Assignment of the human integral transmembrane protein 1 gene (ITM1) to human chromosome band 11q23.3 by in situ hybridization and YAC mapping.
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Chromosome Mapping↗
Biomedical subjects
J Merregaert
Publications and source records attributed to J Merregaert.
Characterization of a processed pseudogene of human FAU1 on chromosome 18.
A member of the human FAU (Finkel-Biskis-Reilly murine sarcoma virus-associated ubiquitously expressed) gene subfamily, encoding the ribosomal protein S30 fused in frame to an ubiquitin-like protein, was cloned, sequenced and analysed. This clone, FAU1P, is a processed pseudogene with a completely intact, although transcriptionally silent, open reading frame of 137 codons. FAU1P exhibits an amplification of the (AAG) triplet repeat present in the S30 coding part of FAU. FAU1P is integrated in an antisense orientation within a sequence homologous to the promoter of the islet amyloid polypeptide (IAPP or amylin)-encoding gene. By means of PCR hybrid panel mapping, FAU1P was assigned to chromosome 18.
Amyloid↗
Molecular cloning, characterization, and genetic mapping of the cDNA coding for a novel secretory protein of mouse. Demonstration of alternative splicing in skin and cartilage.
A novel 85-kDa protein secreted by the mouse stromal osteogenic cell line MN7 was identified using two-dimensional polyacrylamide gel electrophoresis (Mathieu, E., Meheus, L., Raymackers, J., and Merregaert, J. (1994) J. Bone Miner. Res. 9, 903-913). Degenerate primers were used to isolate the cDNA coding for this protein. The full-length cDNA clone is 1.9 kilobases (kb) and codes for a protein of 559 amino acid residues. The DNA and deduced amino acid sequences have no counterparts in public data bases, but a structural similarity involving typical cysteine doublets can be observed to serum albumin family proteins and to Endo16 (a calcium-binding protein of sea urchin). Northern blot analysis revealed the presence of a 1.9-kb transcript in various tissues, and a shorter transcript of 1.5 kb, derived by alternative splicing in tail, front paw and skin of embryonic mice. The gene for the p85 protein, termed Ecm1 (for extracellular matrix protein 1), is a single-copy gene, which was localized to the region on mouse chromosome 3 known to contain at least one locus associated with developmental disorders of the skin, soft coat (soc). Alternative splicing may serve as a mechanism for generating functional diversity in the Ecm1 gene.
Alternative Splicing↗
The mouse Fau gene: genomic structure, chromosomal localization, and characterization of two retropseudogenes.
The Fau gene is the cellular homolog of the fox sequence of the Finkel-Biskis-Reilly murine sarcoma virus (FBR-MuSV). FBR-MuSV acquired the Fau gene by transduction in a transcriptional orientation opposite to that of the genomic Fau gene. The genomic structure of the mouse Fau gene (MMFAU) and its upstream elements have been determined and are similar to those of the human FAU gene. The gene consists of five exons and is located on chromosome 19. The first exon is not translated. The promoter region has no well-defined TATA box but contains the polypyrimidine initiator flanked by regions of high GC content (65%) and shows all of the characteristics of a housekeeping gene. The 5' end of the mRNA transcript was determined by 5' RACE analysis and is located, as expected, in the polypyrimidine initiator site. Furthermore, the sequences of two retropseudogenes (Fau-ps1 and Fau-ps2) are reported. Both pseudogenes are approximately 75% identical to the Fau cDNA, but both are shorter due to a deletion at the 5' end and do not encode a functional protein. Fau-prs is interrupted by an AG-rich region of about 350 bp within the S30 region of the Fau cDNA. Fau-ps1 was localized on chromosome 1 and Fau-ps2 on chromosome 7.
Animals↗
Establishment and characterization of two clonal cell lines derived from murine mandibular condyles.
