Two divergent cellular src genes are expressed in Xenopus laevis.

Genomic and cDNA clones of the X. laevis src gene have been isolated and characterized by hybridization and DNA sequence analyses. The haploid genome of X. laevis contains two src genes, which can be distinguished from one another by virtue of sequence divergence in the 3' untranslated regions. Both of the genes are functional as indicated by the fact that oocytes contain RNAs transcribed from each of the genes. The two genes each encode an RNA which is 3.3 kb in length, or twice the length required to encode the 60,000 dalton src protein (pp60). Sequence analysis of the cDNA clones revealed that nearly all of the non-coding sequence is located at the 3' end. The availability of sequence data from cDNA clones has also made it possible for the first time to identify with certainty the carboxyl terminal sequence of a cellular pp60 molecule.     

Two heparanase splicing variants with distinct properties are necessary in early Xenopus development

Heparanase is an endoglycosidase that cleaves heparan sulfate (HS) side chains from heparan sulfate proteoglycans (HSPGs) present in extracellular matrix and cell membranes. Although HSPGs have many functions during development, little is known of the role of the enzyme that degrades HS, heparanase. We cloned and characterized the expression of two heparanase splicing variants from Xenopus laevis and studied their function in early embryonic development. The heparanase gene (termed xHpa) spans over 15 kb and consists of at least 12 exons. The long heparanase (XHpaL) cDNA encodes a 531-amino acid protein, whereas the short splicing variant (XHpaS) results in a protein with the same open reading frame but missing 58 amino acids as a consequence of a skipped exon 4. Comparative studies of both isoforms using heterologous expression systems showed: 1) XHpaL is enzymatically active, whereas XHpaS is not; 2) XHpaL and XHpaS interact with heparin and HS; 3) both proteins traffic through the endoplasmic reticulum and Golgi apparatus, but XHpaL is secreted into the medium, whereas XHpaS remains associated with the membrane as a consequence of the loss of three glycosylation sites; 4) overexpression of XHpaS but not XHpaL increases cell adhesion of glioma cells to HS-coated surfaces; 5) XHpaL and XHpaS mRNA and protein levels vary as development progresses; 6) specific antisense knock-down of both XHpaL and XHpaS, but not XHpaL alone, results in failure of embryogenesis to proceed. Interestingly, rescue experiments suggest that the two heparanases regulate the same developmental processes, but via different mechanisms.     

Germin-like genes are expressed during somatic embryogenesis and early development of conifers

Germins and germin-like proteins (GLPs) are members of a superfamily of proteins widely distributed in plants. Their localization within the extracellular matrix and in some cases their hydrogen peroxide-producing activity suggests that these proteins are involved in cell wall metabolism during stress responses and developmental processes. Several very highly conserved conifer GLPs have been identified in somatic embryo tissues. In order to gain more knowledge on their potential involvement in the development of this particular tissue, we have characterized a new GLP gene, LmGER1 in hybrid larch. Anti-GLP immunserum and in-gel activity analyses suggested the presence of superoxide dismutase activity in apoplastic proteins from larch somatic embryos. These results could indicate a possible role for LmGER1 in this physiological process. The expression of LmGER1 has been followed during the maturation of somatic embryos and in different organs of young plantlets by homologous transformation with a promoter-gus construct. This promoter was activated in the root cap of young embryos and, later on, in the cotyledons and in the vascular procambium and xylem. Furthermore, the importance of this gene in embryo development was evaluated by transforming embryonal masses with a gene construct encoding a hairpin RNA leading to gene silencing. The potential role of LmGER1 in cross-linking of cell wall components is discussed.     

Xenopus hsp 70 genes are constitutively expressed in injected oocytes.

