聚球藻RF-1細胞中具有一分子量為97-kDa的蛋白質，以45Ca2+標識法鑑定以後發現，其為該藻最主要之鈣結合蛋白。經以SDS萃取液或Tween-20萃取液抽取，發現其含量相當恆定，但培養基中缺鈣會造成其含量降低。經缺鈣 (加EGTA) 處理後，可在native PAGE觀察到氧化及還原兩種型態，而含鈣處理 (不加EGTA) 則可見到多個蛋白質帶，推測它可能具有多個鈣結合位。為鑑定合成此蛋白的基因，利用其N端胺基酸序列合成的退化性寡核甘酸 (degenerated oligonucleotides) 作為探針，與限制酵素切割過的聚球藻RF-1的DNA 進行南方轉漬分析，可偵測到一段2.2 kb EcoRI-HindIII DNA片段含有雜合訊號，由DNA序列分析之結果證明此段DNA含部份N端開始之鈣結合蛋白基因。以PCR技術合成含鈣結合蛋白部份基因的DNA探針，並以pUC19或λZAPⅡ為載體，無法偵測到含完整鈣結合蛋白基因的選殖體；然而以pUC19為載體選殖HindIII切割聚球藻RF-1 DNA之3 kb片段，則篩選到一個含6 kb插入DNA的選殖體，此段插入DNA包含部份鈣結合蛋白基因的DNA序列。以pUC19及pBR322重新選殖此選殖體的插入DNA，發現6 kb之選殖體是由二段3 kb片段接合後插入pUC19中，其中一段為部分鈣結合蛋白基因，另一段不是。實驗亦證明含部份鈣結合蛋白基因之3 kb DNA片段似乎無法單獨選殖於pUC19，而需一段其他的DNA片段作為輔助。此段3 kb DNA片段所含鈣結合蛋白基因之DNA序列較先前2.2 kb DNA片段長，若以此延伸之部份DNA作為探針，應可選殖到含此蛋白質C端部份之DNA序列，並進而得到完整鈣結合蛋白基因之DNA序列。

In cyanobacterium Synechococcus RF-1 a 97-kDa protein was identified by 45Ca2+ autoradiography being the most predominant calcium-binding protein. This protein was extractable in both the SDS sample buffer and the Tween-20 buffer. Under the implemented culture condition, the abundance of the 97-kDa protein in Synechococcus RF-1 remained constant but was reduced upon depletion of calcium ion in the medium. With the presence of EGTA, the 97-kDa calcium-binding protein migrated into a reduced isoform and an oxidized isoform in a native gel. With the absence of EGTA, the 97-kDa calcium-binding protein was separated into several distinct bands in the native gel, suggesting that it might reside multiple calcium binding sites. To identify the gene encoding the 97-kDa protein, the Synechococcus RF-1 genomic DNA was restriction enzyme-digested and analyzed by Southern blotting using a degenerated oligonucleotide probe corresponding to the N –terminus amino acid sequence of the 97-kDa protein. A 2.2 kb EcoRI-HindIII cut DNA fragment has been cloned and sequenced. It was identified to contain the DNA sequence of the first 108 amino acids from the N-terminus of the protein. Attempts to clone a 6 kb Xba I cut fragment of I cut DNA fragments into the expression vector of pUC19 and lZAPII have not been successful and this is plausibly incurred by the lethal effect of the expressed calcium binding protein to the host cells. So another strategy was used. Based on the restriction map, the DNA sequence of the complete gene can be identified by cloning two overlap DNA fragments. A 3 kb HindIII-cut DNA fragment was cloned into pUC19, and one clone containing the target DNA has been identified. However, besides the target DNA fragment, the clone seemed to contain a different insert DNA of 3 kb. The suggestion was proved when the insert DNA was subcloned into a non-expression vector pBR322. When the target DNA fragment is sequenced, it will be used as a probe to clone the C-terminal region of the calcium-binding protein gene. Further sequencing experiment would help unravel the complete genetic sequence of the 97-kDa calcium binding protein.

