I. Genomic Organization and Promoter Analysis of the Mouse AIE1 Protein Kinase Gene II. The Expression and Functional Analysis of the Nuclear Mitotic Apparatus Protein (NuMA) During Early Mouse Embryonic Development

• Main Authors: Hu, Hui-mei, 胡惠美
• Other Authors: Tang, Tang K.
• Format: Others
• Language: zh-TW
• Published: 2000
• Online Access: http://ndltd.ncl.edu.tw/handle/86586989699922791788

博士 === 國防醫學院 === 生命科學研究所 === 89 === I. Genomic Organization and Promoter Analysis of the Mouse AIE1 Protein Kinase Gene We previously described two novel testis-specific protein kinases, AIE1 (mouse) and AIE2 (human), that share high amino acid (a.a.) identities with the kinase domains of fly Aurora, yeast Ipl1 and frog Eg2 (Tseng et al., 1998). Here we report the entire intron/exon organizations of the AIE1 gene and analyze the expression patterns of AIE1 mRNA during testis development. The cis-acting elements in the promoter region, and the transcription factors that may affect AIE1 expression, are also investigated. By screening a mouse genomic library, I have obtained the entire 18.5 kb AIE1 genomic sequence. The mouse AIE1 gene spans ~8 kilobases (kb) and contains seven exons. The results from primer extension assay indicated that the AIE1 gene has two transcription initiation sites: PE1 (minor form, -34 bp from 103d cDNA clone) and PE2 (major form, -18 bp from 103d cDNA clone). The sequences of the exon-intron boundaries of the AIE1 gene conform to the consensus sequences (GT/AG) of splicing donor and acceptor sites of most eukaryotic genes. Comparative sequence analysis revealed that the gene structure is highly conserved between mouse AIE1 and human AIE2. However, much less homology was found in the sequence outside the kinase domain. I have also mapped the AIE1 gene to mouse chromosome 7A2-A3 by fluorescent in situ hybridization (FISH). RT-PCR analysis has demonstrated that AIE1 mRNAs are predominantly expressed in testis, kidney, and sperm; but are not present in egg and in the embryonic tissues in early developmental stage. Northern blot analysis showed that AIE1 mRNA is expressed at a low level at day 14 and reaches its plateau at day 19 in the developing postnatal testis. RNA in situ hybridization indicated that the expression of AIE1 transcripts were restricted to meiotically active germ cells with highest levels detected in late pachytene spermatocytes. These findings suggest that AIE1 may play a role in spermatogenesis, particularly during the stages of meiosis. I have transfected a series of 5''-end deletion constructs of AIE1 promoter that fused with a luciferase reporter gene (AIE1p-Luc) into a mouse testis cell line to define the minimum region for AIE1 expression. My results showed that the promoter region (nt. -659 ~ -583) is essential for PE1 transcription initiation, while the region (nt. -442 ~ -343) is important for for PE2 transcription initiation. I also demonstrated that the Sp1/hsp70 binding site is necessary for PE2 transcription initiation and the CRE-like element (nt. -1093 ~ -1103) is essential for AIE1 expression. The influence of other transcription factors or protein kinases are analyzed by cotransfected with various deleted AIE1p-Luc constructs. My preliminary results showed that (1) PKA can avtivate, but MEKK-1 inhibit the AIE1p activity; (2) the l-TZFP is a repressor, but the s-TZFP is an activator. Furthermore, several specific transcription factors, such as TZFP (testis zinc finger protein) and CRE binding protein, that bind to the promoter region of AIE1 gene have been identified by the electrophoresis mobility shift assay (EMSA). The competition assay demonstrated the binding specificity. These results will help us to understand the control mechanism of AIE1 gene expression. II. The Expression and Functional Analysis of the Nuclear Mitotic Apparatus Protein (NuMA) During Early Mouse Embryonic Development The nuclear mitotic apparatus protein (NuMA) was first described by Lydersen and Pettijohn (1980) as a predominantly nuclear protein that is present in the interphase nucleus and is concentrated at the spindle pole of mitotic cells. Recently, cDNA clones that cover the entire coding region of human NuMA have been isolated and sequenced (Compton et al., 1992; Yang et al., 1992). Structure analysis reveals that NuMA is composed of a long a-helical central core flanked by two globular domains. We have recently shown that human NuMA is composed of at least three isoforms that differ mainly at the carboxy terminus. Multiple NuMA isoforms are generated by alternative splicing of a common mRNA precursor from a single NuMA gene (Tang et al., 1993). The NuMA-l (T33 / p230) was present in interphase nuclei and was concentrated at the polar regions of the spindle apparatus in mitotic cells. NuMA-m (U4 / p195) and NuMA-s (U6 / p194) were present in the interphase cytosol and appeared to be mainly located at the centrosomal region. When cells entered into mitosis, NuMA-m and -s moved to the mitotic spindle pole (Tang et al., 1994). In the mouse oocytes, the centrioles were reported to appear first in blastocysts (Magnuson and Epstein, 1984). Therefore, early mouse embryos offer an opportunity to elucidate the mechanisms of microtubule nucleation and spindle morphogenesis in the acentriolar centrosome. To investigate the expression patterns of different NuMA proteins or mRNAs during early mouse embryogenesis, the second part of my thesis is to clone and sequence the mouse homologue of human NuMA cDNA. By screening a mouse spleen cDNA library, I obtained the cDNA clones that cover the entire NuMA coding region. I also used the primer extension assay to define the transcription initiation site of NuMA mRNA. Furthermore, I have characterized the expression pattern of different NuMA isoforms in mouse tissues and during early stage of mouse embryonic development by RT-PCR method. My results showed that NuMA-l is present in all mouse tissues and in the 1.5 d.p.c. to 4.5 d.p.c. embryos. Interestingly, NuMA-m is expressed in all examined tissues except sperm, while NuMA-s is not detectable. Furthermore, the +42 bp NuMA isoform was detected in all tissues, while the -42 bp NuMA isoform was detected in testis and kidney only. To analyze the functional effect of NuMA on embryonic development, I injected mouse embryos with either antisense oligonucleotides (complementary to NuMA mRNA) or polyclonal antibodies against NuMA. Our results showed that injection of anti-NuMA antibody did significantly interfer the cell division during embryo-genesis. It suggested that NuMA may play a role in the cell division during early embryonic development.