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dictyNews Volume 03 Number 02
CSM News
Electronic Edition
Volume 3, number 2
July 9, 1994
Please submit abstracts of your papers as soon as they have been
accepted for publication by sending them to CSM-News@worms.cmsbio.nwu.edu.
Back issues of CSM-News, the CSM Reference database and other useful
information is available by anonymous ftp from worms.cmsbio.nwu.edu
[165.124.233.50], via Gopher at the same address, or by World Wide Web
through www.nwu.edu.
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Abstracts
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Detection of specific microorganisms in environmental samples using
flow cytometry.
Graham Vesey, Joe Narai,Nicholas Ashbolt, Keith Williams & Duncan Veal
School of Biological Sciences, Macquarie University, Sydney, NSW 2109,
Australia
Methods in Cell Biology, in press.
Summary
Flow cytometers are technologically advanced instruments which combine
laser interogation of a fluid stream with sophisticated data handling
technology for obtaining information about, and potentially isolating,
particles that pass through the laser beam. Traditionally they are
amongst the most expensive of laboratory instruments and require
highly skilled personnel to operate them. This combined with the fact
that samples must be particulate and of reasonably uniform size has
limited their application in biology to well funded laboratories in
biomedical research where they are used to analyse blood cell sub
populations (immunology, AIDS, cancer) or separate chromosomes. The
reduced cost and ease of operation of analytical flow cytometers
(which collect data but do not sort particles) means that applications
in other areas of biology are now envisaged. The applications of flow
cytometry to clinical microbiology have been described by Shapiro
(1990). In this chapter we discuss the use of flow cytometry within
the environmental microbiology laboratory. In particular we focus on
flow cytometric methods for the detection of low numbers of, and even
single, specific microorganisms within environmental samples. Flow
cytometric analysis performed in environmental microbiology
laboratories is often more stringent than that required for the
analysis of mammalian cells and can push sensitivities close to limits
of operation. This is because the volume, nucleic acid and protein
content of bacteria are approximately 1000x less than in mammalian
cells Since detection involves identification of light scatter,
specific proteins or DNA the signals produced by bacteria are
generally several orders of magnitude lower than those from eukaryotic
cells. However, recently flow cytometers have been used to great
effect for microbiological diagnosis and even more recently they have
been applied in environmental microbiology. Developments in both
biological techniques and instrumentation, described in this chapter,
will considerably increase the range of applications of flow cytometry
within environmental microbiology laboratories. Furthermore, these
developments result in greatly simplified protocols allowing not only
research laboratories but also routine environmental testing
laboratories to perform these analyses. We envisage that within the
foreseeable future small, robust, relatively cheap and simple to
operate flow cytometers will be available for the detection of a vast
range of microorganisms in environmental samples.
NOTE TO: Dictyologists - In this paper we show a novel use of
Dictyostelium in setting up assays for detecting very low numbers of
samples in environmental waters. Using two monoclonal antibody tags
to spores, we detected a single spore in a water sample!
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Mitosis in amoebae of the cellular slime mold (mycetozoan)
Acytostelium leptosomum.
Bruno Guhl and Urs-Peter Roos
Institute of Plant Biology, University of Zurich, Zollikerstr. 107,
8008-Zurich, Switzerland
European J. Protozol., in press
SUMMARY
We investigated mitosis in amoebae of Acytostelium leptosomum,
grown in liquid culture, by video microscopy of live cells, by
indirect immunofluorescence with antibodies against tubulins, and by
transmission electron microscopy of ultrathin sections. Amoebae in
interphase contain a single microtubule-organizing center (MTOC) at
each nucleus, from which microtubules (MTs) radiate into the
cytoplasm. These disappear as the intranuclear spindle forms.
Concomitantly, the nucleolus disperses, and the chromosomes that are
visible in phase contrast congress to the spindle equator. The spindle
is closed except for polar fenestrae occupied by broad, amorphous
spindle pole bodies (SPBs). The chromosomes at metaphase are joined to
form several blocks, each attached to several kinetochore MTs.
Anaphase was accomplished within 2.2 min (s.d.=0.5 min, n=11).
