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Utilization of a PCR-based strategy for the isolation of cDNA clones encoding the human α3(IX) chain
Fifth International Conference on the Molecular Biology and Pathology of Matrix for JK-132 could be observed along the sinusoidal wall sporadically. Over 10 months of CCI 4 intoxication, the sinusoidal wall was stained intensely by JK-132. Electron microscopically, continuous endothelial cells with lamina densa were lined along the sinusoidal wall. Collagen fibrils were also increased in number in the perisinusoidal space as well as fibrotic septa to form bundles. Flocculent material filled the perisinusoidal space. Immunoelectron microscopically, dense reaction products for JK-199 were observed on the collagen fibrils. The concentration of BCC in serum increased again 2 to 3 times as high as that in normal condition. Taking account of these findings, a deposit of BCC in the perisinusoidal space in liver appears to be the first step in the development of hepatic fibrosis.
U t i l i z a t i o n o f a P C R - B a s e d Strategy for t h e
Isolation o f c D N A Clones Encoding t h e Human 0~3(IX) Chain
357
nucleotide primers were designed and used to amplify a 213-bp PCR product from human chondrocyte first strand cDNA. The identity of the 213bp PCR product was confirmed by DNA sequencing and the PCR product was subsequently used to screen a human chondrocyte cDNA library constructed in ~.UniZAP XR. Two overlapping cDNAs of 847 and 913 bp that extend from COL2 to the poly A tail were isolated and sequenced. A 1061-bp PCR product was generated by designing a sense primer based on bovine 0t3(IX) amino acid sequences within COL2 and pairing this primer with the antisense primer used to generate the PCR product 213. A 5' PCR product of 545 bp was generated by pairing a primer based on the signal peptide and NC4 domain of chicken 0t3(IX) with a primer based on bovine 0t3(IX) amino acid sequences from the 5' end of COL2. A third PCR product of 625 bp was then generated that overlaps the PCR products 1061 and 545 and closes the gap between these sequences. PCR products 545 and 625 were then used to rescreen the human chondrocyte cDNA library and a 1934-bp insert was identified and characterized that hybridized to both probes.
R. Brewton, B. Wood, Z-X. Ren, B. Lee, W. Horton, J. Baker and R. Mayne Department of Cell Biology and Department of Biochemistry and Molecular Genetics, University of Alabama at Birmingham, Birmingham, AL, 35294; Department of Pediatrics, Baylor College of Medicine, Houston, TX; and Shriner's Hospital for Crippled Children, Portland, OR, USA
Abnormal Processing of Procollagen VII --) Collagen VII underlies Dystrophic Epidermolysis Bullosa in some Families
Leena Bruckner-Tuderman, Dieter R. Zimmermann, Maria T. Dours-Zimmermann, Type IX collagen is located on the surface of Jan-Olof Winberg, Ulrike Kalinke and Tobias collagen fibrils in hyaline cartilage and vitreous Gedde-Dahl, Jr. humor and is stabilized by covalent crosslinks to type II collagen. In order to complete the primary Departments of Dermatology and Pathology, University of structure of human type IX collagen, we have uti- Ziirich, Ziirich, Switzerland; Department of Dermatology lized the polymerase chain reaction (PCR) to obtain University of Miinster, MOnster, Germany; and overlapping PCR products and cDNA clones that Department of Biochemistry, University of Troms6, cover the coding region of the 0t3(IX) chain. Norway Pepsin-resistant fragments of type IX collagen (called HMW and LMW) were isolated from The major constituent of the anchoring fibrils in human newborn sterna and costal cartilage the skin, collagen VII, is synthesized as a procollaobtained at autopsy. Unreduced HMW and LMW gen which has been presumed to be processed to were separated by molecular sieve chromatog- collagen by proteolytic cleavage of the C-terminal raphy. Reduced and alkylated LMW peptides were globular NC-2 domain, but details of this process then separated by C18 reverse-phase HPLC. The have remained unknown. We have investigated 0t3(IX) L M W peptide was digested with the processing of procollagen VII in cultured kerTPCK-trypsin, the tryptic peptides were separated atinocytes and in the skin. For this purpose, bacteby reverse-phase HPLC and select fractions were rial fusion proteins containing unique sequences of subjected to N-terminal amino acid sequencing. A the NC-2 domain of collagen VII were prepared. contiguous stretch of 123 amino acids extending Polyclonal antibodies were raised against the fusion from COL1 into NC1 was obtained from six over- proteins and affinity purified. Immunoblotting lapping peptide sequences. Degenerate oligo- showed that the antibodies reacted with procolla-
