CONTENTS
Main Page Dynamic Development
The Foundations of Developmental
Biology
Gametogenesis
From Sperm and Egg to Embryo
Genetic Regulation of Development
Organizing the Multicellular
Embryo
Generating Cell Diversity
Dynamic Development at a
Glance
Learning Resources
Research Resources
The Developmental Biology Journal
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Developmental Biology Tutorial |
Gene Expression during Amphibian Oogenesis
How does the oocyte nucleus make so much stuff?
One distinction of amphibian oogenesis is the formation of lampbrush
chromosomes during the major phase of oocyte growth and differentiation.
Meiosis I is suspended at the diplotene stage, and the homologs remain attached
by chiasmata. Each homolog is comprised of parallel strands of sister chromatids.
At intervals, the chromatin is compacted into chromomeres, and chromatin
loops extend laterally from the chromomeres.
(Browder et al., 1991, Fig. 3.27, Gilbert, 1997, Fig. 22.25;
Kalthoff, 1996, Fig. 3.12; Shostak, 1991, Fig. 3.13)
A ribonuceoprotein matrix accumulates on the loops, which consists of proteins
and nascent RNA (i.e., RNA that was transcribed on the loop chromatin).
The matrices on sister loops are identical, but those on different loop
pairs are often quite distinctive. The matrix is frequently asymmetrical,
being thin at one end and thick at the opposite end .
(Browder et al., 1991, Fig. 3.29).
This asymmetry suggests directionality in transcription, with the thickness
of the matrix reflecting the lengths of nascent transcripts. This is confirmed
by electron microscopic examination of loops.
(Browder et al., 1991, Fig. 3.30A)
Loops may have more than one transcription unit with opposite polarity.
Note how closely packed the polymerases are on the loop chromatin. This
suggests that the loops are quite active in transcription. Considering the
duration of the lampbrush phase, a tremendous amount of RNA can be synthesized
during oogenesis in these organisms.
The image shown below is from Dr. Ulrich Scheer's entry on the University
of Wuerzburg Web site. It shows a lampbrush chromosome of the salamander
Pleurodeles waltlii, visualized by immunofluorescence microscopy.
The fluorescence is due to a fluorescent-labeled antibody that recognizes
proteins associated with the nascent transcripts on the chromosome.
Transcription on lampbrush loops has been demonstrated by in situ
hybridization. Why does the labeled probe bind only to the nascent
RNA and not to the DNA?
(Browder et al., 1991, Fig. 3.31; Kalthoff, 1996, Fig. 15.1)
Alpha-amanitin is a selective inhibitor of RNA polymeras activity: low concentrations
(0.5 µg/ml) inhibit polymerase II transcription. Incorporation of
label into RNA on lampbrush loops is abolished by this treatment. Incorporation
of label on short stretches of loop axes is retained; this is due to polymerase
III transcription of 5S RNA genes and is abolished by treatment with 200
µg/ml alpha-amanitin.
(Browder et al., 1991, Fig. 3.32)
Further evidence for polymerase II in transcription on loops is provided
by treatment with antibodies to polymerase II, which causes rapid termination
of transcription and retraction of lampbrush loops.
(Browder et al., 1991, Fig. 3.33)
Although they have been studied most intensively in amphibians, they are
quite widespread in the animal kingdom. This suggests that they play a significant
role in synthesis and accumulation of RNA during oogenesis.
Messenger RNA production during oogenesis has been studied extensively by
monitoring the accumulation of polyadenylated RNA. This category of RNA
is representative of a large fraction of the messenger RNA population. Poly(A)
is thought to play two primary roles: protection from 3' exonucleases and
promotion of translation. The polyadenylation status of RNA made during
oogenesis can change dynamically at later stages, and this has functional
significance.
Isolation of poly(A)+ RNA is facilitated by use of oligo (dT), to which
the poly(A) tracts bind reversibly.
The majority of poly(A)+ RNA molecules in the full-grown Xenopus
oocyte are unusual: they contain interspersed single-copy and repeat sequences
that are covalently linked. This suggests that transcription during oogenesis
may ignore termination signals and proceed past them into contiguous repetitive
sequences.
Perhaps this is an explanation for the large sizes of lampbrush transcripts
(which may be up to several kb in length).
Although the interspersed poly(A)+ RNA is not translatable, much of the
RNA in full-grown oocytes is potentially translatable. However, the
majority of this RNA is not translated in oocytes. Only 20% of the translatable
poly(A)+ RNA is in polysomes. Why is 80% of it not being utilized?
The synthesis of poly(A)+ RNA begins early in oogenesis. There is net accumulation
of this class of RNA until the beginning of vitellogenesis, when it plateaus.
Although transcription persists, there is no net quantitative or qualitative
change in poly(A)+ RNA throughout the remainder of oogenesis. The absence
of qualitative changes has been demonstrated by use of cloned probes. The
constancy of the RNA population indicates that the continued synthesis of
poly(A)+ RNA is counterbalanced by degradation. The maintenance of a constant
level of RNA is apparently the task of the lampbrush chromosomes with their
closely-packed polymerases.
Ribosome Components
The rate of ribosome accumulation during oogenesis is very impressive.
It exceeds that of the most active somatic cells by at least 1000-fold.
