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A Stacey

Publications and source records attributed to A Stacey.

30 records · Page 2Linked to original sources
PubMed1990

Generation of collagenase-resistant collagen by site-directed mutagenesis of murine pro alpha 1(I) collagen gene.

Collagenase (matrix metalloproteinase 1) cleaves type I, II, and III collagen helices at a specific site between Gly-Ile or Gly-Leu bonds (residues 775 and 776, P1-P1'). To understand the mechanism of collagen processing, mutations around the cleavage site have been introduced into the cloned murine pro alpha 1(I) collagen (Col1a1) gene. These mutant constructs have been transfected into homozygous Mov13 fibroblasts that do not express the endogenous Col1a1 gene due to a retroviral insertion. Secreted triple-helical type I collagens containing substitutions of Pro for Ile (position 776) (P1') were not cleaved by human rheumatoid synovial collagenase, whereas those containing substitutions of Met for Ile (position 776) were cleaved. Type I collagens containing double substitutions of Pro for Gln-774 (P2) and Ala-777 (P2') were not cleaved regardless of whether they contained the wild-type residue Ile at position 776 or the substitution of Met for Ile at position 776. The wild-type alpha 2(I) chains derived from the endogenous Col1a2 gene were also resistant to enzyme digestion when they were complexed with the mutant alpha 1(I) chains, indicating that the presence of normal alpha 1(I) sequences is critical for cleavage of the alpha 2(I) chains in the type I heterotrimer.

Amino Acid Sequence↗
PubMed1989

Collagen protein abnormalities produced by site-directed mutagenesis of the pro alpha 1(I) gene.

Site-directed mutagenesis of collagen genes offers a powerful new approach for studying structure-function relationships. The construction of engineered mutant collagen genes coding for glycine substitutions and their expression giving rise to the osteogenesis imperfecta type II phenotype in cells and transgenic mice has recently been achieved. This paper further defines the molecular abnormalities of collagen and bone pathology resulting from the expression of the mutant genes.

Animals↗
PubMed1988

Structure of a full-length cDNA clone for the prepro alpha 1(I) chain of human type I procollagen.

A full-length cDNA clone for the human prepro alpha 1(I) chain of type I procollagen was characterized. Nucleotide sequencing of the first 1500 nucleotide residues of the 5'-end of the cDNA clone provided 729 nucleotide residues and the codons for 243 amino acid residues not previously defined from any species. The data made it possible, for the first time, to compare completely codon usage for the human alpha 1(I) and alpha 2(I) chains.

Amino Acid Sequence↗
PubMed1988

Perinatal lethal osteogenesis imperfecta in transgenic mice bearing an engineered mutant pro-alpha 1(I) collagen gene.

Substitutions of single glycine residues of alpha 1(I) collagen have previously been associated with the inherited disease osteogenesis imperfecta type II. Transgenic mice bearing a mutant alpha 1(I) collagen gene into which specific glycine substitutions have been engineered show a dominant lethal phenotype characteristic of the human disease, and demonstrate that as little as 10% mutant gene expression can disrupt normal collagen function.

Amino Acid Sequence↗
PubMed1987

Differential expression in murine somatic and germinal tissues of transcripts homologous to an abundant embryonal-carcinoma-cell mRNA.

EC1, a clone from a cDNA library of embryonal-carcinoma (EC) cells, was used to study the expression of the gene(s) homologous to this sequence in murine germ cells and somatic tissues. Northern-blot analysis was used to determine the size and relative abundance of ECl transcripts in EC cells, ovary, testis, liver, spleen, brain, thymus, and bone marrow. The stage-specific expression of EC1-related transcripts in meiotic cells was examined in enriched populations of testicular cells (meiotic prophase spermatocytes, early and late spermatids, and residual bodies). All tissues and cells examined contained mRNAs of approximately 1.3 and approximately 3.6 kb which were homologous to EC1. In RNA from liver and thymus, a faint, lower-molecular-weight transcript of 0.75 kb was seen. A unique pattern of expression was observed in male germ cells. In cells that had completed meiosis, two additional transcripts of 1.0 and 4.4 kb were visualized, as well as high levels of expression of the 1.3- and 3.6-kb species. Only the 1.3- and 3.6-kb transcripts were detected in ovarian RNAs. The high level and stage specificity of expression of EC1-related mRNAs in germ cells suggest that this gene may be important in meiosis or in germ-cell maturation.

Animals↗
PubMed1987

A temperature-sensitive mutant of the myeloproliferative sarcoma virus, altered by a point mutation in the mos oncogene, has been modified as a selectable retroviral vector.

