GENE223 FINAL

Bacillus sigma F control

A high fidelity system ensures that correct temporal and spatial control of sigma F activity occurs

Shuttle vectors

Shuttle vectors contain both E. coli and S. cerevisiae genetic elements for ease of use in both organisms. Can contain selectable yeast marker (e.g. LEU2), cloned region of interest, selectable bacterial marker (e.g. ampicillin resistance) and bacterial replication origin. These factors allow the propagation in E. coli, can then be put into yeast. In yeast there is no origin of replication, therefore must integrate into chromosome to propagate, this is a simple yeast vector called a yeast integrative plasmid (YIp)

Old school prokaryotes

Prokaryotes (old school) was thought of as homogeneous static structures, good models for eukaryotic molecular biology, poor model for cell biology, small size lacking organelles, no organized internal structure and usually single celled

Sequence of decisions for fate

A sequence of decisions that are progressively more restrictive, resulting in different cell types β cell starts out as an embryonic cell, determined by inheritance of a factor to become part of the endoderm layer. The anterior endoderm progresses onto pancreatic endoderm, becoming an islet cell then a β cell. Fate is hard to change, easier if closely related

Sigma E activation

After sigma F is activated in prespore, sigma E becomes active in the mother cell (synthesized as an inactive pre-protein, activated by proteolysis). Sigma E functions to prevent asymmetric division in the mother cell (that the second FtsZ ring dissolves), triggers engulfment of prespore, initiate spore coat assembly and directs transcription of sigma K. Sigma E is activated by SpoIIGA (phosphatase, removes a pre protein) and SpoIIR (activates SpoIIGA, synthesized by sigma F and secreted into membrane of mother cell)

Bacillus asymmetric division (first commitment to sporulation)

Asymmetric cell division is the first committed stop on the path to sporulation Normally, in the division plane a tubulin-like protein (FtsZ) forms a ring around the center of the cell that constricts. Initial sporulation signals lead to the production of a protein (green/DivIVA protein) that forms at each cell wall. RacA protein coats and guides the chromosomes to the poles in an axial filament formation. Chromosomes are anchored at their origin of replication end to each of the cell poles, the ring forms and a signal causes it to spiral to each end of the cell, ending up with two FtsZ rings at each quarter pole. One of these poles is chosen to form the septum and the other one disintergrates (a lot to do with SpoIIE protein concentration). Results in septum with part of chromosome, as the pore is closing, a protein forms around the closing pore (SpoIIIE, DNA pump), pumping the chromosome into the spore (15 - 20 mins)

Sporulation conditions of Bacillus

Bacillus subtilis endospore formation, sporulation depends on the level and phosphorylation state of a critical regulator Spo0A (two component regulatory protein). Spo0A that is fully phosphorylated and present at a high level will cause sporulation to occur. No single effect acts as a trigger, but high cell density and starvation is required. When the mass of cell culture increases (because each cell excretes small quantum of small peptides into the medium), cells sense increased concentration of small peptide over threshold (quorum sensing) and results in the activation of Spo0A. When cells exhausts all nutrients (starvation), Spo0A phosporelay is initiated (passing of phosphate through different regulatory proteins to Spo0A. e.g. Phosphate from KinaseA to Spo0F to Spo0B to Spo0A). Cells also monitor the intracellular conditions, including chromosome integrity, chromosome replication (one full copy for spore and one full copy for mother cell) and Krebs cycle, essential in making sure once sporulation starts that it can be completed

Bacillus cell cycle and endospore formation

Bacillus subtilis normally doubles in length and divides down the middle, chromosomes normally anchoring at mid cell. Metabolic, environmental and cell cycle signals are integrated and a decision is made on another vegetative division cycle or sporulation. The sporulation process starts off with activation of Spo0 genes (stage 0 genes) to initiate sporulation for polar division. At stage II (there is no stage I), there is asymmetric cell division. The smaller compartment differentiates into the prespore/forespore and the larger compartment is the mother cell that feeds the spore, provide coats etc. Each sister chromosome is anchored to opposite cell poles, simultaneously the polar septum forms with a pumping mechanism for the chromosome to enter the forespore compartment. Development occurs and at stage III, engulfment occurs. Following polar septation, the membrane of the larger mother cell migrates around the prespore until it is completely enclosed (spore is released into cytoplasm/inside mother cell). At stage IV, the prespore differentiates internally (inside mother cell), mother cell builds layers. Sporulation process takes ~7 hours at 37°C, with 500 genes expressed in spatial and temporal gradient

Bacillus growth

Bacillus subtilis usually growing by vegetative growth (doubles in length, splits down the middle), but can develop into endospores resulting in asymmetric cell division and altruism, after the spore has formed, the mother cell will disintegrate to feed the formation of the spore before committing altruism. These spores can be viable for millions of years, resistant to all types of stresses

Bacteria range in size

Bacteria can range in size, largest known bacteria is Epulopiscium fishelsoni that is found in sturgeon fish guts (600 μm (visible to the naked eye), 120,000 copies in genome, daughter cells grow inside mother cells) and Thiomargarita namibiensis that is found in african waters ("Sulfur Pearl of Namibia," just under 1 mm in length)

Congenital adrenal hyperplasia (CAH)

CAH is an autosomal recessive disorder occurring in 1:15,000 births. CAH has defective 21 hydroxlase due to abnormal gene conversion, resulting in no production of aldosterone and cortisol, shunting pathway to produce testosterone. CAH in females cause ambiguous genitalia (small penis)

S. cerevisiae implications

Implications for S. cerevisiae studies are cancer cell growth and understanding cell cycle. Yeast as a model organism for cancer development has advantages of yeast containing nucleus, chromosomes and checkpoints, but there a limitations of absent p53 and immune system in yeast

Proteins involved in S. cerevisiae cell cycle

CDC28 is the key proteins for starting cell cycle (start/G1), also involved in G2/mitosis, S phase and G0. Start of cell cycle in yeast, called the restriction point in animals, G1 is the checkpoint. CDC28 is a cyclin dependent kinase (cdk), functioning to add phosphates to proteins, involved in regulation of protein function. CDC28 is cyclin dependent, there are 3 cyclins (CLN1, CLN2, CLN3), these genes were not detected by mutant (because they are redundant). CLN1 protein levels cycle due to cyclic proteolysis, but transcript (mRNA) levels do not vary. Cln1p is critical in G1 to S phase transitions, therefore protein is present at a higher level during this transition

