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Fig. 1. Effects of eyestalk ablation on guanylyl cyclase expression in land crab
Gecarcinus lateralis tissues. Total RNA from intact and ES-ablated
animals was DNase-treated, reverse-transcribed, and PCR-amplified using
sequence-specific primers (see Materials and methods). PCR products after 35
cycles were resolved on 2% agarose gels (for Gl-GC-Iß, Gl-GC-III and
Gl-EF2) or 10% polyacrylamide gels (for Gl-GC-II), stained with Ethidium
Bromide, and quantified by scanning densitometry. (A) Reversed images of gels.
(B) Quantitative analysis from triplicate measurements for each tissue and
condition (mean ±1 s.e.m., N=3). Both Gl-GC-1ß (
0N
and 32N isoforms were not distinguished; primers were directed to a
common sequence in the C terminus) and Gl-GC-III were expressed at high levels
in ES ganglia (EG), gill (Gi), ovary (Ov) and testis (Ts) from intact animals.
Tissues from intact animals differed in relative expression of the three
Gl-GC-II isoforms. Gl-GC-II(+9) was expressed at varying levels in all tissues
and was the dominant isoform in Y-organ (YO), gill, hepatopancreas (HP) and
gonad (Ov and Ts). Gl-GC-II(+18) was expressed at highest levels in heart
(Ht), claw muscle (CM) and thoracic muscle (TM). Gl-GC-II(+0) was expressed at
low levels in most tissues except ES ganglia, in which it was the major
isoform. ES ablation had no significant effect on Gl-GC-Iß (GC1),
Gl-GC-II (GC2) and Gl-GC-III (GC3) expression in most tissues (B). The only
exception was the increased expression of the Gl-GC-II(+18) isoform in claw
muscles from 3 days and 7 days post-ESA animals. Gl-EF2 was expressed at
similar levels in all tissues. Significant differences between means were
determined by one-way ANOVA post-hoc test (Fisher's PLSD); P
values are indicated with brackets (a, P<0.034; b,
P<0.013; c, P<0.023).
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