Molecular cloning of the mouse S-adenosylmethionine decarboxylase cDNA: specific protein binding to the conserved region of the mRNA 5'-untranslated region.

The nucleotide sequence of a cDNA encoding the proenzyme of mouse S-adenosylmethionine decarboxylase (AdoMetDC) including 257 nucleotides of the 5' untranslated region has been determined. Comparison of the nucleotide sequence of the mouse 5' untranslated region with those of other mammals shows it to be highly conserved. The 52 nucleotides upstream from the translation initiation codon are identical in human, rat, bovine and mouse. The polyamines, spermidine and spermine, have been shown to inhibit AdoMetDC mRNA translation. An RNA gel retardation assay demonstrated that a cytoplasmic extract from mouse brain forms an RNA-protein complex with the completely conserved 5' untranslated sequence and that the complex formation is highly dependent on the presence of spermine. Crosslinking by UV irradiation shows that the complex contains a 39-kDa protein interacting with the 5' untranslated sequence. These data demonstrate spermine-dependent specific protein binding to a highly conserved 5' untranslated region of an mRNA translationally regulated by polyamines.     

Molecular characterization of the mouse mannose-binding proteins. The mannose-binding protein A but not C is an acute phase reactant.

Mannose-binding proteins play a role in first line host defense against a variety of pathogens. We report the molecular cloning of two mouse mannose-binding proteins designated A and C based on their close identity with their rat homologues. The deduced amino acid sequence of the mouse mannose-binding proteins, as with rat and the human forms, have an NH2 terminus that is rich in cysteine that stabilizes a collagen alpha helix followed by a carboxyl- terminal carbohydrate binding domain. We further show that the mouse mannose-binding protein A mRNA, as with the human, is induced like the acute phase reactant serum amyloid P protein, yet the expression of mouse mannose-binding protein C mRNA is not regulated above its low baseline level. The expression of both mannose-binding proteins A and C mRNA is restricted to the liver under basal and stress conditions.     

Influence of subunit transcript and protein levels on formation of a mitochondrial multienzyme complex

Constitutive expression of nuclear genes encoding mitochondrial proteins raises the question of whether these proteins are present in similar amounts in mitochondria of different tissues. We report that amounts of a single multienzyme complex can vary on a per mitochondrion basis depending on the number of mitochondria per cell. Human branched-chain α-keto acid dehydrogenase (BCKD) expression is used as a paradigm in these studies. Expression is compared and contrasted in HepG2 and DG75 cells in which mitochondrial content is twofold higher in the hepatocarcinoma line than in the lymphoblastoid line. Per cell, BCKD activity is equal in the two cell types, but BCKD protein concentration per mitochondrion is twofold higher in DG75 cells. Steady-state mRNA levels do not appear to be directly related to amounts of protein in the two cell lines. To test whether one subunit is limiting in formation of complex, overexpression of each BCKD subunit was elicited by plasmid transfection of the DG75 cells. Only overexpression of the β-subunit of the decarboxylase component induced more BCKD activity without apparent increase in mRNA for the other endogenously expressed subunits. This implies that free BCKD subunits exist in a cell and can be recruited into an active complex when the limiting subunit becomes available. © 1996 Wiley-Liss, Inc.     

Gene expression for a novel protein RGPR-p117 in various species: the stimulation by intracellular signaling factors

The presence and expression for the gene encoding a novel regucalcin gene promoter region-related protein (RGPR-p117) in various species was investigated by using Southern "zoo blot" and Northern hybridization analyses. A "zoo blot" analysis demonstrated that RGPR-p117 gene was widely conserved in various species including human, rat, mouse, dog, cow, pig, rabbit, chicken, fish, C. elegans and yeast. The gene was not found in Xenopus. Northern blot analysis showed that RGPR-p117 mRNA was expressed in the liver of human, rat, mouse, and rabbit as a single mRNA of approximately 4.5 kb, respectively. However, homologous mRNA was not found in the liver of Xenopus. The expression of RGPR-p117 mRNA in liver was clearly enhanced 5 h after a single intraperitoneal administration of CaCl(2) (5 mg Ca(2+)/100 g body weight) to rats. The RGPR-p117 mRNA is also expressed in the cloned H4-II-E rat hepatoma cells, although this expression was weak as compared with that of liver tissues. Moreover, the RGPR-p117 mRNA expression in H4-II-E cells was stimulated in the presence of dibutyryl cAMP, PMA, insulin, 17beta-estradiol, or serum in culture medium. The present study demonstrates that the RGPR-p117 gene is conserved in various species, and that its expression is stimulated by intracellular signaling factors.     

Characterization of an alternative exon of the murine T cell receptor beta-chain. Pattern of expression and evolutionary conservation

In this report, we characterize an alternate gene element of the murine TCR beta-chain. First, we have looked at the expression of the alternate exon, C beta 0, in normal T cell clones, as well as in fetal vs adult whole thymus. The C beta 0 exon is expressed in only 1% or less of TCR-beta messages in four of four mature T cell clones examined. C beta 0 is found at 10-fold higher levels in both fetal and adult thymus mRNA. Thus C beta 0 is developmentally regulated by T cells, although expression of the alternate exon is relatively constant from the fetal thymus to the adult thymus. Second, evolutionary conservation of the C beta 0 gene element was studied in both the rat and the human. The rat beta-locus contains a gene element highly homologous to the mouse C beta 0 gene, but the rat C beta 0 gene contains mutations in both splice sites that probably prevent the gene element from being spliced into mRNA. We have also sequenced the first exon of rat C beta 1, and find that the C beta 0 exon and the intron around C beta 0 are conserved between rat and mouse to the same level as the C beta 1 coding region. The intron around C beta 1, in contrast, shows the decrease in conservation between the two species that is expected for a noncoding region. Analysis of the putative C beta 0-containing region in the human reveals no sequences homologous to the C beta 0 gene element. Because the mouse is the only species that has conserved a functional C beta 0 gene, we conclude that the C beta 0 exon does not play a general role in T cell development.     

