BIOC19: LECTURE 7
BIOC19: LECTURE 7 Slide 2: • Skeletal muscles a.k.a voluntary muscles • Myofilaments are composed of muscle specific proteins: actin and myosin. Slide 3: • Pink diagram shows the development of the neural tube. The ectoderm folds-in, forms the neural groove, and then the neural tube pinches out. This diagram shows the cross-section of the neural tube. • Black diagram shows the top view of the neural tube. The early neural groove is not zipped and the neural tube on its right is zipped up or closing neural tube in its late phase. Its starts to zip at the brain and then to the posterior end of the embryo. Neural tube zippers up before the development of muscles. • The diagram on the lower left also shows a cross-section of a neural tube. In that diagram, neural tube is pinched. Right under the neural tube is notochord and the red part is mesoderm, which eventually develops into different regions. The red mesoderm cells proliferate and surround the embryo. There is also endoderm in the middle and epidermis on the outside. Slide 4: • Diagram with labels show locations or action sites of mesoderm regions. Note: the missing label is "Axial Mesoderm" • Somites form into muscles • The 3 main regions are found on both sides of the central neural tube (refer to the diagram) Slide 5: • Upper right diagram shows a microscopic picture. It shows the neural tube in the middle and sausage-shaped paraxial mesoderm on both its side • Paraxial mesoderm starts diving into somites (the bumps in the upper right picture), starting from head and moving down to the tail sequentially • Different species of animals has different number of somites • All vertebrates have division of paraxial mesoderm • By counting the number of somites, you can tell what stage of development a particular animal is at because as development progresses, the number of somites increase Slide 6: • In the first diagram, you see the pre-segmented stage. Pre-segmentation stage is before the division of paraxial mesoderm into somites when the neural plate is still open, the neural groove is still there, notochord is present, and there is unsegmented mesoderm (particularly paraxial mesoderm)
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Then, in the second diagram, you can see that the neural tube closes and pinches off. The unsegmented mesoderm starts cutting off individual somites. As you can ssee, somite is a mass of cells Then, in the third diagram, you can see that the somite differentiates into two components. The part closer to notochord and the bottom of the neural tube differentiates into sclerotome. The part of somite that is more closer to the surface or more dorsal differentiates into dermamyotome. Dermamyotome subdivides into 2 further regions: dermatome and myotome. Dermatome is closest to ectoderm and it forms the dermis layer of skin or the deep layer of skin. The more internal, larger mass of dermamyotome subdivides into myotome which forms the skeletal muscles.
Slide 7: • This experiment proves that the cells in the somites form muscles of the limb/chicken wing • Note: The blue thick double line on the far right of the diagram is Neural Tube • Diagram on the far right shows the limb bud of the chicken that forms the wing • Surgically remove chick somites and replace them with quail somites which have a different pigmentation than chick somites. Then look at what develops. Result: a wing develops and we can see that the wing's cells have striated muscle which all have the pigmentation of quail somite. So, skeletal muscles of the limb originate from somites because as shown in the experiment, limbs of the chicken were derived from cells that originated from the quail somites Slide 8: • Myogenesis is the development of skeletal muscle tissue. It can be divided into 3 stages • Commitment/Determination is when pluripotent commit to the myogenic pathway because otherwise pluripotent cells can differentiate into many other things • Differentiation steps involves turning on genes encoding skeletal muscle protein genes • Skeletal muscles are the only type of cellular body that can contract. Their ability to contract is controlled and triggered by nerve action. That's why nerves must grow into the developed muscle cells to form a neuro-muscular junction. The firing of the motor neuron controls, through the neuro-muscular junction, when the muscle is going to contract. Slide 9: • #1 sentence refers to the diagram on the far left. In fact, each sentence refers to a diagram on the top • Note: On the diagram that is on the far left, label the blue line 'Neural Tube' and the red circle 'notochord' • Shh is an inducing factor, given off by neural tube and notochord, which triggers step# 2 • In step #2, Shh transforms myotome cells to myoblasts, triggering commitment to the myogenic pathway
• • • • • •
•
In step #3, myoblasts undergo rapid cell division and when they reach the right number, they begin to align and undergo cell fusion so lots of myoblasts end up in one cell, giving the cells its contractile ability and multi-nucleated characteristic Muscle specific proteins include actin and myosin Final step is when muscle contracts in a organized manner Bottom picture is of muscle fiber cells. See how the many nuclei get pushed to the edge. Slide 10: There are very distinct series of steps in muscle differentiation, which can be recognized at the morphological with a microscope. They are also recognizable the individual steps at molecular level because of muscle specific proteins actin and myosin that form the myofilaments (the contractile element). At the molecular level, you can see when genes of actin and myosin are turned on and when those genes make the actin and myosin proteins. Certain features of muscle cell differentiation are self-stimulating and we can look at the them in tissue culture without having to add external inducer AS LONG AS WE START WITH MYOBLASTS. In the last slide, we mentioned that myotome cells are pluripotent and Shh transforms them into myoblasts and triggers commitment to the myogenic pathway. That is why we would not have to add an external factor/inducer if we start with those committed myoblasts because we know that they are already committed/restricted to the myogenic pathway.
