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R L Chisholm

Publications and source records attributed to R L Chisholm.

At least 37 records · Page 2Linked to original sources
PubMed1994

Expression of a myosin regulatory light chain phosphorylation site mutant complements the cytokinesis and developmental defects of Dictyostelium RMLC null cells.

In a number of systems phosphorylation of the regulatory light chain (RMLC) of myosin regulates the activity of myosin. In smooth muscle and vertebrate nonmuscle systems RMLC phosphorylation is required for contractile activity. In Dictyostelium discoideum phosphorylation of the RMLC regulates both ATPase activity and motor function. We have determined the site of phosphorylation on the Dictyostelium RMLC and used site-directed mutagenesis to replace the phosphorylated serine with an alanine. The mutant light chain was then expressed in RMLC null Dictyostelium cells (mLCR-) from an actin promoter on an integrating vector. The mutant RMLC was expressed at high levels and associated with the myosin heavy chain. RMLC bearing a ser13ala substitution was not phosphorylated in vitro by purified myosin light chain kinase, nor could phosphate be detected on the mutant RMLC in vivo. The mutant myosin had reduced actin-activated ATPase activity, comparable to fully dephosphorylated myosin. Unexpectedly, expression of the mutant RMLC rescued the primary phenotypic defects of the mlcR- cells to the same extent as did expression of wild-type RMLC. These results suggest that while phosphorylation of the Dictyostelium RMLC appears to be tightly regulated in vivo, it is not essential for myosin-dependent cellular functions.

Actins↗
PubMed1993

The highly divergent alpha- and beta-tubulins from Dictyostelium discoideum are encoded by single genes.

As a step in the characterization of the microtubule system of Dictyostelium discoideum, we have isolated and sequenced full-length cDNA clones that encode the Dictyostelium alpha- and beta-tubulins, as well as the Dictyostelium alpha-tubulin gene. Southern blot analysis suggests that Dictyostelium is unusual in that its genome contains single alpha- and beta-tubulin genes, rather than the multi-gene family common in most eukaryotic organisms. The complete alpha-tubulin cDNA contains 1558 nucleotides, with an open reading frame, that encode a protein of 457 amino acids. The complete beta-tubulin cDNA contains 1572 nucleotides and encodes a protein of 456 amino acids. Analysis of the deduced protein sequences indicates that while there is a significant degree of sequence similarity between the Dictyostelium tubulins and other known tubulins, the Dictyostelium alpha-tubulin displays the greatest sequence divergence yet described. Single alpha- and beta-tubulin transcripts are detected by northern blot analysis during all stages of Dictyostelium development. The highest levels of message accumulate late in germinating spores and vegetative amoebae. Despite changes in alpha- and beta-tubulin mRNA levels, protein levels remain constant throughout development. We have expressed the carboxy-terminal two-thirds of the alpha- and beta-tubulins as trpE fusions in Escherichia coli and used this protein to produce polyclonal antisera specific for the Dictyostelium alpha- and beta-tubulins. These antisera recognize one alpha- and two beta-tubulin spots on western blots of 2-D gels and, by indirect immunofluorescence, both recognize the interphase and mitotic microtubule arrays in vegetative amoebae.

Amino Acid Sequence↗
PubMed1992

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.

Actins↗
PubMed1992

The role of myosin I and II in cell motility.

It 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. 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.

Actins↗
PubMed1992

Collagen and elastin gene expression in aortic aneurysms.