The myeloproliferative sarcoma virus (MPSV) is a mos-oncogenic retrovirus which induces an acute myeloproliferative disease in adult mice. The isolation and molecular cloning of two mutants of MPSV temperature sensitive (ts) for mos transformation (Kollek et al., J. Virol. 50:717-724, 1984) have been described previously. In this report, we describe the biological activity of these clones, the molecular basis of the ts lesion of one clone, and the construction of a selectable vector based on the MPSV ts genome. Both molecular clones, ts159 and ts124, proved to have retained the ts phenotype, the former being tighter for the induction and maintenance of the transformed phenotype. A single transition (G----A) at position 1888 in the mos coding region, resulting in the change of Gly to Arg at position 307, was responsible for the ts phenotype of clone ts159. Substitution of sequences carrying this mutation with the corresponding sequences of the wild-type virus generated a virus that was ts for transformation. Insertion of the dominant selectable marker gene for geneticin resistance (neor) into ts159 did not disrupt mos expression or its ts phenotype. neor-ts159 facilitates the study of mos action by allowing the selection of infected cells at the nonpermissive temperature before mos transformation has been induced. Furthermore, infected cells which show no obvious phenotype alteration due to mos expression can be identified by their Neor phenotype.

Base Sequence↗
PubMed1987

Rescue of type I collagen-deficient phenotype by retroviral-vector-mediated transfer of human pro alpha 1(I) collagen gene into Mov-13 cells.

A full-length cDNA clone corresponding to the human pro alpha 1(I) collagen gene was isolated and inserted into a retrovirus vector. Cell lines were obtained which produced recombinant viruses transducing the collagen cDNA (HUC virus). To test whether the transduced cDNA was functional, Mov-13 mouse cells were infected with the virus. These cells do not produce any type I collagen due to an insertional mutation of the pro alpha 1(I) gene which blocks transcription. While normal amounts of pro alpha 2(I) RNA were synthesized, no alpha 2(I) collagen chains were detectable in the mutant Mov-13 cells. Infection with HUC virus, however, resulted in the production of stable type I collagen, which was secreted into the medium. Analysis of pepsin-resistant proteins indicated that interspecies heterotrimers consisting of human alpha 1(I) and mouse alpha 2(I) collagen chains were secreted by the infected Mov-13 cells. Our results show that pro alpha (I) collagen chains from species as distant as human and mouse can associate to form stable type I collagen. The availability of a retrovirus vector transducing a functional pro alpha 1(I) collagen gene combined with the Mov-13 mutant system should enable us to study the effect of specific mutations on the synthesis, assembly, and function of type I collagen, not only in tissue culture but also in the animal.

Animals↗
PubMed1984

Comparison of myeloproliferative sarcoma virus with Moloney murine sarcoma virus variants by nucleotide sequencing and heteroduplex analysis.

The myeloproliferative sarcoma virus (MPSV) was derived by passage of Moloney sarcoma virus (Mo-MuSV) in adult mice. Mo-MuSV variants transform fibroblasts. However, MPSV also affects erythroid, myeloid, and hematopoietic stem cells. The MPSV proviral genome, two temperature-sensitive mutants derived from it, Mo-MuSV variant M1, and Moloney murine leukemia virus (Mo-MuLV) were compared by heteroduplex mapping. MPSV wild type was found to have 1 kilobase pair deleted from the pol gene and to contain v-mos-related sequences. The 3' end of MPSV, including the oncogene-helper junctions, the v-mos gene, and the 3' long terminal repeat, was sequenced and compared with sequences of Mo-MuLV, MSV-124, and the mouse oncogene c-mos. From these data, MPSV appears to be either closely related to the original Mo-MuSV or an independent recombinant of Mo-MuLV and c-mos. Five possible explanations of the altered specificity of MPSV are considered. (i) The MPSV mos protein has properties inherent in c-mos but lost by other Mo-MuSV mos proteins. (ii) The MPSV mos protein has altered characteristics due to amino acid changes. (iii) Due to a frameshift, MPSV codes for a mos protein truncated at the amino terminal and also a novel peptide. (iv) A second novel peptide may be encoded from the 3' env region. (v) MPSV has long terminal repeats and an enhancer sequence more like Mo-MuLV than Mo-MuSV, with a consequently altered target cell specificity.

Animals↗
PubMed1977

Coronary xeroradiography.

Xeroradiography is shown to be especially helpful in coronary arteriography. Comparatively low concentrations of contrast medium are sufficient to provide adequate diagnostic details in the image. Selection injection techniques yield exceptional resolution of small vessels. The possibility of obtaining adequate coronary arteriograms by aortic root injections, with its inherently lower risk to the patient, is examined.

Angiography↗