Rex L. Chisholm

The two alpha-tubulin genes of Chlamydomonas reinhardi code for slightly different proteins.

Full-length cDNA clones corresponding to the transcripts of the two alpha-tubulin genes in Chlamydomonas reinhardi were isolated. DNA sequence analysis of the cDNA clones and cloned gene fragments showed that each gene contains 1,356 base pairs of coding sequence, predicting alpha-tubulin products of 451 amino acids. Of the 27 nucleotide differences between the two genes, only two result in predicted amino acid differences between the two gene products. In the more divergent alpha 2 gene, a leucine replaces an arginine at amino acid 308, and a valine replaces a glycine at amino acid 366. The results predicted that two alpha-tubulin proteins with different net charges are produced as primary gene products. The predicted amino acid sequences are 86 and 70% homologous with alpha-tubulins from rat brain and Schizosaccharomyces pombe, respectively. Each gene had two intervening sequences, located at identical positions. Portions of an intervening sequence highly conserved between the two beta-tubulin genes are also found in the second intervening sequence of each of the alpha genes. These results, together with our earlier report of the beta-tubulin sequences in C. reinhardi, present a picture of the total complement of genetic information for tubulin in this organism.

A type III intermediate filament gene is expressed in mature neurons

A cDNA (199E) specific for the 57 kd neural IF protein has been isolated from a PC12 cell lambda gt11 library. Antibody eluted from the fusion protein produced by 199E recognizes the 57 kd protein on immunoblots and, in PC12 cells, labels a pattern of fibrillar structures identical to that seen with 57 kd antiserum. In situ hybridization using antisense RNA transcripts labels areas of the nervous system known to contain the 57 kd protein. 199E hybridizes with a single mRNA species of approximately 2.0 kb from PC12 cells. A 199E-reactive message can be detected as early as E10 in rat embryos. Southern analyses suggest that there is only one gene for this protein. Amino acid sequence predicted from 199E indicates that the 57 kd protein is a type III IF protein like vimentin and desmin. Thus, expression of IF structural genes in neurons is not limited to the type IV neuronal IF triplet proteins.

The dictyostelium essential light chain is required for myosin function

A Dictyostelium mutant (7-11) that expresses less than 0.5% of wild-type levels of the myosin essential light chain (EMLC) has been created by overexpression of antisense RNA. Cells from 7-11 contain wild-type levels of the myosin heavy chain (MHC) and regulatory light chain (RMLC). Myosin isolated from 7-11 cells consists of the MHC with the RMLC associated in reduced stoichiometry, and binds to purified actin in an ATP-sensitive fashion. Purified 7-11 myosin displays calcium-activated ATPase activity with a Vmax about 15%-25% of that of wild type, and a Km for ATP of 27 +/- 5 microM versus 83 +/- 30 microM for wild type. At actin concentrations as high as 17 microM, 7-11 myosin displays greatly reduced actin-activated ATPase activity. Phenotypically, 7-11 cells resemble MHC mutants, growing poorly in suspension and becoming large and multinucleate. When starved for multicellular development, 7-11 cells take several hours longer than wild-type cells to aggregate. Although multicellular aggregates eventually form, they fail to develop further. The cells are also unable to cap receptors in response to Con A treatment. Since cells expressing the EMLC are phenotypically similar to MHC null mutants, the EMLC appears necessary for myosin function, at least in part because it is required for normal actin-activated ATPase activity.

Dictyostelium discoideum myosin: isolation and characterization of cDNAs encoding the essential light chain.

We used an antibody specific for Dictyostelium discoideum myosin to screen a lambda gt11 cDNA expression library to obtain cDNA clones which encode the Dictyostelium essential myosin light chain (EMLC). The amino acid sequence predicted from the sequence of the cDNA clone showed 31.5% identity with the amino acid sequence of the chicken EMLC. Comparisons of the Dictyostelium EMLC, a nonmuscle cell type, with EMLC sequences from similar MLCs of skeletal- and smooth-muscle origin, showed distinct regions of homology. Much of the observed homology was localized to regions corresponding to consensus Ca2+-binding of E-F hand domains. Southern blot analysis suggested that the Dictyostelium genome contains a single gene encoding the EMLC. Examination of the pattern of EMLC mRNA expression showed that a significant increase in EMLC message levels occurred during the first few hours of development, coinciding with increased actin expression and immediately preceding the period of maximal chemotactic activity.

