博士 / 國立臺灣大學 / 獸醫學研究所 / 92 / 英文摘要
Heparin is a negatively charged and sulfated glycosaminoglycan. In order to form the proteoglycan, by using the covalently bond, heparin could bind to the serine of core protein. Heparin was also play an important role in the growth and development of many organisms.
This experiment was done by micro-injected the exogenous heparin into the fertilzated egg of zebrafish (Danio rerio) at one cell stage. After cultured at 28�aC for 48 hrs, we observed the effects of the developed phenotype and related hematopoiesis genotype. In order to explore the effects of the heparin, heparin was injected with or without Heparin-binding protein (HBP) mRNA, after 48 hrs, the mortality was around 3~5%. 11.59% of the mutants were heart disorder with swelled, no blood in hearts, and no circulation. In order to characterized the mechanism of the heparin, we analysis the mRNA expression level of the 31 hematopoiesis related genes. We found that the expression level of elb, hhex, ikaros and spt were reduced or near stop. By co-injection heparin with the mRNA of plei-1, plei-2, or FGFBP, show these genes could reduce the damage from the heparin. We also found that the FGFBP protein could increase the damage in the embryos. Further more injected Morpholino of plei-1, plei-2, and FGFBP into yolk sac of zebrafish embryo during 1 – 4 cell stage, shown the same result. All these data shows that plei-1, plei-2, and FGFBP could reduce the mutation and mortality from the heparin.
We cloned the full-length HBNF (Heparin-binding neurotrophic factor) cDNA from zebrafish and determined its genomic structure by bioinformatics analysis. Zebrafish HBNF gene is comprised of five exons and four introns spanning approximately 82 kb. RT-PCR analysis revealed that zebrafish HBNF transcript was highly expressed in adult brain and intestine tissues while less in other tissues. During embryogenesis, zebrafish HBNF transcript was observed to moderately express at earlier stages with a gradual decline. Higher expression level was observed after hatching and maintaining this level into adulthood. The overall amino acid sequence of zebrafish HBNF shows 60% identity to human HBNF, but with approximately 40% identity to other midkine proteins. Like mammalian homolog, zebrafish HBNF could induce significant neurite outgrowth in PC12 cells without NGF stimulation. In addition, zebrafish HBNF was able to enhance extensive neurite outgrowth in zebrafish embryos during embryogenesis. In summary, a feasible in vivo assay for neurite outgrowth was established in zebrafish.
Hip/L29 can bind to heparin/heparan sulfate and is identical to ribosomal protein, L29. In this study, we cloned the gene encoding pufferfish Hip protein and characterized the promoter region of this gene. The pufferfish Hip (TfL29) gene consists of 4 exons and 3 introns spanning 1.7kb and has one intron in the 5’-untranslated region. The 5-upstream 2.2kb of the first exon contains ten E-boxes and many putative binding motifs for transcription factor GATA-1, AML-1a, Oct-1, CdxA and NRF-2. The full-length pufferfish Hip cDNA encodes a protein of 68 amino acid residues, which is 91 amino acids shorter than that of human Hip protein. The pufferfish Hip protein shows 75%, 75%, 49%, and 45% identity to the Hip protein from the catfish, zebrafish, C. elegans, and Drosophila, respectively. One putative heparin/heparan sulfate binding region, from amino acid residues 43 to 58 with the sequences of MRFAKKHNKKGLKKMQ, was found in pufferfish Hip protein. Hip mRNA was ubiquitous expressed in all adult tissues from the round-spotted pufferfish. We isolated 2.6kb DNA fragment containing the promoter region of the pufferfish Hip gene. But there are no TATA or CAAT boxes within a 300-bp region upstream from the transcription initiation site. The different size fragment promoter region were tested either in COS-1 cells or in zebrafish embryos by using Luciferase or GFP as reporter genes, respectively. All of promoter fragment had transcriptional activity, non-introns I fragment was stronger than intron I fragment. Beside the 0.3 kb, the transcriptional activities in vitro and in vivo according to the fragment size.
| Identifer | oai:union.ndltd.org:TW/092NTU05541003 |
| Date | January 2004 |
| Creators | Ming-Huang, Chang, 張銘煌 |
| Contributors | Tzong-Fu, Kuo, 郭宗甫 |
| Source Sets | National Digital Library of Theses and Dissertations in Taiwan |
| Language | zh-TW |
| Detected Language | English |
| Type | 學位論文 ; thesis |
| Format | 134 |
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