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W F Loomis

Publications and source records attributed to W F Loomis.

At least 19 recordsLinked to original sources
PubMed1992

Tagging developmental genes in Dictyostelium by restriction enzyme-mediated integration of plasmid DNA.

Introduction of restriction enzyme along with linearized plasmid results in integration of plasmid DNA at genomic restriction sites in a high proportion of the resulting transformants. We have found that electroporating BamHI or EcoRI together with pyr5-6 plasmids cut with the same enzyme stimulates the efficiency of transformation in Dictyostelium discoideum more than 20-fold over the rate seen when plasmid DNA alone is introduced. Restriction enzyme-mediated integration generates insertions into genomic restriction sites in an apparently random manner, some of which cause mutations. About 1 in 400 of the Dictyostelium transformants displayed arrested or aberrant development. The integrated plasmid, along with flanking genomic DNA, was excised from some of these mutants, cloned in Escherichia coli, and used to transform other Dictyostelium cells. Homologous recombination within the flanking sequences resulted in the same phenotypes displayed by the original mutants, directly demonstrating that the affected genes were responsible for the specific morphological phenotypes. This method of insertional mutagenesis should be useful for tagging, and subsequent cloning, of many developmentally important genes that can be identified by their mutant phenotypes.

Animals↗
PubMed1992

Physical mapping of genes to specific chromosomes in Dictyostelium discoideum.

Cloned genes were used to probe a highly redundant library of large cloned fragments of the Dictyostelium discoideum genome carried in yeast artificial chromosomes (YACs). Each gene recognized several independent YAC clones, thereby grouping them into a contig. Individual YACs were arranged within the contig by positioning genes relative to rare restriction sites and the YAC ends. Genes that had been previously assigned to one of the six linkage groups by parasexual genetics were used to establish physically mapped regions on specific chromosomes. Previously unmapped genes were assigned to specific chromosomes when they recognized members of a mapped contig. Linkage was confirmed by congruence of large-scale restriction maps centered on either the previously mapped or the newly mapped genes. At present, the chromosome-assigned map segments comprise approximately 50% of the genome. About half of each map segment is covered by overlapping YACs.

Animals↗
PubMed1992

Disruption of the sporulation-specific gene spiA in Dictyostelium discoideum leads to spore instability.

The spiA gene of Dictyostelium is expressed specifically in prespore cells and spores during culmination, the final stage of development during which prespore and prestalk cells undergo terminal differentiation to form spores and stalk. We have used homologous recombination to delete this gene and have characterized the resulting phenotype. The spiA- strains develop normally and produce spores that are indistinguishable from those of wild-type strains by transmission and scanning electron microscopy. Mutant spores have normal viability when assayed soon after the completion of development, but, as the spiA- spores age, they lose viability more rapidly than those of the spiA+ parent. The drop in viability is more pronounced when spores are submerged in dilute buffer at a concentration that does not allow germination; after 11 days submerged, the viability of spiA- spores is 10(5)-fold reduced, whereas that of the parent is decreased only 10-fold. Reinserting an intact copy of the spiA gene into a spiA- strain restores the stability of its spores. The product of the spiA gene, Dd31, was identified on Western blots as a 30-kD protein using an antibody raised against a fusion protein containing a portion of the coding sequence. Dd31 is associated with the inner face of spore coat fragments in a detergent-resistant manner. This location is consistent with its observed role in maintaining stability of the spores.

Animals↗
PubMed1991

Coordinate regulation of the spore coat genes in Dictyostelium discoideum.

Genomic clones of the genes coding for the three major spore coat proteins, SP60, SP70, and SP96, were used to measure the accumulation of their respective mRNAs in mutant and wild-type cells allowed to develop under a variety of conditions. These prespore-specific mRNAs were found to be both temporally and quantitatively coordinate under all conditions indicating that they may be subject to identical regulatory processes. Accumulation of the spore coat mRNAs is dependent upon the function of both cAMP receptors and G alpha 2 proteins during the aggregation stage as well as upon concomitant protein synthesis. When cells are dissociated from aggregates at 10 hr of development and rapidly shaken in 0.1 mM EDTA they form clumps but do not accumulate any of the prespore-specific RNAs assayed. However, if either 0.1 mM Ca++ or 20 microM cAMP is added to these cells, the spore coat mRNAs accumulate. Lower concentrations of either Ca++ or cAMP had no effect. These results suggest that expression of the spore coat genes normally involves a Ca+(+)-dependent process, but the Ca++ requirement can be overcome by adding high concentrations of exogenous cAMP. Addition of 50 nM DIF to dissociated cell blocks the accumulation of the spore coat mRNAs even when cAMP or Ca++ is present. The upstream regions of the spore coat genes were compared to those of another gene, D19, that codes for the prespore-specific protein SP29. Short sequences related to CACCCAC were found at about the same position relative to the transcriptional start sites of these coordinately regulated genes.