Caulobacter crescentus division

Caulobacter crescentus has two cell types at each division, stalk cells and swarmer cells. The stalk cell has a stalk, stalk cell divides and gives rise to a swarmer cell. The swarmer cell has a flagellum, can swim away and find somewhere to reside, forming a stalk to divide again. Caulobacter undergoes one round of chromosomal replication for each division, which makes it a good model for understanding cell cycle regulation. These inhabit fresh water and lakes

Caulobacter crescentus cell cycle (starting as swarmer)

Caulobacter crescentus starts off as a swarmer cell at G1 phase, prior to initiation of DNA replication (single non-dividing chromosome). Finds new home (lives in oligotrophic environments with low nutrient levels, therefore cannot all survive in high population densities), swarmer cell differentiates into stalk cell and stalk glues itself to surface (often found in fast flowing rivers), this is at the S phase (DNA replication process). The cells elongate, constriction begins, this is the predivisional stage (S stage finishes with chromosomes are divided). G2 phase is where there is no DNA synthesis, phase prior to cell division where cell differentiation finishes off (new pole grows flagella). Eventually two cells separate and swarmer cell starts cycle again

Caulobacter developmental program regulation

Caulobacter developmental program is regulated and enforces by temporal and spatial changes in gene expression (gene expression is coupled with cell cycle over time), spatial positioning of proteins and other signal molecules, protein phosphorylation and proteolysis

Caulobacter cell cycle

Caulobacter has a proper cell cycle, unlike E. coli, only replicates its chromosome once during the process of cell growth and division. It gives rise to the mother stalk cell and the progeny swarmer cell, these are differentiated by the poles. Only the stalk cell can divide, swarmer cell is non-divisional and has to differentiate into a stalk cell to divide. Caulobacter is a great model system and has huge applicability to eukaryotes because it has a differentiation step There is only one chromosome cycle per cell division, there is a differentiation process strongly coupled with the cell cycle and the differentiation events occur at different phases of the cell cycle producing two different cell types

Caulobacter using poles

Caulobacter uses the fact that there is an old pole and a new pole to be able to differentiate between the ends of the cell, e.g. always a flagella at the new pole and chemotactic apparatus at one of the two poles along the way

Caulobacter cell cycle and polar morphogenesis

Cell cycle and polar morphogenesis is controlled by "just in-time transcription," meaning the genes (products of which are involved in a specific cell-cycle event) have a peak in expression immediately before or coincident with the timing of the event. E.g. transcription of the genes that are involved in DNA replication is induced and reaches a maximal level at the G1-S transition, whereas transcription of the genes that are involved in chromosome segregation is maximal in late S phase

S. cerevisiae checkpoints

Cell cycle checkpoints have been defined by yeast mutants, checkpoints conserved in other species (e.g. humans). The G1/S checkpoint is the start or restriction point, the commitment to DNA synthesis. Checkpoints further in cycle check DNA being properly replicated. Spindle checkpoint checks chromosome alignment on spindle in mitosis (key checkpoint, refer to lab), faulty checkpoints result in nondisjunction (produces one 2N cell and one 0N cell). There are differences in yeast and man, there is a multi functional tumor suppressor p53 protein in key checkpoints in humans (this protein is mutated in over 50% of cancers). p53 is a DNA binding protein involved in several checkpoints in human cells, checkpoint components need to fail for cancerous cells to occur

Yeast cell cycle

Cell cycle has different phases, most cells we see are in G1 (not dividing). S phase (DNA replication) is the commitment to cell replication, critical part is the G1 to S transition. G2 is another gap phase, and mitosis is replication of cells (producing daughter cells)

Cell division in bacteria

Cell division in bacteria (e.g. E. coli) is asymmetric. The daughter cells exhibit polarity, many species exploit this polarity to support directional motility (e.g. Caulobacter) or to produce daughter cells with distinct fates. The cell that inherits the old pole exhibits signs of aging (proteins begin suffering oxidative stress and eventually die of old age), the old pole cell should be considered an aging parent repeatedly producing rejuvenated offspring

Slime model mechanism

Cell migration into collection of cells (pre s***), the cell cycle phase at which the cells respond to the signal will bias the cells to take on different roles. Cells of a particular phase of the cell cycle form the prestalk cells (leading the migration of the s***), while another particular phase forms the prespore cells (survivors). The commitment of the cells are labile (easily changed) at the s*** stage e.g. if all prestalk cells were cut off, the prespore cells can turn into prestalk cells to ensure species survival. Cells are stably committed and differentiated when fruiting body structure is made

Worm - apoptosis

Cell suicides/apoptosis important in development for shape and complex morphology in functional adults, first discovered in C. elegans mutants (ced3). This was the first time there was a gene mutation association with cell suicide, ced3 has 131 extra cells (that should've died) with consequences. Apoptosis is conserved in humans (Cells between cartilage elements die to free up the digits)

Early sporulation phase

Cells that enter the early phase of sporulation use cannibalism as a survival strategy (Spo0A-P low levels). Spo0A-P at low levels, because it binds at high affinity, can bind to repress AbrB promoter (transition state protein between exponential and stationary phase). AbrB normally represses three genes (skfABCDEFGH, sdpABC, sdpRI), these three operons make a killing factor (bacteriocin, not well understood) along with immunity factors. Low levels of Spo0A-P causes Bacillus to produce killing factor by direct activation of operon (skfABCDEFGH) and indirectly through AbrB (repressing a repressor). This process obtains energy and whether to continue the sporulation process

