Faculty of Science - Mahidol University

Mahidol University Annual Research Abstracts, Vol. 33 291
in vivo screening system to select for functionally active proteins
with increased solubility by using Plasmodium falciparum
dihydrofolate reductase (pfDHFR), a by using Plasmodium
falciparum dihydrofolate reductase (pfDHFR), a well-know malarial
drug target, as a model. Prior to the dual selection process, pfDHFR
was fused to green fluorescent protein (GFP), which served as a
reporter for solubility. The fusion gene was used as a template for
construction of mutated DNA libraries of pfDHFR. Two amino acids
with large hydrophobic side chains (Y35 and F37) located on the
surface of pfDHFR were selected for site-specific mutagenesis.
Additionally, the entire pfDHFR gene was randomly mutated using
error-prone PCR. During the first step of the dual selection, mutants
with functionally active pfDHFR were selected from two libraries
by using bacterial complementation assay. Fluorescence signals of
active mutants were subsequently measured and five mutants with
increased GFP signal, namely Y35Q + F37R, Y35L + F37T, Y35G
+ F37L and Y35L + F37R from the site–specific mutant library and
K27E from the random mutant library, were recovered. The mutants
were expressed, purified and characterized as monofunctional
pfDHFR following excision of GFP. Our studies indicated that all
mutant pfDHFRs exhibited kinetic properties similar to that of the
wild-type protein. For comparison of properties similar to that of
the wild-type protein. For comparison of protein solubility, the
maximum concentrations of mutant enzymes prior to aggregation
were determined. All mutants selected in this study exhibited 3- to
6-fold increases in protein solubility compared with the wild-type
protein, which readily aggregated at 2 mg/ml. The dual selection
system we have developed should be useful for engineering
functionally active protein mutants with sufficient solubility for
functional/structural studies and other applications.
(Published in Protein Eng Des Sel 2005; 18: 457-64.)
ELECTRON MICROSOPIC STUDIES ON
LOCALIZATION OF LEAD IN ORGANS
OF TYHPA ANGUSTIFOLIA GROWN ON
CONTAMINATED (NO. 757)
Thanawan Panich-pat 1 , Peerasak Srinives 2 ,*, Maleeya
Kruatrachue 3 , Prayad Pokethitiyook 3 ,Suchart Upatham 4 , and
Guy R. Lanza 5
1 Faculty of Liberal Arts and Science, Kasetsart University,
Kamphaeng Saen Campus, Nakhon Pathom 73140, Thailand; 2
Department of Agronomy, Faculty of Agriculture, Kasetsart
University, Kamphaeng Saen Campus, Nakhon Pathom 73140,
Thailand; 3 Department of Biology, Faculty of Science, Mahidol
University, Bangkok 10400, Thailand. 4 Faculty of Science,
Burapha University, Chonburi 20130, Thailand; 5 Environmental
Sciences Program, University of Massachusetts, Amherst, MA
01003, USA. * E-mail: agrpss@ku.ac.th
Key words : Typha angustifolia, narrow-leaved cattail, lead
accumulation
A greenhouse study was conducted to observe the
localization of lead in narrow-leaved cattail, Typha angustifolia.
Light and transmission electron microscopic studies were performed
on root, rhizome and leaf of the cattail grown in control (75 kg dry
weight of soil with no added lead) and in the same weight of soil
amended with 20,000 mg lead nitrate. At 15 and 90 days after
planting, most lead was accumulated in root cells around vacuoles
and slowly transported to leaves. In the lead-contaminated soil, parts
of the root cell wall were damaged at the end of the experiment.
Lead was deposited in the rhizome near the cell wall. Similar deposits
were observed in the roots and rhizomes suggesting that lead was
transported and localized in a similar area, whereas the leaf cells
accumulated lead in the chloroplasts.
(ScienceAsia, 2005, 31, 49-53;ADB)
INVASIVE PHYTOPHAGOUS PESTS ARISING
THROUGH A RECENT TROPICAL EVOLU-
TIONARY RADIATION : THE BACTROCERA
DORSALIS COMPLEX OF FRUIT FLIES (NO. 758)
Anthony R. Clarke, 1 Karen F. Armstrong, 2 Amy E. Carmichael,
1 John R. Milne, 3 s. Raghu, 4 George K. Roderick, 5 and David K.
Yeates 6
1 School of Natural Resource Sciences. Queensland University
of Technology, Brisbane, Qld 4001, Australia; E-mail:
a.clarke@qut.edu.au; ae.cannichael@qut.edu.au ; 2 Soil, Plant
and Ecological Sciences Division, Lincoln University,
Canterbury, New Zealand; E-mail: annstron@lincoln.ac.nz ; 3
Department of Biology, Faculty of Science, Mahidol University,
Bangkok 10400, Thailand; E-mail: frjnn@mucc.mahidol.ac.th
; 4 Alan Fletcher Research Station, Queensland Department of
Natural Resources & Mines and CRC for Australian Weed
Management, Sherwood, Qld 4075, Australia; E-mail:
raghu.s@nnn.qld.gov.au ; 5 Environmental Science, Policy and
Management, Division of Insect Biology, University of
California, Berkeley, California 94720-3112. E-mail:
roderick@nature.berkeley.edu ; 6 Australian National Insect
Collection, CSIRO Entomology, Canberra ACT 2601, Australia;
E-mail: david.yeates@csiro.au.
Key words diagnostics, larval host range, invasion biology.
The Bactrocera dorsalis complex of tropical fruit flies
(Diptera: Tephri- tidae: Dacinae) contains 75 described species,
largely endemic to Southeast Asia. Within the complex are a small
number of polyphagous pests of international signifi- cance,
including B. dorsalis sensu stricto, B. papayae, B. carambolae, and
B. philip- pinensis. Most species within the complex were described
in 1994 and since then substantial research has been undertaken in
developing morphological and molecular diagnostic techniques for
their recognition. Such techniques can now resolve most taxa
adequately. Genetic evidence suggests that the complex has evolved
in only the last few million years, and development of a phylogeny
of the group is considered a high priority to provide a framework
for future evolutionary and ecological studies. As model systems,
mating studies on B. dorsalis S.s. and B. cacuminata ha! ve substantially
advanced our understanding of insect use of plant-derived
chemicals for mating, but such studies have not been applied to
help resolve the limits of biological species within the complex.
Although they are commonly regarded as major pests, there is little
published evidence documenting economic losses caused by flies
of the B. dor- salis complex. Quantification of economic losses
caused by B. dorsalis complex species is urgently needed to prioritize
research for quarantine and management. Although they have been
documented as invaders, relatively little work has been done on the
invasion biology of the complex and this is an area warranting further
work.
(Annu. Rev. Entomol 2005, 50:293-319)
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