Structure of a genomic clone encoding biologically active human relaxin

Relaxin is a peptide hormone synthesized in the corpora lutea of ovaries during pregnancy and is released into the blood stream prior to parturition. Its major biological effect is to remodel the mammalian reproductive tract to facilitate the birth process. Determination of the structure of human relaxin is thus a first step in opening up the possibility of clinical intervention in cases of difficult labour. However, the limited availability of human ovaries during pregnancy has prevented both direct amino acid sequence determination and isolation of cDNA clones obtained from relaxin producing tissue. Our approach has therefore been to screen directly for a human relaxin gene using an homologous porcine relaxin cDNA probe. We report here the successful identification of a genomic clone from which the structure of the entire coding region of a human preprorelaxin gene has been determined. Synthesis of biologically active relaxin has shown that the novel gene structure described herein codes for an authentic human relaxin. We believe this is the first successful synthesis of a biologically active hormone whose structure was predicted solely from the structure of a genomic clone.     

Vesicular restriction of synaptobrevin suggests a role for calcium in membrane fusion

Release of neurotransmitter occurs when synaptic vesicles fuse with the plasma membrane. This neuronal exocytosis is triggered by calcium and requires three SNARE (soluble-N-ethylmaleimide-sensitive factor attachment protein receptors) proteins: synaptobrevin (also known as VAMP) on the synaptic vesicle, and syntaxin and SNAP-25 on the plasma membrane. Neuronal SNARE proteins form a parallel four-helix bundle that is thought to drive the fusion of opposing membranes. As formation of this SNARE complex in solution does not require calcium, it is not clear what function calcium has in triggering SNARE-mediated membrane fusion. We now demonstrate that whereas syntaxin and SNAP-25 in target membranes are freely available for SNARE complex formation, availability of synaptobrevin on synaptic vesicles is very limited. Calcium at micromolar concentrations triggers SNARE complex formation and fusion between synaptic vesicles and reconstituted target membranes. Although calcium does promote interaction of SNARE proteins between opposing membranes, it does not act by releasing synaptobrevin from synaptic vesicle restriction. Rather, our data suggest a mechanism in which calcium-triggered membrane apposition enables syntaxin and SNAP-25 to engage synaptobrevin, leading to membrane fusion.     

Zfy gene expression patterns are not compatible with a primary role in mouse sex determination

The Y chromosome determines maleness in mammals. A Y chromosome-linked gene diverts the indifferent embryonic gonad from the default ovarian pathway in favour of testis differentiation, initiating male development. Study of this basic developmental switch requires the isolation of the testis-determining gene, termed TDF in humans and Tdy in mice. ZFY, a candidate gene for TDF, potentially encodes a zinc-finger protein, and has two Y-linked homologues, Zfy-1 and Zfy-2, in mice. Although ZFY, Zfy-1 and Zfy-2 seem to map to the sex-determining regions of the human and mouse Y chromosomes, there is no direct evidence that these genes are involved in testis determination. We report here that Zfy-1 but not Zfy-2 is expressed in differentiating embryonic mouse testes. Neither gene, however, is expressed in We/We mutant embryonic testes which lack germ cells. These observations exclude both Zfy-1 and Zfy-2 as candidates for the mouse testis-determining gene.     

Expression pattern of zebrafish pax genes suggests a role in early brain regionalization.

In vertebrates the developing hindbrain is organized in segmental units. These units provide the primary grid for differentiation and axonal outgrowth. In the more anterior regions of the brain, however, the subdivisions remain more controversial. Cellular and molecular studies of the embryonic brain in lower vertebrates such as the zebrafish, Brachydanio rerio, may reveal remnants of such subdivisions. We have isolated complementary DNA clones for two zebrafish pax genes related to Drosophila and mouse paired-box-containing segmentation genes. The expression of these two genes is confined to specific regions in the embryonic forebrain and midbrain. Strikingly, the borders of expression of the two pax genes coincide with morphological landmarks corresponding to the primary axon tracts that are generated in the embryonic brain a few hours after the initiation of expression of these genes.     

Structure of a family of rat amylase genes

The sequences of two cloned rat pancreatic amylase cDNAs comprising 95% of the mRNA sequence are reported. Analysis of cloned rat genomic DNA fragments using cloned cDNA probes indicates that the rat genome contains multiple closely related amylase genes in which the cDNA sequences are distributed within a region 9 kilobases in length and are interrupted by at least seven intervening sequences.     

Expression pattern of the mouse T gene and its role in mesoderm formation.

Formation of mesoderm is a crucial event in vertebrate development, establishing many of the important features of the body. Recent studies have implicated molecules that are similar to growth factors in mesoderm formation in Xenopus, but other gene products involved in this process have yet to be identified. Genetic evidence indicates that in the mouse the T gene (Brachyury) has a role in the formation and organization of mesoderm. Mice homozygous for mutant alleles of the T gene do not generate enough mesoderm, and show severe disruption in morphogenesis of mesoderm-derived structures, in particular the notochord. The cloning of the T gene has now allowed us to examine its expression pattern. We report that T-gene expression occurs in both early stage mesoderm and its epithelial progenitor, and then becomes restricted to the notochord. This expression pattern correlates with the tissues affected in the T-gene mutant, and indicates that the T gene has a direct role in the early events of mesoderm formation and in the morphogenesis of the notochord.     

