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R P Boot-Handford

Publications and source records attributed to R P Boot-Handford.

At least 37 records · Page 2Linked to original sources
PubMed1995

Sequence comparison of three mammalian type-X collagen promoters and preliminary functional analysis of the human promoter.

The mechanism(s) controlling the specific expression of the type-X collagen (COL10A1)-encoding gene in the growth plate of developing long bones is not known. In preparation for identifying and characterizing the 5'-regulatory sequences and transcription factors which control mammalian Col10a1 gene expression, we have isolated and sequenced the first exon and 5' flanking promoter regions of bovine Col10a1. Sequence comparisons, including those previously published for mouse Col10a1, highlighted a number of conserved domains within the promoter and upstream elements. Reporter cat gene (encoding chloramphenicol acetyltransferase, CAT) constructs containing 5'-regulatory sequences of human COL10a1 (hCOL10a1) were transfected into primary cultures of foetal bovine growth plate chondrocytes producing COL10A1 and non-producing epiphyseal cartilage chondrocytes. Constructs containing up to 900 bp of promoter sequence exhibited low levels of CAT production in expressing cells and non-expressing cells. Addition of a further 1.5 kb of upstream sequence resulted in a dramatic increase in CAT production in expressing cells only. The results demonstrate the presence of enhancer-like elements between 900 bp and 2.4 kb upstream of the transcription start point(s) of hCOL10a1, which is distinctly different from that reported for the chick.

Animals↗
PubMed1994

Amino acid substitutions of conserved residues in the carboxyl-terminal domain of the alpha 1(X) chain of type X collagen occur in two unrelated families with metaphyseal chondrodysplasia type Schmid.

Type X collagen is a homotrimeric, short-chain, nonfibrillar extracellular-matrix component that is specifically and transiently synthesized by hypertrophic chondrocytes at the sites of endochondral ossification. The precise function of type X collagen is not known, but its specific pattern of expression suggests that mutations within the encoding gene (COL10A1) that alter the structure or synthesis of the protein may cause heritable forms of chondrodysplasia. We used the PCR and the SSCP techniques to analyze the coding and upstream promoter regions of the COL10A1 gene in a number of individuals with forms of chondrodysplasia. Using this approach, we identified two individuals with metaphyseal chondrodysplasia type Schmid (MCDS) with SSCP changes in the region of the gene encoding the carboxyl-terminal domain. Sequence analysis demonstrated that the individuals were heterozygous for two unique single-base-pair transitions that led to the substitution of the highly conserved amino acid residue tyrosine at position 598 by aspartic acid in one person and of leucine at position 614 by proline in the other. The substitution at residue 598 segregated with the phenotype in a family of eight (five affected and three unaffected) related persons. The substitution at residue 614 occurred in a sporadically affected individual but not in her unaffected mother and brother. Additional members of this family were not available for further study. These results suggest that certain amino acid substitutions within the carboxyl-terminal domain of the chains of the type X collagen molecule cause MCDS. These amino acid substitutions are likely to alter either chain recognition or assembly of the type X collagen molecule, thereby depleting the amount of normal type X collagen deposited in the extracellular matrix, with consequent aberrations in bone growth and development.

Alleles↗
PubMed1993

Intron-exon structure, alternative use of promoter and expression of the mouse collagen X gene, Col10a-1.

