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The complete nucleotide sequence of two Xenopus actin genes encoding cytoskeletal protein isoforms has been determined. Transcripts from these genes are remarkably similar in nucleotide sequence throughout their length and code for type-5 and type-8 cytoskeletal actins. Both share some sequence homology with human gamma-actin mRNA within the 3' untranslated region but none with the equivalent region of any vertebrate beta-actin transcript. The promoter regions of the two Xenopus genes are virtually identical from the cap site to the CCAAT box and show extensive homology further upstream. Despite such similarity, the two genes are divergently expressed during embryonic development. The type-5 actin gene is expressed in all regions of the developing embryo whilst the type-8 gene is coregulated with the muscle-specific skeletal actin gene. In common with mammalian and avian cytoskeletal actin counterparts, the Xenopus genes possess a conserved sequence within their promoter that has previously been identified as a transcription-factor-binding site.
Fig. 1. Dot matrix comparison of Xenopus cytoskeletal
actin genes. A 5522 nucleotide sequence from A 42-5 was
compared with a 5610 nucleotide sequence of A C3-8
using the DIAGON programme of Staden (1984) with a
span of 21 and percentage match of 15. The intron/exon
structure of the two actin genes is indicated. Filled boxes
represent exons encoding the actin polypeptide. Hatched
boxes represent exons encoding untranslated regions of
actin mRNA. Open boxes represent introns. The
direction of transcription of each gene is indicated.
Fig. 2. Nucleotide sequence of type-5 and type-8
Xenopus cytoskeletal actin genes. The two gene
sequences have been aligned from their cap site (the first
nucleotide). Intron sequences are omitted but their
locations indicated by arrows. Only nucleotide differences
between the two genes are shown in the type-8 sequence.
Gaps introduced to permit alignment are indicated by (.).
The deduced amino acid sequence encoded by the type-5
gene is shown and numbered. The single amino acid
difference distinguishing the type-8 polypeptide is boxed.
The hexanucleotide polyadenylation signal is underlined
and two polyadenylation sites identified from cDNA
clones indicated (A). A short region flanking the 3' end
of each gene is shown to indicate its divergence between
the two sequences. The entire gene sequences are
available from the EMBL Nucleotide Library or on
request.
Fig. 3. Sequence homology between Xenopus cytoskeletal
and the human y-actin genes. The 3' untranslated region
of the Xenopus laevis type-5 actin gene is compared with
those of the Xenopus borealis type 1, chick type 5, human
/S, and human y-actin genes as indicated using the
DIAGON programme (span 15, match 11). In each
comparison, the Xenopus type-5 sequence is represented
by the vertical axis. The 5'-most 318 nucleotides of the X.
borealis untranslated region is aligned with the 5'-most
340 nucleotides of the X. laevis sequence. All other
comparisons utilize the complete X. laevis type-5
untranslated region (805 nucleotides) and the entire chick
type-5 (399 nucleotides), human /S (600 nucleotides) and
human y (967 nucleotides) 3' untranslated regions
respectively. Similar results were obtained using the
Xenopus laevis type-8 actin gene sequence (data not
shown).
Fig. 4. Comparison of Xenopus type-5 and type-8 cytoskeletal actin gene promoters. The two sequences are aligned and
numbered with respect to their cap sites (+1). Gaps introduced to permit maximum alignment are indicated (.) as are
nucleotides shared by each promoter (*). The CCAAT and TATA boxes are underlined.
Fig. 5. Conserved promoter sequence shared by vertebrate cytoskeletal actin genes. The sequence downstream of the
CCAAT box from various vertebrate cytoskeletal actin genes is shown. (The human y-actin gene promoter sequence is
from Erba (1986). See text for all other references.) Gaps introduced to permit best alignment are indicated (â). A
conserved 20 nucleotide region which is similar to a transcription-factor-binding site (SRE) located 300 nucleotides
upstream of the human c-fos proto-oncogene cap site is shown in bold. The most conserved region comprising ten
nucleotides at the centre of the binding site is underlined. This region is most conserved between the Xenopus laevis
genes and the human y-actin gene. It is also shared by the Xenopus borealis type-1 actin gene which is 'y-like' (see text).
Fig. 6. Expression of type-5 and type-8 actin genes during Xenopus development. Total RNA from Xenopus oocytes,
embryos and adult tissues was assayed for transcripts of the type-5 and type-8 actin genes by RNAase mapping.
Protected fragments representing transcripts of each gene are indicated by arrows as is the size of the RNA probe (P).
Lane 1: 15 fig ovary RNA. Lanes 2-5: 5/ig of blastula, gastrula, neurula and tailbud RNA respectively. Lane 6: 5/jg
HeLa RNA. Lanes 7-10: ljig adult skeletal muscle, kidney, heart and intestine RNA. Lane 11: 5/ig Xenopus A6
cultured renal cell RNA. Molecular weight markers comprising a Hinfl digest of pBR322 (filled in) are shown (M) and
sizes given.
Fig. 7. Expression of type-5 and type-8 actin genes in
embryonic tissues. Total RNA from seven regions
dissected from tailbud (stage 20-23) embryos was assayed
for the presence of type-5 and type-8 actin transcripts by
RNAase mapping as shown in Fig. 6. Lanes 1-7: ventral
mesectoderm, dorsal endoderm, dorsal ectoderm, ventral
endoderm, notochord, myotome, nerve cord. Each lane
contains RNA from the various parts of three dissected
embryos. The dorsal ectoderm sample (lane 3) contained
much less RNA than the others. Longer exposure reveals
the presence of type-5 actin transcripts in this sample.
The protection obtained with RNA from tailbud embryos
(5/xg) is also shown (tb).
