Genes* e.g. cell cycle genes, CDC28 -Ideal as if genes were not conditional, may be fatal. - ONLY ONE GENE made T.S. can find consequences of removing ONE gene product. - 23˚C = permissive (grow) - 37˚C = restrictive (do not grow) (Single AA substitution that disrupts protein folding at high Temps) - synchronise growth phase with Nitrogen Starvation (G1)

BIOTECH: Bulk Protein in Animals e.g. Pigs

- Pigs do not have the necessary enzymes to extract sufficient phosphorous from their feed (in phytate form) *Transgenes* - Added phytase expressed in salivary glands *Enviro Pig* - Manure lower in phosphorous (75% less) - pigs able to utilise almost all phosphorous in feed - no need to add additional phosphorus to their diet - increased feed proficiency = increased protein

BIOTECH: e.g. Antimicrobials in Milk

- Raw milk contains a lot of bacteria - especially staphylococcus aureus - current antibiotic treatments + cownimmune system can not effectively control the bacteria - S. aureus major cause of mastitis *NATURAL ANTIMICROBIALS IN MILK* - usually too low to control S.aureus LYSOZYMES - damage bacterial cell walls LACTOFERRIN - antimicrobial activity *METHOD* - introduce Lysostaph (bacterial origin, peptidoglycan hydrolase) - modify gene for production in cows (not bacteria) - ovine BLG promoter (milk only) - protein secreted into mammary glands

BT CORN: Bacillus thuringiensis + CRY proteins

- Soil bacterium *CRY proteins* - insecticidal crystal protein - protoxin - resides in inclusion bodies produced during sporulation - CRY genes are insect specific (for broader taxanomic groups) - Domain I = membrane insertion + pore formation - Domain II/III = receptor recognition + binding *Protein Activation* - High pH≈12 in insect gut dissolves crystal - target specific cell surface proteins in gut - forms pore in gut lining - degradation of membrane control causes cell to burst - fast acting *Traditional Use* - previously included in an "organic" pesticide spray

MUTANTS: Balancer Chromosomes

- Usually hard to maintain to create homozygous stock. - Can do it on v smol scale by chance. - selective pressures against homozygous lethal mutations = lost from stock *Balancer Chromosomes* Three key elements *Dominant Morphological markers* - a dominant visible character - can trace offspring containing balancer chromosome *Recessive lethal mutations* - homozygous balancer chromosome offspring die *Multiple Inversions* - For recombination suppression. - recombination can disrupt expected inheritance patterns - if recombination occurs, gametes will not be viable

BIOTECH: E.g. Antibodies GE prod in plants!

- could be used for topical application to mucosal applications to prevent infection *Secretory IgA* - 2 x Light chain, variable region + conserved region - 2x heavy chain, Variable region + conserved region - Forms dimer (Small peptide joining chain/ J chain) - Secreted (secretary component protein/ SC) *Transgenes* all inserted into seperate plants w/ agrobacterium - Strong plant promoter + Light chain sequence - Strong plant promoter + heavy chain sequence - Strong plant promoter + J chain sequence - Strong plant promoter + SC sequence *Production of antibody* proof of concept - Cross L chain + H chain plant - Cross LH/IgA offspring w/ J plant - Cross Dimeric IgA offspring w/ SC plant - Final progeny = full Secretory IgA protein *PROS* - High yield - low cost - existing infrastructure for large scale prod - Correct folding, processing + assembly - could produce right where it is needed - stable storage (in the plant) - safe! free from endotoxins, viruses, oncogenic DNA *CONS* - GMA containment issues - possible allergic reactions? (as w/ all antibodies) - slightly different glycosylation - contamination w/ mycotoxins, pesticides, herbicides + 2˚ metabolites - regulatory/approval uncertainty

AMPHIBIAN MODEL: Symmetry breaking

- egg has 2x poles, animal hemisphere and vegetal hemisphere - sperm entry shifts the animal hemisphere = produced "Grey Crescent" where animal hemisphere previously was. - creates dorsal/ventral patterning (entry = ventral/ grey crescent = dorsal) Bilateral symmetry. - in first cleavage, each hemisphere/buttcheck gets some crescent. - Grey crescent contains dorsal determinant. e.g. If cleavage on a different plane (done manually using thread) and one half doesn't get any grey crescent no dorsal or ventral structures form. only centre belly piece.

