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Genes whose expression is restricted to oogenesis and early development may have important functions in these processes. Northern analysis showed that Xenopus B4 mRNA is expressed in oogenesis and embryogenesis through to the neurula stage. Immunocytochemistry with anti-B4 antibodies showed that B4 protein is only detectable in preneurula stages; it is localized to nuclei and is associated with metaphase chromosomes. Immunoblotting revealed approximately constant levels of B4 protein per embryo for the first 2 days of development. Thus, as the number of nuclei increases during early development, the amount of B4 protein per nucleus is diluted out. Sequencing of two B4 cDNA clones revealed that the predicted B4 translation product is a 29-kD protein with 29% identity with histone H1, distributed over the entire length of its sequence. The B4 protein also has certain other H1 protein characteristics--a tripartite structure consisting of a mainly hydrophobic central domain flanked by an amino-terminal segment and a long hydrophilic carboxyterminal tail containing a tandemly repeated amino acid motif. However, in contrast to histone H1 mRNA, B4 mRNA has a classic polyadenylation signal, is polyadenylated, and lacks the histone H1 3' noncoding consensus sequence involved in RNA processing.
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Figure 1. Northern blot analysis of Xenopus RNA probed with
nick-translated clone B4.0 (Lanes 1-7) 10 ~g/lane of total RNA.
(Lane 1) Stage VI oocyte; (lane 2) cleavage (stages 6.5-7); (lane
3) blastula (stage 9); (lane 4) gastrula (stages 10.5-12); (lane 5)
neurula (stages 17-20); (lane 6) early tailbud (stages 24-26);
(lane 7) 3-day-old tadpole (stages 37-41); (lane 8) 0.8 ~g of liver
poly(A) + RNA; (lane 9} 0.4 ~g of muscle polyIA) + RNA.
Figure 2. (See following page.) (A) Relationship between B4.0 and B4.1 cDNA clones. Below the horizontal line representing the
clones are the nucleotide positions of the beginning and end of the separate cDNAs. The nucleotide differences between the two
clones are shown with their respective nucleotide positions. (*) Absence of a nucleotide. (B) Nucleotide sequence of B4 cDNA and
predicted amino acid sequence of B4 protein. The sequence is a composite of the B4.0 and B4.1 cDNA insert sequences. Numbering of
nucleotides starts at the first nucleotide of the B4.0 clone. At five nucleotide positions there was a disparity in sequence between the
two clones; the sequence of the B4.1 clone is given with the B4.0 nucleotide written above. (*) Nucleotide position that is absent from
the B4.0 clone. The predicted amino acid sequence (three-letter code) is shown underneath the major open reading frame. Numbering
of amino acids starts from the first amino acid of the B4 protein. The change in open reading flame caused by the absence of a
nucleotide (*) in the B4.0 insert is shown beneath. {***) Stop codon of the B4.0 reading frame. The three direct amino acid repeats
toward the carboxyl end are indicated by dashed arrows. The polyadenylation signal AATAAA is dot-underlined. The nucleotide
sequence underlined at position 663-682 is that contained within the oligonucleotides used as hybridization probes (the first nucleotide
was only present in the B4.0 type oligonucleotide; Materials and methods).
Figure 3. Differential hybridization of oligodeoxynucleotides
to B4 SP6 transcripts and oocyte poly(A) + RNA. Hybridization
under differential hybridization conditions to SP6 transcripts
(-150 ng/lane) of the B4.1 type (B4.1) or B4.0 type (B4.0) and to
oocyte (O) poly(A) + RNA (3 ~g/lane) using as probes oligonucleotide
1014 (B4.0 type) (A) and oligonucleotide 1015 (B4.1
type) (B) (Materials and methods). (Arrow) Position of the 1.2-kb
B4 mRNA. Both hybridizing bands in the SP6 transcript lanes
are in vitro transcription products that contain the B4 sequence.
The two left lanes in A are 6-hr autoradiographic exposures;
all remaining lanes are 18-hr exposures.
Figure 4. Hydropathy plot of the B4 protein. Numbers along
the abscissa represent the amino acid positions of the B4 protein,
with numbering starting at the amino terminus. The ordinant
shows the hydropathic index. The segment length for
analysis was 9 (Material and methods). A, B, and C are the three
major domains of the B4 protein (Results).
Figure 5. Comparison of B4 protein with Xenopus histone
H1B (HSXL1B). Double dots indicate identity and single dots
indicate conservative replacements.
Figure 6. Immunocytochemistry of Xenopus sections. Paraffin-embedded Xenopus tissue sections were stained With either B4-specffic
antibodies (A and B, -80 ~g/ml), or Xenopus anti-histone HI antiserum (C, 1 : 100 dilution} and visualized with a fluoresceinated
secondary antibody. Left-hand-side panels show antibody staining; right-hand-side panels show counterstaining of the same
section with DAPI. The length of photographic exposure in seconds for each of the antibody : DAPI pairs, respectively, is given in
parentheses (exp.x,x) below. (A) Low-power view of a cross-section of a blastulaembryo (stage 8) with animal hemisphere at the top of
the picture (exp. 60,8). The embryo is approximately 1.2 mm in diameter. (B) High-power view (6.3 x higher magnification than in A)
of: (a) cleavage (stage 6.5), centering on a set of metaphase chromosomes (exp. 60,8);/b) blastula (stage 8)(exp. 60,8); (c) gastrula (stages
11-11.5) (exp. 60,15); (d) neurula (stage 19)(exp. 60,8); (e) 3-day-old tadpole (stages 40-41}, centering on neural tube (exp. 240,4). (C)
High-power view of: (a) blastula (stage 8) (exp. 60,8); (b) 3-day-old tadpole (stages 40-41) (exp. 30,8).
Figure 7. Immunoblotting of an embryonic nuclear extract
and perchloric acid protein extracts. (A) A Western blot of a
nuclear extract from preneurula embryos was probed with B4-
specific antibodies (-15 mg/ml) (lane 1) or f~-galactosidase-specific
antibodies (-15 mg/ml) (lane 2). (B, C) Western blots of perchloric
acid extracts from stage VI oocytes (lane o), unfertilized
eggs (lane e), stage 9 blastulae (lane b}, and stages 26-27 early
tailbud embryos (lane t) were probed with anti-B4 antibodies
(-15 ml, derived from a different rabbit from those in A)(B}; or
Xenopus anti-H1 antiserum (1:500 dilution)(C). (B) Five-day
autoradiographic exposure; (C) 18-hr autoradiographic exposure.
Antibodies were visualized with l~SI-labeled protein A.
The numbers on the left refer to the molecular weight in kilodaltons.