Bold, H.C. & Wynne, M.J. (1985) Introduction to the algae. 2nd ed., Prentice-Hall, Inc., New Jersey.
Brahamsha, B. (1996) An abundant cell-surface polypeptide is required for swimming by the nonflagellated marine cyanobacterium Synechococcus. Proceeding of the National Academy Sciences of USA. 93：6504-6509.
Burgess, W.H., Jemiolo, D.K. & Kretsinger, R.H. (1980) Interaction of calcium and calmodulin in the presence of sodium dodecyl sulfate. Biochimica et Biophysica Acta 623：257-270.
Carrion, A.M., Link, W.A., Ledo, F., Mellstrom, B. & Naranjo, J.R. (1999) DREAM is a Ca2+- regulated transcriptional repressor. Nature 398：80-84
Chen, T.H., Huang, T.C. & Chow, T.J. (1988) Calcium requirement in nitrogen fixation in the cyanobacterium Synechococcus RF-1. Planta 173：253-256.
Chen, T.H., Huang, T.C. & Chow, T.J. (1989) Calcium is required for the increase of dark respiration during diurnal nitrogen fixation by Synechococcus RF-1. Plant Science 60：195-198.
Chen, T.H., Chen, T.L., Huang, L.M. & Huang, T.C. (1991) Circadian rhythm in amino acid uptake by Synechococcus RF-1. Plant Physiology 97：55-59.
Chen, H.M., Chien C.Y. & Huang, T.C. (1996) Regulation and molecular structure of a circadian oscillating protein located in the cell membrane of the prokaryote Synechococcus RF-1. Planta 199：520-527.
Chen, H.M., Huang, T.C., & Chien, C.Y. (1996) Nucleotide sequence of the nifHDK operon in the aerobic nitrogen-fixing unicellular Synechococcus RF-1. Botanical Bulletin of Academia Sinica 37：99-105
Chou, H.M. & Huang, T.C. (1991) Ultrastructure of the aerobic, nitrogen-fixing unicellular cyanobacterium Synechococcus sp. RF-1. Algological Studies 64：53-59.
Dominguez, D.C., Adams, H. & Hageman, J.H. (1999) Immunocytochemical localization of a calmodulinlike protein in Bacillus subtilis cells. Journal of Bacteriology 181：4605-4610
Garrigos, M., Deschamps, S., Viel, A., Lund, S.,Champeil, P., Møller, J.V. & Le Maire, M. (1991) Detection of Ca2+-binding proteins by electrophoretic migration in the presence of Ca2+ combined with 45Ca2+ overlay of protein blots. Analytical Biochemistry 194：82-88
Geisler, M., Koenen, W., Richter, J. & Schumann, J. (1998) Expression and characterization of a Synechocystis PCC 6803 P-type ATPase in E. coli plasma membranes. Biochimica et Biophysica Acta 1368：267-275
Giraldez-Ruiz, N., Mateo, P., Bonilla, I. & Fernandez-Pinas, F. (1997) The relationship between intracellular pH, growth characteristics and calcium in the cyanobacterium Anabaena sp. strain PCC7120 exposed to low pH. New Phytologist 137：599-605
Grobbelaar, N., Huang, T.C., Lin, H.Y. & Chow, T.J. (1986) Dinitrogen-fixing endogenous rhythm in Synechococcus RF-1. Federation of European Microbiological Societies Microbiology Letters 37：173-177.
Grobbelaar, N., Lin, H.Y. & Huang, T.C. (1987) Induction of a nitrogenase activity rhythm in Synechococcus RF-1 and the protection of its nitrogenase against photosynthetic oxygen. Current Microbiology 15：29-33.
Grobbelaar, N., Li, W.T. & Huang, T.C. (1991) Relationship between the nitrogenase activity and dark respiration rate of Synechococcus RF-1. Federation of European Microbiological Societies Microbiology Letters 83：99-102.
