Differential expression of mouse Disabled 2 gene in retinoic acid-treated F9 embryonal carcinoma cells and early mouse embryos.

Using a differential display PCR, we identified a differentially expressed cDNA fragment which was detectable in retinoic acid (RA) treated F9 embryonal carcinoma (EC) cells but not in untreated F9 cells. A homology search of the Gene Bank indicated that the cDNA fragment is part of the mouse homolog of the Drosophila Disabled (mDab2) gene. Aggregate cultures of F9 EC cells grown in the presence of the RA differentiated into nonmalignant cells resembling the visceral endoderm of the mouse embryo. Upon induction of endodermal differentiation with 10(-7) M RA, the gene expression of mDab2 was increased gradually during the first 96 h. Neither undifferentiated F9 cells, nor the undifferentiated aggregate cells without RA expressed mDab2. Whole-mount in situ hybridization and quantitative RT-PCR also showed that the temporal expression pattern of the mDab2 gene coincides with the initiation pattern of RA synthesis that occurs during mouse embryogenesis. Also, two alternative splicing messages of mDab2 were detected in a tissue specific manner. All the data indicate that mDab2 may play an important role in RA-induced signal transduction during mouse development.     

Neuronal and mesodermal differentiation of P19 embryonal carcinoma cells is characterized by expression of specific marker genes and modulated by activin and fibroblast growth factors

We have used the P19 embryonal carcinoma (EC) aggregation system as a model for early mouse development to study induction and modulation of mesodermal and neuronal differentiation. By studying the expression of marker genes for differentiated cells in this model we have shown that there is a good correlation between the differentiation direction induced in P19 EC aggregates and the expression of these genes. Expression of the neuronal gene midkine is exclusively upregulated when P19 EC cells are induced to form neurons while expression of early mesodermal genes such as Brachyury T, evx-1 , goosecoid and nodal is elevated after induction to the mesodermal pathway. In the present study we have further shown that activin A blocks the different directions of differentiation of P19 EC cells induced by retinoic acid (RA) in a dose-dependent way. To understand the mechanism behind this inhibitory action of activin A the expression of several RA-responsive genes, including the three RA receptor genes (RARα, RARβ and RARγ) was determined. Since activin has no clear effect on the expression and activity of the RAR it is very likely that this factor acts downstream of these receptors. In addition to activin, fibroblast growth factors (FGF) were shown to modulate P19 EC cell differentiation. However, in contrast to activin, FGF exclusively blocks the mesodermal differentiation of P19 EC cells by either 10⁻⁹mol/L RA or a factor produced by visceral endoderm-like cells (END-2 factor). The FGF effect is dose-independent. These results suggest an important function for RA and the END-2 factor in the induction and for activin and FGF in the modulation of specific differentiation processes in murine development.     

The cDNA sequence and chromosomal location of the murine GABAA alpha 1 receptor gene.

The murine GABAA/benzodiazepine (GABAA/BZ) receptor alpha 1 subunit cDNA has been isolated from a BALB/c mouse brain library and sequenced. The cDNA is 2665 nucleotides long with an open reading frame of 455 amino acids. It shows significant homology to the GABAA receptor alpha 1 subunit cDNA sequences of other species. Excluding deletions, the murine GABAA alpha 1 receptor exhibits 96% nucleotide and 100% amino acid sequence homology to the rat alpha 1 receptor cDNA and over 91% nucleotide and 98% amino acid sequence homology to the bovine and human alpha 1 receptor cDNAs in the protein coding region. This murine cDNA was used to locate the alpha 1 receptor subunit gene, Gabra-1, to murine Chromosome 11 between Il-3 and Rel. This assignment extends proximally the segment of mouse Chromosome 11 with known homology to human chromosome 5.     

Molecular cloning of a novel cytokine cDNA encoding the ninth member of the fibroblast growth factor family, which has a unique secretion property.