We have established two clonal cell lines, designated SM1/9 and SM25/3 from the mandibular condyles of newborn BALB/c mice by immortalization with the SV40 large T antigen. These cells have a high proliferative activity and have been maintained in culture for over 50 passages. They are polygonal in shape. Electron microscopic studies indicate an immature phenotype for both clones and a lack of prominent intracellular filaments typical of fibroblasts. SM25/3 demonstrates different biological properties as compared to SM1/9, it is tumourigenic in nude mice, has a faster growth rate and exhibits less differentiated features. Both cell lines have low constitutive levels of alkaline phosphatase, and the activity of this enzyme is increased significantly in a dose and confluency dependent manner by retinoic acid and 1,25 (OH)2 vitamin D3. The cells express transcripts for retinoic acid receptors mRAR-alpha and mRAR-gamma but not for mRAR-beta. They also express mRNA for the 1,25 (OH)2 vitamin D3 receptor. They co-express transcripts for collagen types I, II, III. Expression of mRNA for extracellular matrix proteins such as biglycan, osteopontin, PAI-1 is detected. Cultured cells do not express mRNA for osteocalcin and this transcript is not inducible with 1,25 (OH)2 vitamin D3 or retinoic acid. Chondrocyte markers such as link protein and aggrecan are not detected. In vitro assays indicate that the cell lines have a limited capacity for osteogenic or chondrogenic differentiation. Similarly agarose culture experiments and extended treatment with retinoic acid indicate that they do not resemble dedifferentiated chondrocytes. Both the cell lines appear to express a phenotype intermediate to osteoblasts and chondroblasts and possibly represent transitional differentiation stages of the progenitor cells of the mandibular condyle. These cells could serve as useful models in elucidating the pathways of early mesenchymal cell differentiation.
Alkaline Phosphatase↗
Characterization of the stromal osteogenic cell line MN7: mRNA steady-state level of selected osteogenic markers depends on cell density and is influenced by 17 beta-estradiol.
The steady-state mRNA levels of different osteogenic markers and their modulation by 17 beta-estradiol in the murine osteogenic cell line MN7 during proliferation and differentiation in vitro were examined. mRNA of collagen type I, osteopontin, bone morphogenetic protein 2, plasminogen activator inhibitor 1, alkaline phosphatase, and osteocalcin were isolated from MN7 cultures grown for 7, 11, 14, and 17 days. Northern blot analysis revealed steady-state transcript levels depending on MN7 cell density. The order of appearance of Col I, OP, ALP, and OC resembled the pattern of gene expression observed during osteoblast maturation in vitro. Furthermore, PAI-1 steady-state transcript levels peaked during subconfluence (day 11) but BMP-2 RNA levels reached their maximum after the culture had become confluent. 17 beta-Estradiol showed a dose-dependent stimulation of the different osteoblast-related transcripts present in a subconfluent MN7 culture at the time of analysis. Furthermore, the effects of 17 beta-estradiol (17 beta E2) at different time points of MN7 growth varied according to cell density. 17 beta E2 added to subconfluent MN7 cultures modulated the transcript level in a negative way, but RNA levels of the investigated osteogenic markers in confluent cultures were stimulated with 100 nM 17 beta-estradiol. No effect of 17 beta-estradiol on proliferation was detected. The present studies have revealed differential osteoblast gene expression related to MN7 cell proliferation and differentiation in vitro and emphasize the importance of 17 beta E2 in the regulation of growth of this preosteoblastic cell line in vitro.
Alkaline Phosphatase↗
Characterization of the osteogenic stromal cell line MN7: identification of secreted MN7 proteins using two-dimensional polyacrylamide gel electrophoresis, western blotting, and microsequencing.