Slide 11: • In this experiment, we're going to look at the differentiation of myoblasts in mature muscle fiber cells. • We start with embryonic thigh muscle that was isolated at a step when myoblasts were present. • So, we take a piece of the embryonic thigh muscle, cut it into tiny pieces, and then treat with an enzyme called trypsin. Trypsin digests the cell adhesion proteins that are holding the thigh muscle cells together and thus, the thigh muscle tissue is dissociated into individual myoblast cells. • Then you remove trypsin through centrifugation and you are left with single celled myoblasts. • You put these myoblasts on a petri dish (along with culture medium) and they undergo muscle cell differentiation without any external inducer. • In vitro mean tissue culture Slide 12: • What happens, step by step, when you put the myoblasts on that petri dish? (refer to experiment on the previous slide) • With such a tissue culture, you can recognize the different steps of differentiation at the molecular and morphological level • 2) Cell division stops when the cells reach a certain number/density of myoblast cells and through regulation, they stop dividing
• • •
4) Cell fusion signals the change in the pattern of gene expression to turn on genes encoding actin and myosin 5) Increase in muscle-specific proteins that organize themselves in myofilaments 6) Twitching is spontaneous and not in an organized way because motor neurons have not yet grown in the muscles and neuro-muscular junction has not yet formed
Slide 13: • Multinucleated cells form in 6-12 hours which is too little time for many many rounds of DNA replication for nuclear division • No mitotic figures or condensed chromosomes were observed under the microscope which we would expect to find during nuclear division • Multinucleated cells formed even if you put in DNA synthesis inhibitors which prevent nuclear division Slide 14: • Can different types of muscle cells fuse? No. Myoblasts of different organs or origins do not fuse together to make multinucleated cells • Note: the slide should say 'Thigh muscle myoblasts + heart muscle myoblasts = no cell fusion' Slide 15: • In this experiment, we're trying to see whether myoblasts, that are from the same tissue but different species, fuse. • Rat thigh muscle myoblasts labelled with radioactive triated thymidine + Rabbit thigh muscle myoblasts NOT labelled with radioactive triated thymidine = mix of labelled and unlabelled nuclei in muscle fibers • This experiment confirms cell fusion theory because the muscle fiber cells with labelled and unlabelled nuclei looked like this:
•
If the nuclear division theory was right, muscle fiber cells would have looked like this:
Slide 16: • Mintz and Baker developed a technology where you can take embryos of 2 different mice and fuse them together to form Chimeric mice which can be used for developmental experiments • In other words, chimeric mice are formed of cells that are composed of two types of mice. • Isocitrate dehydrogenase (IDH) is formed in muscle cells
•
Mice muscle cells with AA subunits of IDH + Mice muscle cells with BB subunits of IDH = myotubes of AA, BB, and AB (hybrid form) subunits of IDH. This supported the cell fusion theory
Slide 17: • This picture on the left shows what we would expect if nuclear fusion theory was true. The picture on the right shows what we found and what we would expect if cell fusion theory was true • This experiment also tells us that cell fusion occurs during embryonic development Slide 18: • As mentioned before, cell fusion signals the change in the pattern of gene expression to turn on muscle-specific genes that encode actin and myosin (slide 12). But how do we know this? If we want to analyze the time course of message induction, we can do that in 2 ways: Northern blotting and In site hybridization Slide 19: • Diagram shows how one would do Northern Blotting - used to find out the stage in muscle cell differentiation when myosin message first appears • A) Isolate RNA from all the stages of differentiation in our tissue culture system. Run RNA of all the stages on a gel subjected to electrophoresis to separate messages of different sizes • B) Place gel on wet filter paper between two spacers • C) Place nitrocellulose filter over the gel and blotting paper over the filter. Transfer separated RNA to filter by capillary action after you add weight • D) Take filter/membrane with transferred RNA that has been separated with all the lanes of different stages and put it in a scalable bag and add radioactive single-stranded cDNA probe that's complementary to myosin message so that the radioactive probe hybridizes with RNA of interest wherever it finds the myosin message. • E) After incubating cDNA probe, wash unreactive/unbound cDNA probe and identify where the radioactive cDNA complementary to myosin is bound by putting x-ray film over it to prepare auto-radiograph. Develop a film and you'll be able to see a series of bands. Identify the stage myosin is turned on. • (F) does not apply here. Forget this step. Slide 20: • We use In Situ hybridization to identify the cells in embryo where myosin gene appears, using the same cDNA that we use in Northen Blotting. But this time, instead of hybridizing with cDNA probe with mRNA, we hybridize it with a tissue section, under condition where it binds to the target RNA. • On the slide, the tissue section is from a mouse Slide 21: • We'd also like to correlate the timings when the myosin message comes on with when the myosin protein profile appears.
