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THE CHARACTERIZATION OF
ANTIZYME IN DEVELOPING
Xenopus laevis EMBRYOS
Alexandra Silveira and Charles Toth
Department of Biology
Providence College, Providence, RI
Protein Degradation
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Cell cycle and proliferation
Differentiation and development
Stress response
Genesis of neural networks
Modulation of cell surface receptors
Regulation of immune response
Apoptosis
Transcriptional regulation
Pathogenesis
– Cancer, cystic fibrosis, neurodegenerative diseases
Charles Toth, Providence College
Protein degradation by the 26S
proteasome
http://www.rpi.edu/dept/bcbp/molbiochem/MBWeb/mb2/part1/protease.htm
Protein degradation by the 26S
proteasome
ODC degradation – a unique protein
degradation pathway
John Mitchell, University of Northern Iowa
John Mitchell, University of Northern Iowa
ODC and Polyamine Biosynthesis
http://www.bios.niu.edu/mitchellab/general.html
Polyamines
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Organic compounds found bound
to RNA and DNA in cells
High levels are toxic
Necessary for cell growth and
survival
– gate currents of ion channels
– have effects on chromatin
condensation and
transcriptional regulation
– neutralize negative charges of
RNA and DNA
– regulate the levels of AZ
– provide a necessary
postranslational modification
to eIF-5a protein
http://www.biol.lu.se/zoofysiol/Cellprolif/Research/research_area_1.html
AZ Functions
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ODC degradation
Anti-proliferative
effect on cells
Blocks uptake of
additional polyamines
when polyamine
levels are too high
High levels of
polyamines in turn
regulate AZ through a
ribosomal frameshift
http://www.bios.niu.edu/mitchellab/general.html
Ribosomal Frame-Shifting
High levels of polyamines cause a
ribosomal frame shift that produces an
active form of AZ.
 Without the ribosomal shift a truncated and
inactive AZ is formed.
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Why study antizyme?
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Studying the unique ODC degradation pathway
can lead to a better understanding of
– ubiquitin mediated protein degradation
– the many roles of polyamines in the cell
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AZ is a member of an ancient gene family
– AZ1 and AZ2 widely expressed and involved in ODC
degradation
– AZ3 is testis specific
Why study antizyme?
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AZ has been cloned from various organisms
– vertebrate-man, chicken, rat, mouse, zebrafish,
Xenopus, electric ray
– invertebrate-Drosophila, S. pombe, C. elegans, silk
worms, gray mold, etc.
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AZ knockout in mice (Matsufuji & Noda)
– viable and morphologically normal
– high rate of prenatal fatality from high levels of ODC
– phenotype complicated by additional family members
Xenopus As A Developmental Model
Ideal model because:
– easily cared for in lab
– in vitro fertilization
– the developing
embryos are easily
cared for
– the embryos develop
quickly
http://www.xenopus.com/products.htm
Xenopus life cycle
Wolpert et al., Development 2000
Methods
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In vitro fertilization
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Northern Blot
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Immunohistochemical Staining
In Vitro Fertilization
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Female frogs are
injected with HCG to
induce ovulation
Male frogs are
sacrificed in order to
obtain internal
testicles
Eggs are fertilized in
vitro
http://www.fundamentalbiology.arc.nasa.gov/Image1.jpg
Northern blot analysis
www.columbia.edu/.../c2005/handouts/ northernforweb.gif
Northern blot results
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AZ is expressed as a
maternal and zygotic
transcript
Size of message increases
post-MBT
– MBT is the point where
zygotic genes are expressed
instead of maternal genes
and the cell cycle becomes
asynchronous indicating the
beginning of differentiation
Charles Toth, Providence College
Conclusions
Different sizes of antizyme mRNA are
present during different times in embryonic
development.
 A larger mRNA message is present postMBT.
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Possible Implications
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The larger messages can indicate a
– a closely related familial gene
– different post-transcriptional modifications that
ensure zygotic gene expression
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Results are inconclusive because of the
ribosomal frame-shift needed to produce an
active form of AZ.
Immunohistochemical Staining
Antibodies against
antizyme are used to
detect and stain
antizyme in the
developing embryo.
http://www-celanphy.sci.kun.nl/Bruce%20web/Bruce%20Gifs/immunocyto.gif
Immunohistochemical Staining
Results
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Stage 10 embryos
There is a darkly
staining ring around
the blastopore from
which the mesoderm
will develop.
The animal cap is
almost uniformly
stained.
Immunohistochemical Staining
Results
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Stage 18/19
Top image:
– staining along the neural
groove and at both the
anterior and posterior ends
of the embryo, some
staining at the ventral side
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Bottom image:
– dorsal side shows dark
staining at the posterior and
anterior ends and a ring
pattern of staining in the
area that will eventually
give rise to the mesoderm
Immunohistochemical Staining
Results
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Stage 33/34
Staining is
primarily focused
around the somites
and the anterior end
of the tadpole
These locations are
where the muscles
and nerves are
developing
Conclusions
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There is clearly a differential expression of
antizyme during the various stages of embryonic
development.
– staining of ring around the blastopore and animal cap
– staining of the neural groove, anterior and posterior
ends, ventral, and dorsal lateral staining
– staining of the somites and anterior portion of the
tadpole
Possible Implications
The staining around the blastopore ring, the
dorsal-lateral staining of the neurula, and
the somite staining of the tadpole implicate
a role of antizyme in muscle development.
 The staining of the animal cap and the
anterior end of the developing embryo
implicate a role of antizyme in the
development of the central nervous system.
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Future Endeavors
In situ
 Western
 ablation studies
 forced
expression
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Acknowledgements
Dr. Charles Toth
 Dr. Senya Matsufuji
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– Jikei University, Tokyo
Tiffany LaFortune
 My family
 My roommates
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