Subject(s)
Bacteriophage T7/genetics , Proteoglycans/biosynthesis , Proteoglycans/genetics , Vaccinia virus/genetics , Amino Acid Sequence , Animals , Base Sequence , Cell Line , Chromatography, Affinity , DNA, Recombinant/genetics , DNA-Directed RNA Polymerases/genetics , Gene Expression , Genetic Vectors , Humans , Mammals , Molecular Sequence Data , Polymerase Chain Reaction , Recombinant Proteins/biosynthesis , Recombinant Proteins/genetics , Recombination, Genetic , Transfection , Viral Plaque Assay , Viral Proteins , Virus CultivationABSTRACT
Two recent studies have suggested that the absolute cellular RNA and/or DNA levels in Down syndrome (DS) may be unusual compared to normals and may be linked to phenotypic expression (Pash et al., 1990; Dahl et al. 1988). We have extended these two studies by directly quantitating and comparing the total cellular mRNA, rRNA, and DNA levels in fibroblasts and lymphocytes derived from normal and DS individuals. The assay methods used, which allowed us to present the various nucleic acids levels in picograms per cell, did not reveal any significant difference between the two groups.
Subject(s)
DNA/metabolism , Down Syndrome/metabolism , RNA/metabolism , Adult , Fibroblasts/metabolism , Humans , Lymphocytes/metabolism , RNA, Messenger/metabolism , RNA, Ribosomal/metabolismABSTRACT
A cDNA encoding osteonectin was isolated from a human bone cell cDNA library and used to examine osteonectin protein structure, mRNA structure and expression in human tissue. The deduced protein sequence shows complete identity with a recently isolated placental form and extensive homology to mouse and bovine counterparts. The protein is rich in cysteine residues, which are conserved between species except for cys 194 which is only present in the bovine. In the human, osteonectin mRNA is of two sizes, 2.3 and 3.0 kb, the former being dominant in all tissues studied. Human mRNA was detected in the Ewing sarcoma and in non-bone cell and tissue sources. The potential folded structure of osteonectin mRNA was estimated, based on computer predictions, and indicates the presence of a bulge at the 5' end of the message which includes the start of translation. Southern analysis of human genomic DNA using radiolabeled osteonectin cDNA as probe demonstrates a simple banding pattern confirming earlier studies that the osteonectin gene is present in one copy per haploid human genome.
Subject(s)
Bone and Bones/analysis , Osteonectin/genetics , RNA, Messenger , Amino Acid Sequence , Animals , Base Sequence , Blotting, Northern , Blotting, Southern , Cattle , DNA/analysis , DNA/genetics , Gene Library , Genome, Human , Humans , Mice , Molecular Sequence Data , RNA, Messenger/biosynthesis , Species SpecificityABSTRACT
A complementary DNA clone for bovine osteonectin was used to isolate the osteonectin gene from two libraries of bovine genomic DNA fragments. Two overlapping clones were obtained whose relationship was determined by restriction mapping and sequence analysis. The two clones contain the entire osteonectin coding region spanning approximately 11 kilobases of genomic DNA. The coding region of the gene was determined, by electron microscopy and DNA sequencing, to reside in nine exons. In addition, there is at least one 5' exon interrupted by an intron in the 5'-nontranslated sequence of the gene. Excluding this 5' exon and the 3'-terminal exon, the exons are small and approximately uniform in size, averaging 130 +/- 17 base pairs. Three of the exons at the 5' end of the gene were sequenced and appear to encode discrete protein domains. For example, the putative exon 2 contains the coding region for the leader peptide of the molecule. The amino-terminal protein sequence was determined for osteonectin extracted from human, rabbit, and chicken bone and compared with those for bovine, mouse, and pig osteonectin. These data suggest that osteonectin is highly conserved between species, interspecies changes being seen primarily at the amino terminus of the protein and specifically in the region encoded by putative exon 3 in the bovine gene.
Subject(s)
Carrier Proteins/genetics , DNA/isolation & purification , Genes , Amino Acid Sequence , Animals , Base Sequence , Cattle , Cloning, Molecular , DNA/genetics , DNA/ultrastructure , DNA Restriction Enzymes , Molecular Sequence Data , Osteonectin , Species SpecificitySubject(s)
Cloning, Molecular , DNA , Dental Enamel Proteins/analysis , Amelogenin , Animals , Base Sequence , Cattle , Molecular Sequence DataABSTRACT
The cDNA for the full-length core protein of the small chondroitin sulphate proteoglycan II of bovine bone was cloned and sequenced. A 1.3 kb clone (lambda Pg28) was identified by plaque hybridization with a previously isolated 1.0 kb proteoglycan cDNA clone (lambda Pg20), positively identified previously by polyclonal and monoclonal antibody reactivity and by hybrid-selected translation in vitro [Day, Ramis, Fisher, Gehron Robey, Termine & Young (1986) Nucleic Acids Res. 14, 9861-9876]. The cDNA sequences of both clones were identical in areas of overlap. The 360-amino-acid-residue protein contains a 30-residue propeptide of which the first 15 residues are highly hydrophobic. The mature protein consists of 330 amino acid residues corresponding to an Mr of 36,383. The core protein contains three potential glycosaminoglycan-attachment sites (Ser-Gly), only one of which is within a ten-amino-acid-residue homologous sequence seen at the known attachment sites of related small proteoglycans. Comparisons of the published 24-residue N-terminal protein sequence of bovine skin proteoglycan II core protein with the corresponding region in the deduced sequence of the bovine core protein reveals complete homology. Comparison of the cDNA-derived sequences of bovine bone and human embryonic fibroblast proteoglycans shows a hypervariable region near the N-terminus. Nucleotide homology between bone and fibroblast core proteins was 87% and amino acid homology was 90%.