Slide 22: • 1) Isolate protein from the different stages of the muscle cell differentiation. • 2) Load the protein from the different stages onto different lanes on the gel. Run the gel to separate proteins into different molecular weight size class. • 3) Then, put the gel with separated proteins next to the nitrocellulose filter and transfer the proteins from the gel to the filter. • 4) Then take the filter and interact proteins with an myosin antibody. They survey the blot and bind to the myosin proteins wherever they find them. • 5) Wash away the primary antibody. Add a secondary antibody with fluorescent or a stain tag on it to visualize. • 6) Wash out the excess and we can identify a band which shows the stage at which myosin protein appears. • Immunocytochemistry is used to identify the cell stage in embryo where myosin proteins are turned on. Slide 23: • Myosin mRNA is first detected in the newly formed myotubes and myosin protein is first detected in the myotube right before fiber formation Slide 24: • This image is a microscopic slide of stained liver tissue. As you can see, liver is not complicated. It is mostly composed of one cell. Slide 27: • 1) Process of detoxification • 3) Process of exertion Slide 28: • There two ways to determine the time when hepatic or liver cell differentiation is initiated: in number embryonic days or in number of somites. • E8 means Embryonic day 8 • Hepatic is another word for liver Slide 29: • During frog development, there is a change in liver function at metamorphosis when frog goes from aqueous environment to outside of water • In mammals, one of the changes in the liver is correlated with changes in the nutritional source from fetus to newborn stage. • Xanthine dehydrogenase (XDH) is involved in metabolism of xanthine Slide 31: • Note: Add an arrow after H2O and before urate • XDH
•
xanthine + NAD+ + H2O
urate + NADH + H+
Slide 32: • In this diagram, we're looking at XDH enzyme activity versus days after hatching • Note: Change the axis label to 'XDH Enzyme Activity' • A low, steady level of XDH is present in chicken before hatching • So we must find out the molecular basis for the drastic increase in XDH upon hatching • Maximum activity is obtained by 8 days and then it remain constant throughout life in chickens Slide 33: • Transcription control would be that XDH gene is not turned on before hatching and its turned on right at hatching and at that point, the message is immediately translated. • Translational control would be that XDH gene is turned on before hatching. In other words, the message is there but its not extensively translated into protein yet. And the hatching is the cue when the message is translated. • Post-translational control would be that XDH protein is present before hatching but it's not enzymatically active because it needs something supplied at hatching. Maybe it needs a cofactor or maybe the enzyme that it needs is made before hatching but gets degraded quickly and at hatching it stops getting degraded. Or maybe the enzyme is made but its not active because it must undergo post-translational modification like phosphorylation or glycosylation to be enzymatically active.. Slide 34: • XDH immunochemistry on liver tissue to see if number of XDH protein molecules increase after hatching • As we can see in the images, there are no and very low level of XDH protein molecules in embryo and its number then increases after hatching, reaching its maximum number 8 days after hatching • So it tells us that it can't be the mechanism of post-translational control, which says that the proteins are present before hatching but not active, because this experiment shows the protein is just not there. Slide 35: • Is XDH protein synthesis needed for XDH enzyme activity to increase after hatching? Yes, because no increase in XDH enzyme activity was observed when puromycin (which inhibits protein synthesis) was added Slide 36: • Is the presence of mRNA needed for there to be increase in XDH enzyme activity after hatching? Yes, because no increase in XDH enzyme activity was observed when RNase (which degrades RNA) was added
Slide 37: • Is mRNA synthesis needed for there to be increase in XDH enzyme activity after hatching? No, because an increase in XDH enzyme activity was observed even after actinomycin (which inhibits mRNA synthesis) was added • The mRNA/message is needed, not the synthesis of message which means the message is already there. • So this tells us that it can't be the mechanism of transcriptional control. The mechanism behind the drastic increase in XDH enzyme activity at hatching is translational control. Slide 39: • Purify XDH enzyme and radioactively label them. Then isolate cell extracts from all developmental stages: 14 days old embryonic stage and 1 day, 4 days, and 8 days after hatching and add the same amount of radioactive XDH to all of them • Note: Correct 'After Birth' to 'After Hatching' on the slide • Run the extracts on the gel and look at their sizes. Results are shown on the graph on the slide • The level of radioactive XDH at 14 days old embryo and 1 and 4 days after hatching equals the size we put in originally. So nothing in the liver cells is degrading XDH. • 8 days after hatching, ability to degrade XDH can be observed.