Comparative genomics of related strains of S. cerevisiae

Comparative genomics with closely related strains allows prediction of genes, regulatory elements etc. Different saccharomyces strains and species that were compared showed conserved genes (shows common function or important), reject gene prediction (predicted in one but not in the other) and conserved noncoding sequences (regulatory regions). Regulatory region conservation implies function e.g. GAL4 protein has conserved blocks across species that constitutes the binding site for a transcription factor (in a non-coding region), comparative genomics helps identify common binding sites, conserved blocks can be used as genetic elements (e.g. put in front of another gene, and GAL4 transcription factor will turn gene on)

Yeast strains and wine

Crossing strain that makes high amounts of 4MMP thiol with a lab strain that produces no 4MMP thiol (4MMP thiol has a particular aroma in sauvignon blanc, most S. cerevisiae have disrupted IRC7 gene). Mated two mating types together to form a diploid, induced meiosis for crossing over to occur. Looking at the progeny yeast, looked at amount of produced 4MMP and common gene sequences. The gene region IRC7 encodes a β-lyase, thought to be gene that confers 4MMP producing phenotype, found to be on chromosome 6. To prove the functional role, gene was knocked out (no 4MMP produced) and knocked in (4MMP produced). Genome analysis and using PCR to sequence IRC7 show IRC7 was likely horizontally transferred from S. paradoxus to YJM450 strain (this strain we are talking about). Applications of this include crossing of desirable traits with strains with IRC7 to increase aroma, or using a mixture of S. cerevisiae to get right amount of compound produced (breeding better wine yeast)

Developmental genetics

Developmental genetics studies the genetic control of cell growth, differentiation and morphogenesis. There are a number of different bacterial that can give rise to a number of different looking types and differentiated types of form. How can one bacterial cell, and a single division, give rise to two genetically identical but morphologically different cells, and functionally specialized

Asymmetric septum

Formation of asymmetric septum is the key event in development. It triggers a cascade of changes in gene expression, involving different programs of gene expression in the mother cell and prespore. Sigma factors play an integral role in this process, produced in an inactive form (needs to be activated). Signal factors are transcription initiation factors that target RNA polymerase to specific promoter sequences. Some sigma factors are active all the time, while others may be activated in response to specific environmental conditions

Bacillus forms endospore

Endospores are produced by members of the genera Bacillus and Clostridium. Endospores are induced by cell crowding and starvation for C, N and phosphate (conditions where it cannot grow). Endospores are resistant to many stresses including high temperature, ionizing radiation, chemicals/ solvents, detergents, and enzymes. This dormant state is a long term strategy for survival (maybe 100 years?), with reports of reviving 25 - 40 million year old spores preserved in extinct bee gut trapped in amber and 250 million year old spores isolated from brine trapped in salt crystal

Experiments with S. cerevisiae

Experiments involved ordering S. cerevisiae mutants in cell cycle. They looked at different mutants, putting them in different complementation groups, looking at the different stages of the cell division cycle (cdc) the mutants arrested at. cdc28 mutants cannot initiate DNA synthesis. Mutations in cdc genes results in arrest of cell at those stages (cannot progress along in cell cycle Building up complementation order of genes, through the cell cycle to understand the cell cycle

Sensing mates - yeast

Extracellular signal (pheromone) binds to receptor, signaling pathway to nuclear membrane effects transcription of ~30 genes involved in cell shape and fusion (also negative feedback inhibition at the receptor stage). The activation and inhibition is through specific protein-protein interactions and modifications (involving kinases for specific phosphorylations). Two proteins (Dig1, Dig2) are inhibited, these inhibit Ste12, resulting in reduced inhibition of transcription factors (inhibiting inhibitors). There is specific DNA binding by Ste12p (Ste12 protein) in the nucleus (transcription factor) that activates genes. STE are sterile genes (wildtype) while ste are mutant genes (ste mutants were first isolated)

Fate map

Fate maps can show that cells, which appear the same, are actually not In most embryos, there is some degree of bias early on e.g. tunicate at 64-cell stage shows bias, certain cells consistently end up at specific locations. The fate of a cell is what it becomes if it is left alone. E.g. In a frog blastula fate map, cells that look alike can form different things

Fish and frog model - contributions

Fish and frog models have given us developmental patterning genes, regeneration models (frogs can even regenerate spinal cord) and toxicity screens Fish models have given us developmental and disease models (e.g. melanoma studies) and frog models have given us pregnancy test (lays eggs when HCG conc. is over threshold), Spemann's organizer, and nuclear transfer for cloning and genomic equivalence (test if genes are lost over time, does our cells contain the right amount of genes)

Fission yeast (not S. cerevisiae)

Fission yeast divides symmetrically (S. cerevisiae divides asymmetrically), dividing by fission (similar to mammalian cells) and was used in cell cycle studies. Cell cycle mutants (using UV, chemicals) can cause mutants that elongate without dividing and mutants that arrest without budding

Gene deletion of URA3

Gene deletion uses gene disruption/knockout and positively selects for URA+. The different is the identical sequence flanking the URA3 gene, this marker allows for negative selection against the URA3 gene. Adding FOA in the media results in strains with homologous deletions (due to identical sequence flanks) of URA3 to survive (abc1-Δ). This results in loss of function by deletion

Gene disruption of URA3

Gene disruption or knockout can be done by linear PCR product that yeast can take up (identical sequence flanking gene of interest), inserting a gene in the middle of the chromosomal gene (e.g. ABC1 has URA3 gene inserted into it, resulting in non functional ABC1 gene (abc1::URA3)). Can be positively selected by URA+ phenotype (will grow without Ura in media). This results in the loss of function of original gene by insertion

Gene replacement via homologous recombination

Gene replacement by homologous recombination (reverse genetics) can be done by making a specific mutation on the same gene in a plasmid (e.g. E. coli), then by homologous recombination, causes mutant gene on mutated plasmid to transmit onto genomic chromosome. This introduces a specific mutation on the chromosome

Gene replacement of URA3

Gene replacement with altered version (knock in) uses gene disruption to insert URA3. A linear PCR product DNA (e.g. abc1-1, construct with desired mutation to be incorporated into chromosome) is inserted by homologous recombination and FOA selection is used as negative selection against URA3. This results in a specific mutation that may/may not lose function (e.g. changing one certain amino acid to another)

