MCDB 423 Lecture Notes - Lecture 1: Oncogene, Myc, Sox2

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purplebeaver856

5 May 2017

School

U of M

Department

Molecular, Cellular and Developmental Biology

Course

MCDB 423

Professor

John Kuwada

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Document Summary

Bodies are made up of a lot of cell types that arise from stem cells. A stem cell is unique because of the way it divides. When it divides, it gives rise to two progenies like any other cell. But one of those progenies has to be a stem cell. The other can be a stem cell or a more specialized cell. A cell that gives rise to every cell type is a fertilized egg. The fertilized egg is not really a stem cell because it is not self rege(cid:374)erati(cid:374)g (does(cid:374)"t gi(cid:448)e a fertilized egg in return) To test the two statements above: take an early embryo to get embryonic stem cells and assay to see what the cells will give rise to following division. You start with early embryo and dissociate the cells and then mark them in some way (molecularly, genetically, or physically with dye) The point is that this identifies the cells.

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Summary

please!

Multipotent versus restricted

Morphologically detectable neural crest cells usually are first observed as the cells individualize and delaminate upon emigration from the neural tube, following their epithelial to mesenchymal transition (EMT), in which they convert from a tightly adherent sheet of cells to a dispersed and more individual mesenchymal population. Prior to EMT, however, it is difficult or impossible at most axial levels and in most species to distinguish presumptive neural crest cells from cells that will form dorsal neural tube derivatives. However, there are exceptions, such as a subpopulation of cranial neural crest cells in chick and mouse that appears to be set aside and morphologically distinct at midbrain levels. In addition, some species such as axolotl have a clearly segregated population of neural crest cells that exists as a ridge on the dorsal neural tube.

A long-standing debate in the neural crest literature has been whether neural crest cells are multipotent and/or restricted in their developmental potential. In other words, can a single neural crest precursor form only one type of derivative or are the cells multipotent and able to produce multiple derivatives. Single cell lineage experiments (Bronner-Fraser and Fraser, 1988), in which individual cells within the chick dorsal neural tube are labeled with vital dye, show that some of the labeled clones contribute to multiple differentiated cell types in the periphery, including melanocytes, sympathetic and sensory ganglion cells. Thus, the original precursor was âmultipotentâ in its developmental potential to form neural crest derivatives. In addition to neural crest derivatives, single dorsal neuroepithelial cell also give rise both to migrating neural crest cells and cells that remain in the dorsal neural tube, such as roof plate cells and dorsal sensory neurons and/or interneurons. This suggests a shared lineage between the neural tube and neural crest, at least at this stage. However, labeling migrating neural crest cells in vivo also produced clones that could contribute to more than one neural crest lineage (Fraser and Bronner-Fraser, 1991), again supporting the idea that some of the migrating population retained multipotency.

Clonogenic culture of neural crest cells cultured shortly after their emigration from the neural tube definitely shows that many early migrating neural crest cells are multipotent in vitro as well (Baroffio et al., 1988, Calloni et al., 2007, Calloni et al., 2009, LeDouarin et al., 2008a, LeDouarin et al., 2008b, Sieber-Blum and Cohen, 1980 and Stemple and Anderson, 1993). Exposure to different growth factors can profoundly influence their lineage decisions (e.g. Lahav et al., 1998). Furthermore, they have a capacity for self-renewal, at least for a few cell divisions (Stemple and Anderson, 1993 and Trentin et al., 2004).

The fact that individual neural crest cells can form multiple derivatives has led to the idea that they have stem cell properties. Stem cells are defined as individual progenitor cells that can generate one or more specialized cell types. A cardinal feature of stem cells is their ability to self-renew, that is, to divide so as to give rise to at least one daughter cell that maintains the multipotent character of its parent. The fact that cloned neural crest cells have a limited ability to self-renew has led to the idea that they are stem-like (progenitors) cells rather than true stem cells. Interestingly, however, neural crest stem cells can be derived from adult tissues (Fernandes et al., 2008 and Shakhova and Sommer, 2010; see Chapter Dupin and Sommer), suggesting that they can remain quiescent for long periods of time or maintain long term self-renewal ability when left in situ.