Glia-activating factor (GAF) is a novel heparin-binding growth factor purified from the culture supernatant of a human glioma cell line. It shows a spectrum of activity slightly different from those of other known growth factors. We have isolated the cDNA which encodes human GAF. A homology search revealed that GAF would be the ninth member of the FGF family, and we therefore call it FGF-9. The human FGF-9 cDNA cloned by using oligonucleotide probes encoded a polypeptide consisting of 208 amino acids. Sequence similarity to other members of the FGF family was estimated to be around 30%. Two cysteine residues and other consensus sequences in family members were also well conserved in the FGF-9 sequence. FGF-9 was found to have no typical signal sequence in its N terminus like those in acidic FGF and basic FGF. Acidic FGF and basic FGF are known not to be secreted from cells in a conventional manner. However, FGF-9 was found to be secreted from cells after synthesis despite its lack of a typical signal sequence. It could be detected exclusively in the culture medium of cDNA-transfected COS cells. The amino acid sequence of proteins purified from culture supernatant of the CHO cell line, which was cDNA transfected and selected as a high producer of FGF-9, showed that no peptides were cleaved from the N terminus except the initiation methionine. The rat FGF-9 cDNA was also cloned, and the structural analysis indicated that the PGF-9 gene is highly conserved. Expression of the FGF-9 gene could be detected in the brain and kidney of the adult rat. Restricted gene expression in organs and the unique secretion nature of the protein suggest that FGF-9 plays a physiological role which differs from those of well-characterized acidic FGF and basic FGF.     

果蝇程序化死亡基因5(PDCD5)同源cDNA的克隆和序列分析

 为了解人类白血病细胞凋亡相关新基因 TFAR1 9(PDCD5,programmed cell death5)在不同种属间的序列同源性 ,利用 EST(expression sequence tag)拼接、RT- PCR、DNA序列测定技术及计算机分析技术 ,首次成功地进行了果蝇 PDCD5同源 c DNA编码区基因克隆和序列分析 .发现果蝇与小鼠及果蝇与人 PDCD5在核苷酸水平上分别有 57.5%和 57.1 %的同源性 ,在氨基酸水平上分别有 46.8%和 46.4%的同源性 .功能区分析发现 ,果蝇 PDCD5c DNA编码 1 33个氨基酸 ,计算机预测可能是一种核蛋白 ,含 5个可能的酪蛋白激酶 (casein kinase )磷酸化位点 ,2个可能的 PKC磷酸化位点 ,与人 PDCD5的功能区类似 .因而果蝇 PDCD5是与人 PDCD5同源的新基因 ,可能都与细胞程序化死亡相关 .     

The octamer binding factor Oct6: cDNA cloning and expression in early embryonic cells.

We have cloned a cDNA encoding a novel octamer binding factor Oct6 that is expressed in undifferentiated ES cells. Expression of the Oct6 gene is downregulated upon differentiation of these cells by aggregate formation. Furthermore the gene is transiently up regulated during retinoic acid induced differentiation of P19 EC cells, reaching maximum levels of expression one day after RA addition. Sequence analysis of the cDNA encoding the Oct6 protein indicated that the Oct6 gene is a member of the POU-HOMEO domain gene family. The gene expresses a 3 kb mRNA encoding a 449 amino acid protein with an apparent molecular weight of 45 kD. The sequence of the Oct6 POU domain is identical to that of the rat SCIP (Tst-1) gene. The Oct6 expression pattern suggests a role for this DNA binding protein in neurogenesis as well as early embryogenesis.     

Nucleotide sequence of mouse L19 ribosomal protein cDNA isolated in screening with tre oncogene probes.

A cDNA library was prepared from cytoplasmic poly(A)RNA from mouse NIH-3T3 cells carrying a transfected human tre oncogene. Screening with tre gene probes identified a tre cDNA clone 11-4 and a co-purifying weakly hybridizing cDNA clone 11-5. The 11-5-specific RNA was expressed in both nontransfected and tre-transfected NIH-3T3 cells, showing it is of mouse rather than tre gene origin. Its nucleotide sequence was 717 bp long and contained, starting from the first nucleotide, an open reading frame of 588 bp followed by a 3' noncoding region and 26 A residues at the 3' terminus. Comparison with the GenBank data base revealed 93.7% homology with cDNA encoding the rat L19 ribosomal protein. Furthermore, the 196-amino-acid polypeptide deduced from 11-5 was of the same length and contained only one amino acid difference compared with the rat L19 protein. Comparison with the weakly hybridizing tre gene probe showed stretches of homology that were, however, too short to be taken into consideration. We conclude that the 11-5 sequence encodes the mouse L19 ribosomal protein.     