Genetic analysis approach

General approach to genetic analysis (studying a gene) is making mutants in haploid strain (e.g. using UV, chemicals and mutagens to change phenotype to select the mutants e.g. selecting for tryptophan auxotrophs/trp1). If genome is known, can make specific knockouts or editing using homologous recombination or using CRISPR-Cas9 (direct system). After mutation, characterize mutants using complementation with cDNA expression library to isolate cDNA of wildtype (TRP1), crossing to test for complementation of known mutants (e.g. trp2, trp3...), or map to genome by linkage analysis by crossing over during meiosis

Genetic manipulation of yeast

Genetic manipulation techniques that are available to S. cerevisiae include standard mutagenesis (chemicals, radiation, transposons), transgenesis (integrative plasmid, replicative plasmid, yeast artificial chromosome, shuttle vector) and targeted gene knockouts (gene replacement, knock-in) Integrative plasmid inserts by homologous recombination. Replicative plasmid can replicate autonomously using an autonomously replicating sequence (ARS) or 2μ plasmid. Yeast artificial chromosome replicates and segregates as a chromosome. Shuttle vector can replicate in yeast or E. coli (there are tools in E. coli that can be transferred into S. cerevisiae). Gene replacement and knock-in results in homologous recombination to replace wild-type allele with a null copy

Genomic equivalence

Genomic equivalence is a theory that all cells descended from the zygote should all have the same genome, since mitosis gives each daughter cell a copy of the genome. Our cells interpret the genome differently, how you access it, what proteins are made that determines what role the cell takes. For example, metaplasia is a phenomenon where cells of the wrong type occur in tissues as a result of injury or mutation, suggesting that all cells retain the information needed to make any kind of cell. Amphibian cloning were the first nuclear transplantation experiments to show that differentiated nuclei can retain the ability to direct development of an animal, the ability is reduced with the age of the cell

Sigma E mechanism - 2

High concentrations of sigma E in mother cell (unknown reasons, sigma E degraded in prespore). Pro-sigma E is cleaved by SpoIIGA (serine protease), cleaving inhibitory amino terminal domain to give active sigma E in mother cell. SpoIIGA is activated by SpoIIR, which is made by sigma F and secreted into the mother cell. This cross compartmental communication leads to the activation of sigma E

Frog model

However Xenopus is allotetraploid (~2x as many chromosomes) and Danio has also undergone genome duplication, they have long life cycles, >9 months for Xenopus and 4 months for zebrafish, large housing space is required for aquaria and fish are not tetrapods (no limbs)

Worm - relation to humans

However, only 43% of C. elegans genes have human homologues, it is the most diverged from humans of all metazoan models and the embryos are not easy to manipulate and not easy to study gene expression (embryos are small) C. elegans has given genome sequencing (as trial for larger genomes), apoptosis pathway, knowledge on survival (some were found alive after space shuttle Columbia disaster in 90s), longevity research (daf2, homologue to insulin receptor, these two linked to oxidative stress resistance and longevity) and micro-RNAs

BUB1b gene (human) and BUB1 (yeast)

Human BUB1B compared to S. cerevisiae BUB1 (or MAD3) have differences in size and intron number but key amino acids are conserved. S. cervisiae has genome size of 12 Mb, 16 linear chromosomes containing 6000 protein coding genes (mainly intronless genes, also contains ~1000 non-protein coding genes). 25% of genes have human homologs (can study splicing). The average gene size is 1.5 kb (0.03 intron/gene), there is a small proportion of transposons in the DNA. S. cerevisiae genome sequences in 1996 On a phylogenetic tree, most of the wine yeast from different parts of the world fall into one region of the tree (different yeast strains for sake or rice wine etc.)

Bacillus endospore

In a spore, chromosome is covered with proteins (wrapping it and inert, making it impervious to oxidative stress). There are multiple cell layers, providing barriers to external stresses. The inside spore needs to undergo differentiation for DNA to become resistant. The mother cell lays down layers of protective coatings (then lysis (commits altruism) and releases spore)

a/alpha cell mating type S. cerevisiae

In the a/α cell, the combination of a1, α1 and α2 genes activated results in repression of a-specific genes, no activation of α-specific gene and repression of haploid specific genes Haploids can switch between the mating types. The translocation (in active loci regarding mating type) is a gene conversion initiated by the HO endonuclease (facilitates gene conversion). Lab yeast strains lack HO nuclease for stable haploid phases (ho)

Negative regulators of sporulation Bacillus

In the initiation of sporulation, there are multiple negative regulators, intracellular and extracellular signals e.g. PEP5 indicator of cell density and cell-cell communication (peptide based quorum sensing and control phosphoryl signaling/flux). Phosphorelay must integrate different extracellular and intracellular signals (at different levels) to ensure that sporulation only occurs under appropriate conditions (phosphates can be taken off at different levels if conditions aren't right for sporulation). It is complex because there are a large number of survival strategies

Alpha mating type S. cerevisiae

In the α cell, the two α genes have been copied into the active loci's to be expressed, resulting in α1 and α2 to be made. There is also a constitutively expressed MCM1 transcription factor. α1 activates the α-specific genes, α2 represses the a-specific genes and a1 activates the haploid-specific genes

Kinase A Bacillus sporulation pathway

KinA is subject to two types of regulation, inhibition by Sda and activation through one of its PAS domains. Expression of Sda is activated by impaired DNA replication or DNA damage, thereby preventing sporulation under these circumstances. By contrast, a PAS domain of KinA is thought to sense energy potential or the redox status of the cell and promote sporulation accordingly. High levels of GTP/GDP in the cell results in CodY-GTP to repress KinB and Phr A, C, E activity (GTP/GDP fall at time of sporulation). Cell density (quorum sensing) is sensed by Phr peptides, PEP5 that is secreted, processed and imported back into the cell as pentapeptides. Over threshold, PhrA, C, E will inhibit Rap proteins (Rap A, B, E phosphatases) that cause dephosphorylation of Spo0F-PO4 (Spo0F-phosphate), causing phosphorelay to continue. Rap proteins are usually active to prevent sporulation. Spo0A can be dephosphorylated by Spo0E, YisI and YnzD (these not well understood)

