Biomedical subjects

W B Upholt

Publications and source records attributed to W B Upholt.

At least 55 records · Page 3Linked to original sources

PubMed1990

Cloning and analysis of the 5' region of the rat bone/liver/kidney/placenta alkaline phosphatase gene. A dual-function promoter.

The rat bone/liver/kidney/placenta (BLKP) alkaline phosphatase (ALP) gene is expressed at high level in these particular tissues and at low levels in many other tissues. To study the mechanisms underlying the complex regulation of the rat BLKP ALP expression, we isolated a genomic clone, containing a 10.5-kb insert, which includes the promoter of the BLKP ALP gene with 2 kb of 5' flanking region, its first exon (84bp), and over 7 kb of the first intron. The promoter of the rat BLKP ALP displays features of a "housekeeping" gene promoter: an atypical TATA-box (TTCATAA); 3 potential Spl binding sites; high GC content (82% in positions-134 to -14); and a high CpG to GpC ratio (60:89 in the 0.85 kb promoter region), indicating an abundance of potential methylation sites. Likewise, transient transfection of CAT fusion genes into ROS 17/2.8 osteoblast-like cells reveals weak expression from the promoter and proximal 5' flanking sequences, which can be elevated by an SV40E enhancer. The homologous human bone/liver/kidney (BLK) ALP promoter, which demonstrates a similar combination of tissue-specific and housekeeping characteristics, shares close similarity (184 bp of 79% similarity excluding gaps) with the rat BLKP ALP promoter. The human placental ALP is encoded by a separate gene and its promoter, on the other hand lacks significant similarity to the rat BLKP ALP promoter despite their common expression in the placenta. This lack of similarity appears to reflect the close evolutionary relationship of the human placental ALP gene to the intestinal ALP gene. Significant sequence similarity was found between the rat and human BLK/BLKP ALP promoters and the human and mouse adenine deaminase promoters, and together they may represent a class of dual-function promoters, allowing both constitutive low-level, and tissue-specific higher levels of expression. A pentanucleotide with the consensus sequence 5'-GGCTC-3' is present in these promoters and in the promoters for the human fibronectin and the human alpha 1(II) procollagen genes in the region of maximal similarity with the rat BLKP ALP promoter, and in the vicinity of the Sp1-binding sites.

Alkaline Phosphatase↗

PubMed1990

Regulation of alkaline phosphatase and alpha 2(I) procollagen synthesis during early intramembranous bone formation in the rat mandible.

We have studied intramembranous bone formation in the developing rat mandible. In this system discrete developmental stages can be readily distinguished: mesenchymal condensation, osteoid deposition, and mineralization. In mandibles of 14-day rat embryos avascular condensed mesenchymal cells can be discerned in a region lateral to Meckel's cartilage and anterior to the first molar bud. In 18-day embryos primary bone structures with mineral deposition are evident, and at 2 days postnatally the mandible is extensively mineralized. In the developing mandible we investigated the pattern of bone/liver/kidney/placenta (BLKP) alkaline phosphatase (ALP) and alpha 2(I) procollagen expression in the differentiating osteoblasts. The level of ALP activity in loose mesenchymal tissue is close to background levels. In contrast, the condensed mesenchymal cells in 14-day embryos, which will subsequently form bone, display intense ALP activity prior to discernible osteoid or mineral deposition. ALP activity in the condensed mesenchymal cells can be inhibited by levamisole, indicating activity of the BLKP gene product. We could not detect a corresponding increase in transcript level for either ALP or alpha 2(I) in the condensed mesenchyme in 14-day embryo using in situ hybridization, probably due to low message abundance. At 18 days, cells throughout the developing mandible express ALP activity, and intense in situ hybridization to BLKP ALP probes is evident in cells lining the developing bone trabeculae. Alpha 2(I) procollagen transcripts have accumulated in cells of the developing mandibular bone, but are not specifically localized to osteoblastic cells. Our results demonstrate that ALP activity is a very early marker of differentiation of cells of the osteogenic lineage, since a marked increase in ALP enzyme activity is clearly detectable in condensed mesenchymal cells prior to osteoid or mineral deposition. In contrast, Wright and Leblond, using the same model system and immunohistochemistry, could not localize type I collagen to preosteoblastic cells surrounding the developing bone trabeculae, and demonstrated localization of type I collagen to osteoblasts bordering developing trabeculae, indicating a substantial increase in type I collagen expression (at least at the protein level) during preosteoblast to osteoblast differentiation. These results indicate a discrete pattern of regulation for both the ALP and alpha 2(I) genes during osteogenic differentiation, which may involve both transcriptional and posttranscriptional regulation.