Proteins secreted by the osteogenic stromal cell line MN7 were analyzed using two-dimensional polyacrylamide gel electrophoresis (PAGE), western blotting, immunodetection, and microsequencing. Trichloroacetic acid-precipitated proteins from the conditioned medium of MN7 cell cultures, harvested at different times of growth, were dissolved in denaturing and reducing sample buffer and separated in the first dimension according to isoelectric point and in the second dimension according to molecular weight. Protein patterns were visualized using silver staining. Among the 350 separated protein spots, we identified type I collagen, bone sialoprotein, osteonectin, and cathepsin B by western blotting and immunodetection using polyclonal antibodies. Osteocalcin could not be detected in the conditioned medium of MN7 cells. Furthermore, 15 MN7-specific protein spots were localized after comparison with two-dimensional PAGE patterns from the conditioned medium of the nonosteogenic stromal cell lines MM1 and MV1. Microsequencing of the internal peptides of five selected spots revealed three known proteins, namely the carboxyl-terminal propeptide of the alpha 2 chain of collagen type I, cathepsin L, and the tissue inhibitor of metalloproteinases-2, an 18 kilodalton peptide fragment from osteopontin that has not previously been described, and a novel glycosylated 85 kD protein with an average isoelectric point of 5.7. All identified proteins did not vary in presence between the different time points analyzed by two-dimensional PAGE. The use of two-dimensional PAGE to investigate the secreted proteins of MN7 cells will enable us to establish a complete protein data base of extracellular osteoblast-specific proteins. Furthermore, two-dimensional PAGE in combination with other techniques is a fast and accurate method for the identification of novel proteins that could function as markers in osteoblast differentiation and/or bone formation.
Amino Acid Sequence↗
Molecular mapping of the chromosome 11 breakpoint of t(11;17)(q13;q21) in a t(11;14)(q13;q32)-positive B non-Hodgkin's lymphoma.
The FAU gene is the cellular homologue of the viral FOX sequences in the genome of the Finkel-Biskis-Reilly murine sarcoma virus (FBR-MuSV); the viral FOX sequences have been shown to increase the transforming capacity of FBR-MuSV in vitro. The human FAU gene has recently been isolated, characterized, and mapped to chromosome band 11q13. Here, we report results of fluorescence in situ hybridization (FISH) analysis which indicate that the FAU gene maps proximally to the putative oncogene BCL1 at 11q13. Furthermore, we identified a t(11;17)(q13;q21) translocation in tumor cells of a t(11;14)(q13;q32)-positive B-cell non-Hodgkin's lymphoma patient by FISH analysis using a FAU containing cosmid clone as molecular probe and by double-colour chromosome painting analysis using chromosome 11- and chromosome 17-specific painting probes. The position of the chromosome 11 breakpoint of the t(11;17) translocation was pinpointed to a human DNA region around the FAU gene of about 40 kbp.
Chromosome Mapping↗
Assignment of the human FAU gene to a subregion of chromosome 11q13.
The FAU gene is the cellular homologue of the fox sequence in the Finkel-Biskis-Reilly murine sarcoma virus (FBR-MuSV). FAU (for FBR-MuSV associated ubiquitously expressed gene) encodes the ribosomal protein S30 fused to a ubiquitin-like protein. A cosmid clone containing the human FAU gene was used for fluorescence in situ hybridization to metaphase chromosomes. The obtained localization to 11q13 was confirmed by hybridization against a panel of somatic cell hybrids containing different parts of chromosome 11 on a hamster background. FAU was then further mapped, both on a panel of radiation-reduced somatic cell hybrids designed to carry different parts of the 11q13 region and by pulsed-field gel electrophoresis. This fine mapping assigned FAU close to the skeletal muscle glycogen phosphorylase gene (PYGM), in a region that contains several oncogenes as well as the putative tumor suppressor genes MEN1 and ST3.
Animals↗
Retroviruses and oncogenes associated with osteosarcomas.
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Animals↗
Exclusion of FAU as the multiple endocrine neoplasia type 1 (MEN1) gene.