Base Sequence↗
PubMed1991

A prespore gene, Dd31, expressed during culmination of Dictyostelium discoideum.

During culmination of Dictyostelium fruiting bodies, prespore and prestalk cells undergo terminal differentiation to form spores and a cellular stalk. A genomic fragment was isolated by random cloning that hybridizes to a 1.4-kb mRNA present during culmination. Cell type separations at culmination showed that the mRNA is present in prespore cells and spores, but not in prestalk or stalk cells. After genomic mapping, an additional 3 kb of DNA surrounding the original 1-kb fragment was cloned. The gene was sequenced and named Dd31 after the size of the predicted protein product in kilodaltons. Accumulation of Dd31 mRNA occurs immediately prior to sporulation. Addition of 20 mM 8-Br-cAMP to cells dissociated from Mexican hat stage culminants induced sporulation and the accumulation of Dd31 mRNA, while 20 mM cAMP did not. Dd31 mRNA does not accumulate in the homeotic mutant stalky in which prespore cells are converted to stalk cells rather than spores. Characterization of Dd31 extends the known temporal dependent sequence of molecular differentiations to sporulation.

Amino Acid Sequence↗
PubMed1991

Antisense RNA inhibition of expression of a pair of tandemly repeated genes results in a delay in cell-cell adhesion in Dictyostelium.

Two tandem genes code for a protein recognized by antibodies to gp24, a small surface protein implicated in cell-cell adhesion of Dictyostelium discoideum. However, these genes do not appear to code for gp24 itself. We have transformed cells with a cloned vector that directs the synthesis of antisense RNA from one of the pair of tandemly repeated genes during the first 4 hr of development. During this period, gp24 protein fails to accumulate and the cells do not become mutually adhesive. Antisense RNA encoded by this vector is unstable and rapidly disappears after synthesis stops. gp24 protein then accumulates and the cells acquire cell-cell adhesion and proceed through development.

Actins↗
PubMed1990

Cell-free N-glycosylation in Dictyostelium discoideum: analysis of wild-type and mutants defective in lipid-linked oligosaccharide biosynthesis.

N-glycosylation was measured in wild-type cell lysates of Dictyostelium discoideum and in two mutant strains that synthesize a truncated lipid-linked oligosaccharide, Man6GlcNAc2 lacking terminal mannose and glucose residues. Endogenous lipid-linked oligosaccharide (LLO) was transferred to octanoyl-Asn-[125I]Tyr-ThrNH2 by membrane fractions. About 50% of the glycopeptide product remained associated with membranes. Taurocholate and saponin promoted and preserved glycosylation, but NP-40 and Triton X-100 did not. Using this artificial assay, the rate and extent of transfer of the truncated lipid-linked oligosaccharide in extracts of the two mutant strains, HL241 and HL243, was reduced 5-10-fold relative to that of wild-type. The low activity found in the mutant strains appears to result from either reduced affinity of the truncated LLO for the transferase or from its improper topological localization in the membrane. When protein N-glycosylation is measured in living cells it is nearly normal in HL241, but it is 3-4-fold decreased in HL243. Although the results of the in vitro and in vivo assays differ, they are not in conflict. Rather, they suggest that the static in vitro assay may be capable of revealing subtleties in the productive positioning of LLO and the oligosaccharyl transferase. The decrease in glycosylation seen in intact HL243 cells may be a consequence of the pleiotropic effects of the primary mutation rather than a direct result of the altered LLO structure. Genetic analysis showed that the mutation in HL241 is recessive, while the mutation in HL243 is dominant and prevents normal development. Thus, the two mutants share a lesion in lipid-linked oligosaccharide biosynthesis and in cell-free glycosylation, but differ in their in vivo glycosylation. Their primary defects are probably different.

Cell-Free System↗
PubMed1990

Similarities in eukaryotic genomes.

1. The degree of overlap between the human genome and that of other eukaryotes is considered. Biochemical and molecular studies have shown that all eukaryotic organisms evolved from a common progenator that lived several billion years ago. 2. From a geneological point of view, all eukaryotes are related and their genes are all descended from common ancestors. 3. However, most of the DNA in eukaryotic genomes is not transcribed and has been free to drift in nucleotide sequence. Therefore, the question of overlap can only be applied meaningfully to the few per cent of the genome that is expressed. 4. During the last billion years many genes have duplicated and diverged and new genes have been formed by accretion of domains copied from other genes (exon shuffling). 5. The rate of genetic divergence has been such that only a few portions coding for pieces of highly conserved proteins are still shared by all eukaryotes including those that diverged over 600 million years ago. 6. On the other hand, a fairly large number of shared genes can be recognized among species that separated within the last few hundred million years. 7. Human genes have a high degree of identity with homologs in closely related organisms such as other mammals and a decreasing level of identity with their homologs in more distantly related species.