Xenopus heat-shock genes are transiently heat-inducible in somatic cells, but they are also subject to a long-term developmental control in oogenesis and early embryogenesis. In order to understand whether different genes or different promoter elements are involved in the two types of control, several genomic clones coding for Xenopus heat-shock proteins, hsp 70 and hsp 30, were isolated, characterised and tested for expression in oocytes and COS cells. Three isolated hsp 70 genes are nearly identical in their promoter and mRNA leader sequences, indicating that there is only one type of hsp 70 gene. These promoters contain a consensus sequence element (CT-GAA--TTC-AG) upstream of the TATA-box, which is presumably required for their transient heat-inducibility. The two isolated hsp 30 genes show 5'-flanking sequences similar to each other, except that one of them shows a homology disruption precisely around the consensus sequence element. The same gene contains a frameshift mutation in the protein coding part and, since it cannot be expressed after introduction into oocytes or COS cells, it is probably a pseudogene. The other hsp 30 gene is strongly heat-inducible in injected oocytes or transfected COS cells. In contrast, the hsp 70 genes are strongly heat-inducible in COS cells, but their expression is highly efficient in injected oocytes at the normal temperature and is not increased during heat shock. This represents correct cell type-specific regulation of a cloned reintroduced gene, since the endogenous hsp 70 genes are constitutively activated during oogenesis, leading to the accumulation of stored hsp 70 mRNA in oocytes.     

Two novel Xenopus frizzled genes expressed in developing heart and brain.

A family of genes related to the Drosophila wingless receptor frizzled have been found in vertebrates. We have cloned full length cDNAs of two novel frizzled genes from embryonic Xenopus tissue. We are calling them Xfz7 and Xfz9 (for Xenopus frizzled) because their deduced peptide sequences show extensive similarity to other vertebrate frizzled molecules. Xfz7 is closely related to human, chick and mouse frz-7 and Xfz9 is most related to human FZD9 and mouse fzd9. Xfz7 is expressed in a broad, complex and dynamic pattern beginning at gastrulation. At later stages Xfz7 expression is found in neural crest, neural tube, eye, pronephric duct and the heart. Xfz9 expression in contrast is more restricted to the neuroectoderm and, at later stages of development, to the dorsal regions of the mid- and hindbrain.     

The RNA-binding protein fragile X-related 1 regulates somite formation in Xenopus laevis

Fragile X-related 1 protein (FXR1P) is a member of a small family of RNA-binding proteins that includes the Fragile X mental retardation 1 protein (FMR1P) and the Fragile X-related 2 protein (FXR2P). These proteins are thought to transport mRNA and to control their translation. While FMR1P is highly expressed in neurons, substantial levels of FXR1P are found in striated muscles and heart, which are devoid of FMRP and FXR2P. However, little is known about the functions of FXR1P. We have isolated cDNAs for Xenopus Fxr1 and found that two specific splice variants are conserved in evolution. Knockdown of xFxr1p in Xenopus had highly muscle-specific effects, normal MyoD expression being disrupted, somitic myotomal cell rotation and segmentation being inhibited, and dermatome formation being abnormal. Consistent with the absence of the long muscle-specific xFxr1p isoform during early somite formation, these effects could be rescued by both the long and short mRNA variants. Microarray analyses showed that xFxr1p depletion affected the expression of 129 known genes of which 50% were implicated in muscle and nervous system formation. These studies shed significant new light on Fxr1p function(s).     

Activin receptor mRNA is expressed early in Xenopus embryogenesis and the level of the expression affects the body axis formation.

Activin is a member of the transforming growth factor beta (TGF-beta) and possesses various activities in cellular control phenomena. During Xenopus embryonic development, activin is thought to act as a natural mesoderm-inducing factor. We isolated here the Xenopus activin receptor cDNA from Xenopus tadpole cDNA library and examined the expression of the Xenopus activin receptor gene during the course of early embryonic development. The Xenopus activin receptor has an 87% homology at the level of deduced amino acid sequence with the mouse activin receptor, and using the cDNA obtained, three bands of mRNA with different lengths were detected in Xenopus embryos throughout early embryogenesis. We synthesized activin receptor mRNA in vitro and tested the effect of the injection of the mRNA into Xenopus fertilized eggs on subsequent development. When the synthetic mRNA was injected into uncleaved fertilized eggs, embryos with reduced trunk structure were formed. However, when the mRNA was injected into the ventral blastomeres at the 16-cell stage, embryos with a secondary body axis were formed. These results indicate the importance of the function of activin receptor in the regulatory mechanism for body axis formation.     

Two distinct families of human and bovine interferon-alpha genes are coordinately expressed and encode functional polypeptides.

