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Biochem Biophys Res Commun
1999 Mar 05;2561:162-9. doi: 10.1006/bbrc.1999.0297.
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Spatiotemporal, allelic, and enforced expression of Ximpact, the Xenopus homolog of mouse imprinted gene impact.
Yamada Y
,
Hagiwara Y
,
Shiokawa K
,
Sakaki Y
,
Ito T
.
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Mouse Impact is an imprinted gene encoding an evolutionarily conserved protein of unknown function. We isolated cDNA for the Xenopus homolog of Impact (Ximpact), since the clawed frog not only provides an excellent model for the study of gene function in early development but also allows the generation of interspecific F1 hybrids required for the examination of allelic expression status. The predicted product of Ximpact shows an extreme sequence similarity to those of mouse Impact and its homologs in nematoda, fission yeast, and budding yeast. The transcript of Ximpact is present in oocytes as well as in early embryos, and its spatial distribution is ubiquitous in both embryonic and adult stages. An RT-PCR-RFLP assay using the reciprocal interspecific F1 hybrids and a DNA polymorphism between X. laevis and X. borealis showed that Ximpact is expressed biallelically when analyzed as a whole embryo. Overexpression of Ximpact by RNA microinjection resulted in a higher than normal rate of gastrulation defects, suggesting the need for tight control of its dosage in early development.
FIG. 1. The primary structure of the Ximpact cDNA. Nucleotide sequence and deduced amino acid sequence are shown. Numbers shown
in the left and right indicate those for amino acid residues and nucleotides, respectively. Initiation codon (ATG), termination codon (TAA),
and polyadenylation signal (AATAAA) are underlined. The nucleotide sequence of Ximpact cDNA is deposited in DDBJ, EMBL, and GenBank
(Accession No. AB020319).
FIG. 2. Alignment of the Ximpact homologs. (A) Comparison of deduced amino acid sequences between Ximpact and other homologs.
Identical and similar positions are shaded in black and gray, respectively. Asterisks indicate the motif [G-X(2)-{LIMV}(2)-X(2)-{LIMV}-X(4)-
{LIMV}-X(5)-{LIMV}(2)-X-R-{FYW}(2)-G-G-X(2)-{LIMV}-G] commonly shared by the members of YCR059c/yigZ family. (B) Schematic presentation
of Ximpact homologs. The gray, black, and white boxes indicate the N terminal, central, and C terminal domains, respectively.
Hatched boxes are nonhomologous regions. Numbers in boxes represent identity and similarity expressed in percentile.
FIG. 3. Temporal and spatial expression of Ximpact. (A) Expression
of Ximpact during embryogenesis. Total RNAs (25 ng) isolated
from embryos at different stages (stages 3, 6, 9, 10, 13, 19, 26, and 33)
were used for RT-PCR assay. (B) Spatial distribution of Ximpact
transcript in embryonic tissues. Embryos at 8-cell stage were dissected
into four portions, namely, animal-dorsal (AD), vegetal-dorsal
(VD), animal-ventral (AV), and vegital-ventral (VV) regions as
shown in the upper left panel. Total RNAs isolated from these portions
were subjected to the RT-PCR assay (upper right panel). Embryos
at the neurula stage were dissected into notochord, dorsal
ectoderm, dorsal endoderm, somite, and belly (lower left), and RNA
from each portion was examined by the RT-PCR assay (lower right).
(C) Distribution of Ximpact transcript in adult tissues. The RNAs
from adult brain, skin, heart; stomach, liver; kidney, lung, intestine,
testis, ovary, and muscle were examined by RT-PCR.
FIG. 4. Allelic expression of Ximpact. (A) The schematic map of
the region containing a polymorphic EcoRI site. (B) RT-PCR-RFLP
assay. RT-PCR products from tailbud and tadpole cDNAs prepared
from X. laevis (LL), reciprocal F1 hybrids between X. laevis and X.
borealis (LB and BL), and X. borealis (BB) were digested with EcoRI
and subjected to 4% PAGE.
FIG. 5. Effects of microinjection of Ximpact mRNA. The embryos injected with Ximpact mRNA at the 2-cell stage were observed at early
neurula (stage 14) (A) and the tailbud stage (B). The control embryos were injected with the same amount of lacZ (b-galactosidase) mRNA
and observed at early neurula (C) and the tailbud stage (D).
