DEV BIO LAB EXAM 1

Wild type Ti Plasmid · The wild type Ti plasmid is too large for genetic manipulation. It is too large to easily purify, and contains too many restriction sites for it to be used as a cloning vector. · For this reason, the two component __________ system was developed. · One component is a large Ti plasmid, that has been "disarmed" This means that it has had the ________ removed. This plasmid is maintained in an Agrobacterium strain that is used for making transgenic plants. The _______ genes are on this disarmed Ti plasmid, the T-DNA is not. · The other component is a smaller plasmid, about 14 kb in size. It contains the ________ region with multiple cloning sites for insertion of ________ and a _________ used as a selectable marker for transformed plant cells. This smaller plasmid can be grown in both Agrobacterium, and E. coli. · Cloning and manipulation in E. coli is much easier than it would be in Agrobacterium. The transgene cloning manipulations are done using E. coli, and the final transgene plasmid is then inserted into Agrobacterium for mobilization into the cells of the leaf disks. · Because transformation involves the integration of the transgene permanently into the plant genome, and an entire plant is regenerated from the transformed plant cell (more on this later), this method of transformation is stable and can be passed on to future generations

"binary vector" T-DNA vir T-DNA transgenes resistance gene

Experimental design · MO will be used to knock down the expression of the ________ gene (SLC24A5) in Zebrafish. · SLC24A5 or "golden" gene encodes a protein that is localized in the membranes of melanosomes or their precursors. · If the injected embryos look like golden's, the morpholino injections will have been said to have ________ the golden mutation. · Phenocopy: A phenotype shown because of environmental effects instead of genotype. Does NOT change the DNA (copy the phenotype of genetic mutation but not the genotype) · The gene SLC24A5 is involved in human development as well. The ancestral human SLC24A5 allele is predominantly in African populations, while a variant allele predominates in European populations and is correlated with lighter skin color. Expected lab results: · Difference in pigment is apparent during the ________ stages of development. · MO golden knockdown (phenocopy) is apparent in young fish, while adult knockdown fish are only slightly different in color than wild-type zebrafish. · Thus, the knockdown leads to a __________ reduction in gene expression.

"golden" "phenocopied" early transient (temporary)

Gene knocks down mechanism B: blocking hnRNA splicing · If we create a morpholino that is complementary to the ________, then the intron can't be spliced out · hnRNA is processed in the _________ so morpholino needs to be in the nucleus · Presence of the intron can lead to: o An early termination codon o A frameshift o Other genetic effects that can produce a non-functional protein

5' splice site nucleus

Methods for introducing RNAi into different organism: · Direct uptake of RNAi expression vectors or siRNAs o Transfection by chemical uptake or electroporation · Feed the RNAi (only works with simple organisms such as Planaria and C. elegans) o Put RNAi into food (liver for Planaria, bacteria for C. elegans). · Microinjection, use micro-needle to directly inject RNAi expression vectors into nucleus, or siRNAs into cytoplasm · Transgenic organism expression vectors: o __________ based vectors for plants o __________ for animals

Agrobacterium Lentiviral vectors

· Tissue types affect photosynthetic genes · In some plants, cell-type affects photosynthetic gene o _________ Photosynthesis improves Carbon Fixation Efficiency by __________ CO2 within _____________ o In C4 plants, _________ is only present in chloroplasts of BS cells, where CO2 concentration is very high. Not found in _________ cells. o__________ is the initial CO2 fixation enzyme in C4 plants, not rubisco. Found only in mesophyll cells

C4 concentrating BS Cells (bundle sheath) Rubisco mesophyll PEPcase

Rubisco · Most abundant protein on earth · Most abundant protein in photosynthetic cells · Incorporates atmospheric __________ into an organic form o Primary enzyme of ________________ · Present in all photosynthetic and chemosynthetic (don't use light) organisms · Rubisco is in the __________ of the chloroplast · Composed of _________ Large subunits (LSU) and _________ small subunits (SSU). _______ total subunits to make complete enzyme (L8S8) · LSU encoded by _________ gene on chloroplast chromosome · SSU encoded by ________ gene on nuclear chromosome · Chloroplasts genes "talk" to nuclear genes and vice versa for coordination of expression (activity)