The FAU gene (FBR-MuSV associated ubiquitously expressed gene) encodes the ribosomal protein S30 fused with a Ubiquitin-like molecule. The FAU gene is expressed in a wide range of tissues, is evolutionarily conserved, and has putative tumour suppressor activity in vitro. The human FAU gene maps to the long arm of chromosome 11 band q13, close to the PYGM locus. This locus is tightly linked to the Multiple Endocrine Neoplasia type 1 (MEN1) locus. The FAU gene properties, together with its chromosomal localisation on 11q13, make it a candidate gene for MEN1. To test this hypothesis we screened 33 unrelated patients with MEN1 for constitutional genetic alterations in the FAU gene by Southern blot analysis, denaturing gradient gel electrophoresis (DGGE) and in two cases complemented by DNA sequencing to confirm the DGGE data. Furthermore, 10 parathyroid and pancreatic tumours from MEN1 patients and 15 each of sporadic parathyroid and pituitary tumours were similarly examined. In addition, we studied the expression of the FAU gene at the RNA level in 9 MEN1-associated tumours by Northern blot analysis. No FAU gene anomalies could be demonstrated by any of these techniques. We conclude that FAU is not likely to be the MEN1 tumour suppressor gene.
Base Sequence↗
fau cDNA encodes a ubiquitin-like-S30 fusion protein and is expressed as an antisense sequence in the Finkel-Biskis-Reilly murine sarcoma virus.
The Finkel-Biskis-Reilly murine sarcoma virus (FBR-MuSV) is capable of inducing osteosarcomas in susceptible mice. This retrovirus transduced sequences derived from the transcription factor c-fos and from an unrelated mouse sequence called fox. Here, we describe the cloning and sequence analysis of human and mouse cellular cDNAs hybridizing to the fox sequence. The cloned cDNAs encode for a single ubiquitin-like (Fubi) protein fused in frame to S30, a protein of the small ribosomal subunit. Fubi conserved amino acid residues known to be involved in the ATP-dependent proteolytic activity of ubiquitin. Moreover, the fau gene is conserved in several species, while its mRNA is ubiquitously expressed in different mouse tissues. Surprisingly, FBR-MuSV transduced the complete but mutated open reading frame (ORF) in its reversed transcriptional orientation. This is the first report about a retrovirus in which an antisense sequence to a cellular gene, which we called fau (FBR-MuSV-associated ubiquitously expressed gene), is discovered. Rat-2 cells transfected with plasmids containing v-fau/fox recombinants of FBR-MuSV revealed a twofold increase of the transformation capacity of FBR-MuSV 'in vitro' because of the fau antisense sequence. Newly formed retropseudogenes were identified in three out of eight primary radiation-induced osteosarcomas. This high incidence of creating retropseudogenes in these 90Sr-induced bone tumours may contribute to the mechanism by which FBR-MuSV, originally isolated from such tumours, acquired the fau gene in its reverse orientation.
Amino Acid Sequence↗
Genomic structure and expression of the human fau gene: encoding the ribosomal protein S30 fused to a ubiquitin-like protein.
The fau gene is the cellular homolog of the fox sequence in the Finkel-Biskis-Reilly Murine Sarcoma Virus (FBR-MuSV). This virus acquired the fau sequence in its reversed transcriptional orientation. Human and mouse fau cDNA's were identified and both encode a new protein of 133 AA. We show that fau (for FBR-MuSV associated ubiquitiously expressed gene) becomes expressed in all different tissues tested as a 600 bp messenger and we report the genomic structure of the human fau gene. The gene consists of five exons and four introns and the 5' untranslated region displays characteristic features for a housekeeping gene. Fau encodes the ribosomal protein S30 fused to a Ubiquitin-like protein.
Amino Acid Sequence↗
Establishment of an osteogenic cell line derived from adult mouse bone marrow stroma by use of a recombinant retrovirus.