Amino Acid Sequence↗
PubMed1990

Molecular phylogeny of Dictyostelium discoideum by protein sequence comparison.

Comparison of the amino acid sequences of eight proteins from the soil amoeba Dictyostelium discoideum to those of their homologs in bacteria, yeast, and other eukaryotes indicates that Dictyostelium diverged from the line leading to mammals at about the same time as the plant/animal divergence. Yeast appear to have diverged considerably earlier. It is argued that previous analyses indicating that D. discoideum diverged before yeast were misleading because of the nature of the small ribosomal subunit rRNA sequences used in these studies. We suggest that amino acid sequences may be more reliable than untranslated nucleic acid sequences for evolutionary comparisons, especially among organisms with significant skewing of their A+T content.

Actins↗
PubMed1990

A pair of tandemly repeated genes code for gp24, a putative adhesion protein of Dictyostelium discoideum.

The glycoprotein gp24 has been implicated in cell-cell adhesion of Dictyostelium discoideum. We have used a cDNA clone that codes for gp24 to screen cloned genomic fragments. Two closely linked genes (GP24A and GP24B) were recognized that generate mRNAs of about 650 base pairs after excision of a small intron and addition of poly(A). They appear to have arisen by tandem duplication of about 800 base pairs, followed by divergence. These genes are expressed within a few hours of the initiation of development; their mRNAs accumulate to a peak at 12 hr and persist until culmination. Both genes have short guanine-rich sequences (G boxes) upstream that have been shown to be involved in transcriptional regulation of other genes expressed during development of Dictyostelium. Their mRNAs code for proteins that are 85% identical. GP24A and GP24B mRNAs code for proteins with a hydrophobic domain followed by a highly charged carboxyl-terminal domain.

Amino Acid Sequence↗
PubMed1989

Spore coat genes SP60 and SP70 of Dictyostelium discoideum.

We cloned and sequenced the genes for two of the major proteins found in spore coats of Dictyostelium discoideum. The predicted translation product of each of these genes starts with a hydrophobic signal sequence that is subsequently cleaved. Expression of these spore coat genes is coordinate in prespore cells.

Amino Acid Sequence↗
PubMed1988

Regulation of SP60 mRNA during development of Dictyostelium discoideum.

The accumulation of mRNA recognized by oligonucleotides coding for a portion of the spore coat protein, SP60, was determined throughout development of Dictyostelium discoideum. The 1.8 kb mRNA first appears at the tipped aggregate stage and accumulates until culmination. This mRNA is present in pre-spore cells but absent from pre-stalk cells. A cDNA clone was selected by the oligonucleotides and found to be homologous to this mRNA. Although the oligonucleotides were designed to match the sequence coding for a hexapeptide repeat at the amino-terminus of SP60, they were able to recognize a similar repeated region at the carboxy-terminus of the protein coded by the cDNA clone. The SP60 gene appears to be subject to temporal and cell-type-specific transcriptional controls that are coordinate with those of SP96, another spore coat gene.

Amino Acid Sequence↗
PubMed1988

Cell-cell adhesion in Dictyostelium discoideum.

Three separate mechanisms of cell-cell adhesion have been shown to appear at different stages of development in Dictyostelium discoideum. During the first few hours of development, the cells synthesize and accumulate a glycoprotein of 24,000 daltons (gp24) that is positioned in the membrane. The time of appearance of gp24 correlates exactly with the time of appearance of cell-cell adhesion in two strains in which temporal control varies by several hours. Antibodies specific to gp24 are able to block cell-cell adhesion during the first few hours of development but not during later development. By 8 hr of development, another glycoprotein, gp80, that is not recognized by antibodies to gp24 accumulates on the surface of cells. This membrane protein mediates an independent adhesion mechanism during the aggregation stage that is resistant to 10 mM EDTA. Antibodies specific to gp80 can block EDTA-resistant adhesion during this stage. During subsequent development, gp80 is removed from the cell surface and replaced by another adhesion mechanism that is insensitive to antibodies to either gp24 or gp80. A lambda gt11 expression vector carrying a Dictyostelium cDNA insert was isolated that directs the synthesis of a fusion protein recognized by antibodies specific to gp24. This cDNA was used to probe a genomic library. A clone carrying a 1.4-kb insert of genomic DNA was recognized by the cDNA and shown to hybridize to a 0.7-kb mRNA that accumulates early in development. This unusually small RNA could code for the small protein, gp24. Southern analysis of restriction fragments generated by various enzymes on Dictyostelium DNA with both the cDNA and genomic clones indicated the presence of two tandem copies of the gene. This may account for the failure to recover mutations resulting in the lack of gp24. Mutations have been recovered that result in the lack of accumulation of gp80, and cells carrying these mutations have been shown to be missing the second adhesion mechanism. These mutant strains are able to complete development because the other adhesion mechanisms are not impaired. Sequential addition of adhesion mechanisms provides a means for the formation of multicellular organisms from previously solitary cells.

