Cell Biology UNIT 3 MODULE 6

Replication of what two things occurs during S phase of interphase?

chromosomes and microtubule organizing centers

The bi-oriented chromosomes with several kinetochore microtubules attached to each kinetochore move to a central point between the spindle poles which is known as ______

congression

During metaphase, kinetochore MT maintain a _____ length so that the chromosomes are aligned in the middle of the cell

constant

Intestinal stem cells are found in the ____ region of the intestine. The ____ layer of the intestine is turned over quite rapidly in about 3-5 days. And it's these stem cells that become very rapidly differentiated and move up the crypt and into the _____ to replace some of these epithelial cells that are on the surface of the villus.

crypt; epithelial; villus

In developing countries, in third world countries, most individuals die of infectious disease or malnutrition, but in developed nations, like our own, the most important diseases are ______ diseases. - possibly treatable with stem cells

degenerative 7/10 of the leading causes of death are degenerative disease - heart disease, cancer, cerebrovascular disease (stroke), chronic lower respiratory disease, Alzheimer, Diabetes, Nephritis)

There are two controversies that have been associated with these embryonic stem cells. 1. To isolate them from the blastocysts, you necessarily _____ the blastocyst. And this is a huge question in in bioethics, whether this is morally acceptable to destroy blastocysts in order to isolate the embryonic stem cells. 2. The other controversy is the growth or the co-growth with these fibroblasts. And some people do a lot of measurements on these embryonic stem cells. And they look at the physiology of them in this condition. And some people question whether you're really looking at the embryonic stem cells or whether you're looking at the fibroblasts. And when you passage embryonic stem cells, are you really passaging some fibroblasts. Or are all the fibroblasts truly irradiated in a way that they're not growing. And so there is a contamination of embryonic stem cells, possible contamination with fibroblast which can confound your interpretation of any kind of data that you get from them. However the problem with the feeder cells has been solved, somewhat, with the use of ______ instead of the feeder cells. But again this has been a problem in embryonic stem cell research.

destroy cytokines

Mitosis promoting factor is a ____ that is made up of a protein called ______ which phosphorylates serine residues and threonine residues on other proteins

dimer; cyclin dependent kinase (Cdk)

What occurs in pro-metaphase?

early stage of metaphase, capture these chromosomes by microtubules

Another source of adult stem cells includes _________. But embryonic carcinoma cells are not ____. These cells are derived from tumors that arise in germline cells, so from sperm and egg. So sometimes sperm cells and egg cells can lose their cell cycle control and they form these tumors. And when these tumors arise from these germline cells, the cells in them are called embryonic carcinoma cells. And when one of these tumors is benign, it's known as a ______. And when or if these tumors are malignant, they're called ____. Within the tumor you get differentiation into different cell types. So you can see in this testicular teratocarcinoma that you actually have hair growing. So these are very unusual tumors. And in this x-ray is an individual that has an ovarian teratocarcinoma, so this is a tumor that has arisen from an egg cell in the ovary and it actually has teeth. So there's a differentiation of teeth.

embryonic carcinoma cells embryos teratoma teratocarcinomas

Dr. Yamanaka demonstrated that there were four genes whose expression was critical to giving an embryonic stem cell its phenotype or its embryonic stem cell "nature". So he took adult cells from both mouse and human and he turned on the expression of these 4 genes listed here OCT3/4, SOX2, KLF4, and c-MYC. And he successfully turn adult cells into ______ cells. Today these cells produced by this technology are known as __________.

embryonic stem induced pluripotent stem cells or IPSCs.

Embryonic carcinoma cells (EC cells) DO NOT develop from _____

embryos

Another source of stem cells is from _____ and these are the embryonic stem cells When you have development of the fertilized egg in about two and a half days you have 8 cells. At about 3 days, you have about 16 cells, and eventually you have the formation of the ______. And inside the blastocyst is a group of cells known as the ______, and these are the embryonic stem cells. So if you want to grow embryonic stem cells in the lab, you would dissociate the cells of the blastocysts and you would collect these inner cell mass cells. And then you would put them in a culture dish.

embryos blastocysts inner cell mass - made of embryonic stem cells

Embryonic stem cells have very fastidious growth requirements. They have to be grown in the presence of other cells. And so usually when scientists are growing embryonic stem cells, they will have a layer of _____ cells. They're usually _____ and they are irradiated to halts the cell cycle. That damages the DNA, and remember we talked about the DNA damage checkpoints. So you have damaged DNA in those fibroblasts, so that their cell cycle is halted. They're alive. They're growing. They are secreting the factors that are necessary to grow the embryonic stem cells, but they're not dividing, so they won't take over the culture. So you have these embryonic stem cells that are growing on this layer of irradiated feeder cells.

