RESUMEN

To obtain a better understanding of the olfactory processes that allow mosquitoes to identify human hosts, a molecular study has been performed to identify and characterize molecules in the olfactory signalling pathway of the African malaria vector Anopheles gambiae. Using cDNA libraries from antennae of females and males, a collection of cDNAs encoding odorant binding proteins and other novel antennal proteins were isolated and characterized, which represent various families of putative carrier proteins with homologues in other insects. Using filter array hybridizations and quantitative RT PCR, regulation and gender specificity of expression of these genes was investigated. Significant differences in steady-state levels of some of these putative carrier protein genes were detected between the sexes and after blood feeding in females.

Asunto(s)

RESUMEN

Homozygous loss of the warts (wts) gene of Drosophila, caused by mitotic recombination in somatic cells, leads to the formation of cell clones that are fragmented, rounded, and greatly overgrown compared with normal controls. Therefore, the gene is required for the control of the amount and direction of cell proliferation as well as for normal morphogenesis. The absence of wts function also results in apical hypertrophy of imaginal disc epithelial cells. Secretion of cuticle over and between the domed apical surfaces of these cells leads to a honeycomb-like structure and gives the superficial wart-like phenotype of mitotic clones on the adult. One wts allele allows survival of homozygotes to the late larval stage, and these larvae show extensive imaginal disc overgrowth. Because of the excess growth and abnormalities of differentiation that follow homozygous loss, we consider wts to be a tumor suppressor gene. The wts gene is defined by the breakpoints of overlapping deficiencies in the right telomeric region of chromosome 3, region 100A, and by lethal P-element insertions and excisions. It encodes a protein kinase that is most similar to human myotonic dystrophy kinase, the Neurospora cot-1 protein kinase, two cell-cycle regulated kinases of yeast, and several putative kinases from plants. These proteins define a new subfamily of protein kinases that are closely related to but distinct from the cyclic AMP-dependent kinases. Although myotonic dystrophy is defined by a neuromuscular disorder, it is sometimes associated with multiple pilomatrixomas, which are otherwise rare epithelial tumors, and with other tumors including neurofibromas and parathyroid adenomas. Our results raise the possibility that homozygous loss of the myotonic dystrophy kinase may contribute to the development of these tumors.

Asunto(s)

RESUMEN

In Drosophila, over 50 genes have been identified in which loss-of-function mutations lead to excess cell proliferation in the embryo, in the central nervous system, imaginal discs or hematopoietic organs of the larva, or in the adult gonads. Twenty-two of these genes have been cloned and characterized at the molecular level, and nine of them show clear homology to mammalian genes. Most of these mammalian genes had not been previously implicated in cell proliferation control. Overgrowth in some of the mutants involves conversion to a cell type that, in normal development, shows more cell proliferation than the original cell type. Thus the neurogenic mutants, including Notch, show conversion of epidermal cells to neuroblasts, leading to the 'neurogenic' phenotype of excess nervous tissue. The ovarian tumor mutants show conversion of the female germ line to a cell type resembling the male germ line, which undergoes more proliferation than the female germ line. Mutations of the fat locus cause hyperplastic overgrowth of imaginal discs, in which the epithelial structure is largely intact. The predicted fat protein product is a giant relative of cadherins, supporting indications from human cancer that cadherins play an important role in tumor suppression. Mutations in the lethal(2)giant larvae and lethal(1)discs large genes cause neoplastic overgrowth of imaginal discs as well as the larval brain. The dlg gene encodes a membrane-associated guanylate kinase homolog that is localized at septate junctions between epithelial cells. This protein is a member of a family of homologs that also includes two proteins found at mammalian tight junctions (ZO-1 and ZO-2) and a protein found at mammalian synaptic junctions (PSD-95/SAP90). Genes in which mutations cause blood cell overproduction include aberrant immune response-8, which encodes the RpS6 ribosomal protein and hopscotch, which encodes a putative non-receptor protein tyrosine kinase. The gene products identified by ovarian tumor mutants do not show clear amino acid sequence homology to known proteins. Drosophila provides an opportunity to rapidly identify and characterize tumor suppressor genes, many of which have mammalian homologs that might also be involved in cell proliferation control and tumor suppression.

Asunto(s)

Asunto(s)

