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C D Boyd

Publications and source records attributed to C D Boyd.

At least 55 records · Page 3Linked to original sources
PubMed1995

Elastin gene mutations in transgenic mice.

We have constructed several rat tropoelastin minigene recombinants encoding the complete sequence of rat tropoelastin, two isoforms of rat tropoelastin and a truncated tropoelastin lacking the domains encoded by exons 19-31 of the rat gene. Coding and non-coding domains in all these recombinants were placed under the transcriptional control of 3 kb of the promoter domain of the rat tropoelastin gene. These minigenes were used to prepare a total of 28 separate founder lines of transgenic mice. A species-specific reverse-transcriptase polymerase chain reaction (RT-PCR) assay was established to demonstrate the synthesis of rat and mouse tropoelastin mRNA in several tissues obtained from both neonatal and adult transgenic mice. Thermolytic digestion of insoluble elastin isolated from several neonatal mouse tissues revealed the presence of rat tropoelastin peptides in progeny from all those founder mice in which detectable levels of rat tropoelastin mRNA were noted. Phenotypic and histopathological assessment of transgenic and non-transgenic animals revealed the development of two diverse elastic tissue disorders. The progeny of two separate founder lines overexpressing the rat tropoelastin isoform lacking exon 33, developed an emphysematous phenotype in early adulthood. In contrast, transgenic mice, in which expression of the truncated rat tropoelastin minigene lacking exons 19-31 had been observed, died of a ruptured ascending aortic aneurysm. Tropoelastin gene mutations, therefore, will result in heritable disorders of elastic tissue. Moreover, different mutations in the tropoelastin gene will be responsible for very different abnormalities in elastic tissue function.

Animals↗
PubMed1995

Elastin morphology in normal and obstructed guinea-pig bladders. Localization of elastin to the trigone.

Elastin is the major component of elastic fibers in aortic tissue and has been suggested to be important for the stretch and recoil of the bladder. We have mapped the histologic distribution of elastin throughout the entire guinea-pig bladder as well as changes in the distribution of elastin during bladder-outlet obstruction. We used an animal model in which a jeweler's ring is placed around the urethra of the young guinea pig. As the animal grows, outlet obstruction is gradually induced, and the bladder gradually increases in total weight to as much as 8-fold that of controls. We localized the elastin histologically in normal and obstructed bladders by serially sectioning entire bladders and staining the sections with an elastin-specific stain, Verhoff-van Gieson stain (VVG). Surprisingly, we found that the elastin was predominantly localized only to the trigone of the bladder and that the elastin in this area was increased during obstruction. These results are consistent with the predominantly mesodermal embryologic origin of the trigone, since mesodermal tissues are more often associated with elastin expression.

Animals↗
PubMed1995

The size heterogeneity of human lysyl oxidase mRNA is due to alternate polyadenylation site and not alternate exon usage.

We have isolated the entire gene coding for human lysyl oxidase. Coding and untranslated domains of human lysyl oxidase mRNA were found in 7 exons, distributed throughout approximately 14 kb of human genomic DNA. The appearance of exon sequences in lysyl oxidase mRNA in several human tissues was determined using a reverse transcriptase - PCR assay. In contrast to a previous report, this analysis has unambiguously shown that the size heterogeneity of lysyl oxidase mRNA was not due to alternate usage of any of the exons of the lysyl oxidase gene. Moreover, DNA sequence analysis of the entire 3.8 kb 3'-untranslated region (UTR) within exon 7 revealed multiple poly-adenylation sites which were shown to be differentially expressed in human skin fibroblasts. This differential usage of polyadenylation sites within the 3'-UTR explains the appearance of multiple lysyl oxidase mRNAs of different sizes.

Adenosine Monophosphate↗
PubMed1995

Extensive alternate exon usage at the 5' end of the sheep tropoelastin gene.

Several overlapping cDNA clones were isolated from a lambda gt10 cDNA library constructed using poly A+ RNA from neonatal sheep lung. DNA sequence analysis of these cDNA recombinants revealed the complete derived amino acid sequence of sheep tropoelastin. A comparison of DNA sequences from individual sheep tropoelastin cDNA also confirmed the presence of several tropoelastin mRNA isoforms in neonatal lung tissue. Coding domains corresponding to exons 13, 14 and 33 were present in several of the sheep tropoelastin cDNA fragments but absent in others. The relative amount of alternate usage of these exons was quantitated by polymerase chain amplification. In confirmation of previous studies in other mammalian species, extensive alternate usage of exon 33 was observed in total RNA isolated from aorta, nuchal ligament and pulmonary artery from neonatal sheep. In striking contrast to all previous studies, however, exons 13 and 14 were shown to be subject to almost the same level of alternate usage as exon 33 in all three neonatal sheep tissues examined.

