WBPaper00003191 0 (SVM confidence: high)
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age2126(1) (potentially new)
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Examples of neuronal remodelling are widespread , from simple addition and elimination of synapses according to usage and age2126 , to major nervous system reorganization during metamorphosis69 .
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WBPaper00003271 0 (SVM confidence: high)
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del8(23) (potentially new)
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Furthermore , 14 % of lacZ : : fem-3 ( del8 ) ; dpy-19 ( lf ) animals express -gal in 20 intestinal nuclei in comparison with 0 % of lacZ : : fem-3 ( del8 ) ; mog-1 ( lf ) dpy- 19 ( lf ) animals .
The importance of Southern analyses of transgenic lines became evident when truncated transgenes with no [ lacZ : : fem-3 ( ) ] or low [ lacZ : : fem-3 ( q96 gf ) or lacZ : : fem-3 ( del8 ) ] levels of X-gal staining were found .
The inserts for wt ( 230 ) , del8 ( 222 ) , sgf ( 230 ) and chg8 ( 230 ) are a ClaIEcoRV fragment containing the entire fem-3 3 UTR .
DNA constructs for gel shift analysis The vector pBSKS II ( Stratagene ) was used for all clones . chg8 and del8 mutations were generated by ligation PCR using the wild-type fem-3 3 UTR as template ( technique described in Ho et al . , 1989 ) .
To assess expression of lacZ : : fem-3 ( del8 ) and lacZ : : fem-3 ( q96 gf ) in a mog-1 mutant background , the same procedure was followed except that recovery of lacZ : : fem-3 ( q96 gf ) ; mog-1 ( lf ) , lacZ : : fem-3 ( q96 gf ) ; mog-1 ( lf ) / and lacZ : : fem-3 ( q96 gf ) ; / was performed at 25C . In addition , heat shock of lacZ : : fem-3 ( del8 ) ; dpy-19 mog-1 ( Table II , line 15 ) and lacZ : : fem-3 ( del8 ) ; dpy-19 ( Table II , line 16 ) were not performed in parallel .
Line Transgene typea Temperature Animals with strong X-gal staining ( % ) in ( C ) 10 1 lacZ : : fem-3 ( ) d 33 81 2 lacZ : : fem-3 ( ) ; mog-1 33 1 3 lacZ : : fem-3 ( ) ; mog-2 33 29 4 lacZ : : fem-3 ( ) ; mog-3 33 15 5 lacZ : : fem-3 ( ) ; mog-4 33 0 6 lacZ : : fem-3 ( ) ; mog-5 33 7 7 lacZ : : fem-3 ( ) ; mog-6 33 9 8 lacZ : : fem-3 ( ) ; gld-1 ( q93 ) 33 88 9 lacZ : : fem-3 ( ) ; fog-1 33 89 10 lacZ : : fem-3 ( ) ; glp-4 33 73 11 lacZ : : fem-3 ( ) ; unc-17 33 98 12 lacZ : : fem-3 ( ) ; tra-2 33 96 13 lacZ : : fem-3 ( q96gf ) 30 81 14 lacZ : : fem-3 ( q96gf ) ; mog-1 30 86 15 lacZ : : fem-3 ( del8 ) ; dpy-19 30 82 16 lacZ : : fem-3 ( ) ; dpy-19 mog-1 30 95 17 lacZ : : tra-2 ( ) 33 100 18 lacZ : : tra-2 ( ) ; mog-1 33 97 aqIs43 [ lacZ : : fem-3 ( ) ] , qIs15 [ lacZ : : fem-3 ( q96 ) ] and qIs44 [ lacZ : : fem-3 ( del8 ) ] integrated transgenes were used in these experiments ( see Table I ) . bSee Materials and methods for definition of strong X-gal staining . cTransgene expression levels : / ( 020 % ) ; ( 2040 % ) ; ( 4060 % ) ; nuclei with strong X-gal staining . dResults represent the combined data from control experiments done in parallel with rows 212 . the soma and mog-1 : : GFP promoter fusion is expressed in somatic it issues , including the intestine ( A .
Among the integrated lines , no lacZ : : fem-3 ( ) animals exhibited strong X-gal staining in 20 intestinal nuclei , in contrast to 92 % of lacZ : : fem-3 ( q96 gf ) and 94 % of lacZ : : fem-3 ( del8 ) animals ( Figure 2B and C ) .
The 3 UTR of lacZ : : fem-3 ( del8 ) was confirmed by sequencing . lacZ : : tra-2 ( ) was constructed as described previously and contains the entire tra-2 3 UTR ( Goodwin et al . , 1997 ) .