A physical gene map of the bacteriophage P22 late region: Genetic analysis of cloned fragments of P22 DNA

A physical gene map of the late region of the P22 chromosome has been constructed by genetic analysis of restriction enzyme fragments of P22 DNA cloned in a plasmid vector. Cleavage sites for restriction endonucleases SalI, SstI, SmaI, XhoI, and BglI were mapped on P22 DNA to provide physical reference points in addition to the EcoRI, HindIII, and BamHI cleavage sites previously mapped. Restriction enzymes KpnI, BgIII, and XbaI were found to have no cleavage sites on P22 DNA. Fragments of P22 DNA produced by cleavage with EcoRI, BamHI, or EcoRI plus BamHI were cloned in Escherichia coli using the plasmid vector pBR322, and the resulting recombinant plasmids were introduced into Salmonella typhimurium. The genes present on a cloned fragment were identified by the ability of the hybrid plasmid to complement or recombine with P22 amber mutations in known genes when mutant phage were used to infect S. typhimurium strains carrying the recombinant plasmids. These experiments place all phage genes required for P22 head morphogenesis except gene 3 on the physical map between coordinates 0.000 and 0.318. The coding capacity of this interval is in close agreement with the molecular weights of the proteins assigned to it. The single gene for the P22 base plate protein is placed between coordinates 0.376 and 0.420 on the physical map. These results also show that distances on the recombination frequency map are significantly distorted relative to the physical gene map of the late region. The recombination frequency map is expanded in the region of the physical gene map where terminally redundant ends of the circularly permuted mature chromosomes fall.

Substitution mutations in the myosin essential light chain lead to reduced actin-activated ATPase activity despite stoichiometric binding to the heavy chain.

Myosin essential light chain (ELC) wraps around an α-helix that extends from the myosin head, where it is believed to play a structural support role. To identify other role(s) of the ELC in myosin function, we have used an alanine scanning mutagenesis approach to convert charged residues in loops I, II, III, and helix G of the Dictyostelium ELC into uncharged alanines. Dictyostelium was used as a host system to study the phenotypic and biochemical consequences associated with the mutations. The ELC carrying loop mutations bound with normal stoichiometry to the myosin heavy chain when expressed in ELC-minus cells. When expressed in wild type cells these mutants competed efficiently with the endogenous ELC for binding, suggesting that the affinity of their interaction with the heavy chain is comparable to that of wild type. However, despite apparently normal association of ELC the cells still exhibited a reduced efficiency to undergo cytokinesis in suspension. Myosin purified from these cells exhibited 4-5-fold reduction in actin-activated ATPase activity and a decrease in motor function as assessed by an in vitro motility assay. These results suggest that the ELC contributes to myosins enzymatic activity in addition to providing structural support for the α-helical neck region of myosin heavy chain.

A repetitive Dictyostelium gene family that is induced during differentiation and by heat shock

Clone pB41-6 (2.5 kb) contains sequences that are repeated 200-300 times in the Dictyostelium genome; about 40 of these sequences are part of a 4.5 kb repeated and apparently transposable genomic element. Clone pB41-6 hybridizes to a large number of cytoplasmic polyadenylated RNAs whose accumulation begins in the first hour of differentiation. In order to understand the regulation of these repeated sequences, we have sequenced pB41-6. It contains three long open reading frames in the sense strand. Remarkably, about 70 bases upstream of the transcription initiation site is a sequence identical to that responsible for induction of the Drosophila heat shock genes. A search of published sequences also generated a similar sequence upstream of one of the Dictyostelium actin genes. Indeed, we found that both pB41-6-related RNAs and actin mRNAs are increased as a result of heat shocking growing cells, and that transcription of pB41-6 sequences is induced by heat shock. Thus Dictyostelium contains a set of genes that are induced as a response to heat shock or to the stresses that trigger the initiation of development. We show here that the principal component of this stress is not amino acid starvation but the high density of the cells.