Alternative Splicing↗
PubMed1995

Ultraviolet radiation increases tropoelastin accumulation by a post-transcriptional mechanism in dermal fibroblasts.

Chronically sun-damaged human skin is characterized by dermal connective tissue damage that includes the massive accumulation of abnormal elastic fibers. The content of elastin, the major protein component of elastic fibers, is increased two- to sixfold in sun-damaged skin. The aim of this study was to determine the mechanism responsible for the increase in elastin levels after ultraviolet (UV) irradiation. Confluent cultures of normal dermal fibroblasts were irradiated with 4.5 mJ/cm2 of UVB; sham-treated cells served as the control group. The accumulation of tropoelastin was determined at 5 d after treatment by measuring the incorporation of 14C-proline into radiolabeled tropoelastin isolated from cell layers and media. UV irradiation increased radiolabeled tropoelastin accumulation approximately twofold without affecting DNA content, the total amount of radiolabeled protein, or tropoelastin secretion. Moreover, the steady-state levels of tropoelastin mRNA, as determined by slot blot hybridizations, were unaffected by UV treatment. However, the translation of tropoelastin mRNA was increased when total RNA from irradiated cells was used in cell-free translation experiments. These results suggest that altered translational efficiency may account for the increase in tropoelastin accumulation after UV irradiation. In support of this hypothesis, nucleotide sequences were derived from tropoelastin mRNA isolated from UV-irradiated and nonirradiated dermal fibroblasts. Almost a 12% substitution rate was observed in nucleotide sequences derived from the 3' untranslated region of tropoelastin mRNA from the UV-treated cells. In contrast, a coding domain of tropoelastin did not contain base-substitution mutations. These multiple base substitutions in a noncoding domain of tropoelastin mRNA may be responsible for the post-transcriptional increase in tropoelastin accumulation after UV irradiation.

Base Sequence↗
PubMed1994

Use of an intron polymorphism to localize the tropoelastin gene to mouse chromosome 5 in a region of linkage conservation with human chromosome 7.

The complete coding sequence for mouse tropoelastin was obtained from overlapping reverse transcriptase polymerase chain reaction (PCR) amplimers. These cDNA fragments were derived from mouse tropoelastin mRNA using PCR oligomers complementary to conserved domains within rat tropoelastin mRNA. A comparison of coding domains of mouse and rat tropoelastin mRNA revealed a greater than 93% homology at the nucleotide level and over 96% similarity in the predicted amino acid sequence. PCR primers complementary to regions of the mouse tropoelastin mRNA were used to define a novel intron length polymorphism (ILP) within intron 8 of the mouse tropoelastin gene (Eln). This ILP proved to be informative in an interspecific backcross in which genomic DNA samples from 75 backcross mice were used to map the tropoelastin gene to a position in the distal half of mouse chromosome 5. The linkage and genetic distances between Eln and the closest molecular markers used in this study are centromere-D5Mit95, D5Mit96-6.7 cM-Gus, Eln-4.0 cM-Zp3-telomere.

Amino Acid Sequence↗
PubMed1993

A restriction fragment length polymorphism results in a nonconservative amino acid substitution encoded within the first exon of the human lysyl oxidase gene.

A cDNA covering most of the coding sequence for human lysyl oxidase was used to screen, by Southern blot analysis, genomic DNA from circulating lymphocytes obtained from unrelated, apparently normal individuals. A heritable restriction fragment length polymorphism (RFLP) within a PstI restriction site was detected in 36% of individuals screened (a total of 72 chromosomes were analyzed). The major allele was represented as a 1.7-kb PstI restriction fragment. The minor allele was detected as 1.4 and 0.3kb restriction fragments. Lambda phage-DNA recombinants were isolated from a human lung fibroblast genomic DNA library using the human lysyl oxidase cDNA clone. DNA sequence analysis of several selected phage recombinants revealed that 83% of the coding sequence of lysyl oxidase was localized in four separate exons. Analysis of the coding sequence within exon 1, the most 5' exon within the lysyl oxidase gene, revealed that the PstI RFLP was due to a G-->A transition resulting in a nonconservative arginine to glutamine substitution proximal to a propeptide cleavage domain encoded by exon 1 of the lysyl oxidase gene.

Alleles↗
PubMed1993

Regulation of collagen gene expression in keloids and hypertrophic scars.