Cloning of a complementary DNA encoding a new mouse B lymphocyte differentiation antigen, homologous to the human B1 (CD20) antigen, and localization of the gene to chromosome 19

The human B1 (CD20) molecule is a differentiation Ag found only on the surface of B lymphocytes. This structurally unique phosphoprotein plays a role in the regulation of human B cell proliferation and differentiation. In order to determine whether this structure is also expressed by murine B cells, cDNA clones that encode the mouse equivalent of the B1 molecule were isolated. The longest murine cDNA clone isolated, pmB1-1, contained a 1.4-kb insert with an 873 base pair open reading frame that encodes a protein of 32 kDa. The predicted mouse B1 protein contains three hydrophobic domains that may span the membrane four times and shares a 73% amino acid sequence homology with the human B1 protein. The pmB1-1 cDNA probe was used to examine mB1 mRNA expression. Northern blot analysis indicated that pmB1-1 hybridized with two mRNA species of 2.3 and 3.0 kb that were expressed only in murine spleen lymphocytes, in B lineage cell lines representing mature B cells, and were weakly expressed in one of two plasmacytoma cell lines. pmB1-1 failed to hybridize with RNA isolated from murine T cell lines, thymus, and nonlymphoid tissues. Southern blot analysis indicated that mB1 was encoded by a single copy gene. In situ hybridization localized the mB1 gene to chromosome 19 band B, a region that also contains the genes that encode the Ly-1, Ly-10, and Ly-12 Ag. These results suggest that only B cells express this heretofore undescribed murine cell-surface protein that is structurally homologous with the membrane-embedded human B1 Ag.     

The complete coding sequence of the human A-raf-1 oncogene and transforming activity of a human A-raf carrying retrovirus.

The complete 606 amino acid sequence of the human A-raf oncogene has been deduced from the 2453 nucleotide sequence of a human T cell cDNA. A cysteine-rich region located near the amino terminus, which is highly conserved in A-raf and c-raf, shows significant homology with protein kinase C. A 5' deleted fragment of the cDNA has been incorporated into a murine retrovirus which endows the virus with the ability to transform cells in vivo and in vitro. Functionally, human A-raf is similar to v-raf and v-mos in that transformation is independent of ras gene function.     

Cloning of cDNA for human T-cell replacing factor (interleukin-5) and comparison with the murine homologue.

We have cloned cDNA for T-cell replacing factor (interleukin-5), which replaces T-cell helper function for normal B cells which secrete immunoglobulin, from human T cell leukemia line, ATL-2, using mouse interleukin-5 cDNA as probe. Total nucleotide sequence of the cDNA (816 base pairs) was determined and compared with that of mouse interleukin-5 cDNA. The cloned cDNA encoded the interleukin-5 precursor of 134 amino acids containing an N-terminal signal sequence. Although the human interleukin-5 precursor is one amino acid longer than the murine homologue, the sizes of the mature proteins appear similar. The nucleotide and amino acid sequence homologies of the coding regions of human and murine interleukin-5 are 77% and 70%, respectively. Human interleukin-5 synthesized by the direction of the cloned cDNA induced immunoglobulin synthesis in human B cells stimulated by Staphylococcus aureus mitogen.     

Plasma cell membrane glycoprotein PC-1. cDNA cloning of the human molecule, amino acid sequence, and chromosomal location

The murine cell membrane glycoprotein PC-1 is a homodimer with restricted tissue distribution, being first characterized in plasma cells. We now describe the isolation of cDNA clones encoding the human homolog of the murine PC-1 protein, its complete amino acid sequence, and its chromosomal location. Overall, the amino acid sequence of the human protein is about 80% identical to the murine protein, although the extent of homology varies in different domains. It had not been possible to assign a definitive amino terminus to the murine protein. Comparison of the murine and human sequence necessitates reassignment of the amino terminus, resulting in a cytoplasmic tail of 24 amino acids rather than 58 amino acids as previously published for the mouse. The sequence of several independently obtained cDNA clones indicates that the 3' end of the mRNA is subject to alternative splicing. Southern blots suggest a single copy gene. In situ chromosomal hybridization localizes the gene for human PC-1 to chromosome 6q22-q23, a common site for deletions in human lymphoid neoplasia.     