TRANSGENICS: other transgenic insects

- have to used different species specific transposons. - P-elements only really work in Drosophila - marker gene usually flouresent protein with expression driven in the eye (not white gene like in drosophila) *Application* - potential use as pest and disease control. - study the genetics of non drosopholid insects

BIOTECH: GE Cancer Example

- introduce gene for cancer cell detection + T cell instruction - removal of natural T Cell protein that may interfere with the process - removal of the protein that identifies T cells as immune cells :. prevent cancer cells disabling T cell action

TRANSGENICS: Transgenic Mice

- introducing new genes - no homology utilised *PRONUCLEAR INJECTION* - DNA inserted randomly into the genome of fertilised embryo (<0.5days): could disrupt other genes etc - implant embryos into host mother - HOPEFULLY transgene will become part of germ line and be passed onto off spring. *EMBRYONIC STEM CELLS* - stem cells from young embryos kept in culture - transformed w/ DNA very easily. DNA incorporates randomly into the genome. - stem cells can be induced to form embryos :. transgenic mice *CONS* -abnormal gene expression, not with its native enhancer - insertion is random, may disrupt another gene - multiple copies can be inserted, which may lead to chromosome rearrangement

BIOTECH: Insulin

- regulates many parts of human metabolism - INCL. uptake of glucose into muscle + adipose - used to treat Type 1 diabetes patients *HUMAN INSULIN PROD* 1. mRNA = pre-proinsulin 2. translocated into ER 3. folded + disulfide bonds formed 4. removal of single peptide = proinsulin 5. transported to Golgi + packed into vesicles 6. IN VESICLE pro-insulin cleaved into active form (Removal of C chain) *OTHER ANIMALS* - Highly conserved throughout all animals - used to source therapeutic insulin from other animals *BACTERIA* - insulin previously produced in GE E. coli - A + B chains produced in seperate bacteria - A + B chains produced w/ fusion protein for efficient isolation - Fusion protein cleaved and A + B mixed to HOPEFULLY form correct insulin *YEAST* - Cheaper production - undergoes same eukaryotic processing as in a human cell *Leader-insulin precursor fusion protein* - signal (pre) peptide - Pro peptide - Endopeptidase cleavage site - Human insulin B - MINI Human Insulin C - Human insulin A *PRO* S. cerevisiae vs Bacteria - Recombinant Protein is Glycosylated - Protein is Secreted - Improved transmembrane expression *CONS* S. cerevisiae vs Bacteria - only Moderate expression yields - Gylcosylation is simple BUT can be over glycosylated DIAGRAM

MUTANTS: e.g. Drosophila Eye colour

- wild type eye = brick red with black spot - Eye colour mutations fall into one of four categories: brown, scarlet, white, yellow/peach *Complementation Test* - white + apricot mutants are allelic: Apricot = hypomorphic. white = null - Brown mutants indicate 6 (B-G) brown causing genes - Scarlet mutants indicate 7 (H-N) scarlet causing genes *Epistasis Test* - Brown mutations are epistatic, each over the next. - Scarlet mutations are also epistatic other the next. - White is epistatic over BOTH brown and scarlet. *Interaction Test* - ea. brown mutation interacts with ea. scarlet mutation, causing white eyes. - combination of both colours important for WT phenotype --- white likely at the end of the pathway, as hypomorphic forms still produce red/brown in the eye a little bit.

PLANTS: Find out Gene Function

1. Mutant Phenotypes, traced back to a gene 2. Cloned gene! provide biochemical info about its gene product -- need both phenotype and biochemical info to understand the role of the gene

DEVELOPEMENT: Genomic Equivalence

All cells have the exact same genome BUT cells become different to each other. - Metaplasia: wrong cell type can occur in a tissue due to injury or mutation e.g. throat cells form stomach cells in response to acid reflux. SHOW all cells retain ALL the information needed to become any cell type - Amphibian cloning: early nuclear transplantation show differentiated nuclei can retain the ability to direct development of an animal BUT the ability is reduced with the age of the cell.

