Search PubMed⌕ Search

Biomedical subjects

W F Loomis

Publications and source records attributed to W F Loomis.

At least 91 records · Page 5Linked to original sources
PubMed1987

Surface glycoprotein, gp24, involved in early adhesion of Dictyostelium discoideum.

A membrane glycoprotein of 24,000 Da (gp24) was purified from developed cells of Dictyostelium discoideum and shown to neutralize a crude antiserum (R695) that blocks EDTA-sensitive cell-cell adhesion during the early developmental stages of this organism. Purified gp24 was used to raise rabbit polyclonal antibodies and mouse monoclonal antibodies. Rabbit antiserum R851 was shown to be highly specific to gp24 by both Western analysis and immunoprecipitation. IgG of R851 is able to block adhesion of dissociated cells swirled in suspension. Adhesion of wild-type cells is blocked by R851 antibodies during the first 8 hr of development but not thereafter when other adhesion mechanisms come into play. The glycoprotein gp80 plays an essential role in the second adhesion system that appears during the aggregation stage of D. discoideum. By adding both anti-gp24 and anti-gp80 antibodies, adhesion of aggregation stage cells could be blocked. Late in development a third adhesion mechanism appears that is not blocked by either antibodies to gp24 or gp80 or both antibodies together. Western analysis and immunoprecipitation with monoclonal antibody mLJ11, specific for gp24, indicated that gp24 is absent in cells growing exponentially on bacteria but is rapidly synthesized and accumulated following the initiation of development. Synthesis of gp24 is maximal during the first 4 hr of development and then continues at a reduced rate throughout the remainder of development. The coordinate appearance of gp24 and EDTA-sensitive cell-cell adhesion as well as the ability of this glycoprotein to neutralize the adhesion blocking activity of R695 and R851 antibodies indicates that it plays a role in early cell-cell adhesion.

Antibodies, Monoclonal↗
PubMed1986

DNA sequences required for expression of a Dictyostelium actin gene.

A 2.8-kb fragment of 5' non-coding DNA from the Dictyostelium actin 15 gene has previously been shown to contain all of the cis-acting DNA sequence elements required for normal developmentally-regulated transcription of actin gene fusion RNAs when reintroduced into the genome by DNA-mediated transformation. Deletion analysis of this promoter fragment indicates that all of the necessary information is contained within a 270-bp fragment of actin 15 5' non-coding DNA. This fragment contains four short G/C-rich repeated sequences that are also found in other co-regulated Dictyostelium actin genes. A 12-bp consensus sequence, AAAAATGGGG/ATT, is present in the regions essential for expression of two different Dictyostelium actin genes, actin 6 and actin 15, but is absent from an actin gene showing a different temporal pattern of developmental regulation. Deletion analysis and DNase I footprinting implicate this sequence as a functional cis-acting element required for transcription of the actin 15 gene.

Actins↗
PubMed1986

Hexapeptide repeat structure in Dictyostelium spore coat protein.

The sequences of the NH2-termini of two spore coat proteins of Dictyostelium discoideum have been determined. One of them (SP60) consists of perfect hexapeptide repeats of the sequence Gly-Asp-Trp-Asn-Asn-Asx-. The sequence has some homology to the parvovirus capsid protein which does not display periodicity. The NH2-terminal sequence of the second protein, SP70, contains a modified amino acid in two positions and like SP60 is highly hydrophilic and acidic.

Amino Acid Sequence↗
PubMed1986

Multigene families and vestigial sequences.

Random duplication and deletion events generate complex genomes carrying a large amount of dispensable sequences. We have simulated such events in a computer model. We followed the evolution of a genome carrying at least one copy of each type of gene. Partial duplications and deletions of genes generated nonfunctional vestigial sequences that were dispensable. The size of the genome stabilized only when the amount of dispensable sequences had increased to the point that most deletions did not affect vital genes. Within such genomes, the number of copies of specific genes fluctuated, thereby generating small multigene families. The parameters of the model were tested over 100,000 events in both simple and complex genomes. The results indicate that when the size of the genome is not critical to survival, as appears to be the case within limits in most eukaryotic organisms, the genome carries vestigial sequences that are no longer functional and that many genes are present in multigene families by chance.