CO2 photosynthetic carbon fixation stroma 8 8 16 rbcL RbcS

RNA silencing was first found when trying to over-express a color-producing gene in transgenic petunia plants · __________ produces pigment in flowers · The scientist linked the CHS gene to Cauliflower Mosiac Virus 35 S (CaMV 35S) promoter to get very high levels of CHS expression in transgenic petunia plants · *CaMV 35S is _________, CSH should be expressed everywhere and at very high levels (makes abundant mRNA) · Why did this decrease pigment in petunia plants? CHS mRNA has a __________ in it's _________

Chalcone synthase (CHS) Constitutive stem-loop (double stranded region) 3/ UTR · Sequences in both endogenous CSH and transgene CSH recognized as viral invader

Components of RNAi · RNAi inducer o _____________ o Must be very ________ · ___________ o Type 3 endonuclease o Cleave precursor to form siRNAs · ____________ o 22 nucleotide small RNAs o Complimentary in sequence to target mRNAs · ____________ o Bind siRNAs to form RISC complex · _____________ o Brings siRNA to target sequence within selected mRNA o Leads to degradation of targeted mRNA · _________________ o Needed for amplification and systemic spread of RNAi

Double stranded RNA (dsRNA) abundant Dicer (DCR) Small interfering RNAs (SiRNAs) Argonaute proteins (AGO) RNA Induced Silencing Complex (RISC) RNA Dependent RNA Polymerase,

For the experiments we will perform in this lab course, we will modify the WNT signaling pathway by using two methods: LiCl treatment (zebrafish) and RNAi (Planaria) o LiCl inactivates ________. Low GSK3 ________ beta-catenin. § Promotes _______ development. This mimics ________ Wnt signaling. § This experiment is being done in zebrafish o RNAi inactivates beta-catenin. Promotes ________ development. This mimics _______ WNT signaling. This experiment is being done in planaria).

GSK-3 increases posterior high anterior reduced

RNAi · There are several ways in getting the double stranded RNA for RNAi o __________ o __________ · this double-stranded RNA is processed in the nucleus by ________ in animals and _______ in plants only if it's abundant · The abundant double-stranded RNA is exported from the nucleus by ________ in animals or _______ in plants. This leads to abundant DS RNA in the cytoplasm · Abundant DS RNA in the cytoplasm can also come from virus or we can feed dsRNA as we do in this lab · This dsRNA is chopped up into small fragments called _________ · This leads to RNA silencing

Hairpin/panhandle RNA Transgene Drosha DCL1 exportin hasty siRNAs

Low WNT activity: § _________ GSK3 § ________ beta-catenin § promotes _________ development. High WNT activity: § _______ GSK3 § ________ beta-catenin § promotes _________ development.

High reduced anterior Low High posterior

Things that affect the activity of photosynthesis genes · _________ is a major factor, photosynthesis is a light-dependent process · Dark grown plants are said to be in the __________ state o Smaller leaves and elongated stems o NOT green BECAUSE photosynthesis genes are turned on by light · Photosynthesis genes are affected by environmental toxins · Genetic mutations that affect ________ in chloroplast can also affect the color of the leaf · Disease can also affect photosynthesis gene · Seasons also affect photosynthesis gene o The chloroplast is converted to __________ which are not photosynthetic § Conversion happens because of cooling temperature and shorter days § The reaction center loses __________ but other pigment that are red/orange remains § The leaf always has these colors, it's just the chlorophyll is masking those colors

Light etiolated ribosome chromoplast chlorophylls

Calvin-Benson cycle · The bio energy produced from the _______ is used to power this cycle · A carbon molecule, __________ is also incorporated into this cycle · The CO2 is combined with a __________ molecule to produce _____________ · This cycle is repeated _________ times, each time incorporating 1 carbon. At the end of the 6 round, a ____________ compound will be produced. · The enzyme responsible for the incorporation of CO2 is called _______________

PET CO2 5-carbon 2 3-carbon molecule 6 6-carbon rubisco

Z-scheme of photosynthesis (light reaction/PET) o _____________ absorbs 2 photon which split water into hydrogen and oxygen leading to the elevation of ____________. § The hydrogen is used for energy capture and is used to power an _________ to convert ADP to ATP o Then the 4 electrons drop in energy state and are transferred to ____________ o Then another 2 photons is used to interact with photosystem I to elevate 4 electrons. o Then electron drops in energy state and the energy are used to convert ___________ to __________

Photosystem II 4 electrons ATPase photosystem I NADP+ to NADPH

Introduction of RNA silencing (RNAi) · Ancient process, present in all eukaryotes · May have evolved as a defense against _________ · Requires two things for activation: o Requires ___________: (examples below) o The double-stranded RNA must be __________ · Whatever the source, any abundant, double-stranded RNA is recognized by the cell as a viral invader and is targeted and degraded.

