spacer gif spacer gif spacer gif spacer gif spacer gif
QUICK SEARCH:   [advanced]


spacer gif
     Home     Help     Feedback     Subscriptions     Archive     Search     Table of Contents    

First published online 2 September 2008
doi: 10.1242/jcs.035550

Journal of Cell Science 121, 3140-3145 (2008)
Published by The Company of Biologists 2008
This Article
Right arrow Summary Freely available
Right arrow Full Text
Right arrow Full Text (PDF)
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrow reprints & permissions
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Webb, T. R.
Right arrow Articles by Jackson, I. J.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Webb, T. R.
Right arrow Articles by Jackson, I. J.
Social Bookmarking
Add to CiteULike   Add to Complore   Add to Connotea   Add to Del.icio.us   Add to Digg   Add to Reddit   Add to Technorati   Add to Twitter  
What's this?

Diphthamide modification of eEF2 requires a J-domain protein and is essential for normal development

Tom R. Webb1,*, Sally H. Cross1, Lisa McKie1, Ruth Edgar1, Lucie Vizor2, Jackie Harrison2, Jo Peters2 and Ian J. Jackson1,{ddagger}

1 MRC Human Genetics Unit, Western General Hospital, Crewe Road, Edinburgh EH4 2XU, UK
2 MRC Mammalian Genetics Unit, Harwell, Oxon OX11 0RD, UK


Figure 1
View larger version (17K):
[in this window]
[in a new window]

  		Fig. 1. (A) Schematic of the genetic cross to screen for mutations in the Pax6 deletion interval. Mutagenized males are crossed with females carrying the Pax6Sey-Neu small-eye point mutation. Small-eye offspring (a fraction of which will be carrying a novel point mutation in the region) are crossed with mice carrying the Pax6Sey-1H deletion. The four resultant genotypes are shown below. ENU, ethylnitrosourea. (B) Gene content in the region that is encompassed by the Pax6Sey-1H deletion.

 

Figure 2
View larger version (28K):
[in this window]
[in a new window]

  		Fig. 2. (A) Mutation of Dph4. Top, wild-type (C57BL6/J) sequence. Bottom, mutant sequence. Large font, exon-4 sequence. Small font, intron-4 sequence. (B) RT-PCR analysis of mRNA from wild-type (WT), heterozygous (het) and homozygous mutant (mut) embryos. The wild-type allele produces a 246-bp fragment and the mutant allele produces one of 177 bp. The heterozygote contains both fragments. (C) Schematic illustrating the splicing pattern of the wild-type (upper) and mutant (lower) Dph4 gene. (D) Western blot to detect DPH4 protein in wild-type (WT), heterozygous (het) and homozygous mutant (mut) E12.5 embryos. The lower band is a non-specific reaction of the antiserum, which serves as a loading control. Tracks are from the same gel but lane order has been edited for clarity. (E) Native gel and western blot, probed to detect eEF2 protein in wild-type (WT), heterozygous (het) and homozygous mutant (mut) E12.5 embryos. Mutant protein has a –1 charge shift relative to that of heterozygotes and wild types, and therefore migrates faster. Tracks are from the same gel but lane order has been edited for clarity.

 

Figure 3
View larger version (22K):
[in this window]
[in a new window]

  		Fig. 3. The activity of DT on mouse embryonic fibroblasts. White bars indicate luciferase activity following transfection of pGL3-P luciferase reporter alone into the cells. Dark-grey bars show luciferase activity after transfection of the luciferase reporter plus pPGK-DTA at a molar ratio of 4:1. Light-grey bars show a co-transfection control, showing luciferase activity following transfection of the luciferase reporter plus pBluescript vector only, at a molar ratio of 4:1. Transfections into six different cell lines are shown, representing one wild-type (WT), two heterozygous (het) and three homozygous mutant (mut) genotypes. For each set of transfections the mean of the luciferase reporter alone is normalized to 100%. Error bars are ± s.d.

 

Figure 4
View larger version (93K):
[in this window]
[in a new window]

  		Fig. 4. Subcellular localization of the DPH4 protein. NIH-3T3 cells transfected with FLAG-tagged DPH4 (all panels) and myc-tagged mouse DPH1 (OVCA1, upper panels) are shown. Cells were subsequently stained with anti-FLAG antibodies to detect DPH4 (green), anti-myc to detect mouse DPH1 (red, upper panels) or with phalloidin to detect microfilaments (middle and lower panels). Right-hand panels are merged images.

 

Figure 5
View larger version (60K):
[in this window]
[in a new window]

  		Fig. 5. The developmental consequences of Dph4 mutation. (A-D) Dph4 homozygous mutants are small in size. (A) E10.5 embryos: left, wild-type embryo; centre and right, two homozygous mutant embryos. (B) E11.5 embryos: left, wild type; right, homozygous mutant. (C) E12.5 and E13.5 embryos: left, E12.5 wild-type embryo; centre, E13.5 wild-type embryo; right E13.5 homozygous mutant embryo, to illustrate the ~1-day developmental delay. (D) E13.5 embryos: left, wild type; centre and right, two homozygous mutant embryos. (E-G) Dph4 homozygous mutants have a preaxial polydactyly. (E) Hindfoot of an E14.5 homozygous embryo. (F) Hindfoot of an E16.5 homozygous embryo. (G) Stain.

 

Add to CiteULike CiteULike   Add to Complore Complore   Add to Connotea Connotea   Add to Del.icio.us Del.icio.us   Add to Digg Digg   Add to Reddit Reddit   Add to Technorati Technorati   Add to Twitter Twitter    What's this?



© The Company of Biologists Ltd 2008