Lab S. cerevisiae strains

Lab S. cerevisiae strains often contain genetic markers and mutations to limit viability outside the lab, wild type or domesticated strains not usually used. In the GENE223 lab strain (PJ69-4A) which is MATa (mating type, haploid), trp-1-901 (mutation at 901st, loss of function, can use in selections), leu2-3, 112 (loss of function), ura3-52 (loss of function), his3-200, gal4Δ (deleted to be used in Y2H for protein-protein interactions), gal80Δ, LYS2::GAL1-HIS3 (GAL1 gene inserted into LYS2 gene, LYS2 gene function not lost), GAL2-ADE2 (GAL2 promoter fused to ADE2 gene to use as reporter gene in Y2H), met2::GAL7-lacZ (GAL7 gene inserted into met2 gene, but met2 was disrupted)

MAT genes in yeast

MATa and MATα sense each other using extracellular pheromone (a or α), which is a secreted chemical factor that triggers a response in the same species. The two mating types grow towards each other and produce a shmoo structure, conjugate and nuclei fuse to produce a zygote. The diploid (a/α) can bud and produce daughter cells

Master regulator cascade of Caulobacter - 3

Master regulatory proteins are synthesized in succession to control expression of at least 200 genes. The dynamic subcellular localization of regulatory proteins during the cell cycle is an essential part of the Caulobacter cell cycle control (e.g. rapidly directed to a pole to be phosphorylated/degraded). There are multiple feedback loops that produce cell cycle dependent proteolysis of the CtrA, GcrA and DnaA master regulators. The regulation of the bacterial cell has to be studied and described as a 3-D integrated system

Master transcriptional regulator for S. cerevisiae

Master transcriptional regulators are a fundamental principle for cell type determination. a1 is a homeobox factor (important in vertebrate development), α1 is the activator, α2 is the repressor, a1/α2 represses α1synthesis in diploid cells, a2 has no known function

Sigma F activation

Preferential activation of sigma F in prespore involves multiple mechanisms and a chain of regulators. Removal of the inhibition by SpoIIAB of sigma F in prespore (excludes mother cell) is determined by its intrinsic instability (intrinsic instability/fast turn over rate) coupled with its transient exclusion from prespore (location in cell) and the level of dephosphorylated SpoIIAA due to SpoIIE activity

Mendelian and non mendelian inherited traits S. cerevisiae

Mendelian inherited traits of S. cerevisiae has ~30% of open reading frames (ORFs) or protein coding genes with unknown function, but often similar to other species (15% or half of genes similar to other species, still unknown function). Petite mutant colonies tend to lack mitochondrial function, with mutations usually in nuclear genes Non-Mendelian inherited traits include mitochondrial DNA (important in human diseases), plasmids, and viruses (important in biotechnology)

Micro array and NGS showing gene regulation of Caulobacter

Microarray data shows 15% of genome is transcribed in a cell cycle-dependent manner that acts like a biological clock. This provides cues for the timing of events involved in morphogenic differentiation Next generation sequencing shows clear cascade of gene regulation from swarmer cell to stalk cell differentiation, around 586 genes are controlled in a cell cycle specific manner (blue = low abundance, yellow = high abundance)

Importance of model organisms

Model organisms are selected because they are easy to work with and useful (using animal models when we can't study in cell lines/cultures e.g. B. subtilis, yeast). Animal model organisms are typically easy to breed and have advantages for the study of developmental genetics (fast life cycle, modeling genetics like phenotype and processes) There is genome information for hundreds of species, genetics of model organisms tells us what the individual genes do (through knock out, mutations, etc.), how genes work together to build an animal or plant, and how genes interact with the environment (we are a product of our genes and the environment). We cannot experiment on humans, animal models used are more or less related to us, in terms of genetic sequence (diverged from mice 90 million years ago)

Mating switch

Most eukaryote genes are off (silent) in their ground state (bacterial genes in ground state are 'on'), mainly by chromatin modification. To turn on the gene, transcription factors bind to specific parts of the DNA (sometimes distantly located from the transcript start site), RNA polymerase then turns on the gene. In S. cerevisiae, there is one active set of genes, either expressing α or a mating type Gene silencing means the gene is switched off by chromatin modification. Gene conversion is the conversion of one allele to another (e.g. one version of α genes to the active locus to be expressed). Gene conversion is a common process in many species (e.g. plants, fungi)

Sigma factor F in prespore

Mother cell contains sigma H and Spo0A, Spo0A gets phosphorylated Sigma F functions to couple prespore and mother cell gene expression and direct transcription of sigma G (prespore). Sigma F is active first and regulated by two distinct mechanisms which work together to ensure sigma F is only activated in the newly formed prespore compartment. Sigma F is inactive when first synthesized, activity prevented by an anti-sigma factor (SpoIIAB)

Myxobacteria

Myxobacteria are the gram-negative social prokaryotes capable of multicellular behavior including social gliding/swarming and fruiting body development (formation of environmentally resistant spores)

Wine and S. cerevisiae

NZ sauvignon blanc usually has added S. cerevisiae commercial strains. The choice of strains produces different desirable traits e.g. flavor, aroma. Most properties of the final aroma of the sauvignon blanc is from the different strains of S. cerevisiae. There a volatile compounds synthesized in different amounts by different strains e.g. thiols, H2S

Comparative genetics S. cerevisiae

New genetic techniques provide ease of manipulation and genetic mapping in yeast, including episomal plasmids or integration via homologous recombination. Yeast is a eukaryotic model organism for studying mitosis, cell cycle, regulator of gene expression, mutagenesis, DNA damage, recombination and mitochondrial function. Single gene studies (old) are being replaced by whole genome studies (new), particularly understanding the whole as a system (systems biology - the study of dynamic biological systems taking into account the interactions of all the key elements e.g. DNA, RNA, proteins and cells with respect to one another). Genes can be built up in a systematic network, able to work out what interacts with what

Methods to understand organisms as a whole

New methods allow understanding of the organisms as a whole system (including yeast biology), include understanding the system by understanding the sum of the parts. The tools used can include DNA sequencing, next generation sequencing (NGS), genome wide sets of mutants and constructs (all 6000 genes knocked out) and microarrays of all genes or all promoters