RNA viruses double-stranded RNA § Long or short double-stranded RNA molecules § Double-stranded regions § Hairpin loops very abundant

photosynthesis: _________ combine with _________ to form 2 3-carbon molecule called _________ via _________ o Net gain of 1 C and biomass increase photorespiration RUBp (5C) combines with _________ to form 1 3-carbon molecule called __________ and 1 2-carbon molecule called __________ via photorespiration o 2-P-Glycolate is toxic to plant o Net gain of 0 carbon and biomass loss o Releases ____________

RUBp (5C) CO2 3-P-glycerate carboxylation O2 3-P-glycolate 2-P-Glycolate CO2

Transient gene expression: biolistic "gene gun" transformation ____________ · a gene that is used to `tag' another gene or a DNA sequence of interest (such as a promoter/enhancer, an encoded RNA, or an encoded protein sequence in a transgenic organism · We use the following reporter genes in this class: o Green fluorescent protein (GfpA) o Red fluorescent protein (Rfp, also a version called mCherry) o β-glucuronidase (UidA, or Gus). · Reporter genes are not normally _________ in the organism being studied, so their expression can be distinguished from endogenous genes in transgenic organisms. · Gfp is from jellyfish · Gus is from bacteria. The plasmid used for transformation in this experiment transcribes a chimeric mRNA (made of regions from different organisms) that consists of the 5' untranslated region (5' UTR) of a photosynthetic gene from Flaveria bidentis. This is linked to the ___________ reporter gene, followed by the 3' UTR of this gene. High level constitutive transcription is driven from the ___________ promoter.

Reporter gene expressed GFP CaMV35S

T-DNA on the "armed" wild type Ti plasmid is about 24 kDa. It contains oncogenes and 25 bp direct repeat sequences at the ends. · The repeat sequences are necessary for the transfer of the ________ into the plant. · Anything between the ____________ can be transferred into the plant, regardless of its size. · Placing cloning vector sequences between the borders allows for the transfer of these transgene sequences into the plant. · For our experiment, we are adding _________ to produce a blue dye in the plants.

T-DNA left and right borders GUS

· Vir genes and T-DNA on same large __________ o These genes are said to operate in ______ meaning in this case on the same genetic element · _________ encoded on the Ti plasmid mobilize the T-DNA into the plant cells · Ti plasmid is about 200 Kb, about 1/10 the size of the bacteria chromosome · ________ enters plant cell nucleus, integrates into plant cell chromosome. Integration is random and can occur at multiple locations throughout the genome

Ti plasmid "cis" Virulence (Vir) genes T-DNA

In our experiment: agrobacterium · __________ and _________ on different plasmids. The vir genes are on the large __________, and the T-DNA is on a smaller "binary vector" plasmid. · In this case, these sets of genes are said to operate in ________ meaning on different genetic elements ·

Vir genes and T-DNA Ti plasmid "trans",

· _________ signaling pathway is critical for regeneration to work in Planaria. Why? o The regeneration of the posterior portion requires a __________ wnt environment o The regeneration of the anterior portion requires a ________ wnt environment o WNT establishes a gradient in the planaria with the ________ concentration at the tip of the tail and _________ as you go towards the head Disrupting canonical WNT signaling affects regeneration · We will disrupt WNT signaling by inactivating Beta-catenin using Schmidtea mediterranea as our experimental system · We will inactivate __________ using double-stranded RNA to induce RNAi (also called RNA silencing)

WNT high low highest decreases beta-catenin

Wnt Signaling pathway: · Name is a fusion of the names for the genes Wingless (Wg) in Drosophila) and its ortholog Integrated (Int) in vertebrates. · An ancient and evolutionarily conserved signal transduction pathway. · Passes information from one group of cells into other cells through receptor on the surface of the target cells. Thus, WNT signaling involves cell to cell communication. · It is initiated by the binding of a _______ protein to its receptor on the cell membrane. · This binding can initiate several different pathways to activate WNT-responsive genes in the nucleus. · This pathway regulates many crucial aspects of cell fate determination. · For this lab, we are focusing on ___________ regulation by WNT signaling. · Canonical and Non-canonical form of Wnt pathway