The entire mouse collagen X gene (Col10a-1) has been isolated. The gene is composed of three exons and two introns spanning 7.0 kb of the DNA sequence. Exons 2 and 3 together encode 15-bp of 5' untranslated sequence, a 2040-bp open reading frame and an 895-nucleotide 3' non-coding region. In the 5' flanking region of the gene, two consensus TATA-box sequences were found. Identification of the first exon by ribonuclease-protection assays and the determination of the 5' end of Col10a-1 mRNA transcripts by primer-extension analyses show that the more 3' TATA box is probably predominantly used and that there are at least three transcription start sites in the exon 1 sequence 3' to this, resulting in 5' untranslated regions of 78, 77 and 55 nucleotides. By means of rapid amplification of cDNA ends by polymerase chain reaction, an additional mRNA species was detected which overlapped the other Col10a-1 transcripts, including the 3' TATA box sequence, giving a 5' untranslated sequence of approximately 235 bases. This latter transcript starts approximately 20 bp 3' to the more 5' TATA box. The data suggest alternative use of promoters and transcription starts for the Col10a-1 gene. Comparison of the combined nucleotide and deduced amino acid sequences of exons 2 and 3 with chicken, bovine and human collagen X genes, showed a high degree of similarity indicating conservation of this gene throughout evolution. Mouse Col10a-1 mRNA was shown to be approximately 3.0 kb and the pepsinized protein, as detected by SDS/PAGE, was approximately 45 kDa. The mRNA and protein sizes correlate with that predicted by the open reading frame. Reverse-transcription polymerase chain reaction assays indicate that the mouse collagen X gene is first expressed at 13.5 days post coitum, temporally preceding the onset of endochondral ossification. In agreement with the generally accepted association of type-X collagen with endochondral ossification, in situ hybridization analyses indicate that Col10a-1 mRNA are restricted to the hypertrophic regions of growth cartilage.

Amino Acid Sequence↗
PubMed1992

The fibrillar collagens, collagen VIII, collagen X and the C1q complement proteins share a similar domain in their C-terminal non-collagenous regions.

A sequence comparison of the C-termini of collagens X, VIII, the collagen-like complement factor C1q, and the fibrillar collagens showed a conserved cluster of aromatic residues. This conserved cluster was in a domain of approximately 130 amino acids that exhibited marked similarities in hydrophilicity profiles between the different collagens, despite a low level of sequence similarity. These data suggest that the 'collagen X-like family' and the fibrillar collagens contain a domain within their C-termini that adopts a common tertiary structure, and that a conserved cluster of aromatic residues in this domain may be involved in C-terminal trimerization.

Amino Acid Sequence↗
PubMed1991

The human collagen X gene. Complete primary translated sequence and chromosomal localization.

We report on the complete primary translated sequence of human alpha 1(X) collagen, deduced from a genomic clone, and the chromosomal localization of the human collagen X gene. The primary translated product of human collagen X is encoded by two exons of 169 bp and approx. 2940 bp. The 169 bp exon encodes 15 bp of 5'-end untranslated sequence, 18 amino acid residues (54 bp) of signal peptide and 33 1/3 amino acid residues (100 bp) of the N-terminal non-collagenous domain. The 2940 bp exon encodes 4 2/3 amino acid residues (14 bp) of the N-terminal non-collagenous domain, the complete triple-helical domain of 463 amino acid residues (1389 bp), the complete C-terminal non-collagenous domain of 161 amino acid residues (483 bp) and 1054 bp of 3'-end untranslated sequence up to and including a potential cleavage/polyadenylation signal. The size of the intron separating the two exons, as estimated by partial sequencing and Southern-blot analyses, is approx. 3200 bp. By a combination of somatic cell hybrid screening and hybridization in situ the human collagen X gene (COL10A1) has been assigned to the distal end of the long arm of chromosome 6 at the locus 6q21-6q22.3.

Amino Acid Sequence↗
PubMed1991

Evidence for shared epitopes within the 'naked' protein domains of human mucus glycoproteins. A study performed by using polyclonal antibodies and electron microscopy.