Exclusive gene mapping of congenital microphthalmia in a Chinese family

Congenital microphthalmia is a developmental ocular disorder and might be caused by the mutations in the genes involved in eye development. To uncover the genetic cause in a six-generation Chinese pedigree with autosomal dominant congenital microphthalmia, we performed genescan and linkage analysis in this family. Fourteen microsatellite markers on chromosomes 3, 11, 14 and 15 were selected as genetic markers according to the five pre-viously reported loci associated with microphthalmia (MITF, SOX2, PAX6, MCOP and NNO2). The genomic DNA of each member in the pedigree was amplified with 14 pairs of fluorescence labeled primers. Genome screening and genotyping were conducted on ABI377 DNA sequencer and linkage analysis was performed with Linkage software package. All two-point LOD scores of linkage analysis between the suggested disease genes and microsatellite markers were <-2, which indicated that none of the five genes were responsible for microphthalmia in this Chinese family. Microphthalmia in this family may be caused by mutation in a new gene which is essential in eye development.     

Segment-specific expression of a homoeobox-containing gene in the mouse hindbrain

The process of segmentation, in which the developing embryo is divided into repetitive structures along its antero-posterior (A-P) axis, as a means of organizing and coordinating the body plan is found in a wide range of organisms. In Drosophila, homoeotic genes are involved in all levels of segmental organization and in determining segment identity. The roles of these genes in segmentation have been found mainly by mutational studies, but also by in situ hybridization, which has shown their domains of expression. In contrast to Drosophila, however, embryonic expression of homoeobox-containing genes in vertebrate organisms has not been found to follow a segmental pattern. Vertebrate segmentation can be clearly seen in the mesodermal somites, but repetitive morphological structures in the central nervous system (neuromeres) have only recently been shown to have developmental significance. Neuromeres in the hindbrain (rhombomeres) have been defined as segmental units by their pattern of nerve formation in the developing chick and by the alternating expression of Krox-20, a gene encoding a zinc-finger DNA-binding protein, in the 9.5-day-old mouse. Here we report that a mouse homoeobox-containing gene, Hox-2.9, is expressed in a segment-specific manner in the developing mouse hindbrain. This expression is in a region which is flanked by the regions of expression of Krox-20, and is precisely contained within a single neuromere, rhombomere 4.     

A gene mapping to the sex-determining region of the mouse Y chromosome is a member of a novel family of embryonically expressed genes

A gene mapping to the sex-determining region of the mouse Y chromosome is deleted in a line of XY female mice mutant for Tdy, and is expressed at a stage during male gonadal development consistent with its having a role in testis determination. This gene is a member of a new family of at least five mouse genes, related by an amino-acid motif showing homology to other known or putative DNA-binding domains.     

Unexpected complexity of the Wnt gene family in a sea anemone

The Wnt gene family encodes secreted signalling molecules that control cell fate in animal development and human diseases. Despite its significance, the evolution of this metazoan-specific protein family is unclear. In vertebrates, twelve Wnt subfamilies were defined, of which only six have counterparts in Ecdysozoa (for example, Drosophila and Caenorhabditis). Here, we report the isolation of twelve Wnt genes from the sea anemone Nematostella vectensis, a species representing the basal group within cnidarians. Cnidarians are diploblastic animals and the sister-group to bilaterian metazoans. Phylogenetic analyses of N. vectensis Wnt genes reveal a thus far unpredicted ancestral diversity within the Wnt family. Cnidarians and bilaterians have at least eleven of the twelve known Wnt gene subfamilies in common; five subfamilies appear to be lost in the protostome lineage. Expression patterns of Wnt genes during N. vectensis embryogenesis indicate distinct roles of Wnts in gastrulation, resulting in serial overlapping expression domains along the primary axis of the planula larva. This unexpectedly complex inventory of Wnt family signalling factors evolved in early multi-cellular animals about 650 million years (Myr) ago, predating the Cambrian explosion by at least 100 Myr (refs 5, 8). It emphasizes the crucial function of Wnt genes in the diversification of eumetazoan body plans.     

Trembler mouse carries a point mutation in a myelin gene.

The autosomal dominant trembler mutation (Tr), maps to mouse chromosome 11 (ref. 2) and manifests as a Schwann-cell defect characterized by severe hypomyelination and continuing Schwann-cell proliferation throughout life. Affected animals move clumsily and develop tremor and transient seizures at a young age. We have recently described a potentially growth-regulating myelin protein, peripheral myelin protein-22 (PMP-22; refs 7, 8), which is expressed by Schwann cells and found in peripheral myelin. We now report the assignment of the gene for PMP-22 to mouse chromosome 11. Cloning and sequencing of PMP-22 complementary DNAs from inbred Tr mice reveals a point mutation that substitutes an aspartic acid residue for a glycine in a putative membrane-associated domain of the PMP-22 protein. Our results identify the PMP-22 gene as a likely candidate for the mouse trembler locus and will encourage the search for mutations in the corresponding human gene in pedigrees with hypertrophic neuropathies such as Charcot-Marie-Tooth and Dejerine-Sottas diseases (hereditary motor and sensory neuropathies I and III).