Alkaline Phosphatase↗

PubMed1990

Spondyloepiphyseal dysplasia congenita: genetic linkage to type II collagen (COL2AI).

Spondyloepiphyseal dysplasia congenita (SEDC) is an autosomal dominantly inherited chondrodysplasia characterized by disproportionate short stature (short trunk), abnormal epiphyses, and flattened vertebral bodies. Manifestations are present at birth. We ascertained a 4-generation family exhibiting the clinical manifestations of the disorder. Previous evidence suggesting defects of type II collagen associated with the SEDC phenotype led us to genotype the family for various COL2A1 gene-associated RFLPs. A total of 17 affected and unaffected members of this family were studied. The family was informative for a recently discovered HinfI RFLP. No recombinants between the marker and the phenotype were found in eight informative meioses. A maximum LOD score of 3.01 was obtained at a recombination fraction of .00. Our results indicate that the SEDC phenotype in this family is caused by mutations in or very close to the COL2A1 locus.

Adult↗

PubMed1989

Expression of chloramphenicol acetyltransferase (CAT) from rodent type I collagen promoters in transfected osteosarcoma cells in vivo.

We examined the osteoblastic phenotype of permanently transfected ROS 17/2.8 cells in culture and in vivo, in order to evaluate their relevance for studies of the regulation of gene expression and gene function in osteoblastic cells. Recent reports indicate that the progeny transfected cells may substantially vary and differ from the parental cell line in their phenotype, particularly in their tumorigenicity. ROS 17/2.8 cells were transfected with genetic constructs expressing the CAT gene from either the rat alpha 1 (I) or the mouse alpha 2 (I) collagen promoters. Forty-four clonal cell lines display a range of CAT expression from the transfected collagen promoters in culture. Four of these cell lines were further characterized. Alkaline phosphatase activity in these four cell lines is higher than in fibroblastic cells. These four cell lines are tumorigenic in immunocompatible ACI rats and form calcified tumors similar to those formed by ROS 17/2.8. CAT expression could be demonstrated in tumor extracts of two of the four cell lines, which also expressed higher CAT levels in culture. We conclude that permanently transfected ROS 17/2.8 derived cell lines maintain their tumorigenicity and their osteoblastic-like phenotype, and thus may provide a useful system for studies of gene function and regulation in osteoblast-like cells and bone-like tissue in vivo.

Animals↗

PubMed1989

Fibronectin gene expression during limb cartilage differentiation.

A critical event in limb cartilage differentiation is a transient cellular condensation process in which prechondrogenic mesenchymal cells become closely juxtaposed and interact with one another prior to initiating cartilage matrix deposition. Fibronectin (FN) has been suggested to be involved in regulating the onset of condensation and chondrogenesis by actively promoting prechondrogenic aggregate formation during the process. We have performed a systematic quantitative study of the expression of the FN gene during the progression of chondrogenesis in vitro and in vivo. In high-density micromass cultures of limb mesenchymal cells, FN mRNA levels increase about 5-fold coincident with the crucial condensation process, and remain relatively high during the initial deposition of cartilage matrix by the cells. Thereafter, FN mRNA levels progressively decline to relatively low levels as the cultures form a virtually uniform mass of cartilage. The changes in FN mRNA levels in vitro are paralleled closely by changes in the relative rate of FN synthesis as determined by pulse-labeling and immunoprecipitation analysis. The relative rate of FN synthesis increases 4- to 5-fold at condensation and the onset of chondrogenesis, after which it progressively declines to low levels as cartilage matrix accumulates. High levels of FN gene expression also occur at the onset of chondrogenesis in vivo. In the proximal central core regions of the limb bud in which condensation and cartilage matrix deposition are being initiated, FN mRNA levels and the relative rates of FN synthesis become progressively about 4-fold higher than in the distal subridge region, which consists of undifferentiated mesenchymal cells that have not yet initiated condensation.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗

PubMed1988

Analysis of type II collagen RNA localization in chick wing buds by in situ hybridization.

Type II collagen is a major component of cartilage extracellular matrix. Differentiation of mesenchyme into cartilage involves the cessation of type I collagen synthesis and the onset of type II collagen synthesis. Solution hybridization of mRNA isolated from chick limb buds with a cDNA probe to type II collagen mRNA showed the presence of small amounts of type II collagen message in mesenchymal chick limbs. We have examined the localization of type II collagen mRNA in mesenchymal chick wing buds by in situ hybridization using single stranded RNA probes. Our results show a small but detectable amount of type II collagen RNA distributed uniformly in early limbs until the first precartilage condensations form at stage 22. This is interesting because it is known that mesenchyme isolated from chick wing buds has the capacity to undergo chondrogenesis in culture, even if taken from nonchondrogenic areas of the limb. At stage 23, type II collagen mRNA is found at significantly increased levels in the cells of the precartilage condensation when compared to the other limb cells. As chondrogenesis proceeds, the amount of type II collagen RNA increases even more in cells of the cartilage elements. The signal in the peripheral tissue is indistinguishable from background. These results show that type II collagen message exists at low levels in cells throughout the mesenchymal chick wing bud, until the formation of the condensation results in an elevation of type II mRNA in the prechondrogenic cells found in the core of the limb.

Animals↗

PubMed1988

Temporal and spatial analysis of cartilage proteoglycan core protein gene expression during limb development by in situ hybridization.

As limb mesenchymal cells differentiate into chondrocytes they initiate the synthesis of a cartilage-specific sulfated proteoglycan, cartilage-characteristic type II collagen, and other cartilage-specific proteins. In the present study, in situ hybridization with a 32P-labeled cloned cDNA probe complementary to mRNA encoding the core protein of cartilage proteoglycan has been used to visualize and localize the accumulation of cartilage proteoglycan core protein mRNA sequences during development of the chick limb bud in vivo. When the probe was hybridized to sections through 7-day (stage 32) limbs, an intense hybridization signal was observed over the well-differentiated cartilage rudiments of the limb, while no signal above background was observed over nonchondrogenic tissues including muscle, loose connective tissue, and epidermis. At early stages of limb development, an accumulation of silver grains representing hybridizable core protein mRNA first became detectable in the proximal central core of the limb where the prechondrogenic condensation of mesenchymal cells that characterizes the onset of cartilage differentiation was occurring. In fact, the pattern of silver grain accumulation closely followed the pattern of mesenchymal cell condensation, and no hybridizable core protein mRNA sequences were detectable in the limb bud prior to condensation. Cartilage-characteristic type II collagen mRNA was colocalized with core protein mRNA in the condensing central core of the limb suggesting that the genes for these two major constituents of cartilage matrix are coordinately regulated at the onset of chondrogenesis. Furthermore, the appearance of hybridizable core protein mRNA was closely followed by the appearance of the protein for which it codes as detected by immunohistochemical staining with monospecific antibody. These observations support the hypothesis that at the initial stages of limb chondrogenesis core protein gene expression is controlled primarily at the transcriptional level.

Aggrecans↗

PubMed1988

In situ hybridization analysis of the expression of the type II collagen gene in the developing chicken limb bud.

