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We have isolated a novel acetyltransferase from Xenopus laevis, named Xat-1. Xat-1 cDNA encodes a predicted protein of 846 amino acids that contains tetratricopeptide repeat (TPR) domains mediating protein-protein interactions and a bipartite nuclear localization signal (NLS). Its apparent molecular mass of 98.8 kDa was determined by SDS-PAGE analysis of Xat-1 recombinant protein in vitro translated in rabbit reticulocyte lysate. Xat-1 is homologous to N-terminal acetyltransferase 1 (NAT1), a gene that was originally discovered in yeast. Furthermore, it has many orthologs from human, mouse, Drosophila, C. elegans, and even Arabidopsis, thereby suggesting that these constitute a novel acetyltransferase family whose functions have been not examined. Xat-1 transcripts are expressed at relatively constant levels throughout early embryonic stages. They also exhibit dynamic expression pattern in brain, somites, branchial arches, pronephros, and otic vesicles.
FIG. 1. Amino acid sequence comparison of Xat-1 and its putative homologues identified in human, mouse, and Drosophila: human
protein (Accession No. AF327722), mouse protein (Accession No. AK005056), and Drosophila protein (Accession No. AE003512). The most
conserved residues are indicated by the shaded background. The TPR regions and bipartite nuclear localization signal (NLS) sequence of
these acetyltransferases are indicated by bold lines and asterisks, respectively. Gaps in the sequence alignment are indicated by dashed lines.
FIG. 2. Analysis of the expression of recombinant Xat-1 protein
in rabbit reticulocyte lysates. In vitro transcribed recombinant Xat-1
RNAs were in vitro translated in rabbit reticulocyte lysates and
analyzed on 10% SDS–PAGE. Lane 1, 50 nM of capped Xat-1 RNA
from pCS21 Xat-1; lane 2, 70 nM of firefly luciferase RNA (Promega;
61 kDa); lane 3, no RNA control. Numbers on the left indicate the
positions of marker proteins and their apparent molecular size (in
kDa). The position of recombinant Xat-1 protein is indicated by an
arrow.
FIG. 3. RT-PCR analysis of the temporal expression of Xat-1
during early embryogenesis. Stages are indicated above the lanes.
ODC serves as a loading control and the -RT lane is a control of
RT-PCR on stage 35 whole embryo RNA in the absence of reverse
transcriptase.
FIG. 4. Expression patterns of Xat-1 in Xenopus early embryo assayed by in situ hybridization. (A) Animal (right) and vegetal (left) view
of early gastrula (stage 10) embryos. An arrow indicates yolk plug. (B) Mid-gastrula stage embryo that was cut along the midline before
hybridization. An arrowhead indicates dorsal lip. (C) A cross section of a mid-neurula stage embryo. SM, somitogenic mesoderm; NO,
notochord; NP, neural plate. (D) Lateral view of a late neurula stage embryo. Anterior is left. An arrowhead indicates anteriorendoderm. (E)
Dorsal view of the same embryo as in D. Anterior is up. An arrow indicates neural plate. (F) Head region of a tailbud stage embryo. ba,
branchial arches endoderm; pn, pronephros; sm, somites; ov, otic vesicle; e, eye. An arrowhead indicates strong Xat-1 message in the
mid-brain. (G) Trunk region of the same embryo as in F. (H) Dorsal view of the brain region of a tailbud stage embryo. Arrowheads indicate
positive Xat-1 staining. (I) Head region of a tadpole stage embryo that was sagittally cut. Two arrowheads to the left indicate highly
concentrated Xat-1 mRNA in the neuroepithelia lining the brain lumen. No signals are visible in the axon-enriched marginal zone of brain
(upper right arrowheads). An arrow shows specific Xat-1 signal in the branchial arch.
naa15 (N(alpha)-acetyltransferase 15, NatA auxiliary subunit ) gene expression in bissected Xenopus laevis embryo, assayed via in situ hybridization, NF stage 11, vegetal down, dorsal right.
naa15 (N(alpha)-acetyltransferase 15, NatA auxiliary subunit ) gene expression in Xenopus laevis embryo, assayed via in situ hybridization, NF stage 28, lateral view, anteriorleft, dorsal up.