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•
Then, in the second diagram, you can see that the neural tube closes and pinches off. The unsegmented mesoderm starts cutting off individual somites. As you can ssee, somite is a mass of cells Then, in the third diagram, you can see that the somite differentiates into two components. The part closer to notochord and the bottom of the neural tube differentiates into sclerotome. The part of somite that is more closer to the surface or more dorsal differentiates into dermamyotome. Dermamyotome subdivides into 2 further regions: dermatome and myotome. Dermatome is closest to ectoderm and it forms the dermis layer of skin or the deep layer of skin. The more internal, larger mass of dermamyotome subdivides into myotome which forms the skeletal muscles.
Slide 7: • This experiment proves that the cells in the somites form muscles of the limb/chicken wing • Note: The blue thick double line on the far right of the diagram is Neural Tube • Diagram on the far right shows the limb bud of the chicken that forms the wing • Surgically remove chick somites and replace them with quail somites which have a different pigmentation than chick somites. Then look at what develops. Result: a wing develops and we can see that the wing's cells have striated muscle which all have the pigmentation of quail somite. So, skeletal muscles of the limb originate from somites because as shown in the experiment, limbs of the chicken were derived from cells that originated from the quail somites Slide 8: • Myogenesis is the development of skeletal muscle tissue. It can be divided into 3 stages • Commitment/Determination is when pluripotent commit to the myogenic pathway because otherwise pluripotent cells can differentiate into many other things • Differentiation steps involves turning on genes encoding skeletal muscle protein genes • Skeletal muscles are the only type of cellular body that can contract. Their ability to contract is controlled and triggered by nerve action. That's why nerves must grow into the developed muscle cells to form a neuro-muscular junction. The firing of the motor neuron controls, through the neuro-muscular junction, when the muscle is going to contract. Slide 9: • #1 sentence refers to the diagram on the far left. In fact, each sentence refers to a diagram on the top • Note: On the diagram that is on the far left, label the blue line 'Neural Tube' and the red circle 'notochord' • Shh is an inducing factor, given off by neural tube and notochord, which triggers step# 2 • In step #2, Shh transforms myotome cells to myoblasts, triggering commitment to the myogenic pathway
• • • • • •
•
In step #3, myoblasts undergo rapid cell division and when they reach the right number, they begin to align and undergo cell fusion so lots of myoblasts end up in one cell, giving the cells its contractile ability and multi-nucleated characteristic Muscle specific proteins include actin and myosin Final step is when muscle contracts in a organized manner Bottom picture is of muscle fiber cells. See how the many nuclei get pushed to the edge. Slide 10: There are very distinct series of steps in muscle differentiation, which can be recognized at the morphological with a microscope. They are also recognizable the individual steps at molecular level because of muscle specific proteins actin and myosin that form the myofilaments (the contractile element). At the molecular level, you can see when genes of actin and myosin are turned on and when those genes make the actin and myosin proteins. Certain features of muscle cell differentiation are self-stimulating and we can look at the them in tissue culture without having to add external inducer AS LONG AS WE START WITH MYOBLASTS. In the last slide, we mentioned that myotome cells are pluripotent and Shh transforms them into myoblasts and triggers commitment to the myogenic pathway. That is why we would not have to add an external factor/inducer if we start with those committed myoblasts because we know that they are already committed/restricted to the myogenic pathway.