Anaphase A was virtually absent, but anaphase B contributed
substantially to chromosome segregation. The mean velocity of pole
separation was 3.2 !m/ min (s.d.=0.8 !m/min) and the mean elongation
factor was 2.8 (range) 1.9 to 3.4). The telophase spindle was a shaft
consisting of a few MTs traversing each incipient daughter nucleus and
joining in the interzone. The amorphous SPBs were reconverted to
compact interphase MTOCs as the chromosomes decondensed and the
nucleolus re-formed during cytokinesis. Duration of mitosis and
velocities of its movements are within values typical for lower
eucaryotes. In most aspects of mitosis A. leptosomum is very similar
to two other dictyostelid cellular slime molds, Dictyostelium
discoideum and Polysphondylium violaceum, and the three lower
eucaryotes are clearly distinct from other myceotozoans.
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Mitosis in amoebae of the cellular slim mold (mycetozoan) Protostelium
mycophaga
Bruno Guhl and Urs-Peter Roos
Institute of Plant Biology, University of Zurich, Zollikerstr. 107,
8008-Zurich, Switzerland
Eur. J. Protozol., in press
SUMMARY
We investigated mitosis in amoebae of Protostelium mycophaga by
video microscopy of live cells, by indirect immunofluorescence with
antibodies against tubulins, and by transmission electron microscopy
of ultrathin sections. Amoebae in interphase usually contain two
microtubule centers (MCs) on opposite sides of the nucleus, from which
microtubules (MTs) radiate into the cytoplasm. During prophase these
MTs shorten to form two asters between which the mitotic spindle
develops during prometaphase. Concomitantly, the nucleolus fragments,
the numerous small chromosomes orient amphitelically in the spindle
and congress to the spindle equator, and the asters diminish further
until metaphase. The spindle is open and acentric, but with complex
spindle pole bodies. Each sister-chromatid is attached to a single MT
by a tiny, layered kinetochore. During anaphase and telophase, asters
develop anew and enlarge to become the elaborate MT cytoskeletons of
the daughter cells. Anaphase lasted 2 min on average (s.d.= 0.6 min,
n=4), during which the chromosomes moved poleward with a mean velocity
of 4.0 !m/min (s.d.=0.8 !m/min, n=5). The intermingling of kinetochore
MTs and the numerous non-kinetochore MTs allows for a sliding
interaction between them, but depolymerisation-driven chromosome
movement is also possible. The spindle elongated at a mean rate of 5.9
!m/min (s.d.= 2.2 !m/min, n=5), and the mean elongation factor was
2.4. in live cells. In immunofluorescence preparations the longest
spindles were 3.5. times longer than the average metaphase spindle.
Spindle elongation thus requires the growth of interzonal MTs, that
assemble as several bundles, from an ample pool of tubulin. At the end
of telophase the nuclear envelope is reconstructed from membrane
vesicles and flattended cisternae that appose to the masses of
decondensed chromosomes and nucleolar material.
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PROGRESSION OF AN INDUCTIVE SIGNAL ACTIVATES SPORULATION IN
DICTYOSTELIUM DISCOIDEUM.
Delwood L. Richardson 1, William F. Loomis 2, and Alan R. Kimmel 1
1 Laboratory of Cellular and Developmental Biology, NIDDK, NIH,
Bethesda, MD 20892
2 Dept. of Biology, UCSD, La Jolla, CA 92093-0322.
DEVELOPMENT, in press.
Summary
spiA, a marker for sporulation, is expressed during the
culmination stage of Dictyostelium development, when the mass of
prespore cells has moved partly up the newly formed stalk. Strains
containing a full- length spiA promoter/lacZ fusion were stained for
beta-galactosidase activity at intervals during development. The
results indicate that expression of spiA initiates in prespore cells
at the prestalk/prespore boundary (near the apex) and extends downward
into the prespore mass as culmination continues. A spatial gradient
of staining expands from the top of the prespore mass and intensifies
until the front of activation reaches the bottom, whereupon the entire
region stains darkly. The spiA promoter can be deleted to within 301
bp of the transcriptional start site with no effect on the relative
strength, timing, or spatial localization of expression. Further 5'
deletions from -301 to -175 reduce promoter strength incrementally,
although timing and spatial expression are not affected. Deletions to
-159 and beyond result in inactive promoters. Treatment of early
developmental structures with 8- Br-cAMP in situ activates the
intracellular cAMP-dependent protein kinase (PKA) and precociously
induces spiA expression and sporulation. The absence of an apparent
gradient of staining in these structures suggests that PKA is
equivalently activatable throughout the prespore region and that all
prespore cells are competent to express spiA. Thus, we postulate that
the pattern of expression of spiA reveals the progression of an
inductive signal for sporulation and suggest that this signal may
originate from the prestalk cells at the apex.
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[End CSM-News, volume 3, number 2]