Molecular cloning of cDNA encoding human Rab3D whose expression is upregulated with myeloid differentiation

To identify genes expressed in myeloid differentiation, we isolated a cDNA fragment by differential display using RNA prepared from HT93A cells, a human cell line capable of differentiating into neutrophil and eosinophil lineages in response to retinoic acid (RA). Evaluation of the full-length clone isolated from an HT93A cDNA library showed that it encoded a 24 kDa protein comprised of several domains conserved in the Ras superfamily. Comparison of the deduced amino acid sequence of this clone with Rab proteins revealed that it had highest homology to a small GTP-binding protein, murine Rab3D. The mRNA expression of human Rab3D was upregulated in the course of myeloid differentiation, and it was preferentially expressed in granulocytes. These results suggest that human Rab3D may play a specific role in granulocytes, for example in exocytosis of neutrophil-specific granules or in degranulation of both eosinophils and basophils.     

Identification of a novel fibroblast growth factor, FGF-23, preferentially expressed in the ventrolateral thalamic nucleus of the brain

We isolated mouse cDNA encoding a novel FGF (251 amino acids). As this is the 23rd documented FGF, we termed it FGF-23. FGF-23 has a hydrophobic amino terminus ( approximately 24 amino acids), which is a typical signal sequence. As expected, recombinant mouse FGF-23 was efficiently secreted by High Five insect cell-infected recombinant baculovirus containing the cDNA, indicating that FGF-23 is a secreted protein. We also isolated human cDNA encoding FGF-23 (251 amino acids), which is highly identical ( approximately 72% amino acid identity) to mouse FGF-23. Of human FGF family members, FGF-23 is most similar to FGF-21 and FGF-19 ( approximately 24% and approximately 22% amino acid identities, respectively). Human FGF-23 gene was localized on the chromosome 12p13 and found to be tandem linked (within 5.5 kb) to human FGF-6 gene. The expression of FGF-23 mRNA in mouse adult tissues was examined by real-time quantitative polymerase chain reaction. FGF-23 mRNA was mainly expressed in the brain and thymus at low levels. The localization of FGF-23 mRNA in the brain was examined by in situ hybridization. FGF-23 mRNA in the brain was found to be preferentially expressed in the ventrolateral thalamic nucleus. Therefore, FGF-23 is expected a unique FGF that plays roles in the function of the ventrolateral thalamic nucleus.     

Mouse cathepsin F: cDNA cloning, genomic organization and chromosomal assignment of the gene

A murine cysteine protease of the papain family was identified by dbEST-database search. A 1.87kb full-length cDNA encoding a predicted polypeptide of 462 amino acids was sequenced. Since the encoded polypeptide shows more than 80% sequence identity with human cathepsin F, it is most likely that this cDNA represents the murine homologue of cathepsin F, and it was therefore named accordingly. Murine cathepsin F exhibits a domain structure typical for papain-like cysteine proteases, a 20 amino acid N-terminal hydrophobic signal sequence followed by an extraordinarily long propeptide of 228 amino acids and the domain of the mature protease comprising 214 amino acids. The mature region contains all features characteristic of a papain-like cysteine protease, including the highly conserved cysteine, histidine and asparagine residues of the 'catalytic triad'. Genomic clones covering the murine cathepsin F gene were isolated. The mouse cathepsin F gene consists of 14 exons and 13 introns and spans 5.8kb. Murine cathepsin F was mapped to chromosome 19, a region with synteny homology to a region of human chromosome 11 to which human cathepsin F has been mapped previously. Northern blot analysis of RNA from multiple tissues revealed a ubiquitous expression of cathepsin F in mouse and man.