feeder fibroblasts

Embryonic carcinoma cells are derived from tumors that arise in ____ cells

germline - in testis or ovary

The number one killer in the US is still _______, but not far behind is cancer.

heart disease

There is successful differentiation of ESCs in vitro due to what?

high pluripotency

And ______, there has been a little bit more success with adult stem cells than the embryonic stem cells.

in vivo Adult stem cells have been successfully used so far to treat spinal cord injuries and with some success to regenerate heart tissue, to treat muscular dystrophy, diabetes, Crohn's disease, Parkinson's, and hearing loss, to regenerate cornea, and hair loss, and some cancers. So more success in vivo with the adult stem cells.

Another job of the G1 cyclin-Cdk complexes is to ____ some of the components of this proteasome because you don't want this proteasome to be active at different parts of the cell cycle. Because it might degrade other cyclins that are not used during mitosis and that could be detrimental to the cell cycle. The G1 cyclin-Cdk dimer can actually ____ expression of other genes.

inactivate activate

The ______ kinetochore does contain centromeric DNA wrapped around histones to form nucleosomes.

inner

CPC associates with the ______ early in mitosis and the Aurora B kinase in the kinetochore region can _____ proteins in the kinetochore and outer kinetochore

inner kinetochore phosphorylate

Attachment of microtubules to centromeres: Centromere DNA is associated with very large protein complex known as the _______ protein complex, and that associates with another large protein complex known as the ________ protein complex, and the ______ end of the microtubules bind somewhere in this outer kinetochore region. There is also another region known as _______ outside the outer kinetochore.

inner kinetochore outer kinetochore plus corona Inner kinetochore and outer kinetochore are not individual proteins (as depicted in this diagram) but they are actually large protein complexes.

The _____ motors (like dual head kinesin 5) are attached to two different polar MTs and their heads try to walk towards the ____ end of the MT, resulting in pushing the polar MTs in ______ directions and eventual _____ of new MT at the (+) end as they stretch.

kinesin (+) opposite polymerization

In Anaphase B, the sliding of antiparallel polar microtubules powered by a _____, which is a _______ motor. The astral microtubules grow towards the cell periphery to stretch the cells. In contrast the ____ motors attached to plasma membrane in the cell cortex try to walk towards the ____ end, the spindle pole and generate the pulling force to stretch the astral microtubules and elongate the cell for division.

kinesin-5 plus (+) end-directed dynein-dynactin minus (-)

The chromosomes with only one kinetochore-pole interact with the ______ also known as CENP-E

kinesin-7

Aurora B kinase-mediated phosphorylation prevents _______ interaction with ______ complex

kinetochore MT NDC 80

What is a stem cell that can form multiple differentiated cells?

multipotent

If embryonic carcinoma cells are treated with retinoic acid, what forms?

neuronal cells - very plastic - useful for studying stem cells

What is the function of the NDC 80 complex?

nuclear division cycle (NDC) complex - connects microtubule to kinetochore from where microtubule connects to outer kinetochore to inner kinetochore - create tension due to rigidity

There are 4 DNA damage checkpoint are very interesting because they are all regulated by the same protein which is known as _____. Interestingly p53 is also known popularly known as "the guardian of the genome" among cell biologists. So here are the 4 check points - 4a is a check point. This checkpoint is checking to see if DNA damage has occurred during G1.4b this is checking for DNA damage just before the entry into S phase. Then you have 4c checking for DNA damage during S phase And then you have 4d which is checking for DNA damage duringG2 phase So what happens is that you get a protein called _____ which will activate p53. p53 will induce the expression of p21 CIP and p21 CIP will inhibit any cyclin-Cdk dimers present and halt the cell cycle until DNA damage can be corrected. So say here at this checkpoint you have ATMR it activates p53. Then p53 induces the expression of p21 CIP and p21CIP will inhibit this cyclin. Likewise, right before S phase, once again you have ATMR activating p53, which induces expression p21 CIP, which inhibits this cyclin. And you also have the same process here ATM/R- p53- p21 CIP and you have it inhibiting this cyclin. And also over here you have the same process. And then you have right before the entry into M-phase. Now what's interesting is that two of these DNA damage checkpoints have two processes that are monitoring whether there's DNA damage. So right before entry into S phase this ATM/R can also activate Chk1/2, which will inhibit Cdc25a, which is normally responsible for activating this cyclin. And likewise you have over here ATM/R can activate Chk1/2, which inhibits Cdc25a, which again would activate this cyclin. And so two of these DNA damage checkpoints have an added level of security to them, and that makes sense for example because this is just when the cell's entering S-phase and when the DNA is actually being replicated. And you don't want to replicate your DNA if you have damaged DNA.