Slide 11: • In this experiment, we're going to look at the differentiation of myoblasts in mature muscle fiber cells. • We start with embryonic thigh muscle that was isolated at a step when myoblasts were present. • So, we take a piece of the embryonic thigh muscle, cut it into tiny pieces, and then treat with an enzyme called trypsin. Trypsin digests the cell adhesion proteins that are holding the thigh muscle cells together and thus, the thigh muscle tissue is dissociated into individual myoblast cells. • Then you remove trypsin through centrifugation and you are left with single celled myoblasts. • You put these myoblasts on a petri dish (along with culture medium) and they undergo muscle cell differentiation without any external inducer. • In vitro mean tissue culture Slide 12: • What happens, step by step, when you put the myoblasts on that petri dish? (refer to experiment on the previous slide) • With such a tissue culture, you can recognize the different steps of differentiation at the molecular and morphological level • 2) Cell division stops when the cells reach a certain number/density of myoblast cells and through regulation, they stop dividing
• • •
4) Cell fusion signals the change in the pattern of gene expression to turn on genes encoding actin and myosin 5) Increase in muscle-specific proteins that organize themselves in myofilaments 6) Twitching is spontaneous and not in an organized way because motor neurons have not yet grown in the muscles and neuro-muscular junction has not yet formed
Slide 13: • Multinucleated cells form in 6-12 hours which is too little time for many many rounds of DNA replication for nuclear division • No mitotic figures or condensed chromosomes were observed under the microscope which we would expect to find during nuclear division • Multinucleated cells formed even if you put in DNA synthesis inhibitors which prevent nuclear division Slide 14: • Can different types of muscle cells fuse? No. Myoblasts of different organs or origins do not fuse together to make multinucleated cells • Note: the slide should say 'Thigh muscle myoblasts + heart muscle myoblasts = no cell fusion' Slide 15: • In this experiment, we're trying to see whether myoblasts, that are from the same tissue but different species, fuse. • Rat thigh muscle myoblasts labelled with radioactive triated thymidine + Rabbit thigh muscle myoblasts NOT labelled with radioactive triated thymidine = mix of labelled and unlabelled nuclei in muscle fibers • This experiment confirms cell fusion theory because the muscle fiber cells with labelled and unlabelled nuclei looked like this:
•
If the nuclear division theory was right, muscle fiber cells would have looked like this:
Slide 16: • Mintz and Baker developed a technology where you can take embryos of 2 different mice and fuse them together to form Chimeric mice which can be used for developmental experiments • In other words, chimeric mice are formed of cells that are composed of two types of mice. • Isocitrate dehydrogenase (IDH) is formed in muscle cells
•
Mice muscle cells with AA subunits of IDH + Mice muscle cells with BB subunits of IDH = myotubes of AA, BB, and AB (hybrid form) subunits of IDH. This supported the cell fusion theory
Slide 17: • This picture on the left shows what we would expect if nuclear fusion theory was true. The picture on the right shows what we found and what we would expect if cell fusion theory was true • This experiment also tells us that cell fusion occurs during embryonic development Slide 18: • As mentioned before, cell fusion signals the change in the pattern of gene expression to turn on muscle-specific genes that encode actin and myosin (slide 12). But how do we know this? If we want to analyze the time course of message induction, we can do that in 2 ways: Northern blotting and In site hybridization Slide 19: • Diagram shows how one would do Northern Blotting - used to find out the stage in muscle cell differentiation when myosin message first appears • A) Isolate RNA from all the stages of differentiation in our tissue culture system. Run RNA of all the stages on a gel subjected to electrophoresis to separate messages of different sizes • B) Place gel on wet filter paper between two spacers • C) Place nitrocellulose filter over the gel and blotting paper over the filter. Transfer separated RNA to filter by capillary action after you add weight • D) Take filter/membrane with transferred RNA that has been separated with all the lanes of different stages and put it in a scalable bag and add radioactive single-stranded cDNA probe that's complementary to myosin message so that the radioactive probe hybridizes with RNA of interest wherever it finds the myosin message. • E) After incubating cDNA probe, wash unreactive/unbound cDNA probe and identify where the radioactive cDNA complementary to myosin is bound by putting x-ray film over it to prepare auto-radiograph. Develop a film and you'll be able to see a series of bands. Identify the stage myosin is turned on. • (F) does not apply here. Forget this step. Slide 20: • We use In Situ hybridization to identify the cells in embryo where myosin gene appears, using the same cDNA that we use in Northen Blotting. But this time, instead of hybridizing with cDNA probe with mRNA, we hybridize it with a tissue section, under condition where it binds to the target RNA. • On the slide, the tissue section is from a mouse Slide 21: • We'd also like to correlate the timings when the myosin message comes on with when the myosin protein profile appears.