BIOTECH: e.g. Flavr Savr

Calgene; independent Biotech start up *Goal* - delay ripening :. provide consumers with consistently high qual + fresh produce *Fruit ripening* - controlled by multiple seperate genetic pathways - Ethylene triggers - Develop colour (Chloroplast -> chromoplast, chlorophyll -> carotenoid), texture (Cell wall degradation), flavour (Cell wall degradation- sweet) + aroma *Ethylene* - Nor mutants = non -ripening - rin gene = receptor Ripening reduced via: Low O2, High CO2 Low T˚ *Normal Industry practices* - harvested + shipped unripe - treated w/ ethylene to promote ripening - needs to be firm enough to repackage - still needs reasonable shelf life - uneven ripening (some green tomatoes) - often under developed flavour *METHOD* Tissue Culture, Plant Regen, Synthetic seeds - reduce production of polygalacturonase (PG) (responsible for cell wall degradation) - inhibit via Antisense RNA *Binary vector* - via agrobacterium - TDNA w/ Kanamycin resistance selectable marker = reduced PG up to 90% = reduced pectin breakdown *ISSUES* PUBLIC CONCERN - possible incorporation of bacterial genes? - kanamycin resistance not removed, possible gene escape - allergen concern due to random insertion process - no perceived value w/ the product ALTERNATIVES - produced via traditional breeding - e.g. long keeper, super life, vine ripened LAW - expensive regulatory process - bigger company, Monsanto, claimed breach of patent - despite the case being dismissed, Calgene went under and sold to Monsanto FARMERS - only produced in lab viable strain, not strains best for farmers - tomatoes still not firm enough for existing processes

BACILLUS: Sporulation Phosphorelay

Complicated to allow multiple inputs to control. *Integrate intracellular + Extracellular signalling* - high cell density: *PEP5*. promotes. - DNA damage/ impaired replication: *Sda*. inhibits KinA. - Energy potential redox state (cytoplasm). promotes. Phosphorylates KinA at PAS domain. *PEP5* promotes Phr Peptides (PhrA/C/E) *Phr* inhibits Spo0F phosphotases RapA/B/E *CodY-GTP* will inhibit Phr peptides transcription and inhibit KinB transcription. *KinA-E* phosphorylates Spo0F *Spo0F* response regulator. phosphorylates Spo0B *Spo0B* Hisitidine phosphotransferase. phosphorylates Spo0A *Spo0E, Yisl, YnzD* inhibit via dephosph of Spo0A. *Spo0A* response regulator. at high density, triggers sporulation. -Low Levels: promote skfABCDEFGH + repress AbrB :. indirect activation of sdpABC + sdpRI (all killing factor proteins) - High Levels: sporulation

MUTANTS: Complementation

Determine which phenotypes are Allelic - can not tell visually - different mutations within one gene can produce different phenotypes - mapping can tell you how close, but won't actually tell you if its the same gene *HOW* - cross two homozygous recessive mutants (X + Y) - Results = heterozygous X/Y - if phenotype rescued = different genes - if phenotype no rescued = same gene = allelic

SACCHAROMYCES: Mating Types

Determined by combinations of transcription factors *a Mating Type* - Default Cell type - Only a1 transcription factors (homeobox factor) + MCM1 - a2 present but has no known function. - MCM1 activates a specific genes - Haploid Genes *alpha Mating Type* - alpha 2 repressor TF, alpha 1 activator TF +MCM1 - alpha 2 represses MCM1: NO a specific genes - alpha 1 alongside MCM1: alpha specific genes - haploid specific genes *alpha/a Mating Type* - alpha 2 repressor TF, a1 TF +MCM1 - alpha2 represses MCM1: no a specific genes - alpha 2 and a1 co-activate diploid genes ALSO co-repress alpha 1

PLANTS: ABC in other plants

Different ABC expression patterns create different looking flowers *E.g. Nuphar (basal angiosperm)* - no strong boundaries - irregular part number - hybrid plant structures *E.g. Tulipa (monocot)* - B overlaps all A expression - Tepals not true petals *E.g. Rumex (core eudicot)* - No B/A overlap - no true petal structures

