In a few well-known cases, the biological consequences of the disruption of lim-1 homeodomain (HD) genes have demonstrated the important roles of these genes in vertebrate development, especially in the nervous tissue, kidney, and gonads.
In the latter context, the LIM containing homeodomain (HD) lim-1 genes have been most extensively studied in a range of animals.
Lim-1 encodes a protein with a pair of LIM domains located N-terminal to the HD.
In invertebrates, lim-1 related genes have been identified in nematodes, fruit flies, and sea urchins.
An alternative approach is the analysis of post-translational Lim-1 expression by immunochemical methods.
These polyclonal rabbit antibodies produced against the C-terminal region of the XLim- 1 downstream of the HD (amino acids 265-403) (Taira et al., 1992) have been characterized and shown to cross-react with Lim-1 proteins of fishes, mice, rats, and humans (Karavanov et al., 1996, 1998).
For a precise comparison of the position of the glycoprotein signal with that of Lim-1 immunoreactivity, the blot membrane was cut (along the direction of electrophoretic separation) at the middle of the run pocket width; one half was treated with Iminun-Blot kit and the other with anti-XLim-1 antibodies (see Fig.
In this study we used rabbit polyclonal antibodies against a C-terminal region (as an immunogen of the Xenopus laevis Lim-1 protein (i.e., anti-XLim-1 antibodies).
Although it is generally accepted that the C-terminal peptide is a good choice for the production of antibodies specific to a protein of interest (Hancock and Evan, 1992), we decided to test, additionally, the degree of "specificity" of the Xlim-1 C-terminal region (used as an immunogen) for Lim-1 proteins.
Using the CLUSTAL W program (Thompson et al., 1994), we performed a multiple sequence alignment of the XLim-1 C-terminal region with that of the Lim-1 and Lim-5 proteins identified from BLAST searches (see above).
Collectively, the data indicated that the Xlim C-terminal sequence (used as an immunogen to generate anti-XLim-1 antibodies) is highly conserved among most of other known lim-1 genes and seems to be diagnostic for their protein products.
Note that the open reading frame of the chicken (Tsuchida et al., 1994) and sea urchin (Kawasaki et al., 1999) lim-1 gene encodes a protein with a predicted MW about of 45 kDa.