The osteocalcin gene encodes a 6-kDa polypeptide, which represents one of the most abundant noncollagenous bone proteins, and the present studies establish that osteocalcin mRNA is detected only in bone tissue. An osteocalcin gene was isolated from a rat genomic DNA library, and sequence analysis indicated that the mRNA is represented in a 953-nucleotide segment of DNA consisting of four exons and three introns. A modular organization of the 5' flanking sequences of the gene is reflected by the presence of at least three classes of regulatory elements, which include the following: (i) RNA polymerase H canonical sequences; (U) a series of consensus sequences for hormone receptor binding sites and cyclic nucleotide responsive elements consistent with physiologic expression ofthe osteocalcin gene; and (Uii) a 24-nucleotide sequence in the proximal promoter region with a CAAT motif as a central element. We have designated this highly conserved sequence as an "osteocalcin box" since only 2 nucleotide substitutions are found in the rat and human osteocalcin genes. We have demonstrated two factors regulating osteocalcin gene expression. First, a 200-fold increase occurs in normal fetal calvaria osteoblasts producing a mineralizing matrix, compared to confluent osteoblasts in a nonmineralizing matrix. Second, contained within the 600 nucleotides immediately upstream from the transcription start site are sequences that support a 10-fold stimulated transcription of the gene by 1,25-dihydroxyvitamin D.There has been much interest in the vitamin K-dependent protein of bone, osteocalcin (bone Gla protein), since its discovery over a decade ago (1). A distinguishing feature of this 5.7-kDa protein (46-50 amino acids, depending on the species), and of functional significance, are 3 residues of the calcium binding amino acid, y-carboxyglutamic acid (Gla). Gla residues are posttranslationally synthesized from selected glutamic acid residues by a vitamin K-and C02-requiring enzyme complex (2). They are located at positions 17, 21, and 24 in all species from swordfish to mammals (1). This highly conserved sequence region from residues 20-34 in the central portion of the molecule, which also includes a disulfide loop (Cys-23-Cys-29), accounts for a structural conformation of the protein in the presence of calcium that promotes a tight binding of the protein to hydroxyapatite (1). The appearance of osteocalcin in embryonic bone coincident with mineral deposition (1), its association with the hydroxyapatite component of the matrix (3), its chemoattractant property for cells capable of bone resorption (4), and its modulated synthesis by the calcitrophic hormone 1,25-dihydroxyvitamin D3 [1,25(OH)2D3] (5-7) suggest a role for the protein in bone turnover. Although many properties of the protein have been identified, the precise function of osteocalcin is still unknown.Osteocalcin is synthesized de novo by osteoblasts as a 10,000-kDa precursor (8). While the majority ofthe processed osteocalcin peptide (5.7 kDa) is deposited in b...
The gene responsible for multiple endocrine neoplasia type 1 (MEN1), a heritable predisposition to endocrine tumours in man, has recently been identi®ed. Here we have characterized the murine homologue with regard to cDNA sequence, genomic structure, expression pattern and chromosomal localisation. The murine Men1 gene spans approximately 6.7 kb of genomic DNA and is comprised of 10 exons with similar genomic structure to the human locus. It was mapped to the pericentromeric region of mouse chromosome 19, which is conserved with the human 11q13 band where MEN1 is located. The predicted protein is 611 amino acids in length and overall is 97% homologous to the human orthologue. The 45 reported MEN1 mutations which alter or delete a single amino acid in human all occur at conserved residues, thereby supporting their functional signi®cance. Two transcripts of approximately 3.2 and 2.8 kb were detected in both embryonal and adult murine tissues, resulting from alternative splicing of intron 1. By RNA in situ hybridization and Northern analysis the spatiotemporal expression pattern of Men1 was determined during mouse development. Men1 gene activity was detected already at gestational day 7. At embryonic day 14 expression was generally high throughout the embryo, while at day 17 the thymus, skeletal muscle, and CNS showed the strongest signal. In selected tissues from postnatal mouse Men1 was detected in all tissues analysed and was expressed at high levels in cerebral cortex, hippocampus, testis, and thymus. In brain the menin protein was detected mainly in nerve cell nuclei, whereas in testis it appeared perinuclear in spermatogonia. These results show that Men1 expression is not con®ned to organs a ected in MEN1, suggesting that Men1 has a signi®cant function in many di erent cell types including the CNS and testis.