Journal Article↗
PubMed1986

Developmental regulation of Dictyostelium discoideum actin gene fusions carried on low-copy and high-copy transformation vectors.

The Dictyostelium discoideum genome contains an estimated 17 to 20 actin genes. We report the identification of a new member of this multigene family, actin 15, and its complete nucleotide sequence and transcription initiation sites. We constructed transformation vectors carrying either the actin 15 promoter fused to the neomycin phosphotransferase gene from transposon Tn903 or the actin 6 promoter fused to the neomycin phosphotransferase gene from Tn5. Cells transformed with the actin 15 vector carried less than five copies of vector DNA, while cells transformed with the actin 6 vector carried more than 200 copies. In both cases, the vector appeared to be integrated into the chromosome as a tandem array. Gene fusion RNAs transcribed from the actin 15 and actin 6 vectors were regulated like endogenous actin genes during D. discoideum development. DNA sequences required for temporal and cell type-specific regulation of these genes were contained within 2.8 kilobases of 5' noncoding DNA for actin 15 and 0.7 kilobases of 5' noncoding DNA for actin 6.

Actins↗
PubMed1985

Absence of a carbohydrate modification does not affect the level or subcellular localization of three membrane glycoproteins in modB mutants of Dictyostelium discoideum.

The accumulation and localization of four developmentally regulated membrane glycoproteins were examined in a glycosylation mutant of the cellular slime mold Dictyostelium discoideum. As judged by immunoblot procedures using antipeptide antibodies, the levels of three of the glycoproteins, WGA80B, SP29A, and SP29B, were unaffected, but their apparent molecular masses were reduced by 14,000, 3,500 and 3,500 daltons, respectively. The level of the fourth glycoprotein, gp80, was reduced to below detectable limits. The reduced molecular sizes were apparently due to the absence of certain carbohydrate structures as judged by labeling Western blots with anti-carbohydrate antibodies and a lectin. Using immunofluorescence labeling of permeabilized and intact cells, the localization of WGA80B, SP29A, and SP29B, in intracellular vesicles and on the cell surface of prespore cells, was observed to be unaffected in the mutant cells. The developmentally regulated oligosaccharide structure(s) affected by the modB locus does not influence the subcellular localization and accumulation of these three glycoproteins in the prespore cells of this phylogenetically primitive organism.

Animals↗
PubMed1985

Molecular characterization of anterior-like cells in Dictyostelium discoideum.

Prestalk cells are found in the anterior 15% of slugs of Dictyostelium discoideum and prespore cells are found in the remaining posterior portions. Neutral red staining as well as immunostaining with antisera prepared against spores or a spore-coat protein has indicated that there are cells in the posterior region which have properties similar to prestalk cells. This cell population, called anterior-like cells, has been isolated by density separation of posterior slug fractions. This anterior-like cell preparation contained approximately 80% neutral red-stained cells, none of which carried a surface antigen specific to prespore cells (MUD-1 antigen). These cells also lacked antigens recognized by antisera to the spore-coat proteins or by a prespore-specific monoclonal antibody isolated for this study (mLJ1). Anterior-like cells were found to modify the lysosomal enzymes acid phosphatase and alpha-mannosidase in a manner found specifically in prestalk cells. They also synthesized two prestalk-specific proteins and contain two prestalk-specific mRNA transcripts. Anterior-like cells also failed to express a large number of prespore-specific characteristics. Although very similar to prestalk cells, anterior-like cells differ from them in that they express a prespore-specific protein PSP59 and the levels of the prestalk-specific mRNA transcripts and one of the prestalk-specific proteins ST430 appear to be lower.

Acid Phosphatase↗
PubMed1985

Developmental changes in the modification of lysosomal enzymes in Dictyostelium discoideum.