WNT anterior/posterior

Wnt pathway key components: · _______: A family of cysteine-rich glycoproteins involved in many aspects of developmental signaling · _________: A transmembrane receptor that binds to Wnt signaling factor · _________: In the absence of WNT it interacts with Frizzled receptor (Inactive in this form.) o In the presence of ______, DSH is released from Frizzled, becomes active o When active, interacts with and inactivates _________ · _________: A kinase that targets Beta-catenin for degradation. o It is active in the absence of DSH (- WNT) o It is inactivated by active DSH (+WNT) · __________: A protein that activates transcription factors that transcribe nuclear genes involved in posterior development

Wnt Frizzled Disheveled (DSH) WNT GSK3 Glycogen synthase kinase 3 (GSK3) Beta-catenin

o High levels of __________ and ________ in the transformed plant cells leads to uncontrolled cell division and cell elongation, producing a tumor made of undifferentiated plant cells from the initial infection site. This mass of undifferentiated plant cells is called a _________. o The transformed cells themselves are called "callus cells" o *High levels of nopaline production within the transformed crown call cells provides a food source for the bacteria growing within the cells, which is catabolized by the __________ genes on the nopaline Ti plasmid o *Note that an octopine type plasmid would have Octopine synthesis genes, which would be catabolized by matching octopine catabolism (Occ) genes on the octopine Ti plasmid. o None of the oncogenes on the T-DNA are expressed in the bacteria cells, they are only expressed in the plant cell once they have been integrated into the genome. This is because they have _________ promoter/enhancer and terminator elements and since bacteria are prokaryotes, they can't use them.

auxin and cytokinin crown gall nopaline catabolism (Noc) Eukaryotic

· Leaf explants require added _________ and ________ to grow, if you put the leaf on a MS media with no auxin or cytokinin, they will eventually die. On the contrary, if you take ________ put them on MS media, the callus cells continue to divide and elongate because they make their own auxin and cytokinin. This means the crown gall show ___________.

auxin and cytokinin crown gall cells hormone-independent growth

The plant hormones ________ and _________ are the basis for regeneration of intact plants from leaf disks, or other plant tissues · __________ media is commonly used for plant tissue culture. It contains basic salts and nutrients needed for plant growth (similar to fertilizer, but more precise and defined). For petri plates, it is solidified with agar or phytogel. · When added to the MS media used for plant tissue culture, auxin and cytokinin can determine plant growth and differentiation · Functionally equal amounts of auxin (usually _________) and cytokinin (usually _________ or ______) in the MS media will stimulate cell ______ and cell ________, respectively. This produces _______, the same as the undifferentiated growing callus cells of a crown gall tumor. Both processes utilize the same plant hormone regulation. Remember, cytokinin = auxin = callus

auxin and cytokinin Murishigee and Skoog (MS) indole acetic acid, IAA kinetin or zeatin elongation division callus

Bring it all together, infection with Agrobacterium binary vector and regeneration of transformed plants. 1. Leaf disks placed in solution with Agrobacterium with ____________. 2. Agrobacterium infects edges of leaf disk, which is __________ tissue 3. Place onto solidified MS media with __________ to stimulate callus growth. 4. At first, grow without any selection (no antibiotics at this stage), to allow the agrobacterium to grow with the leaf disks. This is called ___________ 5. Some of the cells at the leaf edge will be transformed with the binary vector, which will insert _________ resistance and the transgene. In this case, a ___________ gene under the control of a CaMV promoter/enhancer 6. Wash and plate onto new media with antibiotics to kill any remaining Agrobacterium and select for kanamycin-resistant transformed plant cells 7. After washing the leaf disk, the transformed disks are placed onto selective (+Kan) regeneration media. 8. Then we transfer them onto another MS media a. MS media with two key plant hormones cytokinin, and auxin b. *High cytokinin: auxin will stimulate shoot formation c. *Kanamycin to select for transformed cells d. *_________, an antibiotic to kill off all of the remaining Agrobacterium. 9. Shoot formation from the original transformed cells. Any shoots that grow are Kanamycin resistant, and therefore transformed

binary vector wound Plant hormones co-cultivation kanamycin B-glucuronidase (GUS) Timetin

