Differential expression of two skeletal muscle beta-tropomyosin mRNAs during Xenopus laevis development.

A cDNA clone for a Xenopus laevis skeletal muscle beta-tropomyosin (beta-TMad) isoform was isolated from an adult skeletal muscle cDNA library. Sequence analysis revealed that this clone corresponded to a second beta-tropomyosin mRNA distinct from the one that was previously characterized (beta-TMemb). The two skeletal beta-TM mRNAs originate from distinct genes and are differentially expressed during development. Beta-TMemb mRNA is expressed only in the somites of the early embryo while beta-TMad mRNA is expressed in pre-metamorphic tadpoles and adult skeletal muscles. We have isolated the promoter region of the beta-TMemb gene and shown that a DNA construct containing 2.9 kb of promoter region is properly expressed after injection in the embryo.

A novel tropomyosin isoform encoded by the Xenopus laevis alpha-TM gene is expressed in the brain.

A full-length cDNA clone encoding a novel non-muscle tropomyosin (nmTM) termed X alpha TMB5, as yet unidentified in vertebrates, was isolated from a Xenopus laevis (Xl) brain cDNA library. X alpha TMB5 is derived from the XL alpha-tropomyosin gene (X alpha TM), which was previously found to express striated muscle and nmTM isoforms via an alternative splicing mechanism. The deduced amino-acid sequence reveals that X alpha TMB5 contains 248 amino acids. The protein differs from the skeletal muscle alpha-TM isoform only at its NH2-terminal region. The mRNA encoding X alpha TMB5 is expressed mainly in brain and striated muscle. Genomic clone analysis reveals that, unlike mammals and avian, the X alpha TM gene is devoid of the brain-specific exon 9c. The amphibian alpha-TM gene is a complex transcription unit containing 14 exons, including two alternative promoters, two internal mutually exclusive exons, and two alternatively spliced 3' exons that encode two different COOH-terminal coding regions. Therefore, a total of at least five distinct mRNAs are expressed from the X alpha TM gene in a cell-type-specific manner.

Cloning of the Xenopus laevis cdk2 promoter and functional analysis in oocytes and during early development.

CDK2 (cyclin-dependent kinase 2) is a serine/threonine kinase which is involved in regulating S-phase entry in higher eukaryotes. To investigate the transcriptional control of this gene, a 13-kb Xenopus laevis genomic clone containing the 5' flanking sequences was isolated. A 2.7-kb fragment containing the promoter region was sequenced and the transcription start point (tsp) was determined by primer extension. Several putative regulatory elements, such as the E2F-binding site, Y box and octamer-binding site, were localized in this region, but no TATA box was found. When fused to cat, a reporter gene encoding chloramphenicol acetyltransferase, the 5' flanking sequences were shown to function in oocytes and an enhancer activity was found in this region. During early embryogenesis, cdk2 promoter activity was tested and de novo transcription was detected at the mid-blastula transition.

FOSP-1 (frog oviduct-specific protein-1) gene: cloning of cDNA and induction by estrogen in primary cultures of Xenopus oviduct cells.

We describe the isolation of a cloned cDNA from a cDNA library of oviduct of estrogen-treated adult Xenopus. Although the protein encoded by this cDNA is not known, it is designated as FOSP-1 (frog oviduct-specific protein-1). A partial restriction map of FOSP-1 cDNA, which is 1.5 kb in size, is presented. Northern hybridization analysis showed that FOSP-1 cDNA codes for a single species of mRNA of 2.6 kb which is exclusively expressed in Xenopus oviduct. Southern blot analysis showed that the gene was present in only one or two copies. Sequencing of partial FOSP-1 cDNA did not reveal homology with any protein in the sequence data bank. Measurement of steady-state levels of FOSP-1 mRNA in primary cultures of Xenopus oviduct cells by a technique of quantitative slot-blot analysis showed that both 17 beta and 17 alpha stereoisomers of estradiol caused a rapid 5-fold enhancement of accumulation of the mRNA with maximum values obtained at 5 X 10(-8) M estrogen. Progesterone caused only a small increase in FOSP-1 mRNA concentration. This hormone-specific induction of mRNA makes FOSP-1 a valuable candidate for exploring tissue specificity of regulation by estrogen of gene expression.

Urodele egg jelly and fertilization.

Fertilization of urodele amphibians is physiologically polyspermic. These amphibians lack sperm entry blocking mechanisms at the egg surface, such as a cortical reaction or a membrane depolarization. Although, egg jelly is necessary for sperm capacitation, a late block to sperm entry does occur about 30 min after fertilization at a precise interface between jelly layers. The jelly is secreted by oviductal cells. In order to investigate its role in fertilization, we studied some biochemical properties of the oviductal secretions of eight species. In double diffusion experiments on agarose plates, some components secreted by the anterior and the middle parts of the oviduct interacted together and formed precipitin lines. This reaction might be responsible for the formation of the dense zone that delimits the capsular chamber. A hemagglutinating activity was found in the anterior or in the posterior part of the oviduct depending on the species. A 18K or 26K lectin was purified respectively from the oviduct of Ambystoma mexicanum and Pleurodeles waltl. In both species, the site where the late block to sperm entry is operative was spatially related to the location of the lectin in the jelly. However, sperm in contact with the purified lectins did not undergo any visible morphological change.

[The properties of precipitation reactions between secretion products in the oviduct of pleurodeles: identification of a lectin]. / Caractéristiques des réactions de précipitation entre les produits de sécrétion de l'oviducte du Pleurodèle: identification d'une lectine.

The properties of two precipitation reactions occurring between secretory products from the oviduct of Pleurodeles waltl have been studied. It has been demonstrated that a lectin is involved in one of the reactions. This lectin precipitated glycogen and starch and required calcium; the most potent saccharide inhibitors were 2-amino-2-deoxy-D-glucose and D-glucose, respectively. The other reaction was related to glycoproteins (probably sulfated glycoproteins) that contained sulphur. The properties of this reaction were not the same as purely ionic interactions; basic protein-acidic polysaccharide interactions have been compared. A lectin was probably implicated but this could not be demonstrated because no saccharide inhibitor was found. There are several similitudes between this reaction and the lectin-galactoside reaction which occurs in the reaction between cortical granule content and egg jellies in anurans.