Polyclonal antibodies were raised in rabbits towards reduced subunits of human cervical mucus glycoproteins. The reduced subunits almost completely inhibited the antiserum, whereas the intact mucins and the heavily glycosylated fragments obtained after digestion of reduced subunits with trypsin (T-domains) caused only partial inhibition. Periodate oxidation of intact mucins, reduced subunits and T-domains caused no effect on the antibody response, and fragments obtained by more extensive proteolysis of the reduced subunits (P-domains) showed no inhibitory activity. By using electron microscopy, antibodies from T-domain-adsorbed antisera were revealed as bound to cervical mucin reduced subunits, either directly or with colloidal gold-Protein A. Binding sites (100-150 nm apart) were observed at the ends and at internal positions of the reduced subunits. We conclude that the antibodies do not recognize carbohydrate structures but are directed to two kinds of protein epitopes, one shared by whole mucins, reduced subunits and T-domains, and the other specific to the reduced subunit fragment. The latter epitopes are 'cryptic' and are probably shielded within folded protein domains stabilized by disulphide bonds. Human bronchial, cervical, gastric and salivary mucus glycoproteins share some of these cryptic epitopes.

Antibodies↗
PubMed1991

Isolation of cDNAs encoding the complete sequence of bovine type X collagen. Evidence for the condensed nature of mammalian type X collagen genes.

The complete primary structure of the bovine alpha 1(X) collagen chain was determined by nucleotide sequencing of cDNA clones. The overlapping cDNA clones encode 3144 bp with a 5'-terminal untranslated region of 148 bp, a 2025 bp reading frame and a 3'-terminal untranslated region of 971 bp. This represents the first complete sequence of a mammalian type X collagen cDNA and has allowed a number of informative comparisons to be made with the previously published chick alpha 1(X) sequence. The primary translation products of both bovine and chick type X collagen are 674 amino acid residues in length and there is a 73.3% identity at the amino acid level (67.8% at the base level). Sequence analyses reveal that the greatest degree of identity between the two species occurs within the triple-helical domain and the C-terminal non-collagenous domain, whereas the identity within the N-terminal non-collagenous domain is markedly lower. The interchain disulphide-bonding observed previously within the triple helix of bovine type X collagen is explained by the presence of two cysteine residues within an imperfection of the triple-helical domain encoded by -Gly-Xaa-Cys-Xaa-Yaa-Cys-Xaa-Yaa-Gly-. Southern blot analyses of bovine genomic DNA demonstrate that the bovine type X collagen gene is likely to have a condensed structure, similar to that of the chick, with at least 1.3 kb of the coding sequence being contained within one exon.

Amino Acid Sequence↗
PubMed1991

Distribution of type X collagen mRNA in normal and osteoarthritic human cartilage.

The function of collagen X, a unique homotrimer synthesised by hypertrophic chondrocytes, is not known but its localisation and transient expression at sites of calcification suggest that it is likely to be associated with events in the early stages of endochondral bone formation. Osteoarthritis (OA) is a disorder characterised by new bone formation but the role of type X collagen in its pathogenesis is unclear. A 700-bp restriction fragment encoding most of the C-terminal non-collagenous domain and part of the 3'-untranslated region of the human collagen X gene has been used for in situ hybridisation studies on human OA joints removed from hip and knee replacement operations and the results compared with immunohistochemical localisation of type X collagen gene product. Collagen X gene expression was detected in chrondrocytes present in OA tissue in areas where there appeared to be a re-initiation of the endochondral bone formation process including osteophytes and areas of subchondral bone sclerosis.

Antibody Specificity↗
PubMed1990

Molecular composition of type VI collagen. Evidence for chain heterogeneity in mammalian tissues and cultured cells.