BACKGROUND: The decreased elastin concentration found in abdominal aortic aneurysms (AAAs) may result from a differential synthetic response wherein elastin gene expression fails to increase in parallel with type I procollagen (COL I) gene expression. The purpose of this study is to determine tissue mRNA levels for elastin and COL I in AAAs compared with levels in normal, age-matched aorta and to determine the relationship between aging and COL I gene expression. METHODS: Total RNA exacted from normal infrarenal aortic tissue (n = 7) and AAA (n = 10) tissue was subjected to Northern analysis. Mean values for COL I, elastin, and alpha-tubulin mRNA levels were compared by use of the Student t test. Age and COL I mRNA levels were analyzed by regression analysis. RESULTS: COL I mRNA was increased significantly in AAAs (1.18 +/- 0.13) compared with normal aortas (0.14 +/- 0.05). A commensurate increase in elastin mRNA (AAAs, 0.11 +/- 0.02, vs normal aortas, 0.39 +/- 0.2) was absent. There was no correlation between age and COL gene expression. CONCLUSIONS: The decreased elastin concentration relative to collagen in AAAs may be explained, in part, by the changes in message level of elastin and collagen. The enhanced COL I gene expression in AAAs is unrelated to age.

Aging↗
PubMed1991

Dictyostelium discoideum essential myosin light chain: gene structure and characterization.

We have used a Dictyostelium essential myosin light chain (EMLC) cDNA clone to isolate additional cDNA clones which supply a different 3' sequence from that previously described. The revised cDNA sequence encodes a polypeptide of 150 amino acids. Amino acid residues 147-167 of the previously reported sequence are replaced by new residues 147 to 150. The new cDNA encodes a polypeptide with 66% amino acid sequence identity with the Physarum polycephalum EMLC, and approximately 30% identity with mammalian EMLC sequences. These new cDNA clones were used to isolate two genomic DNA fragments which contain the entire EMLC gene. The Dictyostelium EMLC gene contains a single intron located immediately 3' of the translation initiation codon and encodes a product most similar to MLC3 isoform of vertebrates. Primer extension analysis places the transcription initiation site approximately 90 nucleotides upstream of the translation initiation site. A DNA fragment containing 350 bases of sequence upstream of the putative transcription initiation site is sufficient to drive expression of a reporter gene upon reintroduction into growing Dictyostelium cells. In addition, the CAT reporter mRNA produced by this construct showed a pattern of developmental regulation similar to that previously reported for the endogenous EMLC mRNA. Based on comparison with published EMLC sequences from a variety of sources, the Dictyostelium EMLC shows slightly higher similarity to vertebrate EMLCs from striated muscle sources than nonmuscle sources. While Dictyostelium and human nonmuscle sequences display only 28% identity over their entire sequence, the region from residue 88 to 108 shows much higher identity (67%). The high evolutionary conservation of this region of the EMLC suggests it may play an important role in EMLC function, and as such, represents a good target for future mutagenesis studies.

Amino Acid Sequence↗
PubMed1990

A pleiotropic defect in cAMP-regulated gene expression in the Dictyostelium agg- mutant synag 7.

During differentiation of Dictyostelium discoideum, cAMP functions as a diffusible, extracellular signal to direct chemotaxis and regulate developmental gene expression. The availability of signal-transduction mutants of Dictyostelium now makes it feasible to pursue a genetic analysis of cAMP signaling. The synag 7 mutant is defective in receptor-mediated adenylate cyclase stimulation and cannot relay a cAMP signal. To further characterize this mutant, mRNA levels of several cAMP-regulated genes were measured during development. cAMP-regulated gene expression was found to be dramatically altered in synag 7:several different genes which require cAMP for expression in wild-type cells were induced in synag 7 in the absence of cAMP. In addition, the gene-encoding discoidin I, which is normally expressed in starved cells and repressed by cAMP, is expressed at very low levels in starved synag 7 cells, possibly due to precocious repression. These results suggest that a pleiotropic regulator of cAMP-regulated gene expression is uncoupled from its normal controls during development in synag 7.

Actins↗
PubMed1989

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.