p53 ATM/R

Embryonic carcinoma cells are very ____

plastic - can be differentiated into a lot of different tissues including neuronal cells

What is one advantage of embryonic stem cells?

pluripotency

Embryonic stem cells are _______, which means they can form all 200 cell types that are normally found in the human body.

pluripotent

What is a stem cell that can form all of the ~200 differentiated cells?

pluripotent

What are the stages of mitosis?

prophase, metaphase, anaphase, and telophase/cytokinesis

The cell cycle control system is a ____ baed machine

protein kinase

The astral MTs grow towards the ____ end/cell periphery and the ____ motors attached to the plasma membrane try to walk towards the ___ end of the astral MTs, resulting in the cell stretching.

(+) dynein (-)

What are the steps of MPF/CDK regulatory systems in the cell cycle?

1. Cyclins are synthesized and degraded (by proteasomes) in a cyclic manner 2. CDK protein levels remain the same throughout the cell cycle 3. Presence or absence of specific cyclin make the CDKs inactive and active

What types of tissues have seen success in embryonic stem cell differentiation in vitro?

1. Ectoderm (skin, brain, nerves) 2. Mesoderm (blood, heart, bone, kidney, muscle, cartilage) 3. Endoderm (lung, liver, digestive system)

What are the two methods of chromosome capture by microtubules?

1. End capture: "search and capture" model - grow towards chromosomes, come in contact, capture it 2. Side capture: microtubule misses kinetochore, so kinetochore proteins interact with the side of the microtubule and chromosomes migrate to tend of microtubule with help of microtubule motor proteins (chromosomes slide to + end of microtubule)

What are the three different types/functions of microtubules?

1. Kinetochore MT - interact with the chromosomes 2. Polar MT - don't interact with chromosomes but with each other in the center of the spindle 3. Astral MT - form tufts at either end of the mitotic apparatus

Properties of stem cells:

1. Stem cells can propagate themselves. When a stem cell divides, it is sometimes referred to as asymmetric cell division, because one of the cells is another stem cell, so it propagates itself. It keeps itself going. But the other cell - the daughter cell - might be a progenitor cell on its way to becoming a differentiated cell. So the first property is that they can propagate themselves and maintain themselves. 2. They can be differentiated. For a very long time it's was thought that once a daughter cell embarked on this path towards differentiation, it could never go backwards - it could never become the stem cell again.

What is the evidence that supports the idea that embryonic stem cells are pluripotent?

1. Under normal developmental conditions, embryonic stem cells form all the tissues of the body. Of course, if the blastocysts are allowed to develop, to fully develop, you end up with all the tissues of the body. 2. If you take embryonic stem cells and you inject them into immuno-deficient mice you form teratomas. And if you look very carefully at those teratomas you will find all kinds of differentiated cells from different tissue types -from the ectoderm, from mesoderm and from the endoderm. And so in those teratomas that are formed by embryonic stem cells that have been injected into immunodeficient mice, you find all these tissue types, which suggests that the embryonic stem cells were originally pluripotent. 3. In experiments where the embryonic stem cells are isolated from a mouse blastocyst, you transfect the stem cells with a GFP tag and inject into the blastocysts of another mouse and implant that in a surrogate mother - offspring will have GFP fluorescence in practically every tissue in the body

What are the bioethical considerations of cloning?

1. What is the moral status of a human embryo? Scientifically, the embryo is a human being but is the embryo a person or a piece of property? 2. There is a current surplus of frozen embryos. These can be replicated and provide a pool of stem cells and differentiated into other cell types 3. Scientific freedom, scientific stewardship

What are the two parts of the mitotic apparatus?

1. spindle part - football shape in center 2. microtubules arranged in bunches at either end of spindle (astral microtubules)

Over ___ different animals have been cloned.

20 - cows, pigs, horses, rabbits, cats, monkeys

The duration of the cell cycle differs in a cell-specific manner. For example, fly embryos have a cell cycle of _____ while mammalian liver cells have a cell cycle of _____

8 mins; greater than one year - don't divide very much

What is the role of the Rb (retinoblastoma) protein in the G1/S transitions?