The synthesis of type I and III collagens in cultured skin fibroblasts from normal skin, normal scar, hypertrophic scar, and keloids was examined. The ratio of type I/III collagen was significantly elevated in keloids compared to that in the other groups. When mRNA steady-state levels coding for alpha 1(I) procollagen were determined, it was apparent that this increase in the type I/III collagen ratio in keloids was paralleled by a specific increase in alpha 1(I) procollagen mRNA. This specific increase in alpha 1(I) procollagen mRNA in keloids was the result of increased gene expression because the transcription rate of the alpha 1(I) procollagen gene was significantly elevated in keloids, as determined by nuclear runoff transcription. The rate of transcription of the alpha 1(I) procollagen gene was also elevated in hypertrophic scars, although no concomitant increase in alpha 1(I) procollagen mRNA levels or alteration in the type I/III collagen ratio was observed. These data indicate that the rate of gene transcription of alpha 1(I) procollagen is increased in both hypertrophic scars and keloids, but only keloids exhibit increased steady-state levels of alpha 1(I) procollagen mRNA and concurrent increases in type I collagen. These results suggest that at least two distinct mechanisms, one pretranscriptional and one post-transcriptional, regulate type I collagen synthesis. It is possible, therefore, that in keloids, neither mechanism functions efficiently to down-regulate type I collagen. In hypertrophic scars, however, the post-transcriptional mechanisms are able to decrease elevated levels of mRNA coding for alpha 1(I) procollagen that result from increased transcription of the alpha 1(I) procollagen gene.

Blotting, Northern↗
PubMed1993

Alternate exon usage is a commonly used mechanism for increasing coding diversity within genes coding for extracellular matrix proteins.

Extracellular matrix proteins are a diverse family of secreted proteins and glycoproteins that are responsible for a variety of critical functions in different tissues. A large number of multiexon genes encode these proteins of the extracellular matrix. Over the last few years, it has become evident that the processing of the pre-mRNA from several of these genes involves alternative splicing. This review summarizes the known examples of alternative splicing in genes coding for the extracellular matrix and attempts to relate the increase in coding diversity generated by alternate exon usage to the function(s) of individual extracellular matrix proteins.

Alternative Splicing↗
PubMed1993

Increases in type III collagen gene expression and protein synthesis in patients with inguinal hernias.

OBJECTIVE: The aim of this study was to determine if alterations in fibrillar collagen synthesis were associated with the development of inguinal hernias. SUMMARY BACKGROUND DATA: Previous work has suggested that alterations in connective tissue accumulation may play a functional role in the development of inguinal hernias. In particular, several investigators have suggested that alterations in collagen synthesis, causally related to connective disorders such as osteogenesis imperfecta, may also be responsible for the inguinal herniation that is markedly increased in such patients. This study was undertaken therefore to study collagen synthesis in patients with inguinal hernia in the absence of any other connective tissue disease. METHODS: Skin fibroblasts from 9 patients with hernias and 15 control individuals were radiolabeled with 3H-proline. Trypsin-chymotrypsin-resistant type I and III collagens were isolated and analyzed by sodium dodecyl sulfate polyacrylamide gel electrophoresis, and recovery was quantified by laser densitometry. Steady-state levels of alpha 1(I) and alpha 1(III) procollagen mRNAs were also determined by northern and slot-blot hybridization analysis. RESULTS: The alpha 1(I)/alpha 1(III) collagen ratios were shown to be 6.3 +/- 0.34 in fibroblasts from control individuals and 3.0 +/- 0.25 in fibroblasts from patients with inguinal hernias. This statistically significant difference (p < 0.0001) was caused by an increase in the secretion of alpha 1(III) procollagen from the fibroblasts of patients with hernias. A concomitant increase in the steady-state levels of alpha 1(III) procollagen mRNA was observed in total RNA isolated from the patients' fibroblasts. CONCLUSIONS: A constitutive and systemic increase in type III collagen synthesis may result in reduced collagen fibril assembly in the abdominal wall, eventually leading to the development of herniation. Although it is not yet clear what genetic factors are responsible for the elevation in type III collagen synthesis in patients with hernias, this study represents the first attempt to define individuals with an abnormality in collagen production that may be specifically related to herniation. A clearer understanding of the possible genetic factors that influence the pathophysiology of this disease will be important to improve the treatment of patients in whom inguinal hernias develop.

Adolescent↗
PubMed1993

Quantitation of tropoelastin mRNA and assessment of alternative splicing in human skin fibroblasts by reverse transcriptase-polymerase chain reaction.

We have developed a reverse transcriptase-polymerase chain reaction (RT-PCR) assay for the quantitative measurement of levels of tropoelastin mRNA in total RNA preparations from skin fibroblasts. This method facilitates the reproducible detection of low abundance tropoelastin mRNA in the range of 10-1000 copies per cell. The procedure is based on a competitive RT-PCR assay where a tropoelastin cDNA-derived internal RNA standard is cotranscribed and coamplified together with the sample derived-endogenous target mRNA. In addition, RT-PCR of several domains of tropoelastin mRNA, followed by DNA sequence analysis of asymmetric PCR products, revealed a previously unknown pattern of alternate exon usage at the 3' end of the tropoelastin gene in human skin fibroblasts.

