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 changesof human fibroblast growth factor receptor 1 genone during development. Method Southernblot analysis of genomic DNA isolated from adult and fetal tissues. Result AdultFGFR1 gene structure is different from its embryonic counterpart. Conclusion Thedisserence 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] . Thebiological activities of FGFs are mediated through Fibroblast Growth Factor Receptors (FGFRs)whichare 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， FGFR3and FGFR4， transcribed from four independent genes. Selective mRNA splicing of FGFR1and FGFR2 can bring forth various isoforms with different li-gand-binding specificity andaffinity， which greatly increases the variety of FGFRs^[2] . Theselection of two or three Ig domains^[3] and changes of the se-cond part ofthe third Ig-domain^[4] during mRNA splicing reflect changed cellularrequirement for FGFs along with specification of cells and tissues during development^[3] .
FGFR genes have been regarded as important developmental genes^[5] .Theexpression of FGFRs is variable at different stages of development and in differenttissues^[6] and，alternative mRNA spli-cing，up to now，is consideredmajor resaon for the variations.However，since Ig and T cell receptor(TCR)，twomembers of the Ig gene superfamily，undergo gene rearrangement during maturation of Blymphocytes and T lymphocytes，it is rational to speculate that chromosomal generearrangement may also occur in FGFR genes during development.Previous work in our labhas demonstrated that changes of FGFR1 genomic DNA may be involved in development ofmurine heart^[7] .To elucidate the possible relations between FGFR1 genestructure and development of human embryo，we have analyzed FGFR1 genomic DNA in humanfetal and adult tissues using Southern blot.

MATERIALS AND MATHODS

Materials HindⅢ，Xba Ⅰ andEcoR Ⅰ endonuclease from Promega，USA；Protease K and RNase from Sino-America Bio.，China；pRc/CMV-FGFR1recombinant plasmid^[8] is a generous gift from Dr.Wang Liying，School ofBasic Medical Sciences，NBUMS；JM109 strain from Promega，USA；Gene clean kit from BIO101，USA；Digoxin labeling and detection kit，from Boehringer-Mannheim，Germany；Nitrous-Cellulosefilter from Schleicher ＆ Schuell，USA；Human adult and embryonic tissues from FirstHospital of NBUMS，with permission of patients.
Probe preparation pRc/CMV-FGFR1 plasmid is transformed intoJM109 and prepared in a large scale.FGFR1 cDNA fragments are released with Hind Ⅲ andXba Ⅰ，separated in agrose gel electrophoresis，purified with Gene clean kit andlabeled with digoxin hapten using Digoxin Labeling and Detection kit.
Southern blot Genomic DNA extracted from various tissues arecompletely digested with EcoR Ⅰ.The digested fragments separated in 0.7% agarose gelelectrophoresis(30v，18h) are denatured and transferred to Nitrous-Cellulose filterswhich 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 humanadult tissues Genomic DNA extracted from nasal mucosa and peripheral blood ofnormal human adult is digested with EcoR I，separated by agarose gel electrophoresis andtransferred to Nitrous-Cellulose filter which is probed by digoxin-labeled FGFR1extracellular fragment cDNA.Immunolo-gical staining reveals three bands with length of4.2，5.3 and 6.5kb respectively(Fig.1A).
Southern blot analysis of FGFR1 in human embryonic tissues Usingthe same methods as above，the genomic DNA extracted from heart，aorta，skeletal muscleand spleen of a 4-month embryo is analyzed by Southern blot with digoxin-labeled FGFR1cDNA probe.Four bands are detec-ted with length of 3.3，4.2，6.5 and 9.4kbrespectively(Fig.1B).

Fig1 Southern blot analysis of genomic DNAextracted 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 FGFR1EcDNA 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 eventsduring development.It is estimated that more than 90% of the genes in differentiatingcells are quiescent and only a very small part is selectively transcribed and expressed ina strict sequence under certain regulating mechanisms，which lead to formation ofstructurally and functionally different cell masses.Although the rearrangement，amplificationand loss of genes at certain stages of development are considered important，the detailedmechanisms for genomic modulation of gene expression are still obscure.
FGFR genes have been recognized as important developmental genes involved indifferentiation and proliferation of various kind of cells^[5] .Pointmutations of the genes have been demoms-trated to be the genetic reseaons fordevelopmental diseases^[9] .Previous work in our laboratory shows thatFGFR1 gene in the heart of adult mouse differs from that of juvenile ones.Based on this，wespeculate that FGFR1 gene may undergo chromosomal rearrangement or gene loss during thedeve-lopment of heart^[7] .

Fig2 Schematic structure of FGFR1 genomic DNAshowing possible mechanisms for chromosomal gene rear-rangement during development

In this paper，we report that FGFR1 genomic DNAstructure in adult is different from its counterpart in embryo(See Fig.2).Thissuggests that chromosomal changes at certain stages of development may affect therestrictive endonuclease sites in FGFR1 gene.The changes may include loss or fusion of asmall part of chromosome or mutations of certain nucleotides creating a new EcoR I sitewhich alters the DNA fragment between EcoR I digestion sites. Such changes of FGFR genestructure may be crucial for the normal development of human body.■

Foundation item： This work is supportedby Trans-Century Training Programme Foundation for the Talents
by the State Education Commission.(1995(4))
Biography： MIAO Chuan-long(1974-)，male，Shandong， resident，masterdegree 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 duringdevelopment

Received： 1998-08-23

Revised： 1999-08-11 (苗传龙 王平 于永利)