A dimer that found in the cell in the G1 is made up of two proteins. One protein is known as RB (retinoblastoma), and another protein E2F which is a transcription factor. And when RB is bound to E2F in this fashion E2F is inactive. So this is an inactive protein complex. As cyclin D is being synthesized in G1 and as the cyclin D/CDK dimer becomes activated during G1, one of the targets of this kinase is the retinoblastoma protein. And so RB becomes phosphorylated. This creates a conformational change in RB such that it releases the E2F transcription factor which can bind to upstream regions of genes and initiate gene expression of a wide variety of genes. One of the genes that it increases expression of is itself. So it binds to the upstream region of its own gene turns itself on. So you get even more E2F. Another gene, that E2F induces expression of is cyclin E, so you get the synthesis of a second G1 cyclin-Cdk dimer that becomes activated and it can actually feedback into this process and phosphorylate more retinoblastoma to further activate E2F - positive feedback loop affecting gene expression

There's 4 DNA damage checkpoints that are scattered throughout the cell cycle. So let's look at each of these individually. So when the cell comes out of S-phase and enters G2, you would hope that all the DNA is replicated properly. And so there is an un-replicated DNA checkpoint and in other words, the cell is asking itself is all my DNA replicated? And if it is not, a protein known as _____ is activated. And ATR in turn can activate a protein known as ______. CHK1 can inhibit another protein known as ______. And Cdc25c is responsible for activating the ______ entry. So if there is un-replicated DNA, the cell can halt the cell cycle before the entry to mitosis through this system right here. Right before the cell enters M-phase, there is a spindle assembly checkpoint Number 2. So the cell is checking whether the spindle is formed properly, whether all the chromosomes are lined at the center. And if they're not, a protein known as ____ becomes activated. And Mad2 will inhibit the proteasome that is responsible for degrading securin. Remember securin is one of those proteins that keeps the two sister chromatids together. And so if securin cannot be degraded because this proteasome is inhibited, the cell is not going to enter anaphase, because the cell will not be able to pull those sister chromatids apart. Another checkpoint is known as the chromosome segregation checkpoint, which happens right before telophase. And if the chromosomes are not delivered to the poles properly, if they are not segregated properly, then you're going to get inhibition of protein known as _____. And Cdc14 is responsible for activating this proteasome complex which degrades and ubiquitinates some of the mitotic cyclins. So if this checkpoint is activated the cell would not enter telophase at this point.

ATR Chk1 Cdc25c cyclin that mediates G2 to M-phase Mad2 Cdc14

What is the purpose of cloning?

Agriculture: champion livestock, raise animals with favorable phenotype Research: using controls in animal models of disease Human Cloning: reproductive cloning (live-birth cloning), therapeutic cloning

What does the M checkpoint check for?

Are all chromosomes aligned on the spindle? Is the environment favorable?

As a few unphosphorylated NDC 80-associated proteins associate with the MTs and pull the outer kinetochore complex away from the inner kinetochore, ______ is left behind and no longer can phosphorylate the NDC 80 proteins; resulting in a strong association of NDC80 with kinetochore MTs required for bi-orientation

Aurora B kinase

The kinetochore region of the DNA is marked by a specific histone and this histone is known as ______

CENP-A - marks the kinetochore region of the mitotic apparatus - inner kinetochore protein complex

A major player in the regulation of bi-orientation is a protein complex known as _____ which contains the kinase, ______

CPC - chromosome passenger complex; Aurora B kinase

Why doesn't reproductive cloning make sense?

Could be used as infertility treatment but it is not a good idea because it is 1. extremely inefficient - takes many embryos before have successful birth AND 2. cloned animals may not be normal (Dolly had early onset arthritis and cancer and cloned mice have shortened life spans) - cloning does not perturb the aging process

What occurs in G1?

DNA pre-replication complexes (large protein complexes) are already starting to assemble at the origins of replication, there are many many origin of replication per chromosome - activation of the G1 cyclin-Cdk dimers and when they become activated they actually increase the expression of the S-phase cyclin. So the G1 phase cyclins induce the expression of the S-phase cyclin, which are going to play a role during DNA replication - inhibitor present during G1 so that S phase cycling CDK dimer remains inactive until S phase - G1 cyclin-Cdk dimer phosphorylates this inhibitor - signal for ubiquitination - ubiquitin groups are added to inhibitor by ubiquitin ligase SCF - targeted by proteosome - inhibitor is degraded, cell enters S phase

How do MPFs regulate the cell cycle?