The classical human interferon-alpha (HuIFN-alpha) gene family is estimated to consist of 15 or more nonallelic members which encode proteins sharing greater than 77% amino acid sequence homology. Low-stringency hybridization with a HuIFN-alpha cDNA probe permitted the isolation of two distinct classes of bovine IFN-alpha genes. The first subfamily (class I) is more closely related to the known HuIFN-alpha genes than to the second subfamily (class II) of bovine IFN-alpha genes. Extensive analysis of the human genome has revealed a HuIFN-alpha gene subfamily corresponding to the class II bovine IFN-alpha genes. The class I human and bovine IFN-alpha genes encode mature IFN polypeptides of 165 to 166 amino acids, whereas the class II IFN-alpha genes encode 172 amino acid proteins. Expression in Escherichia coli of members of both gene subfamilies results in polypeptides having potent antiviral activity. In contrast to previous studies which found no evidence of class II IFN-alpha protein or mRNA expression, we demonstrate that the class I and class II IFN-alpha genes are coordinately induced in response to viral infection.     

Xenopus brevican is expressed in the notochord and the brain during early embryogenesis

A complete cDNA encoding the Xenopus laevis homologue of the aggrecan/versican family member, brevican (Xbcan) was cloned from an embryonic stage 42 cDNA library. In the deduced amino acid sequence, 1152 in length, similarity to the hyaluronan-binding (link) domains of brevicans from other species were present in the N-terminal region as well as EGF-, lectin- and complement regulatory protein-like domains in the C-terminal part, the latter three being characteristic for brevican found within the extracellular matrix (J. Biol. Chem. 269 (1994) 10119). Indeed, Xbcan was secreted into the extracellular space as a soluble protein when expressed in oocytes. No cDNAs encoding a GPI-anchored bcan variant could be isolated from that cDNA library. During embryonic development, the expression of this gene was first observed in the notochord of neurula stage embryos. In addition to this, in tailbuds, Xbcan was also found to be expressed within the fifth and sixth rhombomere of the hindbrain. In tadpole stage embryos, expression was furthermore observed in periventricular regions of the developing brain and the rostral part of the spinal cord.     

Multiple nodal-related genes act coordinately in Xenopus embryogenesis.

Four nodal-related genes (Xnr1-4) have been isolated in Xenopus to date, and we recently further identified two more, Xnr5 and Xnr6. In the present functional study, we constructed cleavage mutants of Xnr5 (cmXnr5) and Xnr6 (cmXnr6) which were expected to act in a dominant-negative manner. Both cmXnr5 and cmXnr6 inhibited the activities of Xnr5 and Xnr6 in co-overexpression experiments. cmXnr5 also inhibited the activity of Xnr2, Xnr4, Xnr6, derrière, and BVg1, but did not inhibit the activity of Xnr1 or activin. Misexpression of cmXnr5 led to a severe delay in initiation of gastrulation and phenotypic changes, including defects in anterior structures, which were very similar to those seen in maternal VegT-depleted embryos. Further, although the expression of Xnr1, Xnr2, and Xnr4 was not delayed in these embryos, it was markedly reduced. Injection of cmXnr5 had no notable effect on expression of Xnr3, Xnr6, derrière, or siamois. Several mesodermal and endodermal markers also showed delayed and decreased expression during gastrulation in cmXnr5-injected embryos. These results suggest that, in early Xenopus embryogenesis, nodal-related genes may heterodimerize with other TGF-beta ligands, and further that one nodal-related gene alone is insufficient for mesendoderm formation, which may require the cooperative interaction of multiple nodal-related genes.     

Two 5S genes are expressed in chicken somatic cells.

Two 5S RNA species were detected in chicken cells. 5S I RNA has the nucleotide sequence of chicken 5S RNA previously published by Brownlee et al. (1) and 5S II RNA differs from it by 10 mutations. The secondary structure of both species is compatible with that proposed for other eukaryotic 5S RNAs. 5S II RNA represents 50-60% of 5S I RNA. Both species were found in total chicken liver and brain and were present in polysomes in the same relative proportions. Only one 5S RNA species could be detected in rat liver and HeLa cells. Chicken is the first vertebrate described so far in which two 5S RNA genes are expressed in somatic cells.