MUTANTS: Mutagenesis

Different animals = varying ability to mutagenise *Chemical Mutagenesis* - Ethyl methanesulphonate (EMS): component of mustard base, highly mutagenic, point mutations. Amount directly proportionate to number of mutations. - Trimethylpsoralen (TMP): DNA cross linking activated by light, produces small deletions. less direct in number of mutations. *Radiation* - in general all radiation caused single base pair mutations, increased dose = deletions - Ionising radiation: DNA damage and mutation - UV irradiation: slow (6 weeks) - Gamma irradiation: fast! *Genetic* - transposon/ retro viruses

DROSOPHILA PATTERNING: Gap genes

E.g. Krüppel, Knirps Hunchback - mutations = huge deletion of segments - Expression pattern mirrors function - mark out large areas of the embryo *ACTIVATION* *HunchBack* - expression is regulated by maternal gene, Bicoid - expressed at certain bicoid threshold *Krüppel* - expression regulated by hunchback and other maternal genes - repressed at high Hunchback concentration - activated by hunchback at certain, lower, threshold

DEVELOPEMENT: Fate Map

Each cell lineage has a specific fate it will follow to become a specific cell type. - bias of cell type early on in developement - cells always undergo the same pathway and become the same group of cells.

DROSOPHILA PATTERNING: Drosophila Embryo dev.

Egg = single diploid nucleus ½ mm of cytoplasm - nuclei multiply in cytoplasm. - nuclei then migrate to surface and cells form around the nuclei. *Genes are epistatic* Maternal Genes > gap genes > pair rule genes > segment polarity genes (also hox genes) - majority of these genes encode TFs (except some segment polarity genes)

MUTANTS: Forward Genetics

Finding the Genotype associated with a Phenotype *Genetic Screen* - mutagenise organism of choice - collect and maintain stocks of mutants w/ mutation causing disruption in target phenotype - isolate the gene in ea. mutant causing the mutation NEEDS: Scorable Phenotype. Way to produce Homozygous Offspring. Way to keep Mutant Stocks (likely to be unwell/ unhealthy).

SACCHAROMYCES: CELL CYCLE Start/Restriction Point

G1 -> S Transition GENE: CDC28 (homologous to human CDC2, discovered via complementation) discovered via mutants. *CDC28* - Cyclin dependent kinase - requires cyclin protein for activation - CLN1 CLN2 CLN3 - redundant to ea. other - active CLN1 protein levels due to cyclic proteolysis (not change in mRNA levels) - can't measure protein levels via mRNA levels

SACCHAROMYCES: CELL CYCLE Mitotic Spindle Checkpoint

G2 -> M Transition between metaphase and anaphase APC/C protein complex LAB

PATTERNING: Organiser Genes to Control Pattering

Gene Fishing: cDNA + expression Library - Must be secreted - Must inhibit BMP *Goosecoid* - homeobox sequence targeted w/ blot - only expressed in organiser - Ectopic expression: TF, not morphogen. - creates second organiser *Noggin* - Use UV to ventralise embryo/ destroy dorsal determinant (disrupts microtubules and prevents grey crescent formation) - insert all organiser mRNA = can rescue ventralised embryo - split mRNA inserted over and over again UNTIL - noggin the single gene that could rescue the phenotype *Chordin + Follistatin* - also secreted BMP inhibitors - redundant w/ noggin.

MUTANTS: Epistasis

Gene to Pathway *One Mutation Overrides the activity of another* - only 1 of the two mutant phenotypes expressed - overriding mutation = epistatic - hidden mutation = hypostatic - indicates 2x non-allelic genes are in the same pathway - inheritance pattern usually: 9:3:3:1 *e.g. Doggo Colour* B gene in dogs determines black or brown, E gene determines colour expression in the coat. Mutation in E will block phenotype of mutation in B. B = hypostatic. E = Epistatic.

