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Changes of type I fibroblast growth factor receptor gene during development
免疫学杂志 2000年第1期第16卷 基础免疫学
作者:苗传龙 王平 于永利
关键词:成纤维细胞生长因子受体I;基因组;发育;Southern印迹分析▲
MIAO Chuan-long,WANG Ping,YU Yong-li
(Department of Immunology, Norman Bethune Univercity of Medical Sciences, Changchun 130021,China)
Abstract:Objective To find the changes of human fibroblast growth factor receptor 1 genone during development.Method Southern blot analysis of genomic DNA isolated from adult and fetal tissues.Result Adult FGFR1 gene structure is different from its embryonic counterpart.Conclusion The disserence might lead to changes of FGFR1 expression as wel as functions of the cells.
Key words:FGFR1; genomic DNA; development; Southern blot
CLC number:392.12 Document code:A
摘 要:目的 研究人成纤维细胞生长因子受体1(FGFR1)基因在发育过程中的可能变化。方法 采用Southern blot的方法对胎儿及多种组织基因组DNA进行分析。结果 成人FGFR1基因与胚胎期的在基因组水平上是不同的。结论 人FGFR1基因在发育过程中可能发生了重排或丢失,这种变化可能导致FGFR1在胚胎期和成年期的表达水平和分子结构的不同,从而改变细胞的功能状态。
INTRODUCTION
Fibroblast Growth Factors(FGFs) comprise a family, with 18 members so far, of structurally related proteins functioning in mitogenesis, differentiation, tissue repair and organ morphogenesis[1]. The biological activities of FGFs are mediated through Fibroblast Growth Factor Receptors (FGFRs)which are Type IV rceptor tyrosine kinase (PTKs) as well as members of immunoglobulin (Ig) gene superfamily. There are four types of FGFRs, FGFR1/flg, FGFR2/Bek, FGFR3 and FGFR4, transcribed from four independent genes. Selective mRNA splicing of FGFR1 and FGFR2 can bring forth various isoforms with different li-gand-binding specificity and affinity, which greatly increases the variety of FGFRs[2]. The selection of two or three Ig domains[3] and changes of the se-cond part of the third Ig-domain[4] during mRNA splicing reflect changed cellular requirement for FGFs along with specification of cells and tissues during development[3].
FGFR genes have been regarded as important developmental genes[5].The expression of FGFRs is variable at different stages of development and in different tissues[6] and,alternative mRNA spli-cing,up to now,is considered major resaon for the variations.However,since Ig and T cell receptor(TCR),two members of the Ig gene superfamily,undergo gene rearrangement during maturation of B lymphocytes and T lymphocytes,it is rational to speculate that chromosomal gene rearrangement may also occur in FGFR genes during development.Previous work in our lab has demonstrated that changes of FGFR1 genomic DNA may be involved in development of murine heart[7].To elucidate the possible relations between FGFR1 gene structure and development of human embryo,we have analyzed FGFR1 genomic DNA in human fetal and adult tissues using Southern blot.
MATERIALS AND MATHODS
Materials HindⅢ,Xba Ⅰ and EcoR Ⅰ endonuclease from Promega,USA;Protease K and RNase from Sino-America Bio.,China;pRc/CMV-FGFR1 recombinant plasmid[8] is a generous gift from Dr.Wang Liying,School of Basic Medical Sciences,NBUMS;JM109 strain from Promega,USA;Gene clean kit from BIO 101,USA;Digoxin labeling and detection kit,from Boehringer-Mannheim,Germany;Nitrous-Cellulose filter from Schleicher & Schuell,USA;Human adult and embryonic tissues from First Hospital of NBUMS,with permission of patients.
Probe preparation pRc/CMV-FGFR1 plasmid is transformed into JM109 and prepared in a large scale.FGFR1 cDNA fragments are released with Hind Ⅲ and Xba Ⅰ,separated in agrose gel electrophoresis,purified with Gene clean kit and labeled with digoxin hapten using Digoxin Labeling and Detection kit.
Southern blot Genomic DNA extracted from various tissues are completely digested with EcoR Ⅰ.The digested fragments separated in 0.7% agarose gel electrophoresis(30v,18h) are denatured and transferred to Nitrous-Cellulose filters which are then probed by digoxin-labeled FGFR1 cDNA.Immunological staining with AP-NBT-BCIP sytem is used to detect the positive bands.
RESULTS
Southern blot analysis of FGFR1 in human adult tissues Genomic DNA extracted from nasal mucosa and peripheral blood of normal human adult is digested with EcoR I,separated by agarose gel electrophoresis and transferred to Nitrous-Cellulose filter which is probed by digoxin-labeled FGFR1 extracellular fragment cDNA.Immunolo-gical staining reveals three bands with length of 4.2,5.3 and 6.5kb respectively(Fig.1A).
Southern blot analysis of FGFR1 in human embryonic tissues Using the same methods as above,the genomic DNA extracted from heart,aorta,skeletal muscle and spleen of a 4-month embryo is analyzed by Southern blot with digoxin-labeled FGFR1 cDNA probe.Four bands are detec-ted with length of 3.3,4.2,6.5 and 9.4kb respectively(Fig.1B).
Fig1 Southern blot analysis of genomic DNA extracted from human tissues
Genomic DNA was digested by EcoRI and hibridized with DIG-labeled FGFR1 cDNA probes. Arrows indicated are positive bands.
A:Genomic DNA of normal tissues of human adult hibridized with FGFR1E cDNA probe
lane 1:peripheral blood;lane 2 and 3:nasal mucosa of different persons
B:Genomic DNA of human fetal tissues hibridized with FuFGFR1 cDNA probe
lane 1:cardiac muscle;lane 2:aortic artery wall;
lane 3:skeletal muscle;lane 4:spleen
DISCUSSION
Turn on and turn off of specific genes in cells are key events during development.It is estimated that more than 90% of the genes in differentiating cells are quiescent and only a very small part is selectively transcribed and expressed in a strict sequence under certain regulating mechanisms,which lead to formation of structurally and functionally different cell masses.Although the rearrangement,amplification and loss of genes at certain stages of development are considered important,the detailed mechanisms for genomic modulation of gene expression are still obscure.
FGFR genes have been recognized as important developmental genes involved in differentiation and proliferation of various kind of cells[5].Point mutations of the genes have been demoms-trated to be the genetic reseaons for developmental diseases[9].Previous work in our laboratory shows that FGFR1 gene in the heart of adult mouse differs from that of juvenile ones.Based on this,we speculate that FGFR1 gene may undergo chromosomal rearrangement or gene loss during the deve-lopment of heart[7].
Fig2 Schematic structure of FGFR1 genomic DNA showing possible mechanisms for chromosomal gene rear-rangement during development
In this paper,we report that FGFR1 genomic DNA structure in adult is different from its counterpart in embryo(See Fig.2).This suggests that chromosomal changes at certain stages of development may affect the restrictive endonuclease sites in FGFR1 gene.The changes may include loss or fusion of a small part of chromosome or mutations of certain nucleotides creating a new EcoR I site which alters the DNA fragment between EcoR I digestion sites. Such changes of FGFR gene structure may be crucial for the normal development of human body.■
Foundation item: This work is supported by Trans-Century Training Programme Foundation for the Talents
by the State Education Commission.(1995(4))
Biography:MIAO Chuan-long(1974-),male,Shandong, resident,master degree of medical sciences, to works mainly in the field of growth factors.
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MIAO Chuan-long,et al.Changes of type I fibroblast growth factor receptor gene during development
Received:1998-08-23
Revised:1999-08-11