MCM7
4.2.3 cDNA eloning ofnewt SAP155
To isolate a newt SAP155 cDNA, we designed 5'‑ and 3'‑primers according to the sequences of the mouse cDNA (Accession No. AB037890) and cloned through reverse
transcriptase (RT)‑PCR. Screening of a newt testis cDNA library gave rise to a clone and nucleotide sequencing revealed that the clone comained a 2.7‑kbp cDNA with an open reading frame (ORF) encoding 930 amino acids homologous to members ofthe spliceosome associated protein family conserved among mammals and amphibians. The amino acid sequence of newt SAPI55 is nearly the same as mouse SAPI55 and Xkinopus 146‑kDa protein, showing 99% and 94 % identity, respectively, and is also highly similar to those of gene products from human (99%) and rat (98%), which were found in the database (Fig. 4.8). Comparison of the amino acid sequence in the N‑terminal region showed the existence of the TP‑dipeptide motifs and adomain containing RWDETP repeat. C‑terminal stretch of SAP155 is remarkably highly
conserved among the five species. BLAST homology search with the C‑terminal sequence fbund the PR65 subunit of protein phosphatase 2A with significant similarity The PR65 subunit is composed entirely of 15 tandem repeats ofa 39 amino acid sequence termed a HEAT motif and according to a consensus sequence of the HEAT motifs, the panial C‑terminal region of newt SAPI55 was composed of7 tandem HEAT‑like repeats.95
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enupter 4
4.2.4 S7age‑specipc and celbmpe specijib expression ofnewt SL4M55
To examine the expression of newt SAPI55 during spermatogenesis, immunoblotting was perfbrmed using testis extract derived from various spemiatogenic stages, rich in early (3rd ‑ 4th) spermatogonia, late (6th ‑ 7th) spermatogonia and primary spermatocytes. The expression ofnewt SAPI55 was detected in all the spermatogenic stages from early to primary sperrnaticyte (Fig.
4.9A). To show cell‑type specific expression of newt SAP155, somatic and germ cells are fractionated as described previously and immunoblotting was perfbrmed. A single band was observed in germ cells fraction with the expected size of SAPI55, indicating that in newt testis SAPI55 was expressed only in germ cells eig. 4.9B).
97
(]hupter 4
A
150 kDa
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B
Somatic Germ
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Figure 4.9 Expression of SAP155 in diffi:rent spermatogenic stages and cell types A) Cell lysates (10 pg) isolated from cells with 3rd ‑4th generation ofearly spermatogonial (EG), the 6th‑7th generation oflate spermatogonial (LG) and the primary sperrnatoeyte stage (PC) were immunoblotted with the indicated antibodies. B) Fractionated somatic and germ cells were lysed and 10 pg of each cell lysates were immunoblotted with the anti‑SAP155 (upper panel) or anti‑Pactin (lower panel).
(]hapter 4
4.2. 5 interaction between EbbB4 and SAP155 in dij72irent spermajogenic stages
To deterrnine the interaction between ErbB4 and SAPI55 in diffbrent spermatogenic stages, co‑immunoprecipitation and immunoblotting experiments were perfbrmed with the ErbB4 and
SAP155 antibodies, respectively. SAP155 was shown to be precipitated only in primary
spermatocyte stage, while ErbB4 existed in all stages (Fig. 4.10A). lmunoprecipitation with the SAPI 55 antibody was also perfbrmed to confirm the association. ErbB4 protein was detected in SAP155‑preciphated samples only in primary spermatocytes stage eig.4.10B), indicated that ErbB4‑SAP155 specifically interacts only in primary spermatoctes stage in newt testis.lmmunoprecipitations with HA antibody was perfbrmed using the extract derived from the testis containing all spermatogenic stages from early spermatogonia to primary spermatocyte as a control (Fig.4.1OA, B, right lane).
99
enapter 4
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Figure 4.10 Co‑immunoprecipitation with ErbB4 and SAP antibodies in diffi:rent spermatogenic stages FSH‑treated testis extract (500 pg) isolated ffom early spermatogonial (EG), late spermatogonial (LG) and the primary sperTnatocyte stage (PC) was precipitated with anti‑ErbB4 (1pg) or anti‑SAP155 (1pg) and with anti‑HA (1pg) or normal mouse serum (NMS) (1pg) as a control. lmmunoblotting was performed with indicated antibodies.
wnole testis contained all sperrnatogenic stages.
enapter 4