Amino Acid Sequence↗
PubMed1989

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

Phosphorylation of the regulatory light chains (RMLC) of nonmuscle myosin can increase the actin-activated ATPase activity and filament formation. Little is known about these regulatory mechanisms and how the RMLC are involved in ATP hydrolysis. To better characterize the nonmuscle RMLC, we isolated cDNAs encoding the Dictyostelium RMLC. Using an antibody specific for the RMLC, we screened a lambda gt11 expression library and obtained a 200-base-pair clone that encoded a portion of the RMLC. The remainder of the sequence was obtained from two clones identified by DNA hybridization, using the 200-base-pair cDNA. The composite RMLC cDNA was 645 nucleotides long. It contained 60 base pairs of 5' untranslated, 483 bases of coding, and 102 base pairs of 3' untranslated sequence. The amino acid sequence predicted an 18,300-dalton protein that shares 42% amino acid identity with Dictyostelium calmodulin and 30% identity with the chicken skeletal myosin RMLC. This sequence contained three regions that were similar to the E-F hand calcium-binding domains found in calmodulin, troponin C, and other myosin light chains. A sequence similar to the phosphorylation sequence found in chicken gizzard and skeletal myosin light chains was found at the amino terminus. Genomic Southern blot analysis suggested that the Dictyostelium genome contains a single gene encoding the RMLC. Analysis of RMLC expression patterns during Dictyostelium development indicated that accumulation of this mRNA increases just before aggregation and again during culmination. This pattern is similar to that obtained for the Dictyostelium essential myosin light chain and suggests that expression of the two light chains is coordinated during development.

Amino Acid Sequence↗
PubMed1988

Translocation and rearrangement of myeloperoxidase gene in acute promyelocytic leukemia.

Acute promyelocytic leukemia (subtype M3) is characterized by malignant promyelocytes exhibiting an abundance of abnormally large or aberrant primary granules. Myeloperoxidase (MPO) activity of these azurophilic granules, as assessed by cytochemical staining, is unusually intense. In addition, M3 is universally associated with a chromosomal translocation, t(15;17)(q22;q11.2). In this report, the MPO gene was localized to human chromosome 17 (q12-q21), the region of the breakpoint on chromosome 17 in the t(15;17), by somatic cell hybrid analysis and in situ chromosomal hybridization. By means of MPO complementary DNA clones for in situ hybridization and Southern blot analysis, the effect of this specific translocation on the MPO gene was examined. In all cases of M3 examined, MPO is translocated to chromosome 15. Genomic blot analyses indicate rearrangement of MPO in leukemia cells of two of four cases examined. These findings suggest that MPO may be pivotal in the pathogenesis of acute promyelocytic leukemia.

Bone Marrow↗
PubMed1988

The effect of caffeine, adenosine, and buffer ionic composition on the induction of cell-surface cAMP binding during starvation of Dictyostelium discoideum.

We have analyzed the effects of the cAMP relay inhibitor, caffeine, and the receptor antagonist, adenosine, on the regulation of the cell-surface cAMP receptor in suspension-starved Dictyostelium discoideum cells by measuring ammonium sulfate-stabilized binding of [3-H]cAMP to intact cells. When cells were starved in fast (230 r.p.m.) shaken suspension in 10 mM Na+/5 mM K+ phosphate buffer, pH 6.5, plus 1 mM CaCl2 and 2.5 mM MgCl2, and assayed for specific cAMP binding, receptor accumulation peaked at approximately 6 hours, reaching a maximum of 1.5 pmol cAMP bound/10(7) cells (saturation binding). Neither caffeine nor adenosine inhibited the accumulation of cAMP receptors. Similar results were obtained in caffeine-treated, slow shaken (90 r.p.m.) suspension cultures. These results suggest that starvation alone is sufficient stimulus to induce the cAMP receptor. We have also tested the effects of different buffer ionic compositions on the accumulation of cAMP receptors. Elevation of the monovalent ion concentration to 30-40 mM was found to significantly inhibit the induction of cAMP receptors.

Adenosine↗
PubMed1988

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.

Adrenal Gland Neoplasms↗
PubMed1988

Isolation of cDNAs encoding desmosomal plaque proteins: evidence that bovine desmoplakins I and II are derived from two mRNAs and a single gene.

