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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 (Fig. 1).
Figure 1. Lampbrush chromosome from Pleurodeles
oocyte. The chromosome was photographed with phase contrast optics using an inverted
microscope. Reproduced with permission of Dr. Ulrich Scheer,
University of Wuerzburg.
(See also 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.
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 polymerase 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
Isolation of poly(A)+ RNA is facilitated by use of oligo (dT), to which the poly(A) tracts
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
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,
By the way, which polymerase molecule is used for ribosomal gene transcription?
- 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?
- Check out the description of lampbrush chromosomes by Dr. H.C. Macgregor, who studies
Brown, D.D. 1997. E.B. Wilson Award Lecture, 1996. Differential gene action. Mol. Biol.
Cell 8: 547-553.
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,
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.
Shostak, S. 1991. Embryology. An Introduction to Developmental Biology.
HarperCollins. New York.