The presence of some multipotent neural crest precursors cannot, however, rule out the possibility that other precursors may be more restricted in their developmental potential. In fact, experiments in zebrafish suggested that neural crest cells contribute to different sets of derivatives accordingly to their migration order (Raible and Eisen, 1994). However, if the leader cell was ablated, the next cell in line took up the fate that would have been filled by the ablated cell (Raible and Eisen, 1996). Similarly, early migrating neural crest cells normally exhibit a broader range of derivatives than later migrating cells; however, when the early population is ablated and replaced by late migrating cells, the late migrating cells assume a broader developmental potential than that prescribed by their normal fate (Baker et al., 1997). This raises a very important issue: that developmental potential is greater than or equal to a cell's normal fate. Only by challenging the cell by putting it into a new environment can one test for restriction of cell fate. This is best exemplified by experiments in which the potential of neural crest populations was challenged by performing heterotopic transplants between different axial levels (LeDouarin and Teillet, 1974), such as exchanging cranial and trunk, or vagal and trunk populations. The results demonstrate a combination of flexibility in cell fate and some axial level-autonomous characteristics. For example, cranial neural crest cells normally make cartilage and bone of the face whereas trunk neural crest cells do not. Transplantation of cranial neural folds to the trunk results in production of many normal trunk derivatives, as well as the formation of ectopic cartilage nodules (Le Lievre et al., 1980 and LeDouarin and Teillet, 1974). Conversely, transplantation of trunk neural folds to the head results in contributions to cranial neurons and glia of cranial ganglia, but not to cartilage, although some connective tissues and pericytes derive from this graft (Nakamura and Ayer-le Lievre, 1982). This reveals some flexibility in fate, but a more limited ability to form skeletal derivatives (LeDouarin et al., 2004). However, trunk neural crest cells can form cartilage in vitro under appropriate culture conditions (Calloni et al., 2007 and McGonnell and Graham, 2002). Because challenging prospective neural crest fate is a difficult experiment, it is much easier to prove multipotency than restricted cell fate, leaving the question of whether or not there are lineage-restricted neural crest precursors still open to debate (see Krispin et al., 2010).

Exercise #5: The Nervous system and Perception

Task #1: Color vision

Introduction

In the human retina, there are two kinds of light-sensitive cells: rods and cones. These cells are located on the outside of the retina, away from the vitreous humor and against the choroid layer, and are covered by a later of bipolar and ganglion cells as well as neurons. Light enters the posterior chamber through the lens and must pass through several cell layers before it is absorbed by the rods and cones.

Cones are involved in color vision at daylight intensities. Generator potentials in a cone excite one neuron, giving high-level resolution vision. Cones will not cause neural firing at low light intensities and are, therefore, nonfunctional at night.

Rods provoke black-and-white vision at low light intensities. A bipolar cell associates with several rods, so that generator potentials sum in their effect. This assures neuronal firing at low light intensities and provides a highly sensitive vision but with less resolution.

Receptor convergence. A ganglion cell, which receives input from a number of rods, spread out over the retina, cannot pinpoint the source of a light stimulus as well as a ganglion cell, which is stimulated by a single photoreceptor. In the case of a ganglion cell receiving the added input of several rods, light hitting any one of the rods can cause a neural spike in the ganglion cell, but the brain has no way to know which rod was excited. It is therefore harder to pinpoint the location of light stimuli on the retina when the stimulus is detected by rods (as compared to cones). The result is that our ability to see detail, or our resolving power, is considerably lower under dim light conditions than under bright light conditions.

Dark adaptation. If you go from a lighted area to a very dark room, you will not be able to see. After about ten minutes, however, you begin to be able to see again. This occurs because the rhodopsin pigment, which gets broken down when it absorbs light, is continually re-synthesized in the rods. When there is very little light, very little rhodopsin gets broken down and the amount of rhodopsin gradually builds up, making the rod much more sensitive to dim light.

When light strikes a receptor cell, it triggers a photo-chemical reaction that leads to neuronal firing. If a rod or cone receives high-intensity light, the visual pigments will be temporarily bleached and will not absorb any more light. Within a matter of a few seconds, enzyme systems in the cells will restore the visual pigment to its light-absorbing form. This can be easily demonstrated. If, after viewing a bright light, the eye is quickly shut or turned to a dark wall, the bright image will persist as a positive afterimage because the high generator potential causes the continued firing of neurons. If the eye is cast on a lighter background after a few seconds, a negative afterimage, or a dark image of the object will appear. This is because the still bleached cells are not receptive to the light coming from the light background.

According to the Young-Helmholtz theory of color vision, there are three types of cone cells, which respond respectively to red, green, and blue light.

All other colors are perceived as the brain interprets impulses coming from a mix of these receptors. If an object of one color is viewed for a long period of time at high light intensity, the cones for that color will become bleached. The afterimage of the object will be seen in the complementary color.