Alternative Splicing↗
PubMed1992

Elements of the rat tropoelastin gene associated with alternative splicing.

Multiple isoforms of tropoelastin, the soluble precursor of elastin, are the products of translation of splice-variant mRNAs derived from the single-copy tropoelastin gene. Previous data had demonstrated DNA sequence heterogeneity in three domains of rat tropoelastin mRNA, indicating alternative splicing of several exons of the rat tropoelastin gene. Rat tropoelastin genomic clones encompassing the sites of alternative splicing were isolated and sequenced. Two sites of alternative splicing identified in rat tropoelastin mRNA sequences corresponded to exons 13-15 and exon 33 of the rat tropoelastin gene. Furthermore, the variable inclusion of an alanine codon in exon 16 resulted from two functional acceptor sites separated by three nucleotides. DNA sequences flanking exons subject to alternative splicing were analyzed. These exons contained splicing signals that differed from consensus sequences and from splicing signals of constitutively spliced exons. Introns immediately 5' of exons 14 and 33, for example, lacked typical polypyrimidine tracts and had weak, overlapping branch point sequences. Further, a region of secondary structure encompassing the acceptor site of exon 13 may influence alternative splicing of this exon. These results demonstrate that multiple cis-acting sequence elements may contribute to alternative splicing of rat tropoelastin pre-mRNA.

Animals↗
PubMed1992

Abnormalities in the biosynthesis of type III procollagen in cultured skin fibroblasts from two patients with multiple aneurysms.

We examined the synthesis of collagenous proteins by cultured skin fibroblasts taken from 14 patients with an abdominal aortic aneurysm and either an aneurysm at a second site (8 patients) or a first order relative with an abdominal aortic aneurysm (6 patients). Fibroblasts were labeled with [3H] proline and, following pepsin digestion of media proteins, the ratio of type I/III collagen was examined by denaturing polyacrylamide gel electrophoresis (SDS-PAGE). With the exception of two patients, the ratio of type I/III collagen in the media of fibroblasts from aneurysm patients was similar to control values (6 controls). In two of the patients, the type I/III collagen ratio was greater than 3 standard deviations from the mean of both control ratios and those of other aneurysm patients. mRNA levels coding for type III procollagen, however, were normal in both patients. Patient #1 (ME) showed reduced type III procollagen on SDS-PAGE analysis of intracellular proteins. Intracellular and media type III procollagen levels were normal in patient #2 (HR), but media type III collagen was reduced by over 50% after digestion with a combination of trypsin and alpha-chymotrypsin for 5 minutes at 36 degrees C. Control type III collagen was only reduced after digestion at 39 degrees C. These data suggest an altered thermal stability of the type III collagen trimer synthesized by this patient, probably due to a mutation in the amino acid sequence. The data presented in this paper suggest that some forms of common abdominal aortic aneurysms may be caused by mutations in the gene coding for type III procollagen.

Aged↗
PubMed1992

The complete derived amino acid sequence of human lysyl oxidase and assignment of the gene to chromosome 5 (extensive sequence homology with the murine ras recision gene).

Lysyl oxidase catalyzes the oxidation of lysine residues to alpha-aminoadipic-delta-semialdehyde. This is the first step in the covalent cross-linking of collagen and tropoelastin and results in the formation of insoluble collagen and elastic fibers in the extracellular matrix. We have characterized the complete nucleotide sequence of human lysyl oxidase (EC 1.4.3.13) and compared the derived amino acid sequence (417-amino acids) to rat lysyl oxidase and the mouse ras recision gene (rrg). 88% of amino acids and 83% of nucleotides were conserved between human and rat lysyl oxidase. The mouse ras recision gene demonstrated 89% conservation of amino acids with human lysyl oxidase. The sequence conservation was not evenly distributed along the molecule. The carboxy terminus of the protein, which contains the putative copper binding sites and is likely to be the catalytically active domain, was more highly conserved than the amino terminus. The 89% amino acid sequence similarity between the murine ras recision gene and human lysyl oxidase suggests that they are the same gene product. Therefore, in addition to cross linking of extracellular matrix proteins, lysyl oxidase may have a direct role in tumor suppression. Northern blot analysis of poly A+RNA from cultured skin fibroblasts revealed at least three-distinct transcripts, sized 4.8 kb, 3.8 kb and 2.0 kb. In addition, using a panel of human mouse cell hybrids, the lysyl oxidase gene was assigned to human chromosome 5.

Amino Acid Sequence↗