Dimers come together at various points of cell cycle, and cyclin is bound to Cdk, the Cdk becomes activated. So you have Cdk cyclin pairs that operate in mitosis. You have CDK cyclin pairs that operate in G1 and that operate in S phase. And so you have a number of Cdk cyclin dimers that operate throughout the cell cycle. And the reason why they're called cyclins is because the cyclin portion of this dimer goes through a cycle of synthesis and degradation with every single cell cycle. So with every single cell cycle you get synthesis of these new cyclins which combine with CDKs in the cell to activate them at various points in the cell cycle.

The retinoblastoma protein in the G1/S transitions: Rb inhibits _____ but when Rb becomes _____ by cyclin D CDK4/6, E2F is activated and released - drives the expression of other cyclins and other genes - includes genes necessary for ________, some proteins that actually mediate centrosome duplication - produces cyclin E/A CDK2 which phosphorylates even more Rb in a positive feedback loop

E2F phosphorylated DNA replication in S phase

What occurs in telophase?

Formation of a contractile ring of actin and myosin and contraction event of actin and myosin leading to cytokinesis - punching off of the two daughter cells

The DNA pre-replication complex is formed in what phase?

G1

What about S-phase cyclin inhibitor? E2F can induce the expression of the S-phase cyclins, but you don't want the S-phase cyclin-Cdk complexes to be active during G1. You only want it to be active upon entry into S phase. The S-phase cyclin CDK complexes begin to accumulate in ____ but are inhibited by a protein known as _____. So Sic1 is an inhibitor of that cyclin - the S-phase cyclin-Cdk dimer. So eventually one or more of the G1 cyclins will phosphorylate the Sic1 and when Sic1 becomes phosphorylated it becomes a target for ubiquitination. There is the addition of ubiquitin groups which tags the Sic1 for degradation by a proteasome. And so it becomes degraded. Then the S-phase cyclin-Cdk dimer is activated and promotes DNA replication in S phase.

G1 Sic1

there are multiple _____ complexes that are operating during G1 and during the transition from G1 to S phase.

G1 cyclin-Cdk

The un-replicated DNA checkpoint happens at the end of ____ right before the cell enters M-phase.

G2

Explain the phases of the cell cycle

Gap phase 1 - the first growth phase - and during G1 the cell has just come out of mitosis and its growing and its monitoring the environment to make sure the environment is ok, that nutrient sources are OK so that the DNA can be replicated. At that point, after G1, the cell enters the S-phase or Synthesis phase and during S-phase the DNA is replicated, so this is the DNA synthesis phase. After S-phase you have G2 phase and G2 phase really serves as kind of a safety gap so that the cell can make sure that the chromosomes have been duplicated properly or the DNA has been duplicated properly, before the cell enters mitosis. And then finally mitosis or M phase this is when the cell divides.

What occurs in G2?

In G2 the mitotic cyclin-Cdks are synthesized and become activated and then the cell can enter mitosis

What occurs in mitosis?

In metaphase, all the chromosomes are attached to the kinetochore microtubules. As the cell moves from metaphase to anaphase, a second proteasome complex becomes activated and this proteasome complex is responsible for degrading a protein known as securin. And securin is one of the proteins in the cohesion complex that's holding the two sister chromatids together. And of course degrading the proteins in that complex is necessary because during anaphase the result of shortening of the kinetochore microtubules and as a result of the forces that are imparted by the polar microtubules and the astral microtubules, you have the separation of the sister chromatids and their movement towards the poles of the cell. Right after anaphase, you have the activation of another proteasome complex, and that proteasome complex is responsible for degrading all the mitotic cyclin-Cdks that are hanging around. And at this point the cell can enter telophase and finally cytokinesis.

How do polar MTs cause cell elongation?

In the overlap region of the polar microtubules, you have some kinesins, the tails are interacting very tightly with one polar microtubule. The head of that kinesin is walking towards the plus end of the microtubule on the neighboring polar microtubule. And that causes those microtubules to push against each other and that causes the elongation event.

What does the G2 checkpoint check for?

Is all DNA replicated? Is the environment favorable? Is the cell big enough? Is the DNA damaged?

What does the G1 checkpoint check for?

Is the cell big enough? Is the environment favorable? Is the DNA damaged?