Desmoplakins (DPs) I and II (approximately equal to 240 and approximately equal to 210 kDa) are major components of the internal portion of the desmosomal cytoplasmic plaque. Desmosomes play a crucial role in cell-cell adhesion and serve as specific attachment sites for cytoplasmic intermediate filaments. Although DP-I and -II are closely related molecules, their structure (i.e., amino acid or DNA sequence) has not been determined. In addition, it is not known whether these proteins are derived from one or more genes or whether they result from posttranscriptional or posttranslational events. This paper describes the isolation and characterization of eight DP cDNA clones from a bovine lambda gt11 expression library. Fusion proteins from six of these clones selected antibodies that reacted with DP-I and -II and two selected antibodies that reacted with DP-I alone. Antibodies made against fusion protein produced by the DP1A clone reacted specifically with DP-I and -II on immunoblots. When used for indirect immunofluorescence on bovine tongue cryostat sections and cultured mouse keratinocytes, these antibodies produced a typical desmosomal staining pattern. RNA blot analysis demonstrated hybridization of three DP-I/II cDNA probes with two messages of approximately equal to 7.5 and approximately equal to 9.5 kilobases in bovine tongue RNA. In contrast, a cDNA clone that affinity-purified antibodies reacting with DP-I only hybridized exclusively with the 9.5-kilobase band. Southern blots of genomic DNA digested with a panel of restriction enzymes were hybridized with one probe derived from a DP-I/II clone and with one from a DP-I clone. Both probes hybridized with single bands of the same size in each digested sample of DNA. Together, these data suggest that DP-I and DP-II are translated from two separate messages in bovine tongue and that these messages may be derived from a single gene.

Animals↗
PubMed1988

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.

Amino Acid Sequence↗
PubMed1987

Superinduction of the Dictyostelium discoideum cell surface cAMP receptor by pulses of cAMP.

Extracellular cAMP plays a crucial role in regulating the developmental program of Dictyostelium discoideum, functioning as a chemotactic agent, as well as a signal that regulates expression of developmentally expressed genes. These activities appear to be mediated by a cell-surface receptor for cAMP. We have studied the regulation of this receptor in cells developed in starved suspension cultures exposed to 50 nM pulses of cAMP every 6 min. cAMP-pulsed cells display roughly 10-fold higher cAMP receptor levels than cells that developed on filters or that were starved in suspension without cAMP pulses. Based on saturation binding analysis, the superinduced binding activity represents an increase in receptor number, while receptor affinity for cAMP is unaffected. Photoaffinity labeling of superinduced cells results in specific labeling of the same molecules that are labeled in starved cells. This increased cAMP binding activity was also detected in membrane preparations from cAMP-pulsed cells. These results provide evidence for an unusual mode of receptor regulation: autogenous induction of the receptor by its ligand.

Affinity Labels↗
PubMed1987

cDNA cloning of human myeloperoxidase: decrease in myeloperoxidase mRNA upon induction of HL-60 cells.

Myeloperoxidase (MPO), the most abundant neutrophil protein, is a bacteriocidal component of the primary granules and a critical marker in distinguishing acute myelogenous leukemia from acute lymphoid leukemia. A cDNA clone for human MPO was isolated by immunologic screening of human hematopoietic lambda gt11 expression vector libraries with specific anti-MPO antibody. The identity of the cDNA clone was confirmed by finding that epitope-selected antibody against this clone recognizes purified MPO and MPO in human promyelocytic (HL-60) cell lysates by immunoblot analysis, and that hybrid selection of HL-60 mRNA with this cDNA clone and translation in vitro results in the synthesis of an 80-kDa protein recognized by the anti-MPO antiserum. RNA blot analysis with this MPO cDNA clone detects hybridization to two polyadenylylated transcripts of approximately 3.6 and approximately 2.9 kilobases in HL-60 cells. No hybridization is detected to human placenta mRNA. Upon induction of HL-60 cells to differentiate by incubation for 4 days with dimethyl sulfoxide, a drastic decrease in the hybridization intensity of these two bands is seen. This is consistent with previous data suggesting a decrease in MPO synthesis upon such induction of these cells. The MPO cDNA should be useful for further molecular and genetic characterization of MPO and its expression and biosynthesis in normal and leukemic granulocytic differentiation.

Cell Line↗
PubMed1985

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.

Amino Acid Sequence↗