Wnt signaling: Non-canonical pathway · Wnt signals _________ release (efflux, from membrane or another compartment). · Drugs such as praziquantel (PZQ, a worming medicine for pets) _______ calcium release (causes influx, to the membrane or other compartments). · Affects anterior/posterior development (overall, promotes anterior, reduces posterior). · Possible mechanism: PZQ equalizes calcium gradient. This is antagonistic to beta-catenin. · This mimic (to some extent) reduced WNT activity, promoting anterior development (or, interfering with posterior development).

calcium inhibit

Leaf cross section · The __________ surrounds the plant cell · The _________ is in the middle of the cell and holds waste · Chloroplast is green due to ________ and the nucleus is stained _________. The chloroplast also has blue spots because they also have their own ________. Cytoplasm and vacuole are black · Nucleus encoded genes begin with an ________ case letter while other organelles encoded genes like in the chloroplast start with ________ case letter.

chloroplast vacuole fluorescent blue DNA UPPER lower

Agrobacterium tumefaciens and crown gall tumor disease · A ___________ develops at the wound site · The crown gall is a growing mass of undifferentiated cells called ________ · Callus cells produce excess amounts of a rare amino acid called an _________. Could be either Nopaline, Octopine, or Agropine. · Only Agrobacterium containing the appropriate _________ can utilize the opine. Plant cells cannot utilize the opine. Other than the appropriate Agrobacterium strain, no organisms in nature can utilize the opine · The Agrobacterium cells live and multiply within the crown gall cells and use the opine as to a unique specialized food source. · The crown gall cells also produce excess amounts of the plant hormones called ________ and _________. These cause undifferentiated cell growth that leads to crown gall formation. · The undifferentiated, unregulated growth defines the crown call as a plant tumor. It is caused by the Ti plasmid of Agrobacterium. · Eventually, the gall uses up the plant nutrients/resources. When the plant dies, the gall degrades, and all the Agrobacterium cells are released into the soil to start in the infection cycle again. · Note that the base of the plant stem, near the ground, is called the "crown". Since this is where the tumor is usually found, it is called a crown gall. However, the tumor can actually form anywhere on the plant where a wound occurs.

crown gall (aka callus) callus cells opine Ti plasmid auxin and cytokinin

C. Elegans · C. elegans is a nematode, a small roundworm about 1 mm in length. · It is one of several model organisms used to study developmental processes. · There are several advantages to this organism that make it a highly useful model for developmental research. o It has a rapid life cycle, about 16 hours o Its small size and rapid generation time allow it to be grown and propagated in petri dishes. o C. elegans has a small number of genes for a complex cellular organism, only about 3000. This is 35 times less than in mammals. o It has relatively few cells and cell types. The body of an adult hermaphrodite contains exactly 959 somatic cells, with each cell lineage precisely mapped. Unlike vertebrates, the cell lineage between individuals is identical. · Most individuals are hermaphroditic, which means they produce both eggs and sperm, and are capable of both cross-fertilization and self-fertilization. There are also males. Female can reproduce without male · Like Planaria (see experiment #5), RNAi can be introduced into C. elegans simply by feeding _________ to reduce the expression of a particular gene of interest. Experiment: · C. elegans will be fed double-stranded RNA corresponding to two different developmental genes: o One gene encoding ___________ § RNAi will disrupt normal cuticle development o Another gene encoding _________ § RNAi will disrupt normal body growth and development

double stranded RNA Blistered (Bli-1) Dumpy (Dpy-11)

How does RNAi work? 1. Dicer recognizes abundant ________ in the cytoplasm 2. Dicer cuts the dsRNA into 21-22 nt ________ 3. The siRNAs associate with the _________ to form the RISC complex 4. RISC finds complementary target sequences in __________ or _________ 5. Then the RISC complex cuts it up through ________ cleavage 6. Leads to RNA degradation and gene silencing 7. Some of the siRNAs are used as primers to produce new copies of RNA by __________ a. These are clip by dicer and some goes back into the RISC complex and some interact with __________ to be transported to other cells leading to systemic spread of RNAi

dsRNA siRNAs argonaute protein mRNA or viral RNA endonuclease RNA dependent RNA polymerase transporter proteins

Experiment: · This plasmid is expressed in bacteria. To get RNAi into the planaria, the bacteria expressing the _________ promoter plasmid are mixed with ________ and fed to the planaria Schmidtea mediterranea is a very attractive model organism for studying regeneration. Advantages of using S. mediterranea include: · the low cost to keep, maintain, and expand a sufficient number of animals in the laboratory · a recently sequenced genome · established tools and methods for loss-of-gene-function studies via RNAi.