U t i l i z a t i o n o f a P C R - B a s e d Strategy for t h e
Isolation o f c D N A Clones Encoding t h e Human 0~3(IX) Chain
357
nucleotide primers were designed and used to amplify a 213-bp PCR product from human chondrocyte first strand cDNA. The identity of the 213bp PCR product was confirmed by DNA sequencing and the PCR product was subsequently used to screen a human chondrocyte cDNA library constructed in ~.UniZAP XR. Two overlapping cDNAs of 847 and 913 bp that extend from COL2 to the poly A tail were isolated and sequenced. A 1061-bp PCR product was generated by designing a sense primer based on bovine 0t3(IX) amino acid sequences within COL2 and pairing this primer with the antisense primer used to generate the PCR product 213. A 5' PCR product of 545 bp was generated by pairing a primer based on the signal peptide and NC4 domain of chicken 0t3(IX) with a primer based on bovine 0t3(IX) amino acid sequences from the 5' end of COL2. A third PCR product of 625 bp was then generated that overlaps the PCR products 1061 and 545 and closes the gap between these sequences. PCR products 545 and 625 were then used to rescreen the human chondrocyte cDNA library and a 1934-bp insert was identified and characterized that hybridized to both probes.
R. Brewton, B. Wood, Z-X. Ren, B. Lee, W. Horton, J. Baker and R. Mayne Department of Cell Biology and Department of Biochemistry and Molecular Genetics, University of Alabama at Birmingham, Birmingham, AL, 35294; Department of Pediatrics, Baylor College of Medicine, Houston, TX; and Shriner's Hospital for Crippled Children, Portland, OR, USA
Abnormal Processing of Procollagen VII --) Collagen VII underlies Dystrophic Epidermolysis Bullosa in some Families
Leena Bruckner-Tuderman, Dieter R. Zimmermann, Maria T. Dours-Zimmermann, Type IX collagen is located on the surface of Jan-Olof Winberg, Ulrike Kalinke and Tobias collagen fibrils in hyaline cartilage and vitreous Gedde-Dahl, Jr. humor and is stabilized by covalent crosslinks to type II collagen. In order to complete the primary Departments of Dermatology and Pathology, University of structure of human type IX collagen, we have uti- Ziirich, Ziirich, Switzerland; Department of Dermatology lized the polymerase chain reaction (PCR) to obtain University of Miinster, MOnster, Germany; and overlapping PCR products and cDNA clones that Department of Biochemistry, University of Troms6, cover the coding region of the 0t3(IX) chain. Norway Pepsin-resistant fragments of type IX collagen (called HMW and LMW) were isolated from The major constituent of the anchoring fibrils in human newborn sterna and costal cartilage the skin, collagen VII, is synthesized as a procollaobtained at autopsy. Unreduced HMW and LMW gen which has been presumed to be processed to were separated by molecular sieve chromatog- collagen by proteolytic cleavage of the C-terminal raphy. Reduced and alkylated LMW peptides were globular NC-2 domain, but details of this process then separated by C18 reverse-phase HPLC. The have remained unknown. We have investigated 0t3(IX) L M W peptide was digested with the processing of procollagen VII in cultured kerTPCK-trypsin, the tryptic peptides were separated atinocytes and in the skin. For this purpose, bacteby reverse-phase HPLC and select fractions were rial fusion proteins containing unique sequences of subjected to N-terminal amino acid sequencing. A the NC-2 domain of collagen VII were prepared. contiguous stretch of 123 amino acids extending Polyclonal antibodies were raised against the fusion from COL1 into NC1 was obtained from six over- proteins and affinity purified. Immunoblotting lapping peptide sequences. Degenerate oligo- showed that the antibodies reacted with procolla-