MUTANTS: Gene Interaction

Gene to Pathway *double mutant should produce worse phenotype than each single mutant* - Genes that act in the same process, not necesarily the same pathway - inheritance pattern 9:3:3:1 *E.g. Corn Snake Colour* - two pathway for colour patterns, Black pathway and orange pathway - cross an orange mutant (mutation in black gene) and a black mutant (mutation in orange gene) = albino mutant (no colour produced at all)

SACCHAROMYCES: e.g. Y2H

LAB

BACILLUS: Sporalation Process

Long + energy consuming with severe consequences to the cell. 7hours at 37˚C 500 genes in spatial/temporal gradient. *I. Decision* *II. Asymmetric Division* (Important) - Chromosomes anchored at each pole. - Asymmetric polar septum develops - Chromosome pumped into forespore (smaller division) (Important) *III. Engulfment* - Membrane of larger spore migrates around perspire until completely enclosed *IV. Cortex Development* *V. Spore Coat Development* *VI/VII. Maturation/Cell Lysis*

SACCHAROMYCES: Mating type switching

MAT genes determine Mating Type (Mat A or Mat alpha) *GENE CONVERSION* MAT gene identity can be converted by copies of HMLalpha OR HMRa gene - common in plants and fungi - causative in some human disease e.g. Congenital Adrenal Hypoplasia (no 21 hydroxyls :. no gene conversion :. excess testosterone production) - Translocation/conversion initiated by HO endonuclease - lab strains have no HO endonuclease (control mating type) *GENE SILENCING* chromatin modification silences HMx genes that are not required. - requires facultative/induced expression

AMPHIBIAN MODEL: Morphogens

Morphogen - long range signal, secreted from cells in one location - diffuses to form a gradient - gradient maintained by source (producer) to sink (destroyer) - cells along the gradient develop different fates (dependent on thresholds): different gens will be activated at different thresholds - Must instruct equivalent cells to form multiple fates. - reponse MUST BE DIRECT, not via a relay mechanism

BACILLUS: Endospores

Produced by Bacillus and Clostridium *Resistant to environmental stress:* High Temp, Ionising Radiation, oxidative stress, Chemicals/Solvents, detergents, enzymes etc... *LONG DORMANT STATE:* Survival Strategy not sure exactly how long. Potentially 250mya endospore found. *Alternative Survival Strategies* - Motility + negative Chemotaxis: move to new environment - Induced Competence: new DNA -Antibiotics: kill competitors. provide nutrients.

SACCHAROMYCES: Why a Model?

S. cerevisiae *FAST GROWTH* - Rapid: 90-140min doubling - slower than e. coli but faster than mammalian. - selective media can be used *CHEAP* - grows well on chemically defined media/broth/plates. - inexpensive *HIGH SCREENING* - small dispersed cells (not filamentous) - single colonies - multiple colonies per plate!!!!!! *EUKARYOTIC* - diploid + haploid grow well - can study recessive/dominant

BIOTECH: Yeast

Saccharomyces cerevisiae - Simple eukaryotes - ER, Golgi, Vesicles etc - sugar -> ethanol + CO2 *PROS* - easy to grow in bioreactors - can secrete proteins - eukaryotic glycosylation (N linked Highly conserved) - eukaryotic PTMs - well understood fermentation technology - easy isolation/ mutant selection - small genome size w/ whole sequence known - haploid OR diploid - Host-Vector system established - multiple characterised strains *Traditional Yeast Uses* - Beer + Wine = Ethanol Production - Bread = CO2 production *GE Yeast Uses* - Production of heterologous proteins/ Proteins that differs from any protein normally found in the organism in question (Blood: Albumin, Hirudin, Transferrin. Hormones: Insulin, Glucagon. Antigens: Hepatitis) - combing GE and bioreactor biology - Degrade cellulose in animal feed for more available sugars *Bioreactor v Simple Cell Culture* - much higher efficiency - less labour - faster?

SACCHAROMYCES: Info

Single Celled budding fungi: yeast - classified based on cell, ascospore, colony character and physiology (sugar fermentation) - Common on plants + animals and in soil. - Bakers and Brewers yeast = Saccharomyces - Fermentation = CO2 + Ethanol

BACILLUS: Asymmetric Division

Spo0A-P, sigma H, sigma A *Shifting Cell Division Machinery* 1. Increase FtsZ mediated by ftsZ transcription 2. FtsZ/Zring fotms via spiral intermediate 3. SpoIIE repositions FtsZ into two rings at each pole 4. ONE of these rings = assymetric division (ring with highest SpoIIE) other ring disintegrates (Sigma E) - only ⅓ of chromosome actually in prespore - remaining ⅔ transported through septum via DNA translocase SpoIIIE.