Evidence has been found for a generalized change in the post-translational modification of lysosomal enzymes during development of Dictyostelium discoideum. The physical and antigenic properties of four developmentally regulated lysosomal enzymes, N-acetylglucosaminidase, beta-glucosidase, alpha-mannosidase, and acid phosphatase, have been examined throughout the life cycle. In vegetative cells, a single major isoelectric species is detected for each enzymatic activity on native nonequilibrium isoelectric focusing gels. Between 6 and 10 hr of development, all activities, including the preformed enzyme, become less negatively charged, resulting in a modest but reproducible shift in the isoelectric focusing pattern. This alteration is not detected by native gel electrophoresis at constant pH. As development continues, the specific activity of beta-glucosidase, alpha-mannosidase, and acid phosphatase continues to increase and coincidentally, new, less acidic isozymic bands of activity can be observed on both gel systems. Some of these new isozymes accumulate preferentially in anterior cells, while others accumulate preferentially in posterior cells of migrating slugs. N-Acetylglucosaminidase does not increase in specific activity late in development and no new isozymic species appear. Using a monoclonal antibody that reacts with sulfated N-linked oligosaccharides shared by vegetative lysosomal enzymes in D. discoideum, the antigenicity of the developmental isozymes has been characterized. All of the enzymatic activity present during vegetative growth and early development is immunoprecipitable. However, the less negatively charged isozymes that accumulate after aggregation are not recognized by the antibody. Nonantigenic acid phosphatase and alpha-mannosidase are found in both anterior and posterior cells from migrating pseudoplasmodia. Since each enzyme is coded by a single structural gene, these results suggest that the isozymes present late in development arise from the synthesis of the same polypeptides with altered post-translational modifications. The appearance of anterior and posterior specific isozymes is likely to be the result of cell type specific changes in the glycoprotein modification pathway for newly synthesized proteins.

Acetylglucosaminidase↗
PubMed1985

Adhesion mutants of Dictyostelium discoideum lacking the saccharide determinant recognized by two adhesion-blocking monoclonal antibodies.

A mutant of Dictyostelium discoideum, strain HL260, was isolated based on its failure to bind d-41, a monoclonal antibody that blocks developmentally regulated cell-cell adhesion. The mutant fails to normally acquire cell-cell adhesion as assayed with cells shaken in 10 mM EDTA, but aggregates and and constructs fruiting bodies. Other mutant strains, HL216 and HL220, previously shown to have impaired cell-cell adhesion, also lack the determinant that binds d-41. The three strains all carry mutations in a gene designated mod B, which directs a post-translational modification of several developmentally regulated D. discoideum glycoproteins. Diploids formed between independent mod B mutant haploid strains also lack this determinant and show marked impairment of cell-cell adhesion in EDTA, indicating that mutations in mod B, rather than other mutations not shared by the haploid strains, are related to the adhesion defect. The results are consistent with other evidence that an oligosaccharide carried on several developmentally regulated glycoproteins plays an essential role in EDTA-resistant cell-cell adhesion in D. discoideum. However, this type of adhesion is not essential for morphogenesis in that the only defect detected thus far in mod B mutant strains is that they construct relatively smaller fruiting bodies that contain fewer spores.

Animals↗
PubMed1985

Regulation of cell-type-specific differentiation in Dictyostelium.

During development of Dictyostelium, there are at least a dozen discrete stages of differentiation that can be distinguished by the expression of specific genes. The early stages are triggered by amino acid starvation and are dependent on a small heat-stable effector secreted by the cells to indicate a critical cell density. After development has proceeded for 12 hours, late genes are expressed that are dependent on the conditions found in multicellular aggregates. Cells monitor these conditions and appear to respond by raising their internal cAMP levels to act as a second messenger. Multicellularity can be bypassed as an essential condition if high levels of cAMP are added to the environment. The EDTA-resistant cell-adhesion mechanism that develops by 12 hours is not a required aspect of multicellularity. A final set genes can be induced in 18-hour-developed cells by lowering the pNH3. The spore coat proteins are well-characterized markers for prespore differentiation; their genes are first expressed at 12 hours. Prestalk cells do not express these genes. A small number of prestalk cells become redistributed in the posterior during slug migration and appear to undergo respecification when their position is changed. Prestalk genes become repressed in these "anterior-like" cells and prespore genes are activated. These results clearly indicate that a fieldwide system of positional information regulates cell-type differentiation in Dictyostelium.