In situ hybridization with [32P]- or [35S]-labeled double-stranded DNA or single-stranded RNA probes was used to investigate the temporal and spatial distribution of cartilage-characteristic type II collagen mRNA during embryonic chick limb development and cartilage differentiation in vivo. When the type II collagen probes were hybridized to sections through embryonic limb buds at the earliest stages of their development (stages 18-25), an accumulation of silver grains representing type II collagen mRNA first became detectable in the proximal central core of the limb coincident with the prechondrogenic condensation of mesenchymal cells that characterizes the onset of cartilage differentiation. At later stages of development (stage 32; 7 days) intense hybridization signals with the type II collagen probes were localized over the well differentiated cartilage rudiments, whereas few or no silver grains above background were observed over the non-chondrogenic tissues. In contrast, sections hybridized with a probe complementary to mRNA for the alpha 1 chain of type I collagen exhibited an intense hybridization signal over the perichondrium and little or no signal over the cartilage primordia. At all stages of development examined, [32P]-labeled double-stranded DNA probes or single-stranded RNA probes labeled with either [32P] or [35S] provided adequate hybridization signals. Several experimental protocols were employed to control for the potential cross-hybridization and non-specific hybridization of the type II collagen probes. These included the utilization of labeled noncomplementary "sense-strand" type II collagen RNA as a control probe for nonspecific background, and prehybridization with a large excess of appropriate unlabeled RNA to block sequences in heterologous collagen RNAs that might cross-hybridize to the specific labeled probe.

Animals↗

PubMed1986

Exon/intron organization of the chicken type II procollagen gene: intron size distribution suggests a minimal intron size.

Overlapping genomic clones have been isolated that contain the alpha chain and COOH-terminal propeptide coding regions of the chicken type II procollagen gene. All type II procollagen exon sequences present in these clones have been identified and mapped by DNA sequencing. These include 43 exons coding for the alpha-chain triple helix, 1 exon coding for the junction between the COOH-terminal propeptide and the alpha-chain region, and 3 exons coding for the COOH-terminal propeptide and 3' noncoding sequences. With the exception of one additional intron between 2 exons coding for amino acids 568-585 and 586-603, exon-intron boundaries have been conserved when compared with genes for all other characterized genes for fibrillar collagens. The chicken type II procollagen gene differs from most other collagen genes in having introns of considerably smaller average size. The size distribution of the introns suggests that approximately equal to 80 base pairs may be a minimal functional size for introns in this gene. This size of intron may be necessary in a gene with a very large number of small exons to prevent aberrant splicing from removing exon sequence together with intron sequence.

Animals↗

PubMed1985

Quantitation of type II procollagen mRNA levels during chick limb cartilage differentiation.

A single-stranded DNA probe complementary to chicken type II procollagen mRNA has been used to quantitate levels of that mRNA present in chicken limb mesenchyme during cartilage differentiation. Excess labeled probe prepared from a cDNA template cloned in M13mp9 was hybridized to completion to increasing amounts of total RNA and assayed by protection from S1 nuclease digestion. Estimates of the absolute levels of type II procollagen RNA were determined using the M13mp9 template containing the coding strand as a standard. RNA complementary to the probe increased from 20 copies per diploid genome in stage 24 limb to approximately 2000 copies per diploid genome in stage 24 limb mesenchyme which had differentiated to cartilage in culture. Similar levels were found in cartilage from stage 31 limb. Sternal cartilage from 17-day embryos contained approximately 10,000 copies per diploid genome suggesting that the level of expression of this gene is different in limb growth cartilage compared with sternal cartilage. Low but detectable levels of RNA complementary to the probe were observed in limb at stages 20-24. Since a large fraction of the type II procollagen RNA in these early limbs is associated with polysomes, the type II procollagen gene appears to be expressed at a low level prior to phenotypic differentiation and prior to the accumulation of immunologically detectable levels of type II collagen.

Animals↗

PubMed1985

Copy number of the chicken type II procollagen gene.

