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Biochemical and Biophysical Research Communications 256, 330–332 (1999)

Article ID bbrc.1999.0326, available online at http://www.idealibrary.com on

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wo Homologous Cytochrome b5s Are Expressedn Both Neurons and Glial Cells of the Rat Brain

in Yoo1

epartment of Biology, Keimyung University, Taegu, 704-701, Korea

eceived January 12, 1999

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To avoid the possibility of blood contamination andf gene rearrangement during library construction wesolated total RNA directly from cultured rat embry-nic neuronal cells and glioma C6 cells to be used asemplate for RT-PCR. By using specific primers foroth membrane-bound b5 and soluble b5, DNA bands ofppropriate size were clearly amplified indicatinghat both neurons and glial cells expressed b5s, al-hough soluble b5 seemed to be less expressed in theseells. Nucleotide sequence of the internal exon for sol-ble b5 was reinvestigated and confirmed to be 58 bpontaining genetic codons for His-Ser-Ala-Leu andtop. © 1999 Academic Press

Cytochrome b5 (b5) is a small heme-containing pro-ein, which supplies electrons for many cytochrome450 catalyzed reactions (1). It exists as two distinct

orms. In most mammals liver b5 is a membrane-boundrotein that consists of 134 amino acids, whereasrythrocyte b5 is a soluble protein composed of 98mino acids (2, 3). It has been well demonstrated thathese two forms are derived from a common precursorRNA through alternative mRNA splicing mecha-isms. The mRNAs for soluble b5 in rabbit and humanre larger by 24 bp than ones for membrane-bound b5

ue to the inclusion of internal extra exons (4, 5).Recent advances in molecular genetics have indi-

ated the necessity of b5s in the brain along with theiscovery of cytochrome P450 enzymes (6, 7). Succes-ively, we were able to isolate cDNAs for bothembrane-bound b5 and soluble b5 from the rat brain

DNA library (8). Interestingly, the extra exon of ratrain soluble b5 was somewhat larger (58 bp) compar-ng to other mammalian species although its locationas consistent. As a result rat soluble b5 consisted of00 amino acids instead of 98.However, this report was strongly argued by

teggles et al. (9) because of the following reasons:

1 Fax: 82-53-580-5537. E-mail: ymin@kmucc.keimyung.ac.kr.

330006-291X/99 $30.00opyright © 1999 by Academic Pressll rights of reproduction in any form reserved.

he procedure of brain library construction whichight supply messages for soluble b5. (ii) possibility of

he gene rearrangement during the library construc-ion which could be the reason why the mRNA for ratoluble b5 contained larger extra exon. So, it is prereq-isite to clarify these questions before we take anyurther step for studying the function of b5s in therain. The only way of clarifying these questions is usef cultured rat brain cells as direct source of mRNAs.Brain consists of neurons and glial cells. Most neu-

ons are so specialized for carrying information thathey cannot maintain themselves. They are kept alivey the help of glial cells.The aims of the present study are to determinehich type of brain cells expresses b5 (or b5s) and to

onfirm the structure of extra exon for rat brain soluble5 at cellular level.

ATERIALS AND METHODS

Preparation of the embryonic neurons. Primary neuronal cellsere isolated from the fetal rat brain as described by Brewer et al.

10). Briefly, embryos were recovered by C-section from a pregnantprague–Dawley rat at 15 days of gestation. Hippocampal neuronsere collected after decapitation and washed briefly with Hanks’alanced salt solution (HBSS, GIBCO) supplemented with 1 mModium pyruvate, 10 mM Hepes (pH 7.4). Neurons were then treatedith 0.25% Trypsin in HBSS for 25 min at 37°C followed by washingith HBSS two more times. Individual cells were separated byentle pipetting several times. After centrifugation cell pellet wasently resuspended in 3 ml of culture medium. An aliquot was mixedith an equal volume of 0.4% trypan blue and dye-excluding cellsere counted in a hemacytometer. Initial culture density was 6 3 106

ells/culture plate.

Cell culture. Primary neuronal cells were cultured in poly-D-ysine coated, 6-well tissue culture plates with 3 ml of Neurobasal

edium (GIBCO) supplemented with 2% B-27 (GIBCO), 0.5 mM-glutamine, 25 mM glutamate and 25 ml of 2-mercaptoethanol. Afterdays of initial culturing, one-half of the medium was changed to theedium without glutamate. Rat glioma C6 cells (Dongsan Medicalenter, Taegu, Korea) were cultured in 100-mm tissue culture platesith 10 ml of Dulbecco’s modified Eagle’s medium (DMEM, GIBCO)

upplemented with 10% heat-inactivated fetal bovine serum, 13ntibiotic-Antimycotic (GIBCO). Cultures were maintained at 37°C

n a humidified incubator under an atmosphere of 5% CO2.

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Vol. 256, No. 2, 1999 BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS

Reverse transcription (RT). Total RNA was isolated from theultured cells using RNAzol-B solution (Biotex Laboratories, Inc.).ne microgram of purified total RNA was used as template in RT

eaction. RT mixture contained 4 ml of 53 buffer (250 mM Tris–HClpH 8.3), 375 mM KCl, 15 mM MgCl2, 50 mM DTT), 4 ml of 10 mMNTPs, 100 pmol of oligo(dT)16, 20 units of RNasin, 100 units of-MLV reverse transcriptase and DEPC-treated, RNase-free H2O tototal volume of 20 ml. RT mixture was incubated for 1 h at 42°C for

he synthesis of first-strand cDNA.