In order to characterize fibroblastic colony-forming units (CFU-F) from murine bone marrow in relation to osteogenesis, adherent cells of 7-day-old BALB/c mouse bone marrow cultures were infected with a recombinant retrovirus (N2/ delta fosB) containing the bacterial neomycin resistance gene. One of the G418-resistant clones, MN7, was selected for further analysis on the basis of its high expression of the bone-specific alkaline phosphatase. The cells have now been in culture for more than 1 year and maintain a stable phenotype. The osteogenic nature of the immortalized clone MN7 was demonstrated as follows: (1) Mineralization was detected by 85Sr uptake and with the Von Kossa staining method only after in vitro cultivation on a collagen type I matrix. (2) Osteoblastic phenotype markers, including the synthesis of type I collagen, osteonectin, and the bone-specific isoenzyme of alkaline phosphatase were expressed in vitro. (3) MN7 cells responded to bone effectors such as parathyroid hormone and 1,25-dihydroxyvitamin D3. (4) Intraperitoneal injection of MN7 cells into 1-day-old BALB/c mice produced typical osteosarcomas in all animals. We conclude that MN7, derived entirely in vitro from a stromal CFU-F colony, represents a stable murine osteosarcoma cell line expressing the osteoblastic phenotype and provides the first direct evidence needed to establish adult mouse marrow-derived, nonhematopoietic stromal cells as osteoprogenitors.
Alkaline Phosphatase↗
The open reading frame ORF S3 of equine infectious anemia virus is expressed during the viral life cycle.
The genome of equine infectious anemia virus (EIAV) contains several small open reading frames (ORFs), the importance of which in the development of the virus is not clear. We investigated the possibility that the largest of these ORFs (ORF S3) is expressed during the course of the viral infection. The ORF S3 information was expressed in Escherichia coli, and the antigen was used to raise monospecific antiserum. A 20-kDa protein expressed in cells producing EIAV was identified as the gene product of ORF S3. Furthermore, sera from EIAV-infected animals specifically recognized this protein, indicating that the ORF S3 antigen is expressed in vivo as well. A model for the expression of this new viral antigen is presented. The proposed splicing pattern is similar to that of the VEP-1 protein of maedi-visna-virus, which tempts us to speculate that ORF S3 defines the second exon of the EIAV Rev protein.
Amino Acid Sequence↗
Isolation of human immunodeficiency virus type 2 from a homosexual man in Belgium.
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Belgium↗
Provirus integration and myc amplification in 90Sr induced osteosarcomas of CF1 mice.
In mice, endogenous retroviruses are known to be activated during the course of radiation osteosarcomagenesis. Using the Southern blotting procedure, we have studied the presence of somatically acquired proviruses in genomic DNA isolated from seven primary 90Sr induced osteosarcomas and one osteosarcoma cell line, 0-127a1, of the CF1 mouse strain. Specific hybridization probes demonstrated the presence of newly integrated ecotropic proviruses in four primary tumors. Probably, clonally integrated proviruses were present at distinct locations in different subpopulations of tumor cells, reflecting tumor heterogeneity. Genomic DNA isolated from cultured osteosarcoma cells contained different additional MCF-related proviruses. No proviruses were found integrated in the vicinity of c-myc, but a large domain containing the complete c-myc gene was found amplified in one primary tumor (greater than 22 kbp) and in 0-127a1 cells (greater than 39 kbp). Our data suggest that activated retroviruses are not essential for the development of radiogenic osteosarcomas in CF1 mice, but they might be responsible for the deregulated expression of a growth promoting gene in some bone tumor cells.
Animals↗
Nucleotide sequence of a radiation leukemia virus genome.
The complete nucleotide sequence of an infectious molecular clone of a radiation murine leukemia proviral DNA RadLV/VL3(T+L+) has been determined. The sequence of the RNA genome is 8318 nucleotides long and contains three large open reading frames encoding the gag, pol, and env gene products. With the exception of a xenotropiclike R peptide and the LTR which bears structural similarities to a xenotropic LTR, displaying typical enhancerlike sequences, the remaining sequences are strikingly similar to the endogenous, ecotropic Akv murine leukemia virus. Therefore, it could be postulated that the leukemogenic properties of RadLV/VL3(T+L+) were generated by a recombination event between a xenotropic virus and an Akv-like ecotropic virus.
Base Sequence↗