We have studied the cell cycle-regulated expression of the thymidine kinase (TK) gene in mammalian tissue culture cells. TK mRNA and enzyme levels are low in resting, GO-phase cells, but increase dramatically (10-to 20-fold) during the S phase in both serum-stimulated and simian virus 40-infected cells. To determine whether an increase in the rate of TK gene transcription is responsible for this induction, nuclear run-on transcription assays were performed at various times after serum stimulation or simian virus 40 infection of growth-arrested simian CV1 cells. When assays were performed at 12-h intervals, a small (two-to threefold) but reproducible increase in TK transcription was detected during the S phase. When time points were chosen to span the G1-S interface a larger (six-to sevenfold) increase in transcriptional activity was observed in serum-stimulated cells but not in simian virus 40-infected cells. The large increase in TK mRNA levels and the relatively small increase in transcription rates in growth-stimulated cells suggest that TK gene expression is controlled at both a transcriptional and post-transcriptional level during the mammalian cell cycle. To identify the DNA sequences required for cell cycle-regulated expression, several TK cDNA clones were transfected into Rat-3 TK-cells, and their expression was examined in resting and serum-stimulated cultures. These experiments indicated that the body of the TK cDNA is sufficient to insure cell cycle-regulated expression regardless of the promoter or polyadenylation signal used.
We have examined the sequences required in vivo to promote transcription of a cell cycle-regulated human H4 histone gene. Deletion mutants of the 5' flanking region were assayed in mouse cells or fused with the chloramphenicol acetyltransferase (CAT) gene for assay in HeLa cells. The functional limits of the regulatory sequences were shown to extend at least 6.5 kilobases (kb) upstream. Sequences sufficient for correctly initiated transcription were found in the 70 base pairs (bp) immediately 5' to the cap site. A proximal element located 200-400 bp upstream increased the level of transcription several times above the basal level, although not to maximal levels. Maximal levels of expression were achieved with 6.5 kb of 5' flanking sequence adjacent to the proximal promoter sequences or when a distal enhancer element with both position-and orientation-independent function was moved proximal to the promoter. Our results indicate that a series of 5' cis-acting sequences are functionally related to the fidelity and level of expression of this human H4 histone gene.The human histone genes encode a set of proteins essential for maintaining the integrity of eukaryotic chromosomal structure (1-4). The combined studies of several investigators have now established two basic facts: (i) most histone genes are expressed coordinately during the cell cycle in conjunction with DNA synthesis, and (ii) there are many different copies of the core and H1 histone genes being expressed concomitantly (5-8). This leads one to conclude that histone gene expression on a larger scale is coordinately controlled, but that at the level of the single gene there may be differences in the extent and timing of expression. Our previous results with H4 histone genes support this contention that not all human histone genes are expressed to the same degree during S phase (5). We and others have shown that the regulation of histone gene expression occurs at both the transcriptional and post-transcriptional levels. The two levels of regulation together serve to increase significantly the amount of histone messenger RNA during S phase (6-11). Taking these results into account, we have initiated in vivo studies to identify transcriptional regulatory sequences associated with a human H4 histone gene. The gene we have chosen to study has been previously shown to be one of the most highly expressed human H4 histone genes (5).Many studies have established that there are consensus sequences present in RNA polymerase II promoters that are necessary for expression (12)(13)(14). Sequence analysis of the F0108A H4 histone gene has demonstrated the presence of several such sequences in the 5' flanking region, including two "CAAT" boxes and a "TATA" box (15). In vitro transcription studies of this gene using whole-cell lysates have indicated that only the TATA box is necessary for correct transcript initiation (15).To establish in vivo the functional properties of 5' flanking sequences associated with the F0108 human H4 histone gene, we constructed a seri...
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