The chain composition and relative abundance of type VI collagen synthesized by cells cultured from foetal bovine nuchal ligament and skin were compared with those of the type VI collagen present in these foetal tissues. Immunoprecipitation of intact collagen VI from medium and cell layers of nuchal ligament fibroblasts and skin fibroblasts at confluence revealed collagen type VI molecules with a chain composition consistent with an [alpha 1(VI)alpha 2(VI)alpha 3(VI)] monomeric assembly. Maintenance of cells in a post-confluent quiescent state promoted a marked phenotypic change in these ratios, with increased concentrations of assemblies composed of equimolar ratios of alpha 1(VI) and alpha 2(VI) chains detected in the medium of these cultures. Analysis of steady-state concentrations of mRNA for alpha 1(VI) and alpha 2(VI) chains revealed these species to be present in increased abundance at post-confluence in all the cultures, but no corresponding increase was observed in the alpha 3(VI) mRNA. In order to assess the physiological significance of these observations, the chain composition of the collagen VI content of the corresponding foetal tissues was assessed by Western blotting after extraction in guanidinium isothiocyanate under reducing conditions. Extracts of nuchal ligament revealed a collagen VI chain composition consistent with a heterotrimeric chain assembly. In contrast, the skin extracts revealed an abundance of alpha 1(VI) and alpha 2(VI) chains with only traces of the alpha 3(VI) chain detected. Increased equimolar concentrations of the alpha 1(VI)-chain and alpha 2(VI)-chain mRNAs in skin again reflected the increased concentrations of these polypeptide chains. Type VI collagen was present in greater abundance both in the nuchal ligament and in the corresponding nuchal-ligament fibroblast cultures. The results indicate that the chain composition of type VI collagen is subject to modulation at the level of transcription as a result of variations in the proliferative state of the cells, and demonstrate that different isoforms of collagen VI occur in foetal development.

Animals↗
PubMed1990

Serum esterase activities and hyperlipidaemia in the streptozotocin-diabetic rat.

The levels of activity of four serum esterases were measured in control and streptozotocin-diabetic rats for a period of 6 months. Pseudocholinesterase activity was significantly elevated in the diabetic rats at all time points tested, reaching 250% of the control activity at 6 months. Levels of paraoxonase activity progressively decreased with time in the diabetic rats, being 36% lower than in controls at 6 months. No significant differences in either serum arylesterase or carboxylesterase activity between control and diabetic rats were observed.

Animals↗
PubMed1990

Thrombospondin gene expression by endothelial cells in culture is modulated by cell proliferation, cell shape and the substratum.

Endothelial cells plated on the surface of a two-dimensional substratum (gelatin-coated dishes, dishes coated with native type I collagen or collagen gels) form a cobblestone monolayer at confluence, whereas cells plated within a three-dimensional gel matrix elongate into a sprouting morphology and self-associate into tube-like structures. In this study, we have compared the synthesis of thrombospondin by quiescent endothelial cells displaying (a) the same morphological phenotype (cobblestone) on different substrata (gelatin and collagen) and (b) different morphological phenotypes (cobblestone and sprouting) on the same substratum (collagen). We demonstrate that thrombospondin is a major biosynthetic product of confluent, quiescent cells cultured on dishes coated with either gelatin or collagen, and that the synthesis of this protein is markedly decreased when cells are plated on or in three-dimensional collagen gels. Moreover, we demonstrate that cells plated in gel (sprouting) secrete less thrombospondin than do cells plated on the gel surface (cobblestone). The regulation of thrombospondin synthesis is reversible and occurs at the level of transcription, as steady-state mRNA levels for thrombospondin decrease in a manner comparable with the levels of protein secreted by these cells. We also show that mRNA levels for laminin B2 chains are increased when cells are cultured on and in collagen gels compared with on gelatin-coated dishes, suggesting that the syntheses of thrombospondin and laminin are regulated by different mechanisms. When cells are cultured on gelatin- or collagen-coated dishes, thrombospondin gene expression is directly proportional to the proliferative state of the cultures. By contrast, the synthesis of thrombospondin by cells cultured on collagen gels remains at equally low levels whether they are labelled when they are sparse and rapidly proliferating or when they are confluent and quiescent. Fibronectin synthesis was found to increase with increasing confluency of the cells plated on all three substrata. These results demonstrate that thrombospondin gene expression is modulated by cell shape, cell proliferation and the nature of the substratum used for cell culture.

Animals↗