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H M Warrick

Publications and source records attributed to H M Warrick.

12 recordsLinked to original sources
PubMed1993

Force on single actin filaments in a motility assay measured with an optical trap.

We have used an optical trap to measure or exert a force on single actin filaments via the attachment of polystyrene beads which were coated with NEM-modified HMM. In the simplest experiment, beads were attached to rhodamine phalloidin labelled actin filaments and observed to move on an HMM coated surface in the presence of ATP. Moving beads were steered into the vicinity of the trap using a PZT operated microscope stage. The minimum force needed to stop a moving bead was measured by lowering the trap strength until the bead resumed movement. By aligning the optical trap with the centre of a quadrant detector placed in an image plane of the microscope, it was possible to measure the force exerted on a filament by measuring the displacement of the bead position from the centre of the trap. In each of these experiments, the trap was calibrated by applying a Stokes force to a bead in free solution. The characteristics of the trap were studied, and the displacement of the bead from the centre of the trap was shown to be directly proportional to the applied force over a large part of the total range of the trap. The compliance of the trap could be substantially reduced by the use of feedback control to deflect the laser beam via an acousto-optic modulator. The advantages and limitations of this technique will be discussed.

Actin Cytoskeleton↗
PubMed1991

Quantized velocities at low myosin densities in an in vitro motility assay.

An in vitro motility assay has been developed in which single actin filaments move on one or a few heavy meromyosin (HMM) molecules. This movement is slower than when many HMM molecules are involved, in contrast to analogous experiments with microtubules and kinesin. Frequency analysis shows that sliding speeds distribute around integral multiples of a unitary velocity. This discreteness may be due to differences in the numbers of HMM molecules interacting with each actin filament, where the unitary velocity reflects the activity of one HMM molecule. The value of the unitary velocity predicts a step size of 5-20 nm per ATP, which is consistent with the conventional swinging crossbridge model for myosin function.

Adenosine Triphosphate↗
PubMed1991

An approach to reconstituting motility of single myosin molecules.

Over the last five years, the value of in vitro motility assays as probes of the mechanical properties of the actin-myosin interaction has been amply demonstrated. Motility assays in which single fluorescent actin filaments are observed moving over surfaces coated with myosin or its soluble fragments are now used in many laboratories. They have been applied to a wide range of problems including the study of structure-function relationships in the myosin molecule and measurement of fundamental properties of the myosin head. However, one limitation of these assays has been uncertainty over the number of myosin heads interacting with each sliding filament, that frustrates attempts to determine properties of individual heads. In order to address this limitation, we have modified the conditions of the actin sliding filament assay to reduce the number of heads interacting with each filament. Our goal is to establish an assay in which the motor function of a single myosin head can be characterized from the movement of a single actin filament.

Actins↗
PubMed1989

Multiple actin-based motor genes in Dictyostelium.

Dictyostelium cells, devoid of conventional myosin, display a variety of motile activities, consistent with the presence of other molecular motors. The Dictyostelium genome was probed at low stringency with a gene fragment containing the conserved conventional myosin head domain sequences to identify other actin-based motors that may play a role in the observed motility of these mutant cells. One gene (abmA) has been characterized and encodes a polypeptide of approximately 135 kDa with a head region homologous to other myosin head sequences and a tail region that is not predicted to form either an alpha-helical structure of coiled-coil interactions. Comparisons of the amino acid sequences of the tail regions of abmA, Dictyostelium myosin I, and Acanthamoeba myosins IB and IL reveal an area of sequence similarity in the amino terminal half of the tail that may be a membrane-binding domain. The abmA gene, however, does not contain an unusual Gly, Pro, Ala stretch typical of many of the previously described myosin Is. Two additional genes (abmB and abmC) were identified using this approach and also found to contain sequences that encode proteins with typical conserved myosin head sequences. The abm genes may be part of a large family of actin-based motors that play various roles in diverse aspects of cellular motility.

Actins↗
PubMed1988

Codon preference in Dictyostelium discoideum.