On the shortening side as you can see in the figure _________ stimulates kinetochore microtubule disassembly at the plus (+) end. The dynein-dynactin motor protein complex moves the chromosome toward the spindle pole, the minus end of the microtubule. On the other hand the lengthening side of the kinetochore microtubule, the _____ holds on to the growing microtubule. Note that the chromosome arms still point away from the spindle poles owing to associated _______ also know as chromokinesin. The force pulling to the poles overcomes force pulling the arms towards the center of the spindle.

Kinesin-13 kinesin-7 Kinesin-4

In Anaphase A, the movement of chromosomes to respective poles is powered by microtubule shortening by _____ proteins at the kinetochore and this is happening at the _____ end of the kinetochore microtubule. In addition, Kinesin-13 also aids in kinetochore microtubule _____ at the minus (-) end in the _____. The Kinesin-13 just makes the disassembly faster in addition to normal disassembly of the microtubules at the minus (-) end. The chromosome arms still point away from the spindle poles owing to associated ____ due to the force pulling to the poles overcomes force pulling the arms toward the center of the spindle.

Kinesin-13 plus (+) disassembly spindle poles kinesin-4

During Anaphase A, what are the forces generated by?

Kinetochore MTs that are interacting with the chromosomes

In the crypt region (stem cell region) the intestinal cells are called ____ stem cells. There are also ____ stem cells generated from LGR5+ stem cells and can restore LGR5+ stem cells following an injury. There are ______ cells - divide to produce a variety of differentiating cells like enterocytes, tuft cells, Goblet cells, and enteroendocrine cells - as they proliferate, they move up, migrate up to replace epithelial cells in the villus, but ______ cells are regenerated from transient amplifying cells and migrate downward to the base of the crypt and secrete antimicrobial defense proteins

LGR5+ (leucine rich g protein coupled receptor 5); reserve; transient amplifying; Paneth

What are the advantages of using adult stem cells?

Less fastidious growth requirements; multiply indefinitely (embryonic have only certain lifetime in culture before they stop dividing); no rejection (autologous transplants)

What is the disadvantage of using adult stem cells?

Less potent - less of an issue if turn on four genes necessary to make induced pluripotent stem cells

What occurs in prophase?

Migration of two microtubule organizing centers (spindle poles) towards the pole of the cell, chromosome condensation, nuclear envelope starts to break down

The cell cycle is divided into two stages:

Mitotic (M) phase - shorter Interphase - longer

What are the conditions of those with retinoblastoma?

Mutant Rb is defective Defective mutant Rb cannot bind to E2F transcription factor E2F is ALWAYS ACTIVE to drive transcription leading to cell division (uncontrolled)

How does the NDC 80 complex function in the regulation of bi-orientation?

NDC 80 is a sleeve-like multiprotein complex formed around kinetochore MT; attached to each kinetochore of the sister chromatids (two per chromosome); help in all chromosome movements during mitosis; recruitment side of kinesin and dynein motors for pulling/pushing movement

Are kinetochore MT in metaphase a static structure?

No - they are constantly treadmilling - constant addition on plus end - interacts with kinetochore region and constant subtraction at minus end

Are there only three checkpoints in the cell cycle control system?

No, there are many more than three checkpoints

During Anaphase B, what are the forces generated by?

Polar microtubules that are interacting with each other in the spindle and the astral microtubules on either end of the spindle

How will the S-phase cyclin will promote the new replication? _____ complexes assemble at the origins of replication, and this usually happens in early to mid G1. And some of these are going to participate in DNA replication. Some of these components are inhibitors that are preventing the action of these proteins that are involved in DNA replication Once you get an active S-phase cyclin, which is shown here in step 2, it will phosphorylate a number of the components or proteins of the ______ complex - here in blue. You can see these yellow phosphate groups have been added. In some cases this will cause a conformational change in proteins, such that they fall off the replication origin and you no longer have inhibition of that replication origin. So now the DNA begins to unwind and phosphorylation groups are added directly on the components of the replication origin which activates them, and that will induce the initiation of DNA replication in the cell.

Pre-replication origin replication

What occurs in Anaphase A?

Rapid shrinking of the microtubules - loss at the plus end by Kinesin-13 which destabilizes microtubules for rapid de-polymerization with the plus end that causes those microtubules to shrink or appear to shrink towards the spindle pole which pulls the sister chromatids towards the spindle pole.

What are the effects of fusing a mammalian cell in mitosis with one in interphase?

