dictyNews
Electronic Edition
Volume 29, number 14
November 16, 2007

Please submit abstracts of your papers as soon as they have been
accepted for publication by sending them to dicty@northwestern.edu
or by using the form at
http://dictybase.org/db/cgi-bin/dictyBase/abstract_submit.

Back issues of dictyNews, the Dicty Reference database and other
useful information is available at dictyBase - http://dictybase.org.


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Abstracts
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DNA oligonucleotide-assisted genetic manipulation increases transformation 
and homologous recombination efficiencies: evidence from gene targeting 
of Dictyostelium discoideum

Hidekazu Kuwayama1,2*, Toshio Yanagida2 and Masahiro Ueda2.

1.Graduate School of Life and Environmental Sciences, University of Tsukuba, 
Tsukuba, Tennodai 1-1-1, Ibaraki 305-8572, Japan
2.Graduate School of Frontier Biosciences, Osaka University, Yamadaoka 1-3, 
Suita, Osaka 565-0871, Japan

correpondence should be addressed to Hidekazu Kuwayama 


Journal of Biotechnology, in press

Artificial gene alteration by homologous recombination in living cells, termed
gene targeting, presents fundamental and considerable knowledge of in vivo
gene function. In principle, this method can possibly be applied to any type
of genes and transformable cells. However, its success is limited due to a
low frequency of homologous recombination between endogenous targeted
geneand exogenous transgene. Here, we describe a general gene-targeting
method in which co-transformation of DNA oligonucleotides (oligomers) could
significantly increase the homologous recombination frequency and
transformation efficiency. The oligomers were simply designed such that they
were identical to both the ends of the homologous flanking regions of the
targeting construct. Using this strategy, both targeted alleles of diploid
cells were simultaneously replaced in a single transformation procedure.
Thus, the simplicity and versatility of this method applicable to any type
of cell may increase the application of gene targeting.


Submitted by: Hidekazu Kuwayama [hidekuwayama@biol.tsukuba.ac.jp]
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The role of cGMP and the rear of the cell in Dictyostelium chemotaxis and 
cell streaming

Douwe M. Veltman and Peter J.M. Van Haastert§


J. Cell Science, in press

During chemotaxis, pseudopod extensions lead the cell towards the source 
of attractant. Whereas the role of actin filled pseudopodia at the front of 
the cell is well recognized, the function of the rear of the cell in 
chemotaxis and cell-cell interactions is less well known. Dictyostelium 
cell aggregation is mediated by outward propagated waves of extracellular 
cAMP that induce chemotaxis and cell-cell contacts, resulting in streams 
of cells moving towards the aggregation centre. Wild type cells efficiently 
retract pseudopodia in the rear of the cell during the rising flank of the 
cAMP wave and have a quiescent cell posterior. This polarization largely 
remains during the declining flank, which causes cells to continue their 
chemotactic movement towards the aggregation centre and to form stable 
streams of moving cells. In contrast, mutant cells lacking cGMP signalling 
do fail to retract properly lateral pseudopodia. Dominance of the leading 
edge pseudopod rescues chemotaxis during the rising flank of the wave, but 
cells move in random directions after the peak of the wave has passed. As 
a consequence, cell-cell contacts cannot be maintained, and the cell 
streams break up. The results show that a quiescent rear of the cell 
increases the efficiency of directional movement and is essential to 
maintain stable cell-cell contacts.

Submitted by: Peter J.M. Van Haastert [P.J.M.van.Haastert@rug.nl]
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[End dictyNews, volume 29, number 14]