If lacZ : : fem-3 ( del8 ) ; mog-1 ( lf ) dpy-19 ( lf ) had been derepressed , a percentage 14 % would have been expected .
Instead each was performed in parallel with the same control : lacZ : : fem-3 ( del8 ) ; / .
( C ) Graph of results of a single experiment with qIs43 [ lacZ : : fem- 3 ( ) ] , qIs15 [ lacZ : : fem-3 ( q96 gf ) ] and qIs44 [ lacZ : : fem-3 ( del8 ) ] . n , number of animals examined .
Neither lacZ : : fem-3 ( q96 gf ) nor lacZ : : fem-3 ( del8 ) 6343 become derepressed further when placed in a mog-1 mutant background ( Table II , compare lines 13 and 14 , and lines 15 and 16 ) .
wt ( 81 ) , wt ( 35 ) , sgf ( 35 ) chg8 ( 81 ) and del8 ( 73 ) were constructed by PCR using the above constructs as templates .
Since transgenes do not express well in the germ Table I . Transgenes used Transgene name Transgene type Type of array qEx212 lacZ : : fem-3 ( ) extrachromosomal qEx213 lacZ : : fem-3 ( ) extrachromosomal qIs43 lacZ : : fem-3 ( ) integrated qEx131 lacZ : : fem-3 ( q96gf ) extrachromosomal qEx208 lacZ : : fem-3 ( q96gf ) extrachromosomal qIs15 lacZ : : fem-3 ( q96gf ) integrated qEx387 lacZ : : fem-3 ( del8 ) extrachromosomal qEx388 lacZ : : fem-3 ( del8 ) extrachromosomal qEx389 lacZ : : fem-3 ( del8 ) extrachromosomal qIs44 lacZ : : fem-3 ( del8 ) integrated aTransgene expression levels : / ( 020 % ) ; ( 2040 % ) ; ( 4060 % ) ; nuclei with strong X-gal staining .
On the other hand , if mog-1 functions through a cis-element independent of the PME , additional derepression of lacZ : : fem-3 ( q96 gf ) and lacZ : : fem-3 ( del8 ) should occur .
We justified comparing these two lines since lacZ : : fem-3 ( del8 ) ; / in each case produced similar amounts of X-gal staining .
The fem-3 3 UTR represses a lacZ reporter gene in vivo To ask whether the fem-3 3 UTR is sufficient for PME- mediated regulation , we developed a transgenic reporter assay by fusing a fem-3 3 UTR to a lacZ reporter gene . lacZ : : fem-3 ( ) contains a wild-type fem-3 3 UTR , whereas lacZ : : fem-3 ( q96 gf ) contains a mutant fem-3 3 UTR with a single base change in the PME , and lacZ : : fem-3 ( del8 ) contains a mutant fem-3 3 UTR with an eight-nucleotide ( nt ) deletion , removing the PME ( Figure 2A ) .
If , on the one hand , the q96 and del8 lesions do indeed abolish PME function , lacZ : : fem-3 ( q96 gf ) and lacZ : : fem-3 ( del8 ) should not become derepressed further in a mog-1 mutant background if mog-1 functions through the fem-3 PME .
Animals carrying lacZ : : fem-3 ( ) expressed a low level of -gal whereas animals carrying lacZ : : fem-3 ( q96 gf ) or lacZ : : fem-3 ( del8 ) expressed a higher level of -gal ( Table I ; Figure 2B and C ) .
Heat shock was carried out at 30C since lacZ : : fem-3 ( q96 gf ) and lacZ : : fem-3 ( del8 ) already express a high level of -gal when heat shocked at 33C . The hsp-16 promoter is less active at 30 than 33C ( Jones et al . , 1989 ) .
The 3 UTR of lacZ : : fem-3 ( del8 ) transgene was made by ligation PCR ( Ho et al . , 1989 ) using the lacZ : : fem-3 ( ) construct as template .
The point mutation of lacZ : : fem-3 ( q96 gf ) contains a C to T change ( middle construct ) . lacZ : : fem-3 ( del8 ) contains an 8 nt deletion ( bottom construct ) .
To test whether the PME itself mediates mog repression , we asked whether lacZ : : fem-3 ( q96 gf ) or lacZ : : fem-3 ( del8 ) can become further derepressed in a mog-1 mutant background .
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WBPaper00003496 0 (SVM confidence: low)
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WBPaper00031533 0 (SVM confidence: medium)
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WBPaper00037690 0 (SVM confidence: high)
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