Results in a heterokaryon - fused cell - DNA is double - DNA in nucleus of interphase cell beings to condense - factor in mitotic cell that introduces condensation fo DNA from interphase - controlling factor was termed mitosis promoting factor (MPF)

DNA replication begins in what phase?

S

What occurs in S phase?

S phase cyclin-CDK dimer is active during DNA replication - phosphorylates a number of factors on the replication complex which activates the complex

How do astral MTs cause cell elongation?

The astral microtubules interact with dyneins. The tail of the dyneins are bound very tightly to the plasma membrane. The heads of the dyneins are walking along those astral microtubules towards the minus end. And that causes a tension to be set up and that pulls on the astral microtubules, such that you even get further elongation.

How is the chromosome drawn toward the spindle pole?

The chromosome is drawn toward the spindle pole by the dynein-dynactin motor protein complex that is associated with one of the kinetochores walking toward the minus (-) end of the kinetochore microtubule.

What occurs in Anaphase B?

The polar microtubules interact with each other where they overlap. They actually push against each other and push the poles apart which causes an elongation of the spindle. And then you have the astral microtubules which are attached to the plasma membrane of the cell and will actually pull on that spindle pole, which will further enhance the elongation of that structure.

What is therapeutic cloning?

The process by which you generate embryonic stem cells to treat yourself through somatic cell nuclear transfer. You would take an enucleated egg, and you would take some kind of somatic cell from your own body. And you would remove the nucleus, and you would place that nucleus in this enucleated egg. You would bring that cell or that group of cells to the blastocyst stage; isolate the inner mass cells and culture those embryonic stem cells. Then you can differentiate them into whatever you need for whatever your ailment you have - blood cells, nerve cells, heart cells, liver cells. So say you have some kind of degenerative disease in the muscles or the nerves you might manipulate the genes in these cells to overcome a certain malady and then you would transplant these back into the patient. That's what is meant by therapeutic cloning. In this case you eliminate the disadvantage of transplant rejection, which is one of the disadvantages of embryonic stem cells. But you definitely are still faced with the ethical issue of destroying the blastocyst to generate these embryonic stem cells.

How do CDK levels change in the cell?

They don't -- CDK activities change due to synthesis and degradation of phase-specific cyclins, but CDK protein levels do not change in the cell; they just are switched between active (cyclin present) and inactive (cyclin absent)

How does a somatic cell nuclear transfer occur in cloning?

To do this you take an egg cell and you remove the nucleus or you enucleate that cell. So now you have an egg cell that does not have nucleus. And then you take a donor cell, an adult cell, and you remove the nucleus from that adult cell and you insert that into the egg. Remember normally the egg only has half the amount of genetic material. But this nucleus that's taken from an adult mammalian cell - a diploid organism -will have the complete set of chromosomes and you insert that into the egg and then the egg can continue on its differentiation. And again this was pioneered in frogs, but the first mammal that was cloned was this sheep-Dolly.

What is retinoblastoma?

Tumor development in the eye due to mutant Rb protein - cannot inhibit E2F A child can't make functional Rb protein due to the mutation The absence of the Rb renders E2F transcription factor always active which does not halt the cell cycle at G1/S transition, resulting in uncontrolled cell division

What happens when Aurora B Kinase phosphorylates the NDC 80 complex?

Weakens the interaction between the microtubule and this region - little to no tension BUT When bi-orientation occurs, each of the kinetochore regions on each sister chromatid is bound to an opposing microtubule and tension forms and you get a change in the conformation of the chromosome, such that the kinetochore protein complexes are now pulled away from the CPC so Aurora B kinase cannot phosphorylate the proteins within this kinetochore region and that causes the association of the NDC 80 complex with the kinetochore proteins to become very strong or firmly attached to the microtubule - it stabilizes the chromosome and it creates that tension and it's a very stable structure. And that's really how the cell makes sure that the chromosomes are bi-oriented in metaphase because if they have any other kind of orientation --bound only to one microtubule or bound to microtubules from the same side of the mitotic apparatus -- you're going to have phosphorylated proteins in the kinetochore. And you're going to have a very weak association with microtubules. Whereas if it's properly oriented, you're going to have a very strong association with these microtubules due to the fact that phosphorylation events are no longer being maintained by Aurora B kinase.

