Worm Breeder's Gazette 11(5): 26

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Other Helicases We Have Cloned (Including an eIF-4A-like PCR Product from C. elegans)

Deborah Lynn Roussell, Catherine Christoffersen and Karen Bennett

Figure 1

With one set of primers designed to amplify the Drosophila vasa-
equivalent from C.  elegans, we produced several distinct PCR products.
Because these primer sequences were not unique to the vasa sequence 
but rather were shared by all members of the so-called DEAD family of 
helicases, our results were not unexpected.  We predicted that the 
different PCR products would correspond to different helicase protein 
genes.  The multiple PCR amplified DNA fragments were gel isolated, 
cloned and sequenced.  To date, sequence analyses of our PCR clones 
suggest that C.  elegans may have as many as six different potential 
helicase protein genes that belong to the DEAD family of helicases (
Nature 335:611-617).  Most of these represent potential helicases that 
have not been previously characterized in another organism.  It may be 
possible to characterize a whole family of putative helicase genes in 
Caenorhabditis.One of our PCR clones, called NA9-1, has 61% perfect 
amino acid homology to eIF-4A, the protein synthesis initiation factor 
that has been cloned from mouse (Nielsen et al., NAR 13:6867-6880).  
The identity is 83% with conserved amino acid changes.  Shown below is 
a comparison of NA9-1 predicted amino acid sequence with that of the 
mouse genes eIF-4AI/II and the yeast equivalents Tif1/2 (Linder and 
Slonimski, NAR 16:10359).  There is 54% perfect amino acid homology 
between the C.  elegans clone, NA9-1, and the yeast Tif1/2.  The 
position of an intron whose exact location is conserved between the 
mouse and nematode sequences is also indicated.
The eIF-4A protein is well characterized (for review see Moldave,K., 
Ann.  Rev.  Biochem.  54:1109-1149).  It recognizes the 5' cap 
structure of RNAs, has RNA binding activity, and has an ATPase-
dependent helicase activity upon RNAs.  Some of the different helicase 
motifs that are conserved among this large class of proteins have been 
functionally analyzed with eIF-4A.  The two genes eIF-4AI and II, 
although highly conserved, have different patterns and levels of 
expression in different tissues (Nielsen and Trachsel, EMBO J.  7:2097-
2105).  We are not yet certain if there are multiple Caenorhabditis 
eIF-4A-like genes.  There are two EcoRI fragments of C.  elegans DNA, 
2.4 Kb and 1.3 Kb, that hybridize to the insert of NA9-1 on genomic 
Southerns, but there is also an EcoRI site in our PCR clone.  In 
addition, the small size and unequal hybridization signals of the two 
genomic EcoRI fragments does not distinguish between a single gene or 
multiple highly conserved genes that corresponds to our PCR clone, NA9-
We have also used the NA9-1 insert DNA to isolate a cDNA from a C.  
elegans cDNA library previously constructed in this laboratory.  We 
are currently completing the sequence analysis of this clone.  
Preliminary data show that the cDNA clone, called CEB1, corresponds to 
the amplified PCR product and that its homology at the amino acid 
level with eIF-4A extends outside the region delimited by the PCR 
[See Figure 1]

Figure 1