Amino Acid Sequence↗
PubMed1984

Antigenic determinants shared by lysosomal proteins of Dictyostelium discoideum. Characterization using monoclonal antibodies and isolation of mutations affecting the determinant.

Polyclonal antisera raised against a single lysosomal enzyme from Dictyostelium discoideum will cross-react with other lysosomal proteins due to the presence of common post-translational modification antigens. We have now isolated hybridoma cell lines secreting monoclonal antibodies that also recognize a shared lysosomal determinant. Although the same proteins are recognized by the monoclonal and polyclonal antibodies, the actual determinants differ. Also, the polyclonal serum contains antibodies which recognize at least one determinant not recognized by the monoclonal antibodies. All of the antigenic proteins are minor cell proteins but represent a majority of the proteins secreted from the cells during axenic growth. Anode-directed nonequilibrium two-dimensional gel electrophoresis of cellular and secreted protein indicates that the antigenic proteins have extremely acidic pI values; moreover, most are both sulfated and phosphorylated. Several sulfated polysaccharides, including dextran sulfate, chondroitin sulfate, and heparin inhibit binding of the monoclonal but not the polyclonal antibodies, indicating that the shared determinant recognized by the monoclonal antibodies may involve sulfated oligosaccharides. As a step in analyzing the function of the post-translational modification of lysosomal enzymes, we have screened a mutagenized population of amoeba for those lacking the antigenic determinant recognized by one of the monoclonal antibodies. Five cell lines have been isolated that completely lack the determinant. They appear to fall into at least three different classes based upon physical properties of the enzymes. One strain lacks any detectable sulfation of glycoproteins. Several of the strains are capable of forming fruiting bodies indicating that the antigenic determinant plays no crucial role in morphogenesis under laboratory conditions.

Antibodies, Monoclonal↗
PubMed1984

Biochemical and genetic analysis of pre-stalk specific acid phosphatase in Dictyostelium.

The only known enzymatic marker of pre-stalk cells at the slug stage of development in Dictyostelium discoideum is an isozyme of acid phosphatase, AP2. There is another isozyme of acid phosphatase, AP1, which is present in vegetative cells and is not cell-type specific. We have purified these isozymes and find they differ in Km and thermostability. Both isozymes are affected by mutations in a single locus, acpA. Two mutations in the acpA locus abolished all activity of both AP1 and AP2 while a third mutation reduced the activity and altered the thermostability of both isozymes. It is likely that acpA is the structural gene responsible for both AP1 and AP2. The cell-type specificity of AP2 appears to result from differences in the modification of the acpA gene product between pre-spore and pre-stalk cells. The resulting difference in AP2 provides a useful marker for pre-stalk cells.

Acid Phosphatase↗
PubMed1984

The timing of cell-type-specific differentiation in Dictyostelium discoideum.

We have used two-dimensional gel electrophoresis to identify over 30 proteins which are specific to one or other of the two cell types of Dictyostelium discoideum, either at the slug stage or in mature fruiting bodies. Our results support the idea that there is a continuous developmental program that begins in prespore cells at the hemispherical mound stage (10-12 hr) and results in spore differentiation (24 hr). Prestalk differentiation, on the other hand, appeared largely unrelated to stalk differentiation, which was first detectable at the onset of culmination (18 hr). We have also used this approach to study the differentiation of stalk-only mutants and have found that the cells can switch from spore to stalk differentiation as late as 2 hr before the end of the wild-type developmental program.

Cell Differentiation↗
PubMed1984

Cloning and expression of a cDNA that comprises part of the gene coding for a spore coat protein of Dictyostelium discoideum.

The three major spore coat proteins of Dictyostelium discoideum are developmentally regulated, cell-type-specific proteins. They are packaged in prespore vesicles and then secreted to form the outer layer of spore coats. We have isolated a cDNA clone from the gene coding for one of these proteins, SP96, a glycoprotein of 96,000 daltons. We screened the cDNA bank by the method of hybrid select translation followed by immunoprecipitation of the translation products with SP96-specific polyclonal antiserum. We found that the gene was first transcribed into stable mRNA a few hours before the time of detection of SP96 synthesis and that the mRNA, like the protein, accumulated specifically in prespore cells and spores. SP96 constituted the same proportion of newly synthesized protein as the proportion of its message in polyadenylated RNA. SP96 appeared to be encoded by a single gene as judged by Southern blot analysis of digested genomic DNA hybridized to the cDNA clone.