For the _____ stem cells there has been more success in vitro

adult There are reports of treatments for more than 90 medical conditions and diseases, including sickle cell anemia, multiple myeloma cancer and damaged heart tissue. As you can see here adult stem cells have successfully been differentiated into many other cell types. For example, stem cells from fat tissue have been differentiated in culture into bone into cartilage, into muscle, into neurons. Stem cells from the brain have been differentiated into brain cells, nerve cells, muscle cells. So you can see that there's been quite a bit of success in culturing and differentiating adult stem cells in culture or in vitro.

What occurs in metaphase?

alignment of chromosomes in center of cell

There is an ongoing "tug of war" kind of thing going on with one set of kinetochore microtubule shortening while the others are elongating, and this is known as the _____________ of the chromosomes.

bi-directional oscillations

A kinetochore microtubule from the opposite pole becomes attached to the free kinetochore to _____ the chromosome

bi-orient

Sister chromatids must be ______ during metaphase to prevent nondisjunction from a kinetochore MT only attaching to one side of a chromosome. (must be attached to kinetochore MT from opposite spindle poles)

bi-oriented So the sister chromatids have to be oriented so that when they separate they move to the opposite poles and this is known as bi-orientation and the cell has a certain way to make sure that each sister chromatid pair attaches to the microtubules emanating from opposite spindle poles so that they are bi-oriented before metaphase proceeds into anaphase.

There are stem cells in _______ that can form all of the cell types that you find in the blood. For example, there is a pluripotent hematopoietic stem cell and it's called pluripotent but it's more like a multipotent cell which is probably a more appropriate term. It can be differentiated into the ______ lineage, and give rise to T-cells and B-cells, which are really important cells of the immune system. Or they can be differentiated into _______ lineage and give rise to platelets, red blood cells, eosinophils, macrophages and granulocytes. The last three cell types are also really important in the immune system. So bone marrow is a source of adult stem cells.

bone marrow lymphoid myeloid

p53 is also implicated in _____

cancer

There's a chromosome ______ checkpoint that also happens during mitosis

segregation

The first mammal that was cloned was the sheep shown here known as Dolly via ______

somatic cell nuclear transfer (compared to the normal fertilization process)

John Gurdon who pioneered a technique known as __________

somatic cell nuclear transfer - cloned frogs

There is a _______ checkpoint that happens in mitosis.

spindle assembly

p53 is not _____ so the cell makes it and it becomes degraded quickly unless there is DNA damage in which case p53 becomes stabilized. It can bind to upstream regions of genes like ____ - induces synthesis of this protein which will bind to any ______ that is present during the cell cycle and inhibit the cell cycle

stable p21 CIP cyclin Cdk dimer

A malignant EC cell is a _____

teratocarcinoma

A benign EC cell is a ____

teratoma

In vivo research with embryonic stem cells has had very limited success, so if you inject embryonic stem cells into a mouse, a ___ will result.

teratoma But there was one research group that did an experiment where they differentiated embryonic stem cells in the culture dish into hepatocytes which are liver cells or cells that looked like a liver cell based on physiology and on the genes and proteins that were expressed. This group also added a GFP transgenes so they could track it, and then they injected those differentiated cells into a mouse, and the GFP hepatocyte that had come from embryonic stem cells populated the liver and persisted in the liver and seem to behave like liver cells. But to date the success of embryonic stem cells populating an organ and behaving like the cells in that particular organ have been virtually zero.

What is a stem cell that can form all of the differentiated cells + specialized tissues like placenta?

totipotent

p53 is a _____

transcription factor

What can't you use embryonic stem cells for?

treating infertility

What occurs in anaphase?

two sister chromatids get pulled apart and are distributed to either end of the cell

APC/C a _____ , ubiquitinylates the anaphase inhibiting protein and causes its degradation through proteasome. The activity of APC/C, ubiquitin ligase, increases during ____ to promote degradation of the protein that inhibits mitosis and then decreases afterward

ubiquitin ligase mitosis

What is a stem cell that can form only one type of differentiated cell?

unipotent

There has been some success at differentiating embryonic stem cells to all the different tissues: ectoderm, mesoderm, and endoderm. Note- in _______

vitro or in cell culture.

What cell is permanently in the G0 phase?

your neurons are completely amitotic, and they are permanently in the G0 phase and do not come out of it at all

What are the disadvantages of embryonic stem cells?

•Teratoma formation •Inefficiency in establishment of cell culture (1/10 embryos provide usable ESCs) •Differentiation often results in a mixture of cell types •Limited evidence (right now) ESCs useful in treating degenerative diseases •Transplant Rejection •Unstable Genomes •Ethical Questions