Differences in yeast strains

Ongoing studies have identified variations, including single nucleotide polymorphism (SNPs) and copy number variations (CNVs) within the species S. cerevisiae that affect phenotype. The differences are critical for applications in making specific wines and bread. Genetic studies indicate NZ has unique strains (bc NZ is the best), some of the yeast strains adapt from commercial strains, some are wild strains from NZ and some from import (from barrels)

Complementation of yeast to help understand yeast cell cycle relating to humans

Other experiments that helped the understanding of yeast cell cycle relating to the human cell cycle. Melanie Lee experimented with complementation of yeast mutant with human gene. She found human gene CDC2 is homologous to CDC28 in S. cerevisiae (CDC2 in humans named because complementation first done in S. pombe and was called cdc2) Mutation in particular gene (e.g. cdc28) in a ts mutant cannot grow in high temperatures (no clonies). Adding a random gene will not normally complement phenotype, but adding gene where complementation will occur (e.g. CDC28) causes ts mutants to be able to grow in high temperatures

Caulobacter protein localization

Protein localization (can be coupled to fluorescent proteins to visualize) of Caulobacter proteins include anterior/posterior positioning and dorsal/ventral positioning. Anterior/posterior positioning shows DivJ protein is localized at the stalk cell pole, PleC localized at the new cell (swarmer) pole and ZapA localized involved in the constriction of the division between. Dorsal ventral positioning shows crescentin on one side of the cell, thus able to differentiate left, right, top and bottom. There are chemoreceptors found at the new pole Localizing specific proteins into specific locations in the cell is responsible for the differentiation of different cell types

Awesome power of yeast

S. cerevisiae (yeast) is a key organism for studying genomics, systems biology, genetic control of cell cycle, recombination, mating types, gene interactions (Y2H), mitochondrial inheritance. It is a model for other eukaryotes (humans, plants, animals etc.) S. cerevisiae is a budding yeast (fungus), the species are classified on cell, ascospore, colony characteristics and physiology (sugar fermentation). It loves commonly on plants, animals and in soil. S. carlbergenesis (brewers yeast) isolated in 1888, with the first genetic studies performed ~1935. Yeast fermentation produces CO2 and ethanol, and different strains of S. cerevisiae are used for bread and brewing

Frootzen

S. cerevisiae allows analysis of other Saccharomyces and isolates S. cerevisiae can be mixed with other strains in the wine making process. A non saccharomyces yeast (Pichia kluyveri) was found in sauvignon blanc that gave it it's fruity flavor. Pichia kluyveri (FrootZen, been commercialized) boosts fruit flavors, on a phylogenetic tree, shows it's distantly related to S. cerevisiae. P. kluyveri survives up to ~ 4-5% alcohol, therefore needs to be added at the start

Characteristics of S. cerevisiae for good model eukaryotic organism

S. cerevisiae is eukaryote but has simple unicellular lifecycle (dividing by budding), existing in both diploid and haploid forms that both grow well. This means we can study recessive and dominant mutations Haploid yeast (one copy of each chromosome) can be cultured as a haploid, going through mitosis cell cycle. The mother cell has 'scars' from where previous budding occurred, mother dies after 8-9 bud cycles Two mating types (a - female, α - male) can mate (fuse together) to produce a diploid (two copies of each chromosome), diploids can continually go through mitosis. Diploid cell can be induced into meiosis through starvation or nitrogen, where four haploid spores contained in an ascus is produced (similar to human meiosis). An ascus is a capsule containing four haploid ascospores, these haploid cells within an ascus can be taken and cultured like any other yeast haploid

Signaling in early patterning development

Segregation and bias to different fates can be due to localized determinants, inductive signaling, environment, paracrine and juxtacrine signaling Localized determinants is inheritance of preferentially located substance in only one daughter cell when the cell divides due to asymmetric cell division (e.g. nerve cells) Inductive signaling is communication, one cell communicates with other cells Different environment exposures causes differentiation e.g. blastocyst, inner cell mass and trophectoderm Paracrine signaling is the induction of nearby cells. Signaling cell produces protein signal, responding cell has receptors to receive signal resulting in a response. May result in a change of fate due to different experiences Juxtacrine signaling is the induction of adjacent cells. Signaling cell produces protein signal targeted to membrane (stuck in membrane, cannot diffuse out). Responding cell has receptor that can bind signal. May result in change of fate

Sigma G

Sigma G dependent genes in prespore functions to couple late prespore and mother cell gene expression, protect spore from hazardous conditions, and prepare the spores from germination. Sigma dependent genes in mother cell functions for activation of genes involved in the formation of spore coat and spore maturation. Both sigma G and K are synthesized inactive, kept inactive until a certain developmental stage, activated by signals from other compartments. Sigma G is kept inactive until engulfment by unknown mechanism, sigma K has the precursor processed by a protease. Sigma G activation requires signal from mother cell, and sigma K activation requires signals from prespore

Beginning sporulation sigma factors

Spo0A-P (phosphorylated Spo0A) represses AbrB (a gene that represses the stationary phase and sporulation genes) and activates two crucial transcription factors: SpoIIA (sigma F) and SpoIIG (sigma E). These two are sigma factors, guiding the initiation of the developmental process, sigma factors are subunit of RNA polymerase that directs RNA polymerase to the right subunit

SpoIIAB

SpoIIAB is an anti sigma factor of sigma F, it is co-synthesized with sigma F (located in the same operon) and prevents its activity (has kinase activity on spoIIAA) SpoIIAA is an anti anti sigma factor (antagonist of SpoIIAB), regulated by phosphorylation by SpoIIAB and dephosphorylated by SpoIIE. SpoIIAA releases sigma F from SpoIIAB when dephosphorylated (active). SpoIIE is a phosphatase that dephosphorylates SpoIIAA

SpoIIE

SpoIIE is activated in the prespore, its phosphatase activity becomes greater than the kinase activity of SpoIIAB. The dephosphorylation of SpoIIAA by SpoIIE favors the release of sigma F from SpoIIAB in the prespore (resulting in RNA polymerase)