RESUMEN

Tumor suppressor genes, whose products are required for the control of cell proliferation, have been identified by their mutant phenotype of tissue overgrowth. Here we describe recent work on the molecular identification of tumor suppressor genes that function in two different cell types of the Drosophila larva: the blood cells, and the undifferentiated epithelial cells of developing imaginal discs. Mutations in the aberrant immune response8 (air8) gene lead to overproduction and precocious differentiation of blood cells. This gene encodes the Drosophila homolog of human ribosomal protein S6. The mutant phenotype is consistent with a role for S6 in the control of cell proliferation, and is compatible with findings from mammalian cells where alterations in S6 expression and phosphorylation are associated with changes in cell proliferation. Mutations in the discs large (dlg) gene cause neoplastic overgrowth of imaginal discs in the larva. The mutant discs show loss of septate junctions and of apical-basal cell polarity, and they also lose the ability to differentiate cuticular structures. The dlg protein product (DlgA) is localized at septate junctions between epithelial cells, and cDNA sequencing indicates that the gene product includes a domain with homology to guanylate kinase (GUK). Two mammalian homologs of this gene have been identified, and one of them (PSD-95/SAP90) encodes a component of synaptic densities in the brain; this protein therefore resembles the DlgA protein in being located in a specialized cell junction that functions in information transfer between cells. Mutations in the fat gene cause hyperplastic imaginal disc overgrowth, in which the overgrowing disc tissue retains its epithelial structure and its ability to differentiate. Some of the excess disc tissue is shed as vesicles suggesting a loss of cell adhesion. In support of this hypothesis, the predicted gene product shows homology to cadherins in its extracellular domain. However, the fat protein is much larger than known cadherins. As in human cancer, somatic loss of the normal alleles of tumor suppressor genes can lead to tumor formation in Drosophila; an example of this is provided by the warts (wts) locus. The wts gene was identified by the dramatic overgrowth of mitotic recombination clones that are homozygous for a wts deletion. In these clones the cuticle intrudes between epithelial cells, suggesting an alteration in cell adhesion. The study of these and other tumor suppressor genes in Drosophila is providing new evidence supporting the critical role of cell interactions and specialized apical junctions in controlling epithelial cell proliferation.

Asunto(s)

RESUMEN

A third major, calcium-insoluble component of the sea urchin (Strongylocentrotus purpuratus) hyaline layer has been purified and physically characterized. In the absence of divalent cations, the native, soluble protein has a sedimentation coefficient of 9.6 S and a molecular weight of 4.5 +/- 0.1 x 10(5). These data indicate that this large protein assumes an elongated, nonspherical conformation in solution. Its sedimentation behavior and its mobility on nondenaturing electrophoretic gels distinguish the 9.6 S protein from the 11.6 S and 6.4 S hyalin proteins we have previously characterized. That the 6.4 S, 9.6 S, and 11.6 S proteins are the major calcium-insoluble structural components of the hyaline layer is supported by the fact that we have found them in a variety of hyalin protein fractions prepared by a number of standard approaches. All three proteins are precipitated by calcium ions, thus fitting the operational definition of hyalin. Evidence is presented that the 11.6 S protein may overlie the 9.6 S protein in the hyaline layer.

Asunto(s)

RESUMEN

A major protein component of the sea urchin, Strongylocentrotus purpuratus, cortical granule exudate has been purified and characterized. In the absence of divalent cations, the native, soluble protein has a sedimentation coefficient at infinite dilution of 6.4 S and a molecular weight from sedimentation equilibrium measurements of 2.8 +/- 0.3 X 10(5). These and other data indicate that the protein assumes an elongated, rod-like structure in solution. The protein is greater than 95% homogeneous as judged by agarose- and sodium dodecyl sulfate-gel electrophoresis. In the latter experiments, the protein shows a relative molecular weight of 1.8 X 10(5) and is clearly distinct from the 11.6 S protein described earlier which shows two bands corresponding to 3.2 X 10(5) and 2.1 X 10(5). The 6.4 S protein is the major protein of the calcium-insoluble fraction of cortical granule exudate and contributes to the formation of the extracellular investments of the sea urchin embryo. Using a light-scattering assay, we show that the purified protein retains the ability to aggregate in the presence of divalent cations mirroring its assembly in vivo. Calcium ion alone is able to initiate this reaction and the rate of precipitation increases with calcium concentration. Magnesium alone is ineffective in this regard but, in combination, the two ions act synergistically. Strontium and barium can substitute for calcium, but higher concentrations of the former cations are required to produce an equivalent effect.

Asunto(s)

RESUMEN

The sea urchin fertilization envelope (FE) is formed following initial sperm-egg interaction from the egg surface vitelline envelope (VE) and the paracrystalline protein fraction (PCF), derived from cortical granules. Although mature FEs are physicochemically hardened postinsemination, a major protein fraction consisting of seven major polypeptides was extracted from Strongylocentrotus purpuratus FEs and the major, separated components were immunologically cross-reactive with the principal polypeptides in PCF and isolated cortical granules. Antibodies prepared against extracted, core FEs were immunologically crossreactive with isolated VEs, but not with PCF, suggesting that only VE components are covalently crosslinked. Based on protease inhibitor experiments, our model of FE development is that a benzamidine-sensitive, cortical granule protease cleaves a 200 kD VE polypeptide during initial envelope elevation to set up the morphological change in FE papillae which occurs later. Divalent cations precipitate the PCF and form metal proteinate bridges between the VE and PCF. Based on peroxidase inhibitor experiments, we suggest that the cortical granule peroxidase crosslinks VE polypeptides, beginning at 2-3 min postinsemination, to restrict the permeability of the VE so that normal envelope thickening occurs. A 305 kD VE polypeptide was isolated and appears to be important in sperm-egg interaction based on inhibition of sperm binding and fertilization by antibodies against the purified polypeptide.

Asunto(s)