Grobbelaar, N., Chou, W.M. & Huang, T.C. (1992) Effect of CO2, O2, DCMU, FCCP, and DL-glyceraldehyde on the nitrogenase activity of Synechococcus RF-1. Botanical Bulletin of Academia Sinica 33：167-174.
Gumpel, N.J. & Smith, A.G. (1992) A novel calcium-binding protein from Euglena gracilis. European Journal of Biochemistry 210：721-727.
Harmon, A.C., Putnam-Evans, C. & Cormier, M.J. (1987) A calcium-dependent but calmodulin-independent protein kinase from soybean. Plant Physiology 83：830-837.
Harper, J.F., Sussman, M.R., Schaller, G.E., Putnam-Evans, C., Charbonneau, H. & Harmon A.C. (1991) A calcium-dependent protein kinase with a regulatory domain similar to calmodulin. Science 252：951-954.
Huang, T.C. & Chow, T.J. (1986) New type of N2-fixing unicellular cyanobacterium (blue-green alga). Federation of European Microbiological Societies Microbiology Letters 36：109-110.
Huang, T.C. & Chow, T.J. (1988) The cyclic synthesis of the nitrogenase of Synechococcus RF-1 and its control at the transcription level. Federation of European Microbiological Societies Microbiology Letters 50：127-130
Huang, T.C., Tu., J., Chow, T.J. & Chen, T.H. (1990) Circadian rhythm of the prokaryote Synechococcus sp. RF-1. Plant Physiology 92：531-533.
Huang, T.C. & Chow, T.J. (1990) Characterization of the rhythmic nitrogen-fixing activity of Synechococcus sp. RF-1 at the transcription level. Current Microbiology 120：23-26
Huang, T.C. & Chou, W.M. (1991) Setting of the circadian N2-fixing rhythm of the prokaryotic Synechococcus sp. RF-1 while its nif gene is repressed. Plant Physiology 96：324-326.
Huang, T.C., Lay, K.C., & Tong, S.R. (1991) Resetting the endogenous circadian N2-fixing rhythm of the prokaryote Synechococcus RF-1. Botanical Bulletin of Academia Sinica 32：129-133.
Huang, T.C., Chen, H.M., Pen, S.Y. & Chen, T.H. (1994) Biological clock in the prokaryote Synechococcus RF-1. Planta 193：131-136.
Huang, T.C. & Pen, S.Y. (1994) Induction of a circadian rhythm in Synechococcus RF-1 while the cells are in a “suspended state”. Planta 194：436-438
Huang, T.C. & Grobbelaar, N. (1995) The circadian clock in the prokaryote Synechococcus RF-1. Microbiology 141：535-540.
Huang, T.C., Lin, R.F., Chu M.K. & Chen, H.M. (1999) Organization and expression of nitrogen-fixation genes in the aerobic nitrogen-fixing unicellular cyanobacterium Synechococcus sp. strain RF-1. Microbiology 145：743-753.
Johnson, C.H. & Golden, S.S. (1999) Circadian programs in cyanobacteria：adaptiveness and mechanism. Annual Reviews of Microbiology 53：389-409
Kerovuo, J., Lappalainen, I. & Reinikainen, T. (2000) The metal dependence of Bacillus subtilis phytase. Biochemical and Biophysical Research Communications. 268：365-369
Kretsinger, R.H. (1980) Structure and evolution of calcium-modulated proteins. CRC Critical Reviews in Biochemistry 8：119-174.
Lee, J.Y., Yoo, B.C. & Harmon, A.C. (1998) Kinetic and calcium-binding properties of three calcium-dependent protein kinase isoenzymes from soybean. Biochemistry 37：6801-6809
Ling, V. & Zielinski, R.E. (1993) Isolation of an Arabidopsis cDNA sequence encoding a 22 kDa calcium-binding protein (CaBP-22) related to calmodulin. Plant Molecular Biology 22：207-214.