SpoIIE mechanism

SpoIIE is recruited to the FtsZ rings by a direct interaction with FtsZ. The development of the two rings is asymmetrical and the ring that contains the most SpoIIE protein (upper in this case) usually achieves division first (the other dissolves away). During or following division, it seems that the SpoIIE protein becomes enriched in the prespore compartment, greatly enhancing the likelihood of σF activation in that compartment. SpoIIE phosphatase activity also seems to be regulated and it is possible that this regulation responds in some way to formation of the septum. This high fidelity system ensures correct temporal and spatial control of sigma F activity

Bacillus sporulation

Sporulation is a stress-response pathway of last resort (it's a gamble). It is a long (~7 hours) and energy consuming process, with severe consequences to the cell including death of mother cell and cessation of metabolic activity of daughter cell. There are other survival strategies designed to help the cell maintain growth and negate the need to sporulate. Motility and chemotaxis helps cell to swim to a new environment, induction of competence (lytic proteins that kill non-competent cells) helps scavenging of DNA and production of antibiotics helps to kill competitors and provide nutrients. Cells in the initial stage of sporulation produce and export a killing factor and a signaling protein that act cooperatively to cause sister cells to lyse, thus cannibalizing their siblings and providing nutrients to enable them to delay the final sporulation decision (Bacillus cannibalizes siblings to ensure enough nutrients through sporulation process)

Conditional mutations to study the cell cycle

Temperature sensitive (ts) conditional mutants of S. cerevisiae are permissive (grow) at low temperatures (21°C) and are restrictive (stop growing) at high temperatures (37°C). Ts mutants important in biology and yeast. These ts mutants have a substitution that prevent protein folding at a higher temperature (often due to a amino acid substitution, change in conformation of protein). Using nitrogen starvation, able to synchronize cells in G1 phase (all arresting in the same phase) These two genetic advances (ability to make ts mutants and synchronize cells in particular phase) enable the study of cell cycle

SpoIIAB mechanism

The SpoIIAB anti-σ factor (green circles) is an unstable protein, encoded by a gene located at an oriC-distal part of the chromosome (green boxes). During the period immediately after formation of the polar septum (e), and before the SpoIIAB gene is translocated into the prespore (f). SpoIIAB protein concentrations decrease, allowing the more stable sigma F protein to become active in the small compartment/prespore (gene is not in prespore, not making more SpoIIAB protein in prespore, this is the transient exclusion from last paragraph). This is the chromosome partitioning effect

Chicken model organism

The chicken, Gallus gallus, develops outside the mother, each and cheap to manipulate, large limb buds, retroviral insertion of transgenes, able for gene expression studies (flat embryos) and has amniote meaning closer to humans that fish or frogs. White dot in egg yolk is what develops into the chicken However, there is no genetics, can't be bred in labs and the very early stages are not accessible (developing in mother) The chicken model has given us limb development knowledge, and somitogenesis (formation of segmented vertebrate body parts which go on to form vertebrae and muscles)

Fish and Frog model organisms

The fish (Danio rerio, only part of egg cleaves, yolk sac for nutrients) and frog (Xenopus laevis, whole egg cleaves) can be made transgenic, able for morpholino antisense gene knockdown, develops externally of mother (easier to study), and has large embryos that are easy to manipulate (especially frogs). Able for forward genetic screens on fish models and genome editing in fish and frog models. Fish and frog models are vertebrates, meaning closer physiology and skeleton

Master regulators of Caulobacter

The forward progression of the cell cycle is driven by three master regulators (transcriptional regulators): CtrA, DnaA and GcrA and a methyl transferase (CcrM). The levels of each active protein oscillate in time over the course of the cell cycle/growth phase, successively regulating the transcription of over 200 genes. These regulators interact with one another and switch on gene expression at the required time

Fruit fly - contributions

The fruit fly has given us polytene chromosomes (helped figure out what a chromosome was), genetics as a field of study (not exaggeration), heredity (works in animals), hox genes, linkage and mapping of chromosomes and developmental toolkit (Heidelberg screen)

Fruit fly model organism

The fruit fly, Drosophila melanogaster, has been a genetic model for over 100 years with a life cycle of 2 weeks. The fruit fly is fast breeding, small sized, able for forward and reverse genetics, contains P element (transposons), has balancer chromosomes to maintain homozygous lethal mutations, great for gene expression and 61% of genes have human homologues However, insects diverged from humans 600 million years ago, with different physiology (circulatory system, immune response and skeletal system) and they get everywhere

Mouse model organism

The mouse, Mus Musculus, is mammalian (closest relative), has inbred strains to reduce variation, short life cycle (3 months), sperm able to be frozen and able for forward and reverse genetics (but mostly reverse) However, it is expensive, come in small litters, no micromanipulation able, and hard to get post implantation embryos (once implanted in uterus, must kill mother to get embryo) The mouse model has given us hundreds of disease models, embryonic stem cells, comparative genomics, drug testing, obese mouse (leptin), and iPS cells

Modern view of prokaryotes

The new view of prokaryotes views them as highly order (in terms of metabolism, infrastructure), dynamic cells capable of polarizing (recognizing which pole is which) and differentiating into different cell types (e.g. antibiotic resistance). Prokaryotes cells have intracellular organizations, including cytoskeletons containing homologues of tubulin (FtsZ) and actin (MreB), and protein localization (many proteins now known to be targeted with exquisite precision to specific locations in the cell or to undergo rapid directed changes in localization). Prokaryotes are able to signal each other to coordinate multicellular actions

Slime mould model

The slime model, Dictyostelium discoideum, is a social amoeba with 12,500 genes. Single celled myxamoebae has 3 cell adhesion molecules, activated by starvation and attracted by cAMP. They come together to form a 'multicellular structure'. Front end of structure begins to move, finds good location and forms a Mexican hat, starting to form a stalk with fruiting body. Cells differentiate into stalk cells with big vacuoles that eventually die (to save species), prespores that will be released and cup cells to hold spores in fruiting body until release