Polymerase chain reaction (PCR). Three primers were designedased on our previous report (8). Their sequences were GAGCATC-TGGTGGGGAAGAAGTCCT (sense, primer 1), GGGATCACCC-GTTGGTCCACC (antisense, primer 2) and CTGTGCTAAGAG-AAAACTCA TAGCGC (antisense, primer 3). PCR was performed

n a solution containing 1 ml of RT product, 3 ml of 103 buffer (100M Tris–HCl, pH 8.3, 50 mM KCl, 20 mM MgCl2, 0.1% gelatin), 1 ml

f 10 mM dNTPs, 10 pmol of each primer (sense and antisense), 1nit of Taq DNA polymerase and deionized H2O to a total volume of0 ml. Thirty-five cycles were carried out with a program as followed:enaturation at 94°C for 30 s, annealing at 55°C for 30 s andxtension at 72°C for 30 s.

Southern blot analysis. After electrophoresis of PCR products on.2% agarose gel DNAs were transferred onto the Hybond N1 nylonembrane. Hybridization were carried out at 55°C for 12–18 h usingdigoxigenin-labeled rat b5 cDNA (membrane-bound form).

DNA sequencing. PCR products were ligated into pBluescript KSlasmids and nucleotide sequences were determined by a dideoxyhain termination method.

ESULTS AND DISCUSSION

To avoid the possibility of blood contamination and ofene rearrangement during the library constructione isolated total RNA directly from the cultured brain

FIG. 1. Partial structure of the mRNA for soluble b5 and experielative positions of primers 1, 2, and 3. Black-boxed area represent

FIG. 2. Results of RT-PCR using mRNAs isolated from the cul-ured embryonic neuronal cells (lane 1) and rat glioma C6 cells (lane) as template. Primers were P1 and P2. Lane M represents the sizearker, 100-bp ladder.

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ells and used it as template for RT-PCR. Embryoniceuronal cells were originated from the hippocampalrea of 15 days fetal rat. Rat glioma C6 cells wereerived from astrocytes. The strategy of experimentalrocedure is represented in Fig. 1.From the RT-PCR using primers 1 and 2, about

20-bp bands were strongly amplified for both embry-nic neuronal cells and glial cells (Fig. 2). This was theize that corresponded to the mRNA of membrane-ound b5. Along with these strong bands, we detectedeak amplifications, too. These weak bands were esti-ated roughly to be 280 bp. To investigate the internal

tructure of these weak bands two separate experi-ents were carried out. First, we extracted 280-bp

ands from the agarose gel and used these DNAs asemplate for further PCR amplifications. Combinationf primers 1 and 3 was used at this time. Primer 3 wasoluble b5 specific and designed based on the internalxon of soluble b5 according to our previous report (8).o, if our previous report was wrong due to the geneearrangement no DNA band could be amplified at thisime. However, 195-bp bands were clearly reamplifiedrom these templates (Fig. 3). Again, this was the exactize for soluble b5 and the results were same for bothmbryonic neuronal cells and C6 glial cells. So, it isummarized that both membrane-bound b5 and soluble5 were clearly expressed in the rat brain althougholuble b5 seemed to be less expressed. Both neuronalells and glial cells expressed these b5s.

ntal strategy for PCR amplifications. P1, P2, and P3 represent thee internal extra exon for soluble b5.

FIG. 3. Results of PCR amplifications using 280-bp DNA frag-ents extracted from lanes 1 and 2 of Fig. 2 as template. Primersere P1 and P3. Lane M represents the size marker, 100-bp ladder.

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After subcloning 280-bp DNA bands into pBluescriptS plasmids we determined the nucleotide sequence of

nternal exon for soluble b5. The result of sequencing ishown in Fig. 4. It was exactly 58 bp and the sequenceatched up to what we reported before (8). It contained

enetic codons for His-Ser-Ala-Leu and stop. So, rat isnique in that its soluble b5 consisted of 100 amino

FIG. 4. Detailed sequence of internal extra exon for rat brainoluble b5 mRNA.

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xon.There was absolutely no chance of getting mRNA for

oluble b5 from the blood contamination in these experi-ents because the source of mRNA was the cultured

rain cells. It is very interesting that Steggles et al. wereble to detect only membrane-bound form of b5 in theabbit brain (5). However, he did not exclude the possi-ility that tissue distribution of b5s may differ betweennimal species. We do not know at the present time if rats the only animal to express soluble b5 in the brain.

In conclusion, we demonstrate at cellular level thatoth membrane-bound b5 and soluble b5 are expressedn the rat brain. Both neuronal cells and glial cellsxpress these b5s. It needs to be further investigatedhy soluble b5 is expressed in the rat brain and what itoes in the brain.

EFERENCES

1. Vergeres, G., and Waskell, L. (1995) Biochimie 77, 604–620.2. Mathews, F. S. (1985) Prog. Biophys. Mol. Biol. 45, 1–56.3. Ozols, J. (1989) Biochim. Biophys. Acta 997, 121–130.4. Giordano, S. J., and Steggles, A. W. (1991) Biochem. Biophys.

Res. Commun. 178, 38–44.5. Giordano, S. J., and Steggles, A. W. (1993) Biochim. Biophys.

Acta 1172, 95–100.6. Yoo, M., Ryu, H. M., Shin, S. W., Yun, C. H., Lee, S. C., Ji, Y. M.,

and You, K. H. (1997) Biochem. Biophys. Res. Commun. 231,254–256.

7. Yoshida, S., Yubisui, T., and Takeshita, M. (1984) Arch. Bio-chem. Biophys. 232, 296–304.

8. Yoo, M. (1997) Biochem. Biophys. Res. Commun. 236, 641–642.9. VanDerMark, P. K., and Steggles, A. W. (1997) Biochem. Bio-

phys. Res. Commun. 240, 80–83.0. Brewer, G. J., Torricelli, J. R., Evege, E. K., and Price, P. J.

(1993) J. Neurosci. Res. 35, 567–576.