The Exercise 6 PowerPoint will demonstrate afterimages to you. Use the slide show option to project the square of red light on your entire screen in a dark room. After staring intensely at the square for 30 seconds or so, without shifting your gaze, click enter to change the slide to a soft white. Continue staring at the screen as the change is made. What do you see? What was the color of the afterimage?

Repeat this experiment using a blue square. Before doing the experiment, hypothesize what color the afterimage will be according to the Young-Helmholtz theory.

Hypothesis to be tested: (2 pts)

What did you see when the experiment was conducted? (2 pts)

Was you hypotheses accepted or rejected? (1 pt)

Explain the colors seen in these afterimages using the Young-Helmholtz theory. (5 pts)

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2.Conclusion:

What have you learned from your study?

Interpret your data/ results:

Do your results support of disprove your hypothesis?

Compare and contrast your result with those of previous research.

Significance of the results: âBig Pictureâ

What is the global impact of your results?

What questions remain unanswered?

What kinds of experiments can come out of your study?

i have lab for biology and i need help with the Lab Report

the lab name is Bacterial Transformation Lab

1. Introduction:

Introduce the background and the nature of the problem your experiment is investigating.

Summarize the current knowledge of your subject.

Use examples of other research that have examined the same or a similar topic as your experiments.

Ending your introduction:

State your hypothesis

State your predictions

Give a brief statement about how you tested your hypothesis.

please help me with it is important for my lab class tomorrow

that is the lab i put the info please help me for the intro and for number 2 please be clear please

i would be happy if you help me it is due tommorrow i realy need your help on the intro and number 2 please be clear and show clear work hope you do it because it is due tomorrow

Bacterial Transformation Lab

Genetic engineering is one of the newest applications of basic knowledge in biology. It involves the isolation of specific genes from one organism and the insertion of those genes into a second organism of the same, or even different, species. Remember that a gene is a piece of DNA which codes for a product, usually a protein, which gives an organism a particular trait. The development of these techniques has generated considerable excitement and answered many questions, but it has also raised questions. Many scientists and others see genetic engineering as offering opportunities to make life better, for example, by exploring how genes are regulated, by curing diseases of genetic origin, by adding desirable traits to agricultural crops such as the ability to fix nitrogen in corn and pest resistance in cotton, and by genetically altering bacteria with genes enabling them to digest oil spills.

For almost 100 years, it has been possible to isolate DNA from cells, but only in the last 40 years or so has its function been understood. Your textbook discusses the work of such investigators as Fred Griffith and Oswald Avery, who demonstrated that if DNA was isolated from a pneumonia-causing strain of bacteria and added to cultures of nonvirulent bacteria, the recipient cells were transformed into virulent types that caused pneumonia. These transformation experiments were the first to show that genes could be artificially transferred in the laboratory. Since the time of those experiments (Avery's work was done in the 1940s), our knowledge of gene structure, function and control has increased astronomically. The application of this knowledge has created the field of genetic engineering.

Modern genetic engineering techniques depend on three critical factors:

A suitable host organism in which to insert the foreign gene;

A vector to carry foreign genes into the host; and

A means of isolating the host cells that have taken up the foreign gene.

The host organism

The most commonly used host organism is the gut bacterium Escherichia coli (E. coli). Its chromosome is a single large circular DNA molecule, called the genomic DNA, containing about 4 x 10⁶ base pairs. Not every cell will take up foreign DNA when exposed to it, but E. coli grows rapidly, dividing in less than 20 minutes, so that a single cell can give rise to a billion genetically identical descendants in a matter of 10 hours. Thus, a few transformed cells can provide a large number of offspring within a day. The strain we are using is K-12.

The vector

A vector is a DNA molecule used as a vehicle to carry genes from one organism to another. This DNA molecule can be modified by gene-splicing techniques using restriction enzymes and ligases so that it carries the genes of interest (essentially a cut-and-paste operation). Sometimes viruses are used as vectors, but for E. coli, plasmids are often used. A plasmid is a small circular DNA molecule containing 1000 to 200,000 base pairs. Plasmids exist naturally in many strains of E. coli and other bacteria. Plasmids replicate as the cell grows, independent of replication of the genomic DNA. These plasmids are transmitted to each of the product cells during cell division. Plasmids carrying genetic information that is beneficial to the host cell are maintained in a given population by natural selection. Plasmids frequently carry genes that confer antibiotic resistance, an obvious benefit.