Gene expression in cell cycle of Caulobacter

The swarmer (has flagellum and pili) cell has high levels of CtrA because it is motile and not dividing/chromosomal DNA replication (G1 phase), CtrA disappearing until late S phase. With appropriate environmental cues, the flagellum and pili are shed and grows a holdfast, turning into the stalk cell. Early stalk cell has high levels of DnaA during early S phase, which initiates DNA replication and early gene expression, mainly present in G1 - S transition phase. DnaA is then taken over by GcrA in the stalk cell, dominant for much of the S phase. Predivisional cell has high levels of CtrA and GcrA with a stalk and flagellum and opposite poles, but after division, swarmer cell only has CtrA (other transcriptional regulators degraded by protease) while stalk cell ends up with mainly GcrA (CtrA rapidly degraded). The swarmer cell finds a new home while the stalk cell keeps budding off

Worm - fate map

The worm is able to be fate mapped (each cell progress tracked through time), we already know the lineage or each and every cell and the timing of 131 cell suicides (work done by Brenner, Sulston and Horvitz 2002)

Worm model organism

The worm, Caenorhabditis elegans is a free living microscopic nematode worm that is 1mm long, can be grow on petri dishes (easy to grow and culture). It is transparent, mostly hermaphrodites (XX) with minority males (XO) with a 3.5 day life cycle (egg to adult, able to go through generations rapidly). It has 979 somatic cells (unless it is mutated), with around 2000 germ cells

Master regulator cascade of Caulobacter - 2

There is cyclic accumulation of the global regulators CtrA, DnaA and GcrA. In swarmer cells, CtrA represses initiation of DNA replication (replication occurs when DnaA binds to Ori) and GcrA expression. At the swarmer - stalked cell transition, CtrA is proteolyzed (targeted to a pole then chopped up) resulting in liberation of Ori and PGcrA (GcrA promoter) and DnaA is expressed. Ori and the PGcrA are no longer silenced by CtrA and can be bound by DnaA, initiating DNA replication and GcrA expression. GcrA accumulates in the stalked cell activating genes responsible for DNA segregation, replication elongation and polar development, represses DnaA expression. In the S - G2 transition, the chromosome is almost fully replicated, GcrA starts CtrA expression (part of a two component regulator system, CtrA is phosphorylated by histidine kinase to activate). In turn CtrA shuts down GcrA expression in the late stalked cell (S-G2 transition) and CtrA binds to Ori (Ori silencing). In the predivisional cell, there is CtrA proteolysis in the stalked compartment and GcrA proteolysis in the swarmer compartment

Master regulator cascade of Caulobacter

There is expression of DnaA in the swarmer - stalk cell transition, DnaA, required for DNA replication, acting as a transcriptional activator that expresses around 40 genes required at this early stage. DnaA expresses GcrA genes, which is the next regulatory protein in this cascade, and negatively feedsback on itself (prevents transcription of DnaA). GcrA controls 50 genes, expressing genes required for metabolic processes (membrane and cell wall extension, growth) and acts on CtrA promoter that leads to the activation of CtrA. CtrA controls 95 genes that relate to motility (chemotactic apparatus, pili, and flagellum), represses GcrA (drop in GcrA is enhanced by certain proteases) and becomes dominant. CtrA represses the initiation of replication by directly binding to DnaA promoter to prevent expression. CtrA expresses a methyl transferase, methyl transferases (CcrM) methylates adenines in the DNA, some adenines are in the promoters of genes, depending on the gene the methylation leads to repression or activation. CcrM represses it's own promoter, represses of GcrA and activates DnaA promoters (unknown reason) and starting the cascade again

Mating types S. cerevisiae

There is three states a yeast cell can be, either a, α or diploid (a/α). Each state has different expressions for their different morphologies In the a cell, a-specific genes activated, α-specific genes silent, and haploid-specific gene activated by a1

Bacillus two component regulatory system

Two component regulatory systems (including Spo0A and kinases) discovered in bacteria around 1986 - 87. Two components are sensor histidine kinases (component that recognizes change in environmental condition) and response regulators. Activation of histidine kinase (external input signal) leads to autophosphorylation of histidine residue. Phosphoryl group is transferred to an aspartate residue (D) on the receiver domain of the response regulator to activate it. Response regulators are often transcription factors and now able to bind DNA (activate gene expression). In Bacillus, there are 5 histidine kinase sensors (kinase A - E), phosphoryl group transferred to Spo0F (response regulatory) to Spo0B (histidine phosphotransferase) to SpoOA (final response regulator, activates transcription of crucial sporulation genes)

URA3

URA3 encodes orotodine 5 phosphate decarboxylase (enzyme used in pathway of making uracil, which makes up nucleic acids). ura3 mutant strain requires uracil in the media, this can be used as a positive selection. URA3 strains is killed by 5-fluoro-orotic acid (FOA), which can be added in media for negative selection

Worm - use of cell ablation

We can use cell ablation, which is laser targeted destruction of chosen cells in anytime during development to study. The connections of its nervous system and nerve to muscle connections are all mapped (302 neurons). In worms, forward and reverse genetics (RNAi - interference RNA) is possible, can homozygously (hermaphrodites) mutate without crossing, easy to grow, maintain and can be frozen

YEp

YEp (episomal) contains E. coli genes/segments, yeast genes/segments, replication origins (replicating separate from the chromosome as an episome, can also integrate) and host yeast markers

YIp

YIp (integrative) contains E. coli genes/segments (for propagation in E. coli), yeast genes/segments (selectable marker genes) and host yeast markers (paired with marker gene). YIp doesn't have replication origins (must integrate into chromosome to survive). There is usually one copy that replicates with the chromosome, with a stable integration and expression

Yeast

Yeast has a rapid growth of 90 - 140 minute doubling, slower than E. coli (30 mins) but faster than mammalian cells (~2 days). Yeast grows well on chemically defined media, broth, plates (inexpensive) and defined selective media can be used (e.g. in labs). Yeast has dispersed cells, rather than filaments (most fungus grows as filaments), forming single colonies when plated from broth, with many colonies per place (can screen for mutants and phenotypes)