Cloning, Molecular↗
PubMed1984

Mutations affecting a surface glycoprotein, gp80, of Dictyostelium discoideum.

We isolated two independent mutations in Dictyostelium discoideum that result in the absence of the antigenic determinant recognized by monoclonal antibody E28D8. This antibody reacts with a post-translational modification on the surface glycoprotein gp80 and several other proteins. Both of the mutations occur in the same locus, modB, which was mapped to linkage group VI. The modB mutations result in sufficient alteration of gp80 that it is absent or unrecognizable by two-dimensional gel electrophoresis. Strains carrying modB mutations exhibit "contact sites A"-mediated cell-cell adhesion although more weakly than do wild-type strains and develop to fruiting bodies carrying viable spores. Although gp80 has been implicated in the mechanism of cell-cell adhesion in D. discoideum, it is clear from the behavior of these mutant strains that the determinant on gp80 recognized by E28D8 is not necessary for either morphogenesis or reduced EDTA-resistant adhesion.

Alleles↗
PubMed1983

Spore coat proteins of Dictyostelium discoideum are packaged in prespore vesicles.

Previous studies have shown that Dictyostelium discoideum spore coat proteins are found in prespore cells, which are localized to the posterior region of migrating slugs, and in the coats of mature spores. Prespore vesicles, identified by morphology and by staining with anti-D. mucoroides spore serum, are also localized in the posterior region of migrating slugs. Using antisera specific to the spore coat proteins, we show that the spore coat proteins are packaged in prespore vesicles. They are present in the vesicles as a complex which can be dissociated by denaturation. The anti-D. mucoroides spore serum reacts with at least five proteins in whole spore extracts including the spore coat proteins SP96 and SP70.

Dictyostelium↗
PubMed1983

Loss and resynthesis of a developmentally regulated membrane protein (gp80) during dedifferentiation and redifferentiation in Dictyostelium.

When developing cultures of Dictyostelium discoideum are disaggregated and resuspended in nutrient medium, they lose the capacity to rapidly reaggregate after 90 min, in a rapid and synchronous step referred to as the "erasure event." They then proceed to lose remaining developmentally acquired functions in a program of dedifferentiation culuminating with the loss of EDTA-resistant cohesion roughly 5 hr later. Immediately following the erasure event, cells can be stimulated to reenter the developmental program even though they still possess a number of developmentally acquired functions. These cells therefore appear to undergo dedifferentiation and redifferentiation simultaneously (D. R. Soll and L. H. Mitchell, 1982, Dev. Biol. 91, 183-190). In this report, we have employed an antiserum made against a developmentally acquired membrane glycoprotein, gp80, to examine whether gp80 is lost during dedifferentiation and whether it is either reutilized or resynthesized during redifferentiation. Results are presented which demonstrate that (1) when 9-hr developing cells are disaggregated and resuspended in nutrient medium, gp80 continues to accumulate for several hours after the erasure event, then is lost at roughly the same time as EDTA-resistant cohesion; (2) when cells are stimulated to reenter the developmental program immediately after the erasure event, both gp80 and EDTA-resistant cohesion are still lost according to the program of dedifferentiation, but are then reacquired soon afterwards according to the program of redifferentiation; (3) during redifferentiation, cells do not reutilize gp80 which had been synthesized during initial development; rather they synthesize gp80 de novo; and (4) developing cells of a dedifferentiation-defective variant, HI4, when disaggregated and resuspended in nutrient medium, retain gp80, EDTA-resistant cohesion, and the capacity to rapidly reinitiate aggregation for at least 12 hr. This last result indicates that the loss of gp80 is regulated by the dedifferentiation process and is not an independent response to disaggregation or the reintroduction of nutrients. Together, these results reinforce the conclusion that dedifferentiation and redifferentiation can function independently and simultaneously in the same cells.

Cell Adhesion↗