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The molecular cloning and characterisation of a novel human gene encoding a putative mitochondrial zincin metallopeptidase - PRSM-1
Başlık:
The molecular cloning and characterisation of a novel human gene encoding a putative mitochondrial zincin metallopeptidase - PRSM-1
Yazar:
Scott, Ian C., author.
ISBN:
9780438043251
Yazar Ek Girişi:
Fiziksel Tanımlama:
1 electronic resource (249 pages)
Genel Not:
Source: Dissertation Abstracts International, Volume: 76-08C.
Özet:
The zincins are a superfamily of structurally-related zinc-binding metallopeptidases which play critical roles in a diverse range of biological processes, such as developmental patterning (eg. BMP-1/tolloid), cytokine activation (eg. pro-TNFa convertase) and extracellular matrix (ECM) degradation (eg. MMPs). The procollagen N-proteinases play a role in the regulation of ECM synthesis and biochemical evidence suggests they could be members of the zincin superfamily. A partial cDNA was identified from a human placental library using polyclonal antibodies raised to the type III procollagen N-proteinase, the metalloendopeptidase which specifically removes the N-propeptide of type III procollagen (Halila, R. and Peltonen, L. (1986) Biochem J. 239, 47-52). This thesis describes the isolation, molecular cloning and characterisation of this gene and suggests it encodes a novel mitochondrial zincin metallopeptidase which has structural similarities with the type III procollagen N-proteinase. The gene encodes a 33 kDa protein which contains the zinc-binding HEXXH pentapeptide characteristic of the zincin superfamily. The gene, prsm-1 (PRoteaSe, Metallo, number 1), is represented once in the human genome on chromosome 16 (q24.3) (Scott, I. C. etal. (1996) Gene 174, 135-143). The complete nucleotide sequence of prsm-1 was identified by a combination of cDNA and genomic library screening, PCR-cloning and primer extension. The deduced primary structure of PRSM-1 showed no homology to sequences on public sector databases except for the minimal zincin motif (HEXXH) and a glutamic acid zinc-ligand (20 amino acids C-terminal to the HEXXH motif) analogous to thermolysin-like metallopeptidases. PRSM-1 lacks a secretory signal sequence but does have a putative N-terminal mitochondrial-like targeting signal. The functional significance of a short collagenlike repeat in PRSM-1 was unknown. Northern blot and RT-PCR analyses indicated the wide distribution of the prsm-1 RNA transcript in human tissues and cultured cells. Analysis of sequences upstream of the transcriptional start site showed the prsm-1 gene promoter lacks TATA and CCAAT boxes, which is characteristic of genes which are widely expressed and have growth control-related or housekeeping functions. Anti-PRSM-1 polyclonal antibodies were raised to purified recombinant PRSM-1 and were used in western blot and immunolocalisation studies. The anti-rePRSM-1 antisera recognised a protein of approximately 30 kDa (consistent with the size of PRSM-1) in western blot analysis of human tissue and cell extracts. Immunocytochemical analysis, using the anti-rePRSM-1 antisera, suggested that PRSM-1 was localised in the mitochondria, an observation consistent with the identification of a mitochondrial-like targeting signal in PRSM-1 and the lack of a secretory signal sequence. Western blot analysis of MG63 cell extracts (an osteoarcoma cell line) using the anti-rePRSM-1 antisera identified an additional protein of approximately 70 kDa raising the possibility that PRSM-1 belongs to a family of proteins. Western blot analysis was performed on mouse skin before and after incisional wounding to investigate the possibility that proteins related to PRSM-1 could be involved in processes such as ECM synthesis, cytokine activation and cell migration. The anti-rePRSM-1 antisera detected a 50 kDa protein in both resting and wounded mouse skin. An additional 60 kDa protein was identified in mouse skin 7-days post-wounding. Immunohistological studies of mouse skin were consistent with the peak of expression of the 60 kDa protein 7-days post-wounding, which is the phase of wound repair associated with connective tissue biosynthesis.
Notlar:
School code: 1543
Tüzel Kişi Ek Girişi:
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Yer Numarası | Demirbaş Numarası | Shelf Location | Lokasyon / Statüsü / İade Tarihi |
---|---|---|---|
XX(684262.1) | 684262-1001 | Proquest E-Tez Koleksiyonu | Arıyor... |
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