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We have studied excited-state dynamics of “nonfluorescent” flavoproteins including riboflavin binding protein (RBP), d-amino acid oxidase benzoate complex (DAOB), and others by means of femtosecond fluorescence up-conversion method and have observed ultrafast fluorescence quenching dynamics for the first time. We have interpreted the fluorescence quenching mechanisms of these flavoproteins as due to the ultrafast electron transfer (ET) to flavin chromophore (F) in the excited electronic state from nearby tryptophan (Trp . NH) or tyrosine (Tyr . OH) residues placed in the protein nanospace (PNS), on the basis of their X-ray structures. Extremely fast fluorescence quenching in RBP (τf ∼ 90−100 fs) could be attributed to the compact stacked arrangement, Trp . NH.....F.....Tyr . OH, supremely favorable for the ultrafast ET reaction dynamics. Comparisons of fluorescence time profiles and spectral characteristics of F in solution with those in PNS have indicated the existence of extremely fast FC (Franck−Condon) → Fl (fluorescence) state conversion in PNS within the time resolution of the apparatus. The ultrafast FC → Fl conversion may be a coherent process coupled with intra-chromophore high-frequency modes leading to formation of vibrationally nonrelaxed or only partially relaxed Fl state, from which barrierless ET seems to occur. Fluorescence dynamics of DAOB have indicated faster initial decay in both blue and red sides of the spectrum contrary to other flavoproteins which showed practically wavelength-independent fluorescence dynamics. This result of DAOB is similar to those of photoactive yellow protein and visual rhodopsin although their reaction mechanism (twisting) is different from DAOB (ET). We have proposed a possible mechanism for this fluorescence dynamics of DAOB on the basis of an extremely compact stacked configuration of F...benzoate-...Tyr . OH which seems to undergo moderate frequency intermolecular vibration coupled with intra-chromophore high-frequency modes of F in the course of ET from Tyr . OH to excited F.

The X-ray crystallographic structure of porcine kidney D-amino acid oxidase, which had been expressed in Escherichia coli transformed with a vector containing DAO cDNA, was determined by the isomorphous replacement method for the complex form with benzoate. The known amino acid sequence, FAD and benzoate were fitted to an electron density map of 3.0 A resolution with an R-factor of 21.0%. The overall dimeric structure exhibits an elongated ellipsoidal framework. The prosthetic group, FAD, was found to be in an extended conformation, the isoalloxazine ring being buried in the protein core. The ADP moiety of FAD was located in the typical beta alpha beta dinucleotide binding motif, with the alpha-helix dipole stabilizing the pyrophosphate negative charge. The substrate analog, benzoate, is located on the re-face of the isoalloxazine ring, while the si-face is blocked by hydrophobic residues. The carboxylate group of benzoate is ion-paired with the Arg283 side chain and is within interacting distance with the hydroxy moiety of Tyr228. The phenol ring of Tyr224 is located just above the benzene ring of benzoate, implying the importance of this residue for catalysis. There is no positive charge or alpha-helix dipole near N(1) of flavin. Hydrogen bonds were observed at C(2) = O, N(3)-H, C(4) = O, and N(5) of the flavin ring.

We have cloned cDNAs involved in germ cell-specific expression. For this, a subtracted cDNA library was generated by subtracting cDNAs derived from supporting cells of mutant testis from wild-type testis cDNAs. Detailed analyses of mRNA expression revealed that the genes corresponding to the cloned cDNAs were exclusively expressed in testes and were developmentally controlled.Key words: Testis; Mouse; Protamin; cDNA library; Subtraction; WnV mutant; Actin capping protein IntillctlonMouse spermatogenesis is an excellent model system to study regulation of gene expression during differentiation. Postnatal development of the mouse seminiferous epithelium is a complicated process which finally generates a tissue able to produce functional spermatozoa. The whole process can be subdivided into three parts: (i) a premeiotic phase characterized by an increase in cell number due to mitotic divisions of diploid spermatogonia; (ii) a meiotic prophase, which leads to the formation of haploid round spermatids; and (iii) a post-meiotic phase, which includes the morphogenetic events required for spermatozoa formation (spermiogenesis) [ 1,2]. Specific cells derived from the stem cells in seminiferous epithelium of the adult testis undergo these processes and continuously provide the mature sperms. The precise regulation of such germ cell differentiation requires a strict program of stage-and cell-specific gene expression in germ cells as well as in surrounding somatic cell types [3]. To understand the mechanism of testicular germ cell differentiation, it is of great interest to isolate specific genes and characterize their functions as well as their regulation. Materials and methods Preparation of cDNA libraries carrying directional insertsTotal RNA followed by purification of poly (A)+ RNA was extracted by the guanidme thiocyanate/CsTFA method from the testes of adult wild-type B6 mice and 4-month-old W/W" mutant mice [4]. Their cDNA libraries were prepared as described by Gubler and Hoffmann with some modifications (Kobori et al., manuscript in preparation) [5]. Briefly, cDNA was synthesized in a reaction mixture including 'MedCTP with reverse. transcriptase (Superscript II) from 2-5 pg of mouse testis poly(A)+ RNA and 1.6 pug of oligo (dT) primer carrying a Not1 site. The reaction mixture was treated with RNase H, followed by reaction with DNA polymerase I. Each end was blunt-ended with T4 DNA polymerase and ligated to an unphosphorylated BglII-SmaI adaptor. After digestion with NotI, small DNA fragments of less than 300 bp were removed by a CROMA spin-400 column (Clontech, USA). The cDNA fragments were directionally inserted between the Not1 (dephosphorylated) and BgZII sites of vector pAP3neo (H. Nojima, unpublished). The ligation mixture was electroporated into MC1061A cells as described [q. The complexities of the cDNA libraries used here *Corresponding author. Fax: (81) (6) 879-8339.were 6.0 x lo6 colony forming units (cfu) for the B6 wild-type mouse and 2.8 x lo6 cfu for the Wm mutant mouse. Preparation of a s...

We have isolated a cDNA clone encoding a germ cell specific protein from an expression cDNA library prepared from the mouse testis, using testis-specific polyclonal antibodies. Sequence analysis of the cDNA revealed that the deduced amino acid sequence consisted of 284 residues, including a nominal repeat structure in the N-terminal region. Northern blot analysis revealed the presence of a transcript of 1.3 kb exclusively expressed in the testis and ovary, but at relatively low levels in the ovary. In contrast, no other tissues and organs expressed significant levels of the transcript. Expression of the mRNA in the testis was first detected on day 14 in postnatal development. Western blot analysis showed the presence of the protein with a molecular weight of approximately 40 kDa and an isoelectric point of 4.9. The protein was exclusively found in the testis and ovary, but in a far lesser amount in the ovary as was the case with the transcript. Immunohistochemical examination revealed that the protein was predominantly present in the cytoplasm in pachytene spermatocytes through to round spermatids. However, during the disappearance of the nuclear envelope at both the first and second meiotic divisions, the protein was localized around the metaphase chromosomes and spindles. Because of this, the name meichroacidin which stands for male meiotic metaphase chromosome-associated acidic protein is proposed for this antigen. The highly regulated stage-specific expression of meichroacidin and its specific association with the metaphase chromosomes and spindles suggest that the protein plays important roles in male meiosis.