Identification of a serum-inducible messenger RNA (5B10) as the mouse homologue of calcyclin: tissue distribution and expression in metastatic, ras-transformed NIH 3T3 cells

A mouse mRNA, provisionally designated 5B10, has been cloned based on its inducibility by serum in quiescent murine fibroblasts. Here we report the full-length complementary DNA sequence and a partial characterization. There are about five copies of the gene in the mouse genome. Sequence analysis of the 5B10 coding region reveals 94 and 97% amino acid identity to human and rat calcyclin, respectively. Although the coding region has been highly conserved during evolution of the rodent and human genomes, the untranslated flanking sequences differ significantly. A protein of Mr about 8000 was produced by in vitro translation of the mRNA transcribed in vitro from 5B10 complementary DNA in a riboprobe vector. An antiserum raised against a portion of the predicted human calcyclin protein cross-reacted with this mouse protein. 5B10 mRNA was found in greatest amount in organs containing proliferating cells, e.g., epidermis, skin, stomach, uterus of pregnant mouse, placenta, and decidua. Brain, liver, mature thymus, and skeletal muscle had little or no detectable 5B10 mRNA. 5B10 mRNA levels were higher in cells treated with 7,12-dimethylbenzanthracene and 12-O-tetradecanoylphorbol-13-acetate than in their normal counterparts, suggesting a role in tumorigenesis. In addition, high 5B10 mRNA levels were associated with metastatic ability in a series of ras-transformed cells, in proportion to levels of ras p21 expressed by the cells, implicating 5B10 even more deeply in carcinogenesis.     

Conservation and variation in human and common chimpanzee CD94 and NKG2 genes.

To assess polymorphism and variation in human and chimpanzee NK complex genes, we determined the coding-region sequences for CD94 and NKG2A, C, D, E, and F from several human (Homo sapiens) donors and common chimpanzees (Pan troglodytes). CD94 is highly conserved, while the NKG2 genes exhibit some polymorphism. For all the genes, alternative mRNA splicing variants were frequent among the clones obtained by RT-PCR. Alternative splicing acts similarly in human and chimpanzee to produce the CD94B variant from the CD94 gene and the NKG2B variant from the NKG2A gene. Whereas single chimpanzee orthologs for CD94, NKG2A, NKG2E, and NKG2F were identified, two chimpanzee paralogs of the human NKG2C gene were defined. The chimpanzee Pt-NKG2CI gene encodes a protein similar to human NKG2C, whereas in the chimpanzee Pt-NKG2CII gene the translation frame changes near the beginning of the carbohydrate recognition domain, causing premature termination. Analysis of a panel of chimpanzee NK cell clones showed that Pt-NKG2CI and Pt-NKG2CII are independently and clonally expressed. Pt-NKG2CI and Pt-NKG2CII are equally diverged from human NKG2C, indicating that they arose by gene duplication subsequent to the divergence of chimpanzee and human ancestors. Genomic DNA from 80 individuals representing six primate species were typed for the presence of CD94 and NKG2. Each species gave distinctive typing patterns, with NKG2A and CD94 being most conserved. Seven different NK complex genotypes within the panel of 48 common chimpanzees were due to differences in Pt-NKG2C and Pt-NKG2D genes.     

Identification of Gasz,an evolutionarily conserved gene expressed exclusively in germ cells and encoding a protein with four ankyrin repeats,a sterile-alpha motif,and a basic leucine zipper

To discover causes of infertility and potential contraceptive targets, we used in silico subtraction and genomic database mining to identify conserved genes with germ cell-specific expression. In silico subtraction identified an expressed sequence tag (EST) present exclusively in a newborn mouse ovary library. The full-length cDNA sequence corresponding to this EST encodes a novel protein containing four ankyrin (ANK) repeats, a sterile-alpha motif (SAM), and a putative basic leucine zipper (bZIP) domain. Northern blot and semiquantitative RT-PCR analyses demonstrated that the mRNA is exclusively expressed in the mouse testis and ovary. The expression sites were localized by in situ hybridization to pachytene spermatocytes in the testis and oocytes in the ovary. Immunohistochemistry showed that the novel protein is localized to the cytoplasm in pachytene spermatocytes and early spermatids, oocytes at all stages of oogenesis, and in early preimplantation embryos. Based on its germ cell-specific expression and the presence of ANK, SAM, and basic leucine zipper domains, we have termed this novel protein GASZ. The mouse Gasz gene, which consists of 13 exons and spans 60 kb, is located on chromosome 6 between the Wnt2 and cystic fibrosis transmembrane conductance regulator (Cftr) genes. Using genomic database mining, orthologous genes encoding GASZ were identified in the rat, cow, baboon, chimpanzee, and human. Phylogenetic analyses reveal that the GASZ proteins are highly conserved among these species. Human and mouse GASZ proteins share 85.3% amino acid identity, and human and chimpanzee GASZ proteins differ by only 3 out of 475 amino acids. In humans, the GASZ gene resides on chromosome 7 and is similarly composed of 13 exons. Because both ANK repeats and the SAM domain function as protein-protein interaction modules that mediate signal transduction cascades in some systems, GASZ may represent an important cytoplasmic signal transducer that mediates protein-protein interactions during germ cell maturation in both males and females and during preimplantation embryogenesis.