Lim Domain Protein Function And Gene Kyot Removed Mouse Phenotype Preliminary Analysis And Kyot Molecules,

The Notch receptor is a cell surface molecule conserved from worm through man, and involved in cell fate determination of various cell lineages in many tissues throughout development. The two single-pass transmembrane proteins, Delta and Serrate, have been identified as Notch ligands. The transcription factor Suppressor of Hairless [Su(H)] is the major downstream effector of Notch signaling pathway.RBP-J, the mammalian homolog of Su(H), recognizes the core sequence (C/TGTGGGAA) of DNA. The targeted disruption of mouse RBP-J revealed that homozygous null mutants die before 10.5 days postcoitum (dpc) with various abnormalities, including growth retardation and defects in the central nervous system and somites. It has recently been shown that the intracellular region of the Notch receptor (RAMIC) is proteolytically cleaved by interaction with the ligand and is translocated Into the nucleus. RBP-J directly interacts with RAMIC to activate transcription of downstream target genes that prevent cell differentiation. Thus, RBP-J is a critical effector molecule in Notch signaling pathway.In order to assess the function of RBP-J, a yeast two-hybrid screening using RBP-J as a bait protein was employed and a LIM protein, KyoT, was isolated. Alternative splicing gave rise to two transcripts of the KyoT gene, KyoTl and KyoT2, which encoded proteins with four and two LIM domains, respectively. KyoT2 lacked two LIM domains from the C terminus and had a frameshift in the last exon, creating the RBP-J-binding region in the C terminus. KyoTl had a negligible level of interaction with RBP-J. The binding site of KyoT2 on RBP-J overlaps those of RAMIC and KyoT2 repressed the RBP-J mediated transcriptional activation by RAMIC by competing with them for binding to RBP-J.In order to assess the function of KyoT2, Northern blot, immunohistochemical SABC and in situ hybridization methods were used to find out the expression of KyoT and KyoT knockout mice which were kept in our laboratory were analyzed. Moreover, we employed a yeast two-hybrid screening using KyoT2 as a bait protein and got 13 interacting proteins. The function of a novel protein of them, KBP was investigated.The results were as follows:1 Northern blot, RT-PCR and Western blot showed that KyoT was mainly expressed in lung, kidney and testis of adult mice. During the embryogenesis the level of KyoT mRNA increases in 17 dpc and the distribution is as same as adult mice. Immunohistochemical SABC and in situ hybridization analysis showed KyoT was mainly located in Leydig cells of testis of mice. Northern blot showed that KyoT was expressed in testis in all stages of development and the level of KyoT mRNA began to increase in the adolescence and was the highest in adult. We found the number of sperm of KyoT knockout mice was lower than that of wild type mice. The wall of seminferous tubule of KyoT knockout mice was thinner than wild type mice by hematoxylin-eossin staining, disruption of spermides in the cavity of seminferous tubule of KyoT knockout mice. These results suggest that KyoT plays an important role in the development of testis and spermatogenesis.2 We performed yeast two-hybrid screening of a cDNA library from human lymph nodes using KyoT2 as a bait protein. 42 clones were gotten after 5xl06 were screened by four kinds of nutrition limitation and P -galactosidase assay, 22 clones were got after restriction of positive clones, at last, 13 genes were got by sequence assay including RBP-J known as the interacting protein with KyoT before and two novel genes. PIAS1, a protein that was also located in test's specifically was got PDZ domain protein tight junction protein 2 and PcG (polycomb group) family RING1 and polycomb 2 were included. The results of GST-pulldown and yeast two-hybrid showed KyoT2 interact with them respectively in and yeast and vitro.3 Using the yeast two-hybrid system, we isolated a novel protein, KBP (KyoT Binding Protein). Three types of cDNAs were identified by RT-PCR and were designated as KBP1, KBP2, KBP3, r...