Mandel, G. & Goodman, R.H. (1999) DREAM on without calcium. Nature 398：29-30
Maruyama, K., Mikawa, T. & Ebashi, S. (1984) Detection of calcium binding proteins by 45Ca autoradiography on nitrocellulose membrane after sodium dodecyl sulfate gel electrophoresis. Journal of Biochemistry 95：511-519.
Miroux, B. & Walker, J.E. (1996) Over-production of proteins in Escherichia coli：mutant hosts that allow synthesis of some membrane proteins and globular proteins at high levels. Journal of Molecular Biology 260：289-298
Molinari, M. & Carafoli, E. (1997) Calpain：a cytosolic proteinase active at the membranes. Journal of Membrane Biology 156：1-8.
Norris, V., Chen, M., Goldberg, M., Voskuil, J., McGurk, G. & Holland, I.B. (1991) Calcium in bacteria：a solution to which problem？ Molecular Microbiology 5：775-778.
Norris, V., Grant, S., Freestone, P., Canvin, J., Sheikh, F.N., Toth, I., Trinei, M., Modha, K. & Norman, R.I. (1996) Calcium signalling in bacteria. Journal of Bacteriology 178：3677-3682
Onek, L.A. & Smith, R.J. (1992) Calmodulin and calcium mediated regulation in prokaryotes. Journal of General Microbiology 138：1039-1049.
Onek, L.A., Lea, P.J. & Smith, R.J. (1994) Isolation and characterization of a calmodulin-like protein from the cyanobacterium Nostoc sp. PCC6720. Archives of Microbiology 161：352-358
Roberts, D.M. ＆ Harmon, A.C. (1992) Calcium-modulated proteins：targets of intracellular calcium signals in higher plants. Annual Reviews of Plant Physiology and Plant Molecular Biology 43：375-414
Sambrook, J., Fritsch, E.F. ＆ Maniais, T. (1989) Molecular cloning：a laboratory manual, 2nd ed., Cold Spring Harbor Laboratory Press, New York.
Sanger, F., Nicklen, S. & Coulson, A.R. (1977) DNA sequencing with chain terminating inhibitors. Proceeding of the National Academy Sciences of USA. 74：5463-5467
Sarma, P.V.G.K., Sarma, P.U. ＆ Murthy, P.S. (1998) Isolation, purification and characterization of intracellular calmodulin like protein (CALP) from Mycobacterium phlei. Federation of European Microbiological Societies Microbiology Letters 159：27-34
Stanier, R.Y., Kunisawa, R., Mandel, M. & Cohen-Bazire, G. (1971) Purification and properties of unicellular blue-green algae (order Chroococcales). Bacteriological Reviews 35：171-205.
Suzuki, K. (1987) Calcium activated neutral protease：domain structure and activity regulation. Trends in Biochemical Science 12：103-105.
Van Asselt, E.J. & Dijkstra, B.W. (1999) Binding of calcium in the EF-hand of Escherichia coli lytic transglycosylase Slt35 is important for stability. Federation of European Biochemical Societies Letters 458：429-435
Weber, C., Lee , V.D., Chazin, W.J. & Huang B. (1994) High level expression in Escherichia coli and characterization of the EF-hand calcium-binding protein caltractin. Journal of Biological Chemistry 269：15795-15802
Yoon, G.M., Cho, H.S., Ha, H.J., Liu, J.R. & Lee, H-S.P. (1999) Characterization of NtCDPK1, a calcium-dependent protein kinase gene in Nicotiana tabacum, and the activity of its encoded protein. Plant Molecular Biology 39：991-1001
周雪美 (1987) 二種單胞固氮藍綠藻之形態與生理特性的比較. 師大生物學報 22：33-43.
林榮芳(1995) 縐葉菸草異源染色體添加系的建立與鑑定. 國立臺灣大學植物學研究所博士論文.
陳弘民 (1996) 聚球藻RF-1韻律基因的特性研究. 國立臺灣師範大學生物學研究所博士論文.