The role of myosin I and II in cell motility

SummaryIt has been recognized since the turn of the century that cell motility by non-muscle cells requires virtually continuous restructuring of the cytoskeleton (see refs [1–4]). It is also clear that cell motility requires a mechanism for converting chemical energy into mechanical work [5]. The proteins actin and myosin, two important constituents of the cytoskeleton, have been postulated to act as the chemicomechanical transducer in motile cells. Central to their role as a force generating mechanism in motile cells is the ability of myosin (a) to hydrolyze ATP when it interacts with actin and (b) to form filaments. Recent studies on mammalian cells and on the cellular slime mold Dictyostelium discoideum have shed light and at the same time raised questions regarding the involvement of myosin in cell motility. Moreover, they have demonstrated the presence of two types of myosins, called myosin II and myosin I, that have unique biochemical and regulatory properties and that may play different roles in mediating cell motility. In this chapter we will discuss the properties of these two myosins and then describe what is known about their involvement in Dictyostelium and mammalian cell motility.

Development of Dictyostelium discoideum: Chemotaxis, Cell-cell Adhesion, and Gene Expression

INTRODUCTION AND OVERVIEW The cellular slime mold Dictyostelium discoideum has attracted the interest of developmental biologists for many years. Starvation of this unicellular, eukaryotic organism triggers a program of differentiation during which cells undergo chemotaxis to form a multicellular aggregate, followed by differentiation of two new cell types: spores and stalks. This developmental program exhibits many features seen during development of higher eukaryotic organisms: A homogeneous population of cells differentiate into two new cell types; developing cells communicate with each other via extracellular hormones; specific cell-cell contacts are formed during the multicellular stage of development; and a specific arrangement of spore and stalk cells form during morphogenesis. Yet Dictyostelium is haploid and amenable to mutational analysis; stocks of developmental lethal mutations can be propagated vegetatively and the developmental defects studied in detail. As differentiation occurs only when growth and DNA replication have ceased, one can concentrate on the developmental process in the absence of cell growth. Additionally, the two differentiated cell types and their precursors can be isolated in quantity, allowing biochemical analysis. Here we review our current understanding of the developmental program of Dictyostelium, focusing on the signals that regulate differentiation, the changes in gene expression, and the molecular mechanisms used by Dictyostelium to regulate the levels of gene products during development. Morphogenesis and pattern formation are considered in a separate paper in this volume (MacWilliams and David). Several excellent reviews consider other aspects of Dictyostelium development and, in particular, two books edited by Loomis provide an excellent starting...

Expression and organization of BP74, a cyclic AMP-regulated gene expressed during Dictyostelium discoideum development.

We have characterized a cDNA and the corresponding gene for a cyclic AMP-inducible gene expressed during Dictyostelium development. This gene, BP74, was found to be first expressed about the time of aggregate formation, approximately 6 h after starvation. Accumulation of BP74 mRNA did not occur in Dictyostelium cells that had been starved in fast-shaken suspension cultures but was induced in similar cultures to which cyclic AMP pulses had been added. The BP74 cDNA and gene were characterized by DNA sequence analysis and transcriptional mapping. When the BP74 promoter region was fused with a chloramphenicol acetyltransferase reporter gene and reintroduced into Dictyostelium cells, the transfected chloramphenicol acetyltransferase gene displayed the same developmentally regulated pattern of expression as did the endogenous BP74 gene, suggesting that all of the cis-acting elements required for regulated expression were carried by a 2-kilobase cloned genomic fragment. On the basis of sequence analysis, the gene appeared to encode a protein containing a 20-residue hydrophobic sequence at the amino-terminal end and 26 copies of a 20-amino-acid repeat.