Slide 22: • 1) Isolate protein from the different stages of the muscle cell differentiation. • 2) Load the protein from the different stages onto different lanes on the gel. Run the gel to separate proteins into different molecular weight size class. • 3) Then, put the gel with separated proteins next to the nitrocellulose filter and transfer the proteins from the gel to the filter. • 4) Then take the filter and interact proteins with an myosin antibody. They survey the blot and bind to the myosin proteins wherever they find them. • 5) Wash away the primary antibody. Add a secondary antibody with fluorescent or a stain tag on it to visualize. • 6) Wash out the excess and we can identify a band which shows the stage at which myosin protein appears. • Immunocytochemistry is used to identify the cell stage in embryo where myosin proteins are turned on. Slide 23: • Myosin mRNA is first detected in the newly formed myotubes and myosin protein is first detected in the myotube right before fiber formation Slide 24: • This image is a microscopic slide of stained liver tissue. As you can see, liver is not complicated. It is mostly composed of one cell. Slide 27: • 1) Process of detoxification • 3) Process of exertion Slide 28: • There two ways to determine the time when hepatic or liver cell differentiation is initiated: in number embryonic days or in number of somites. • E8 means Embryonic day 8 • Hepatic is another word for liver Slide 29: • During frog development, there is a change in liver function at metamorphosis when frog goes from aqueous environment to outside of water • In mammals, one of the changes in the liver is correlated with changes in the nutritional source from fetus to newborn stage. • Xanthine dehydrogenase (XDH) is involved in metabolism of xanthine Slide 31: • Note: Add an arrow after H2O and before urate • XDH
•
xanthine + NAD+ + H2O
urate + NADH + H+
Slide 32: • In this diagram, we're looking at XDH enzyme activity versus days after hatching • Note: Change the axis label to 'XDH Enzyme Activity' • A low, steady level of XDH is present in chicken before hatching • So we must find out the molecular basis for the drastic increase in XDH upon hatching • Maximum activity is obtained by 8 days and then it remain constant throughout life in chickens Slide 33: • Transcription control would be that XDH gene is not turned on before hatching and its turned on right at hatching and at that point, the message is immediately translated. • Translational control would be that XDH gene is turned on before hatching. In other words, the message is there but its not extensively translated into protein yet. And the hatching is the cue when the message is translated. • Post-translational control would be that XDH protein is present before hatching but it's not enzymatically active because it needs something supplied at hatching. Maybe it needs a cofactor or maybe the enzyme that it needs is made before hatching but gets degraded quickly and at hatching it stops getting degraded. Or maybe the enzyme is made but its not active because it must undergo post-translational modification like phosphorylation or glycosylation to be enzymatically active.. Slide 34: • XDH immunochemistry on liver tissue to see if number of XDH protein molecules increase after hatching • As we can see in the images, there are no and very low level of XDH protein molecules in embryo and its number then increases after hatching, reaching its maximum number 8 days after hatching • So it tells us that it can't be the mechanism of post-translational control, which says that the proteins are present before hatching but not active, because this experiment shows the protein is just not there. Slide 35: • Is XDH protein synthesis needed for XDH enzyme activity to increase after hatching? Yes, because no increase in XDH enzyme activity was observed when puromycin (which inhibits protein synthesis) was added Slide 36: • Is the presence of mRNA needed for there to be increase in XDH enzyme activity after hatching? Yes, because no increase in XDH enzyme activity was observed when RNase (which degrades RNA) was added
Slide 37: • Is mRNA synthesis needed for there to be increase in XDH enzyme activity after hatching? No, because an increase in XDH enzyme activity was observed even after actinomycin (which inhibits mRNA synthesis) was added • The mRNA/message is needed, not the synthesis of message which means the message is already there. • So this tells us that it can't be the mechanism of transcriptional control. The mechanism behind the drastic increase in XDH enzyme activity at hatching is translational control. Slide 39: • Purify XDH enzyme and radioactively label them. Then isolate cell extracts from all developmental stages: 14 days old embryonic stage and 1 day, 4 days, and 8 days after hatching and add the same amount of radioactive XDH to all of them • Note: Correct 'After Birth' to 'After Hatching' on the slide • Run the extracts on the gel and look at their sizes. Results are shown on the graph on the slide • The level of radioactive XDH at 14 days old embryo and 1 and 4 days after hatching equals the size we put in originally. So nothing in the liver cells is degrading XDH. • 8 days after hatching, ability to degrade XDH can be observed.