dual liver

The chloroplast has an outer membrane covering an inner membrane · Inside the inner membrane are: o Stacks of _________ which are made up of _________ § Thylakoids are disc like to _________ exposure to sunlight o Chloroplast chromosome circular DNA o _________: transcription and translation occur in the chloroplast o ___________: aqueous fluid that fills the lumen of chloroplast · Thylakoid o Within the membrane are photosynthetic proteins that go through a series of __________ reaction also called __________ of photosynthesis because of how they are arranged or ___________ or __________ § This reaction produces __________ that goes into the __________ of the chloroplast and power the _____________ to produce 6 carbon sugars

granum thylakoids increase Ribosome Stroma electron transport Z-scheme light reaction PET bioenergy stroma Calvin cycle (dark reaction)

Advantages of MOs · Because of their structure, and the lack of a negatively charged backbone, they are resistant to __________ and are very stable within the cells. · They do not react with other components of the cell, and therefore, for the most part, are non-toxic and show no side effects. · For experimental organisms without a stable transformation system, they provide a rapid and easy way to selectively inhibit gene expression for developmental studies. Disadvantages of MOs · ___________ is sometimes difficult, due to experimental variability inherent in the microinjection procedure. · As with RNAi (RNA silencing), off-target effects (interacting with an RNA with a similar sequence) can be a problem. This can lead to the unintended inactivation of genes other than the target gene. · Can most easily be used with very early embryos. Usefulness with __________ organisms is currently limited (although protocols for use in cancer and gene therapy are under investigation). · As a transient expression system, repression of gene expression is not permanent or heritable.

nucleases Reproducibility adult

What are morpholinos (Mos) · MOs are synthetic _________ composed of chains of about 25 subunits. · Subunits are like DNA and RNA oligonucleotides; except they have a _________ ring rather than a ribose ring. o Ribose has a _______ charge from the phosphate and the morpholinos are _________ · Morpholinos (MOs) can bind to ________ and _________ · MO bases can base pair with nucleic acids. · Backbone structure is different; thus, cellular enzymes or proteins cannot recognize or interact with the MOs. · MOs do inhibit gene expression but not inhibited by the same mechanism as RNAi (i.e the RISC complex is not involved). o This is entirely due to __________which inactivates RNA o There is no degradation of MOs or the target RNA

oligonucleotides Morpholine negative non-charged ssDNA and RNAs base-pairing Morpholino (MO) Injections · Up to 1 ng of single stranded MO RNA (independent of mRNA length) can be readily injected into a zebrafish without major consequences. · In contrast, 100pg of dsRNA or DNA will kill most zebrafish embryos. · Typically injected into the center of the yolk to reduce chance of secondary effects due to disruption of the early blastomeres. · Usually injected between the 1- and 8-cell stage.

Why can the callus cells grow on hormone-free media? · They can grow because of the __________ that are present on the T-DNA. These are expressed in the transformed plant cells o Note that the T-DNA has 25 nt left and right borders. These borders, and everything in between, is transferred and integrated into the _________ o ______ and ______, = cause high levels of indole acetic acid (IAA), an auxin, in the transformed plant cells. This enhances cell elongation. o _______ = causes high levels of zeatin, a cytokinin, in the transformed plant cells. This enhances cell division o _______ = nopaline synthesis, causes the production of the rare amino acid nopaline in the transformed plant cells

oncogenes plant genome *IaaA and IaaH *IptZ *Nos

Advantages and Applications of C4 Photosynthesis o More efficient carbon assimilation o No photorespiration § Because __________ is not pumped into the BS cells where rubisco is located o Resistant to high temperatures and arid conditions o Highly efficient nitrogen utilization o Increased adaptability to marginal conditions o Reduced 13C to 12C isotope discrimination (Bioarchaeology) · Applications o Introduce C4 characteristics into C3 plants (C4 Rice?) o C4 photosynthesis provides high productivity in plants considered for biofuel production o Adaptation to environmental change brought about by global warming

oxygen

Gene silencing in transgenic plants · The __________ vector is used · Identify the region we want to target then we amplify it by PCR with different _________ on the end · Then theses regions are put into an _________ on side of an intron in the opposite orientation · ________ and ________ strands are on opposite sides of the intron, within a transcription unit · In the plant, the intron is spliced out, leaving a _________ o The intron produces a tight interaction between the sense and antisense strand in plants · Intron enhances the formation of DS RNA, probably allows for more flexibility before it is spliced out

pHannible restriction sites MCS (multi cloning site) Sense and antisense DS hairpin loop