The copy number of the type II procollagen gene has been determined by quantitative hybridization of cloned DNA to Southern blots of total cellular DNA from chicken. Two different DNA probes have been used. One contains the 289 base pair exon coding for the junction between the triple helical domain and the carboxyl-terminal propeptide and the other contains an 162 base pair exon coding for amino acids 712-765 of the triple helical domain. Both fragments hybridize to single bands present at one copy per haploid genome in genomic DNA from chicken liver or sternal cartilage after these DNAs are cleaved with either HinfI of PstI. When the hybridization stringency and washing conditions were modified sufficiently to stabilize hybridization of heteroduplexes with up to 31% mismatch, no additional hybridizing fragments were detected. These results show that there is not a second copy of this gene or a pseudogene which is more closely related to the type II procollagen gene than the alpha 1 type I procollagen gene. These studies suggest that the cartilage 3 alpha collagen chain, which appears to be very similar to the alpha 1 (II) collagen chain, is encoded by the type II procollagen gene but is differently processed than the major type II gene product, and that the alpha 1 (II) Minor chain identified in bovine type II collagen preparations most likely represents an allelic form of type II collagen rather than a product of a different gene.

Animals↗

PubMed1985

Structure of the type II collagen gene.

In summary, the exon/intron structure of the chicken type II collagen gene is identical with that of the chicken alpha 2(I) collagen gene and differs at only one known position from the human and mouse alpha 1(I) genes. However, the chicken type II gene is different from the chicken alpha 2(I) gene in that it is considerably shorter because of a much smaller average intron size and in that the G+C composition of the introns is much higher. The codon usage of the type II genes also shows characteristic differences. There is a single copy of the chick type II gene per haploid genome.

Animals↗

PubMed1984

Structure and sequence of the chicken type II procollagen gene. Characterization of the region encoding the carboxyl-terminal telopeptide and propeptide.

The DNA sequence of two overlapping cDNA clones and a genomic lambda clone covering the region coding for 288 amino acids at the COOH terminus of the chicken type II procollagen gene is reported. This region consists of 4 exons coding for the last 15 amino acids of the triple helical domain and 273 amino acids which correspond to the COOH-terminal telopeptide and COOH-terminal propeptide. The sequence, base composition, and codon usage of this region of the type II procollagen gene show particularly high similarity to those of the chicken alpha 1(I) procollagen gene and differ from those of the alpha 2(I) and alpha 1(III) gene sequences. Two DNA tracts of low sequence similarity were observed. One of these regions spans the telopeptide and COOH-terminal propeptidase cleavage site, although 4-5 amino acids at the actual cleavage site are conserved compared with the alpha 1(I) and alpha 2(I) genes. A region of unusually high nucleotide sequence conservation is present in exon 2 (amino acids 171c - 186c ) consisting of approximately 45 nucleotides with only one or two base substitutions compared with the other procollagen genes. Within this conserved sequence is a site for carbohydrate attachment. The 3' nontranslated sequence of the type II procollagen mRNA is longer than that of either the alpha 1(I) or alpha 2(I) mRNA and contains several unusual long tracts consisting primarily of one or two bases. Although the canonical site for polyadenylation is not present, two related sequences, AACAAA and ATATAAA , are present 32 and 41 bases preceding the end of the major RNA species. The exon/intron structure of the type II procollagen gene is similar to that of other collagen genes which have been described. This DNA sequence provides the first extensive report of the amino acid sequence of chicken type II procollagen.

Amino Acid Sequence↗

PubMed1984

Isolation and characterization of genomic clones corresponding to the human type II procollagen gene.

A recombinant human DNA library was screened using probes corresponding to the chick alpha 1 (II) procollagen gene. This resulted in the isolation of 2 different genomic clones, LgHCol(II)a and LgHCol(II)b. LgHCol(II)a was identified as corresponding to the alpha 1(II) gene by comparative hybridization and DNA sequence analysis. DNA sequence established that LgHCol(II)a extends at least from amino acid 694 of the triple helix through 54 amino acids of the COOH-propeptide. Hybridization with a probe containing only the exon at the 3' end of the chicken gene suggests that the clone contains the 3' end of the human gene. Thus LgHCol(II)a contains approximately 40% of the coding sequences of the human type II collagen gene.