Cell Adhesion↗
PubMed1988

Signal transduction, chemotaxis, and cell aggregation in Dictyostelium discoideum cells without myosin heavy chain.

Dictyostelium discoideum cells have been generated that lack myosin heavy chain (MHC) due to antisense RNA inactivation of the endogenous mRNA or to insertional mutagenesis of the myosin gene. These cells retain chemotactic movement in gradients of the chemoattractant cAMP. Furthermore, cAMP does induce many biochemical and physiological responses in aggregative cells, including binding of cAMP to surface receptors, modification, and down-regulation of the receptor; activation of adenylate and guanylate cyclase, secretion of cAMP; and the association of actin to the Triton-insoluble cytoskeleton. Cells lacking MHC were found to have a requirement for bivalent cations in the medium for optimal chemotaxis and cell aggregation.

Actins↗
PubMed1988

Cell motility and chemotaxis in Dictyostelium amebae lacking myosin heavy chain.

Dictyostelium amebae have been engineered by homologous recombination of a truncated copy of the myosin heavy chain gene (heavy meromyosin (HMM) cells) and by transformation with a vector encoding an antisense RNA to myosin heavy chain mRNA (mhcA cells) so that they lack native myosin heavy chain protein. In the former case, cells synthesize only the heavy meromyosin portion of the protein and in the latter case they synthesize negligible amounts of the protein. Surprisingly, it was demonstrated that both cell lines are viable and motile. In order to compare the motility of these cells with normal cells, the newly developed computer-assisted Dynamic Morphology System (DMS) was employed. The results demonstrate that the average HMM or mhcA ameba moves at a rate of translocation less than half that of normal cells. It is rounder and less polar than a normal cell, and exhibits a rate of cytoplasmic expansion and contraction roughly half that of normal cells. In a spatial gradient of cAMP, the average ameba of HMM or mhcA exhibits a chemotactic index of +0.10 or less, compared to the chemotactic index of +0.50 exhibited by normal cells. Finally, the initial area, rate of expansion, and final area of pseudopods are roughly half that of normal cells. The five fastest HMM amebae (out of 35 analyzed in detail) moved at an average rate of translocation equal to that of normal amebae, and exhibited an average chemotactic index of +0.34. In addition, the average rate of cytoplasmic flow in fast HMM cells was equal to that of the average normal ameba. However, fast HMM amebae still exhibited the same defects in pseudopod formation that were exhibited by the entire HMM cell population. These results suggest that myosin heavy chain is involved in the "fine tuning" and efficiency of pseudopod formation, but is not essential for the basic behavior of pseudopod expansion.

Actins↗
PubMed1988

Developmental consequences of the lack of myosin heavy chain in Dictyostelium discoideum.

Two different Dictyostelium discoideum cell lines that lack myosin heavy chain protein (MHC A) have been previously described. One cell line (mhcA) was created by antisense RNA inactivation of the endogenous mRNA and the other (HMM) by insertional mutagenesis of the endogenous myosin gene. The two cell lines show similar developmental defects; they are delayed in aggregation and become arrested at the mound stage. However, when cells that lack myosin heavy chain are mixed with wild-type cells, some of the mutant cells are capable of completing development to form mature spores. The pattern of expression of a number of developmentally regulated genes has been examined in both mutant cell lines. Although morphogenesis becomes aberrant before aggregation is completed, all of the markers that we have examined are expressed normally. These include genes expressed prior to aggregation as well as prespore genes expressed later in development. It appears that the signals necessary for cell-type differentiation are generated in the aborted structures formed by cells lacking MHC A. The mhcA cells have negligible amounts of MHC A protein while the HMM cells express normal amounts of a fragment of the myosin heavy chain protein similar to heavy meromyosin (HMM). The expression of myosin light chain was examined in these two cell lines. HMM cells accumulate normal amounts of the 18,000-D light chain, while the amount of light chain in mhcA cells is dramatically reduced. It is likely that the light chains assemble normally with the HMM fragment in HMM cells, while in cells lacking myosin heavy chain (mhcA) the light chains are unstable.

Cell Adhesion↗