Transgenic plants produced from Agrobacterium tumefaciens: · Agrobacterium tumefaciens occurs naturally in soil. It is a _________ of a group of higher plants called dicots. · In addition to its chromosome, it contains a very large plasmid called the _____________ · It is a very large plasmid, of more than 200 kilobases (200 kb). This is 1/10 the size of the chromosome. · The TI plasmid contains several genes, including genes involved in pathogenesis · As part of the disease process, the bacterium causes a tumor on the plant. The genes responsible for tumor formation are called _________. The oncogenes are located on a DNA fragment called the ________ located on Ti Plasmid. The T-DNA itself is 24 kb and is transferred from the bacterium into the plant during infection. This is a natural example of genetic engineering. The oncogenes integrate into the plant _________, becoming a permanent part of the genome. · The genes involved in transfer from bacteria to plant are called the ___________. These genes are also on the Ti plasmid. The Vir genes do not transfer into the plant cells, unlike the T-DNA. · The infection process requires ________ plant tissue. The bacteria cannot penetrate intact tissues, it must enter through wounds. · For Agrobacterium strains used for transgenic plants, their Ti plasmid has been modified. The oncogenes have been removed. This is called a _________, and it cannot cause disease symptoms on the plant. · The disarmed Ti plasmid works with another plasmid, which contains the gene of interest that is going to be transferred into the plant. Together, the two plasmids make up a ________

pathogen Tumor inducing plasmid (Ti plasmid) oncogenes T-DNA host chromosome Virulence genes (Vir genes) wounded "disarmed" Ti plasmid "binary transformation system"

What do photosynthesis genes do? · Encode proteins that enable _________ function. photosynthesis consists of 2 different reactions what are they called?

photosynthetic · Photosynthetic electron transport (PET) also called light reactions · Calvin-Benson Reactions also called dark-reactions

Planaria · are group of widely distributed free-living flatworms in the class Turbellaria (phylum Platyhelminthes). They live in freshwater. The word Planaria itself refers to one genus, but the name planarian is used to designate any member of the family Planariidae and related families. For this lab, we use Schmidtea mediterranea · Planaria live naturally as scavengers and predators to small protists at the bottom of ponds or streams and are sensitive to pollution and can only live-in clean waters. · In the lab, they live in Petri dishes filled with spring water · Planaria are often thought of as just simple flatworms. In fact, they are very complex animals. Planaria have organs and organ systems, including a brain with left and right "lobes" that coordinate activities of the animal as a whole. The brain is connected to ventral nerve cords that extend to the posterior of the animal. Planaria also have chemoreceptors, photoreceptors, mechanoreceptors, and a complex digestive system · These animals have evolved a remarkable stem cell system for ____________. · Several research groups are developing planaria as a model system for developmental studies. · This is because of their remarkable capacity to regenerate. · The adult animals have stem cells that are called ___________. The __________ is the chemoreceptor in planaria · These are very abundant in the animal; in fact, about 20% of their cells are neoblasts! Even neoblasts located far from the head can regenerate a brain that then connects functionally to preexisting nerve cords, as well as all of the other complex parts that make up the intact organism. · A long-term goal of planaria research is to understand their remarkable regeneration process, so that someday maybe some of that regenerative ability can be made to work in people. · Can regenerate from different fragments o Head, regenerates new posterior section o Tail, regenerated new anterior section' o Center, regenerates new head and tail

regeneration neoblasts auricle

Gene knocks down mechanism A: blocking translation · The complementary MO binds to the mRNA preventing the movements of _________ from going down the mRNA to stop translation

ribosome initiation complex

Cytokinin stimulates _______ formation, while auxin stimulates ________ formation. · Reducing the amount of auxin relative to cytokinin in the MS media (or increasing the ratio of cytokinin to auxin) leads to shoot differentiation. Remember, cytokinin >auxin = shoots. · Increasing the amount of auxin relative to cytokinin in the MS media (or decreasing the ratio of cytokinin to auxin) leads to root differentiation. Remember, auxin > cytokinin = roots.

shoot root