Mohun TJ and Garrett N (1987), An amphibian cytoskeletal-type actin gene is ex...

XB-ART-27955

Development 1987 Oct 01;1012:393-402.

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An amphibian cytoskeletal-type actin gene is expressed exclusively in muscle tissue.

Mohun TJ , Garrett N .

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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.

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Species referenced: Xenopus laevis
Genes referenced: actg1 actl6a fos thra

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	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 (—). 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).