Tsujimura A et al. (1997), Cloning of Xenopus presenilin-alpha and -beta c...

XB-ART-16893

Biochem Biophys Res Commun 1997 Feb 13;2312:392-6. doi: 10.1006/bbrc.1996.6043.

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Cloning of Xenopus presenilin-alpha and -beta cDNAs and their differential expression in oogenesis and embryogenesis.

Tsujimura A , Yasojima K , Hashimoto-Gotoh T .

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Human presenilin (ps)-1 and -2 genes have recently been shown to be involved in genesis of early-onset familial Alzheimer's disease. By probing with human (H-) ps-1 cDNA, we isolated two types of cDNA clones, named X-ps-alpha and -beta, from a Xenopus brain cDNA library. The encoded proteins, X-PS-alpha and -beta, may correspond to H-PS-1 and -2 with 89.4 and 85.9% similarity, respectively. The strongest expression of these genes was observed in ovaries and in the early stages of oogenesis, although weak or moderate expression was detected ubiquitously for both X-ps-alpha and -beta genes in multiple tissues. Upon oocyte maturation, the X-ps-beta mRNA level was constant even after fertilization until the midblastula transition. Zygotic expression of these genes became evident only at the tailbud stage. We propose that presenilins may function in preventing cells from undergoing apoptotic degeneration particularly prior to embryonic development and in developmentally matured tissues.

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Species referenced: Xenopus
Genes referenced: psen2 uqcc6

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	FIG. 1. Structures of X-ps-a and X-ps-b cDNA clones. The cDNA are schematically shown with some restriction recognition sites. Open boxes represent the protein coding regions. Horizontal arrows indicate directions and approximate positions of oligonucleo- tide primers used for competitive RT-PCR. (AC)34 and polyA indicate the presence of 34 times AC repeats or polyA tail, respectively. A arrow indicates the position of the alternative polyadenyla- and extent of probe sequences used for Northern blot analysis (see Results and Discussion).
	FIG. 2. Protein sequence alignments between H-PS-1/X-PS-a and H-PS-2/X-PS-b. The aa sequences deduced from X-ps-a and X-ps-b cDNA clones are aligned with those from H-PS-1 and H-PS-2. Dashes represent gaps for optimal alignment. ‘‘/’’ and ‘‘s’’ respectively indicate positions for identical or conservedly substituted aa residues between H-PS-1 and X-PS-a (1, a), between H-PS-2 and X-PS-b (2, b), and in all four sequences (1, 2, a, b). Mutated aa residues identified in H-ps-1 and H-ps-2 genes in FAD patients are shown by squares. TM regions I to VII are as defined previously (3).
	FIG. 3. Hydropathy and amino acid substitution profiles. Based on the X-PS-a/H-PS-1 (A) and X-PS-b/H-PS-2 (B) protein sequences, average hydrophobicity values for each amino acid (window = 19) were calculated using the algorithm of Kyte andDoolittle (15). Thick and thin respectively. Amino acid substitution values are defined as the numbers of different amino acids including missing and inserted residues (win- dow 19) on the basis of human versus Xenopus proteins and are presented by shadow profiles. The extent of human PS proteins is shown by open boxes on the top of each panel, in which missense mutations reported so far in FAD patients in PS-1 (A) and PS-2 (B) are indicated by vertical lines. The vertical lines with and without arrows represent changed and conserved positions in X-PS-a and -b, respectively. Amino acid positions are numbered for Xenopus and human proteins above and below the horizontal lines, respectively.
	FIG. 4. Tissue and developmental stage-specificity of X-ps-a and X-ps-b gene expression. Competitive RT-PCR was conducted as described in Materials and Methods. (A) Autoradiograph of competitive RT-PCR products labeled with 32P performed against total RNAs isolated from frog brain (Br), eye (Ey), lung (Lg), liver (Lv), kidney (K), intestine (In), testis (Te), ovary (Ov), skeletal muscle (SM) and heart (Ht). Closed and open arrows indicate positions of amplified DNAs originating from X-ps-a, X-ps-b transcripts and those of corresponding competitor DNAs, respectively. (B) Total RNA (0.2 mg/lane) for competitive RT-PCR used in (A) was separated in an agarose gel and stained with ethidium bromide. (C) Differential expression of X-ps genes was analyzed during oogenesis and embryogenesis as in (A). The oocyte and developmental stage numbers are shown on the top of each lane as defined in Materials and Methods. (D) Total RNA (0.2 mg/lane) used in (C) was treated as in (B).
	FIG. 5. Specific reduction in X-ps-a gene expression upon oocyte maturation induced by progesterone. Oocytes at stage VI were incubated in the absence (lane 1) or presence (lane 2) of 1 mM progesterone for 6 hours at 217C, and total RNAs were then isolated. Competitive RT-PCR was conducted as described in Materials and Methods.