Rae1 alpha, Rae1 beta, and Rae1 gamma cDNAs isolated from retinoic acid-treated mouse embryonal carcinoma F9 cells encode cell surface proteins sharing partial homology with MHC class I molecules, and mRNAs corresponding to these cDNAs were detected exclusively in early mouse embryos, especially in the head region. To initiate studies on their roles, the rae1 alpha gene and the genomic DNAs covering the complete coding regions of the rae1 beta and rae1 gamma genes were isolated and their structures were analyzed. Although the coding regions of the three rae1 genes were highly homologous, the restriction map of the 5'-end region of the rae1 alpha gene differed from that of the rae1 beta and rae1 gamma genes. The rae1 family members were mapped by FISH on mouse chromosome 10A4 region. Genomic DNAs hybridizable with a Rae1 cDNA were not detected in rat and human. Rae1 genes were preferentially expressed in early mouse embryos, preferentially in the brain, and RAE1 proteins were anchored on the cell surface by a glycosyl phosphatidylinositol (GPI)-tail, a feature shared by important cell surface ligands.
Rae-1 cDNA is one of the retinoic acid (RA) inducible cDNA clones in mouse embryonal carcinoma F9 cells. Rae-1 mRNAs were detected in mouse early embryos, but not in various tissues of adult mice. RAE-1 protein apparently consists of 253 amino acids and is likely to be a glycoprotein consisting of a leader sequence, an extracellular domain, a serine, threonine, proline-rich domain, and a transmembrane domain. Interestingly, it has a weak, but significant homology with major histocompatibility complex (MHC) class I molecules and was immunocytochemically identified as a cell surface protein. By determining partial nucleotide sequences of 17 Rae-1 cDNAs isolated from the RA-induced F9 cells, at least three different kinds of Rae-1 cDNAs were identified and were named Rae-1 alpha, Rae-1 beta, and Rae-1 gamma cDNAs, respectively. As the overall nucleotide sequence homology among these three cDNAs was about 98%, they constitute a novel gene family which is likely to be involved in early mammalian embryogenesis.
As a first step to catalogue mRNAs present in mouse embryonal carcinoma F9 cells, 879 clones corresponding to low-abundance mRNAs were selected from among 2,896 randomly picked up clones of undifferentiated F9 cDNA libraries, using DNA probes complementary to poly(A)+RNAs prepared from undifferentiated F9 cells and to ones prepared from mouse fibroblast L cells. Five-hundred and eighty-two of the 879 clones were partially sequenced, and the subsequent homology search revealed that 201 corresponded to 180 known genes or known DNA sequences, which include not only housekeeping genes but also various tissue-specific genes. Interestingly, at least 24 of the 180 genes are development-related genes in mammals. Among these 24, those for midkine (growth and/or differentiation factor) and interferon-beta are reportedly up-regulated, and those for ECA39 (target for c-Myc regulation), REX-1 (zinc finger protein), and OCT-3 (POU-domain transcription factor) are down-regulated during the development of mouse embryonal carcinoma cells. Thirty-seven of the 582 clones matched the 36 previously reported unidentified ESTs (expressed sequence tags) and the remaining 344 corresponded to 329 novel ESTs. Therefore, partial sequencing of F9 cDNA clones corresponding to low-abundance mRNAs in F9 cells not only provides valuable information concerning development-related genes in mammals, but also many novel ESTs useful for studying mammalian genomes.
The Polycomb group of (Pc-G) genes and trithorax group of genes are known to play a crucial role in the maintenance of the transcriptional repression state of Hox genes, probably through modification of the chromatin configuration. The rae28/mph1 gene is a mammalian homologue of the Drosophila polyhomeotic gene, which belongs to the Pc-G genes. As reported previously, we established mice deficient in the rae28/mph1 gene and showed that these homozygous animals displayed the developmental defects compatible with a human congenital disorder, CATCH22 syndrome. In this study we analyzed the structural organization of the human counterpart of the rae28/mph1 gene (RAE28/HPH1) and its processed pseudogene (psiPH), which are located on, respectively, human chromosome 12p13 and 12q13. The HPH1 gene consists of 15 exons spanning approximately 26 kb and its structural organization is well conserved between mouse and human. These genetic information of the RAE28/HPH1 gene may provide an important clue for further examination of its involvement in human congenital disorders related to CATCH22 syndrome.
We used expressed sequence tags (ESTs) to identify genes expressed in mouse embryonal carcinoma F9 cells and prepared 2132 ESTs from undifferentiated F9 cDNA libraries: 1026 were prepared after randomly selecting clones from one of the libraries and the remaining 1106 ESTs were prepared after classifying 2896 clones of the libraries into four classes, according to the levels and patterns of expression. Among the former 1026 ESTs, 797 (78%) matched known genes, 61 (6%) matched database sequences of uncharacterized cDNAs, and 168 (16%) represented novel genes. The ESTs matching known genes were catalogued according to putative structural and cellular functions. As many as 53% were related to transcription and translation, and 19% were related to energy metabolism, including transcripts of mitochondrial DNA. These percentages were significantly higher in F9 cells than in the human heart and brain, and a human liver cell line, HepG2. We found that approximately 7% of the ESTs corresponding to low-abundance mRNAs are either related to retinoic acid-regulated genes or mammalian development- and/or differentiation-related genes. Cataloguing of the genes expressed in the F9 cells paves the way for isolating genes involved in early mammalian development.
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