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We have previously described the isolation of several genes expressed exclusively in the nervous system of adult Xenopus laevis and activated in the embryo shortly after neural induction. The sequence of one of these cDNAs, 24-15, identifies the corresponding protein as an isotype of the beta subunit of Na+,K(+)-ATPase [ATP phosphohydrolase (Na+/K(+)-transporting); EC 3.6.1.37]. This form is distinct from the previously described beta 1 subunit of Xenopus, and the protein sequence comparison suggests that it is not the frog homolog of the mammalian beta 2 subunit; therefore, we refer to the 24-15 protein as the beta 3 subunit of Na+,K(+)-ATPase of Xenopus. Antisera directed against beta 3-subunit fusion protein detected a protein in adult brain extracts with the size and properties expected for a Na+,K(+)-ATPase beta subunit. In Xenopus the beta 1 and beta 3 subunits are expressed as maternal mRNAs at similar levels; during embryogenesis rapid accumulation of beta 3 mRNA begins at stage 14 (early neurula), and the rapid accumulation of beta 1 mRNA begins at stage 23/24. In situ hybridization of antisense RNA probes to tadpolebrain sections indicates that beta 3 subunit is expressed throughout the developing brain. We suggest that beta 3 is a major Na+,K(+)-ATPase beta subunit present during early nervous system development in the frog.
FIG. 6. In situ hybridization of antisense Na+,K+-ATPase ,B3 subunit mRNA to a sagittal section of tadpolebrain viewed with dark-field optics; anterior is at left, and dorsal is at top.
FIG. 2. Primer-extension analysis of (83 mRNA. A labeled antisens6
oligonucleotide (5'-GAGTCCGGCGAGGAATCCAT, nt
195-176 of the cDNA sequence, Where the coding region starts at nt
37) was hybridized to 10 ,ig of RNA and extended with reverse
transcriptase. The extension products were electrophoresed on
urea/6% polyacrylamide gel and exposed to x-ray film. Lanes: 1,
control reaction with only carrier tRNA; 2, kidney RNA; 3, stage 8
RNA; 4, stage 19 RNA; and 5, stage 25 RNA. Migration of molecular
size markers (lane M) is indicated at left. Asterisk and triangle at right
represent locations of the endpoint of cDNA 24-15 and the AUG
initiation codon, respectively.
FIG. 3. RNA accumulation of (31 and (3 mRNAs during early
development. Total RNA (10 pAg) isolated from staged embryos was
analyzed by Northern blots probed either with a Xenopus (31 (a) or
(33 (b) coding region probe. The lanes are labeled with the stage, and
migration of 28S and 18S rRNA it indicated at right. Both films were
exposed for 12 hr.
FIG. 4. Induction of Xenopus j33 mRNA in animal caps. Animal
cap explants were isolated from stage-8 to -9 embryos and treated
with XTC-conditioned medium containing MIF. RNA was isolated
and analyzed by Northern blotting for expression of (33 mRNA. As
control, one set of caps was treated with fresh medium; in addition,
total-embryo RNA was isolated at corresponding stages. (a) RNA
from treated animal caps. Lanes are labeled with the equivalent stage
of RNA isolation; + and - refer to treatment with MIF or control
medium, respectively. (b) RNA from whole embryos at indicated
stages. Location of 28S and 18S rRNA is indicated at right.
FIG. 5. Immunoblot of membrane proteins treated with anti-
Xenopus Na+,K+-ATPase /33 subunit fusion-protein antisera. Membrane
proteins (40 ,ug) from different tissues were electrophoresed on
SDS/10% polyacrylamide gel and transferred to Immobilon-P membrane
(Millipore). After incubation with rabbit antisera and 1251[
labeled protein A, immunoreactive proteins were visualized by
autoradiography. Br, brain; Ki, kidney; Li, liver. The lane labeled Br
+ Ki contained a 1:10 mixture of brain and kidney proteins.
Migration of prestained molecular mass markers is indicated at right.
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