Amino Acid Sequence↗

PubMed1984

Cell-free translation of messenger RNA for chondroitin sulphate proteoglycan core protein in rat cartilage.

Total RNA was extracted from the cartilage tissues rat Swarm chondrosarcoma, neonatal-rat breastplate and embryonic-chicken sterna and translated in wheat-germ cell-free reactions. The core protein of the chondroitin sulphate proteoglycan subunit was identified among translation products of rat mRNA by its apparent Mr of 330 000 and by its immunoprecipitation with specific antisera prepared against rat or chicken proteoglycan antigens. The apparent Mr of the rat proteoglycan core protein is 8000-10000 less than that of the equivalent chicken cartilage core-protein product.

Animals↗

PubMed1984

Construction and partial characterization of recombinant cDNA clones for chicken type I collagen messenger RNAs.

Messenger RNAs for the alpha 1 and alpha 2 chains of type I procollagen were partially purified from total embryonic chicken calvaria using gel chromatography on Sepharose 4B and used to construct recombinant cDNA clones corresponding to both mRNAs. Restriction site mapping, nucleotide sequencing and hybridization to RNA blots were used to show that clones pCAL1 and pCAL2 contain inserted sequences corresponding to the mRNAs for chicken alpha 1 and alpha 2 procollagen chains, respectively.

Animals↗

PubMed1983

Identification of genomic DNA coding for chicken type II procollagen.

A segment of the type II procollagen gene has been isolated by screening a lambda Charon 4A library containing fragments of chicken genomic DNA. The specific clone, LgCOL(II), was selected by hybridization using overlapping inserts from two cDNA clones which are specific for a cartilage procollagen (Vuorio, E., Sandell, L., Kravis, D., Sheffield, V. C., Vuorio, T., Dorfman, A., and Upholt, W. B. (1982) Nucleic Acids Res. 10, 1175-1192). DNA sequence analysis of LgCOL(II) in the COOH-telopeptide region of the protein, shows conclusively that this DNA corresponds to the chicken type II procollagen gene. Hybridization of cDNA probes to restriction fragment gel blots together with DNA sequence analysis have established the orientation and position of the procollagen gene within the lambda Charon 4A vector and indicate that LgCOL(II) contains approximately 6 kilobase pairs of the type II procollagen gene plus additional DNA flanking the 3' end of the gene. DNA sequence analysis shows directly that LgCOL(II) contains DNA sequences identical with those in the cDNA clones. The portion of the gene from amino acid 578 of the triple helical region to the COOH-terminal end of the protein (approximately 700 amino acids) is contained within the clone, corresponding to approximately 50% of the amino acid coding sequence of the gene. This region of the chicken alpha 1 (type II) procollagen gene is encoded within a shorter segment of the chicken genome than is the corresponding region of the alpha 2(type I) procollagen gene.

Amino Acid Sequence↗

PubMed1982

Construction and partial characterization of two recombinant cDNA clones for procollagen from chicken cartilage.

Type II procollagen mRNA has been partially purified from embryonic chick sternal cartilage by guanidine hydrochloride extraction, sucrose gradient sedimentation and Sepharose 4B chromatography. Double stranded cDNA was synthesized using AMV reverse transcriptase and E. coli DNA polymerase I, tailed using terminal transferase and inserted into the Pst I site of pBR322. Two putative type II procollagen cDNA clones have been characterized. Both plasmids hybridize to 2 sternal RNA species, a major species of 5.3 kb and a minor species of 7 kb. These RNAs are present in total RNA from sterna and differentiated limb bud cultures but are not detected in RNA from stage 24 limb bud which has not yet differentiated to cartilage or in RNA from calvaria. The time of appearance of these RNAs during the differentiation of limb mesenchyme in culture parallels the appearance of translatable type II procollagen mRNA.

Animals↗