Thus, the ribosomal components (5S, 18S and 28S RNA and ribosomal proteins)
are produced in massive amounts.
5S RNA is synthesized early in Xenopus oogenesis - before the other
components are made. It is then stored in 7S and 42S ribonucleoprotein particles
and later utilized in ribosome assembly. 5S RNA accounts for ~45% of the
total RNA in previtellogenic oocytes.
The genome contains two sets of 5S RNA genes: oocyte-type and somatic-type.
There are 24,000 tandemly-repeated oocyte-type coding units per haploid
genome. How many templates are there in an oocyte nucleus?
Unlike polymerase II genes with which you are familiar, the 5S genes (which
are transcribed by polymerase III) have an internal promoter. Transcription
is initiated as the result of an interaction between the promoter site and
three oocyte transcription factors. This interaction specifies that transcription
will be initiated upstream; i.e., at the transcription start site.
TFIIIA has been especially well studied. TFIIIA is the prototypical zinc
finger protein (of which many are now known). This protein has nine
of these structures, which are loops formed by periodic folding of the protein
due to binding of amino acids within each repeat structure to a zinc ion.
(Browder et al., 1991, Fig. 3.37, Gilbert, 1997, Fig. 10.30)
Binding of TFIIIA to the promoter has been demonstrated by DNase footprinting
analysis. DNA that has been labeled at one end is digested with the enzyme
deoxyribonuclease I under conditions in which the enzyme makes a single
cut in the DNA. Fragments of different lengths are produced, which are separated
by electrophoresis and visualized by autoradiography. Bands will be seen
that correspond to cleavage at each nucleotide position unless protein
protects the site and prevents the enzyme from cutting the DNA. The gap
in the sequence is referred to as a "footprint".
(For an example of this technique, see Browder et al., 1991,
Fig. 18.16A)
TFIIIA is a bifunctional molecule in that it is also capable of binding
to 5S RNA. What consequences would this have for regulation of 5S gene transcription?
The abundance of TFIIIA changes during oogenesis. It is abundant during
previtellogenesis and declines in amount thereafter. The abundance of the
protein reflects the abundance of the messenger RNA. Its abundance declines
5-10x during postvitellogenesis (Pfaff and Taylor, 1992). How does
this differ from most messenger RNA molecules? Trace the consequences of
regulation of TFIIA gene transcription for accumulation of 5S RNA.
5S RNA synthesis is no longer detectable after oocyte maturation. Egg extracts
will only support 5S gene transcription if exogenous TFIIIA is added, whereas
oocyte extracts will support transcription without it addition. How
do you interpret these results? Embryos resume 5S RNA synthesis,
but only from the somatic genes. This is an excellent example of stage-specific
gene expression.
18S and 28S ribosomal RNA are the most abundant components of the oocyte
RNA population. Their massive synthesis during oogenesis is facilitated
by amplification of the ribosomal RNA genes. Although this phenomenon has
been studied most intensively in amphibians, it is also found in some species
of insects, mollusks and fish.
(Browder et al., 1991, Fig. 3.39)
Nuclei of somatic cells of Xenopus contain two nucleoli, but hundreds
of nucleoli line the inner surface of the nuclear envelope of the germinal
vesicle. It has been estimated that it would take 400 years for a frog to
produce the amount of ribosomal RNA found in the full-grown oocyte if amplification
did not take place.
Transcription of ribosomal RNA is very efficient during vitellogenesis,
with polymerase molecules being tightly packed on the chromatin.
(Browder et al., 1991, Fig. 3.40; Gilbert, 1997, Fig. 22.26;
Kalthoff, 1996, Fig. 3.13)
By the way, which polymerase molecule is used for ribosomal gene
transcription?
Learning Objectives
- Correlate formation of lampbrush chromosomes with the events of meiosis.
- How do we know what the matrix of lampbrush chromosomes is composed
of?
- How has transcription been demonstrated on lampbrush chromosomes?
- What is known about poly(A)+ RNA in oocytes? Why do we care?
- What is unusual about the ribosome content of oocytes?
- How many 5S RNA genes are present in the genome?
- Review the organization of 5S RNA genes.
- Review the role of TFIIIA in 5S RNA synthesis.
- How does the oocyte make so much 18S and 28S RNA?
Digging Deeper:
Lampbrush Chromosomes
- Check out the description of lampbrush chromosomes by Dr.
H.C. Macgregor, who studies them.
Ribosome Components
Recent Literature
Brown, D.D. 1997. E.B. Wilson Award Lecture, 1996. Differential gene
action. Mol. Biol. Cell 8: 547-553.
References
Browder, L.W., Erickson, C.A. and Jeffery, W.R. 1991. Developmental
Biology. Third edition. Saunders College Pub. Philadelphia.
Gilbert, S.F. 1997. Developmental Biology. Fifth Edition. Sinauer.
Sunderland, MA.
Kalthoff, K. 1996. Analysis of Biological Development. McGraw-Hill.
New York.
Pfaff, S.L. and Taylor, W.L. 1992. Characterization of a Xenopus
oocyte factor that binds to a developmentally regulated cis-element
in the TFIIIA gene. Develop. Biol. 151: 306-316.
Shostak, S. 1991. Embryology. An Introduction to Developmental Biology.
HarperCollins. New York. |