When E. coli cells are exposed to plasmids or any other foreign DNA, some of the cells, called competent cells, will take up the DNA. We can induce cells to become competent by treating them with divalent cations, such as Ca⁺⁺, followed by a brief exposure to high temperature, called a heat shock treatment. Exactly how this treatment causes cells to become competent is not understood, but it is described as making the membranes "leaky" so that large DNA molecules (like plasmids), which would not normally pass through the cell membrane, can enter the cell. This treatment does not make all cells competent, but it greatly increases the number of competent cells. Once in a cell, the plasmids replicate and are passed to the product cells during cell division.

The selective medium

The last requirement for genetic engineering is a means of isolating host cells that have taken up the gene of interest. The techniques used depend on the growth requirements of the host organism. For example, each species of bacteria has particular nutrient requirements. The composition of the growth medium (agar or broth) can be modified to allow growth of only certain bacteria -- for example, by adding or removing nutrients or other chemicals from the medium mixture. This modified medium is called a selective medium, because it selects for the growth of only some species. By introducing an antibiotic such as ampicillin into the medium in which the bacteria are grown, you will select for growth of only those bacteria carrying the antibiotic-resistance gene on newly acquired plasmids. In a matter of days, billions of antibiotic-resistant cells, all with the plasmid, can be grown.

Genetic engineers use a modification of this technique to insert a foreign gene into E. coli. In simplified terms, the plasmid (already carrying an ampicillin resistance gene) is modified by inserting the gene of interest. The gene of interest has been isolated from another source; for example, the gene for human insulin has been isolated from the human genome. The modified plasmid, carrying the inserted gene, is then mixed with competent E. coli and the bacteria are placed in a medium containing ampicillin. Only those cells that have the plasmid will grow in the medium, and they are also the cells carrying the insulin gene (in their plasmids). These selected cells can now be used to manufacture insulin, which can be used for insulin therapy by diabetics.

The genes

In the following experiment, you will insert a plasmid into E. coli. The plasmid you will use carries the gene bla, which codes for a protein (beta-lactamase) that is secreted by the bacteria, inactivating the ampicillin in the agar and allowing for bacterial growth. The plasmid also carries an indicator gene. The indicator gene we are using was isolated from a bioluminescent jellyfish, Aequorea victoria. It codes for Green Fluorescent Protein (GFP).

The GFP gene has been modified to include a regulator system that can be used to control the expression of the gene. Gene expression in all organisms is carefully regulated to allow for adaptation to differing conditions and to prevent wasteful overproduction of unneeded proteins. The genes involved in the breakdown of different food sources are good examples of highly regulated genes. For example, the sugar arabinose is both an energy source and a carbon source for bacteria. The bacterial genes that make digestive enzymes to break down arabinose for food are not expressed when arabinose is not in the environment. But, when arabinose is present, these genes are turned on. When the arabinose runs out, the genes are turned off.

Arabinose initiates transcription of these genes by promoting the binding of RNA polymerase. In the genetically engineered pGLO DNA, some of the genes involved in the breakdown of arabinose have been replaced by the jellyfish gene GFP. When bacteria with this plasmid are grown in the presence of arabinose, the GFP gene is turned on and the bacteria glow green in UV light. When arabinose is absent form the growth medium, the GFP gene remains turned off and the colonies appear white. This is an example of the central molecular framework of biology in action: DNA ? RNA ? protein ? trait.

In summary, you will render E. coli cells competent to take up plasmid DNA. The plasmid carries two identifiable phenotypic markers:

The beta-lactamase or bla gene codes for resistance to the antibiotic ampicillin. We will use this to select for bacteria that have taken up the plasmid.

The pGLO gene codes for Green Fluorescent Protein (GFP), which is switched âONâ if arabinose is present.

Safety Procedure

For your own safety and the safety of others, it is essential that you follow all instructions about handling bacteria and disposal of materials. When you finish work for the day, swab your table with disinfectant. WASH YOUR HANDS thoroughly with soap before leaving the lab for any reason, and at the end of the day after you have cleaned your table. Report any spills to your instructor.

List of Materials

Prepared agar plates (2 LB, 2 LB/amp, 1 LB/amp/ara)

Transformation solution (50 mM CaCl₂, pH 7.4)

LB broth

1 pack of inoculation loops

Sterile transfer pipets

1 foam microtube holder w/ tubes

E. coli starter plate

1 red biohazard bag for loop disposal (common use â Loops/pipets only - NO PAPER!!!)

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MCDB 423 Lecture Notes - Lecture 1: Oncogene, Myc, Sox2

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