CAULOBACTER: Oscillating Master Regulatory Proteins

Three master regulators. Levels oscillate throughout cell cycle. Regulate >200genes. Cyclical Genetic Circuit: Three Dimensional integrated system *CtrA* G1 swarmer, end of S, new pole G2 - Represses GcrA transcription. - Represses Replication (silences ori) - 95 genes: Late differentiation of swarmer, Flagella, pili etc *DnaA* Initates S - Initiates Replication - 40 genes - Activates GcrA expression *GcrA* During S - 50 Genes: cell wall, membranes etc - Genes for replication elongation (DNA segregation) and polar development - activates CtrA transcription - Represses DnaA *CcrM* - methylytransferase

BIOTECH: Pichia pastoris

alternative yeast for GE - Methyltrophic:. methyl as the sole carbon source - High expression (>30% of total soluble protein) *Inductible promoter* - AOX - induced by exposure to methanol - desired protein inserted down stream of AOX promoter - Protein produced as fusion product to secretion signal of alpha mating factor in S. cerevisiae *PROS* vs S. cerevisiae - High expression - not over glycosylated - easy to scale up + grow at high densities (inducible promoters) - simple cell recovery *CONS* vs S. cerevisiae - Simple glycosylation - Not always very effective ... Harder to work with than S. cerevisiae

DROSOPHILA PATTERNING: Pair-rule genes

e.g. Even-skipped, odd-skipped, paired, runt - mutations = every few segments deleted - Expression pattern mirrors function - mark out every other segment - First genes to be expressed in a segmented way - adjacent segments defined by expression in one, absence in the next. *ACTIVATION* - regulated by the overlap of gap genes: interaction at the enhancer *eve Stripe 2* - regulated by a specific enhancer: multiple TF can bind to the enhancer, some activating, some repressing - :. specific genes only expressed at certain overlaps of gap genes - activated by hunchback and bicoid - repressed by giant and krüppel

DROSOPHILA PATTERNING: Segment Polarity Genes

e.g. gooseberry, patched - mutations = deletion of portion of EVERY segment - actually define the segments from each other *ACTIVATION* - activated by pair rule genes - so exact that each segment in each embryo has the same pattern of segment polarity - indirectly regulated by gap-gene overlap

SACCHAROMYCES: Systems Biology

high throughput versions of old technology = focusing on THE WHOLE genome rather than single genes. -- Next Generation Sequencing -- Genome wide sets (of mutants and gene constructs) -- Microarrays of all genes/ all promoters (robot analysed arrays) - Comparative Genetics! conserved genes likely to have important functions e.g. Gal4 gene binding sites HIGHLY conserved, required for sugar hydrolysis

TRANSGENICS: Knock out/ Knock in mice

targeted approaches *KNOCK OUT* - deleting exisiting genes - ideal to test gene function *EMBRYONIC STEM CELLS* *1. Homologous recombination w/ targeting Vector* - linear DNA - regions homologous to target gene - marker gene A to replace target gene e.g. neoR - marker gene B on the end of the vector. e.g. tk+ POSSIBLE OUTCOMES 1. target gene replaced by vector marker gene 2. Linear DNA randomly inserted into the gene 3. No insertion *2. Selective Culture* - Positive selection for marker gene A (neomycin) - Negative selection for marker gene B (ganciclovir) - selects for outcome 1 - induce to form embryo, insert into host mother *CHIMERIC MOUSE* - Introduce mutated stem cells (as above) from Brown mice, into Blastocyst stage embryo from a black mouse - Successfully mutated mice will have both black and brown marking = Chimeric Mice - Mate mature off spring with Black mouse. Select brown offspring. - Continue crossing until homozygous mutation present. *KNOCK IN* - instead of gene deletion. - can introduce point mutation - e.g. If A Human Disease is caused by hyper/neomorphic gene *Targeting Vector* - target gene with alterations/ new gene - lox P sites either side of Neo R - selection as in knockout - utilise Cre recombinase to remove Neo R gene

PLANTS: Mutagenesis

~ 40,000 seeds mutagenised - plant seeds + grow (M1 generation) - M1 generation: heterozygous mutations not present in every cell (would not want too many mutations as it would inhibit plant development) - Self M1 = M2 - M2 Generation: ¼ = homozygous for the mutant phenotype