Dictyostelium discoideum is of increasing interest as a model eukaryotic cell because its many attributes have recently been expanded to include improved genetic and biochemical manipulability. The ability to transform Dictyostelium using drug resistance as a selectable marker (1) and to gene target by high frequency homologous integration (2) makes this organism particularly useful for molecular genetic approaches to cell structure and function. Given this background, it becomes important to analyze the codon preference used in this organism. Dictyostelium displays a strong and unique overall codon preference. This preference varies between different coding regions and even varies between coding regions from the same gene family. The degree of codon preference may be correlated with expression levels but not with the developmental time of expression of the gene product. The strong codon preference can be applied to identify coding regions in Dictyostelium DNA and aid in the design of oligonucleotide probes for cloning Dictyostelium genes.

Codon↗
PubMed1986

Conserved protein domains in a myosin heavy chain gene from Dictyostelium discoideum.

The 2116-amino acid myosin heavy chain sequence from Dictyostelium discoideum was determined from DNA sequence analysis of the cloned gene. The gene product can be divided into two distinct regions, a globular head region and a long alpha-helical, rod-like tail. In comparisons with nematode and mammalian muscle myosins, specific areas of the head region are highly conserved. These areas presumably reflect conserved functional and structural domains. Certain features that are present in the head region of nematode and mammalian muscle myosins, and that have been assumed to be important for myosin function, are missing in the Dictyostelium myosin sequence. The protein sequence of the Dictyostelium tail region is very poorly conserved with respect to the other myosins but displays the periodicities similar to those of muscle myosins. These periodicities are believed to play a role in filament formation. The 196-residue repeating unit that determines the 14.3-nm repeat seen in muscle thick filaments, the 28-residue charge repeating unit, and the 1,4 hydrophobic repeat previously described for the nematode myosin are all present in the Dictyostelium myosin rod sequence, suggesting that the filament structures of muscle and Dictyostelium myosins must be similar.

Adenosine Triphosphate↗
PubMed1984

The nucleotide sequence of the yeast ARG4 gene.

The complete nucleotide sequence of a 2296-bp DNA fragment containing the yeast (Saccharomyces cerevisiae) ARG4 gene has been determined. This gene specifies the synthesis of the arginine biosynthetic enzyme, argininosuccinate lyase (EC 4.3.2.1). The sequence contains one major open reading frame of 463 codons, giving a calculated Mr of 52 010 for the protein, in good agreement with the experimentally determined value of 53 000. The sequence upstream from the ARG4 gene shares structural features in common with other yeast genes subject to general amino acid control.

Amino Acid Sequence↗
PubMed1979

Electron acceptor taxis and blue light effect on bacterial chemotaxis.

Salmonella typhimurium and Escherichia coli from anaerobic cultures displayed tactic responses to gradients of nitrate, fumarate, and oxygen when the appropriate electron transport pathway was present. Such responses were named "electron acceptor taxis" because they are elicited by terminal electron acceptors. Mutant strains of S. typhimurium and E. coli were used to establish that functioning electron transport pathways to nitrate and fumarate are required for taxis to these compounds. Aerotaxis in S. typhimurium was blocked by 1.0 mM KCN, which inhibited oxygen uptake. Similarly, a functioning electron transport pathway was shown to be essential for the tumbling response of S. typhimurium and E. coli to intense light (290 to 530 nm). Some inhibitors and uncouplers of respiration were repellents of S. typhimurium. We propose that behavioral responses to light or electron acceptors involve electron transport-mediated perturbations of the proton motive force.

Anaerobiosis↗
PubMed1977

Chemotactic mechanism of Salmonella typhimurium: preliminary mapping and characterization of mutants.

Some new, generally nonchemotactic mutants of Salmonella typhimurium were isolated and they, together with previously isolated mutants and some from other investigators, were mapped. Most of the mutants were classified in nine complementation groups, which are probably individual genes. Of these, five map at the end of the flagella region and appear in the order motB-(cheWcheP)-cheX-cheQ-cheR-flaC. Two of the mutations, cheU and cheV, map in the flaQ and flaAII genes, respectively. The remaining genes, cheS and cheT, have not yet been mapped. Most of the mutants are phenotypically smoothly swimming, but some are constantly tumbling. Two of the groups show dominant behavior as recipients in genetic crosses; the rest are recessive. The mutants vary in their responses to stimuli but, since their responses to all chemoeffectors are abnormal, the central processing, rather than individual, receptors must be impaired. The two mutations that coincide with genes for flagella probably involve the locus of the final delivery of sensing signal to the flagella.

Chemotaxis↗