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. 2002:3:5.
doi: 10.1186/1471-2164-3-5. Epub 2002 Feb 11.

Cloning and characterization of the rat homologues of the Inhibitor of Apoptosis protein 1, 2, and 3 genes

Martin Holcik  1 , Charles A Lefebvre, Keiko Hicks, Robert G Korneluk
Affiliations

Cloning and characterization of the rat homologues of the Inhibitor of Apoptosis protein 1, 2, and 3 genes

Martin Holcik et al. BMC Genomics. 2002.

Abstract

Background: Inhibitor of Apoptosis (IAP) proteins are key intrinsic regulators of apoptosis induced by a variety of triggers. We isolated the rat Inhibitor of Apoptosis genes 1, 2 and 3 and characterized their tissue distribution and expression.

Results: Rat iap-1 encodes a protein of 67.1 kDa with 73 % and 89.2 % homology to human and mouse iap-1 respectively. Rat iap-2 encodes a protein of 66.7 kDa with 81.6 % and 89.3 % homology to human and mouse iap-2 respectively. Rat iap-3 encodes a protein of 56.1 kDa with 89.5 % and 93.1 % homology to human and mouse iap-3 respectively. We have generated rabbit polyclonal antibodies against all three rat IAP genes. Northern and Western blot analysis detected rat IAP transcripts and proteins in majority of the tissues examined. In addition, a shorter, alternatively spliced transcript corresponding to iap-2 was found in testes.

Conclusions: We have identified three rat homologues of the IAP genes. The elevated expression of rat iap-1 and iap2 in testes suggests that these two genes play an important antiapoptotic role in spermatogenesis.

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Figures

Figure 1
Figure 1
(A) Northern blot analysis of rat iap mRNA expression in adult rat tissues. A rat multiple tissue northern blot (Clontech) containing 2 μg/lane poly(A)+ RNA per lane was probed sequentially with [32P] dCTP (Amersham) labeled, random primed (Amersham Rediprime) DNA probes derived from the coding regions of rat iap-1, iap-2, iap-3, and β-actin (control). Blots were hybridized overnight in 5 × SSPE/10 × Denhardt's solution/100 μg/ml salmon sperm DNA/ 50% formamide/ 2% SDS and then washed with 0.2 × SSC/ 0.1% SDS at 50°C. The position and sizes of the markers are indicated on the left. The tissues represented on the blot are as follows: (1) heart, (2) brain, (3) spleen, (4) lung, (5) liver, (6) skeletal muscle, (7) kidney, and (8) testis. (B) Northern blot analysis of the expression of iap-2 in testes. A rat multiple tissue Northern blot (Clontech; same as in (A)) was hybridized sequentially with [32P] dCTP labeled DNA probes specific to iap-2 coding region, BIR domain and the RING zing finger. The blots were processed as in (A). The position of testis-specific transcript is indicated by an asterisk (*).
Figure 2
Figure 2
Western blot analysis of the rat IAP proteins distribution in adult rat tissues. The protein samples prepared from fresh adult rat tissues, or bacterially expressed GST-fusion recombinant proteins, were separated on 10% SDS-PAGE (12 μg total protein per lane) and transferred onto a PVDF membrane using standard techniques [20]. Rabbit polyclonal antibodies against rat iap-1 (top), iap-2 (middle), and iap-3 (bottom) were used at 1:1,000 dilution followed by secondary antibody (horseradish peroxidase-conjugated goat anti-rabbit IgG; Amersham) used at 1:1,500 dilution. Antibody complexes were detected using the ECL system (Amersham). Position and size of the markers is indicated on the left. (Note that the recombinant GST-fusion proteins are larger due to the presence of N-terminal GST tag.) Tissues used are as follows: (1) brain, (2) heart, (3) intestine, (4) kidney, (5) liver, (6) lung, (7) spleen, (8) testis, and (9) thymus.
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