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taining and storing hyperthermophilic cultures hopefully will contribute to the rapid advancement of developing genetic systems for hyperthermophiles, which is a prerequisite for biotechnological applications and to understanding how these unique creatures are capable of ‘‘taking the heat and loving it’’ (12). ACKNOWLEDGMENTS The authors are indebted to Drs. Michael W. W. Adams (Department of Biochemistry, University of Georgia, Athens, GA) and M. Cox (Anaerobe Systems, San Jose, CA), for helpful discussion and encouragement.

REFERENCES 1. Stetter, K. O. (1982) Nature 300, 258–260. 2. Woese, C. R., and Wolfe, R. S. (1985) The Bacteria, Vol. 8, Academic Press, New York. 3. Adams, M. W. W., Perler, F. B., and Kelly, R. M. (1995) Biotechnology 13, 662–668. 4. Saiki, R. K., Gelfand, D. H., Stoffel, S., Scharf, S. J., Higuchi, R., Horn, H. G., Mullis, K. B., and Erlich, H. A. (1988) Science 239, 487–491. 5. Wiegel, J. (1986) in Thermophiles: General, Molecular, and Applied Biology (Brock, T. D., Ed.), pp. 17–37, Wiley, New York. 6. Deming, J. W., and Baross, J. A. (1986) Appl. Environ. Microbiol. 51, 238. 7. Blo¨chl, E., Burggraf, S., Fiala, G., Lauerer, G., Huber, G., Huber, R., Rachel, R., Segerer, A., Stetter, K. O., and Vo¨kl, P. (1995) World J. Microbiol. Biotechnol. 11, 9–16. 8. Lin, C. C., and Casida, L. E., Jr. (1984) Appl. Environ. Microbiol. 47, 427. 9. Smidsrod, O., and Skjak-Braek, G. (1990) TIBTECH 8, 71–78. 10. Neuner, A., Jannish, H. W., Belkin, S., and Stetter, K. O. (1990) Arch. Microbiol. 153, 205–207. 11. Fiala, G., and Stetter, K. O. (1986) Arch. Microbiol. 145, 56–61. 12. Rees, D. C., and Adams, M. W. W. (1995) Structure 3, 251–254.

Quantitative Polymerase Chain Reaction Using Homologous Internal Standards Seymour J. Garte*,† and Sabya Ganguly* *Department of Environmental Medicine and †Kaplan Cancer Center, New York University Medical Center, 550 First Avenue, New York, New York 10016 Received April 23, 1996

Quantitative measurement of the expression or the degree of amplification of specific genes is a critically important tool in understanding basic mechanisms both in molecular biology and in a number of applications related to the effects of various agents on cell function. Often traditional approaches for such meaANALYTICAL BIOCHEMISTRY ARTICLE NO.

surements such as blot hybridization are not feasible due to the absence of a sufficient amount of material to extract enough DNA or RNA for analysis. For example, in order to determine the level of expression or amplification of a gene in archived tissue blocks, pathology slides, or other samples containing small amounts of possibly degraded DNA and RNA, only PCR techniques are useful. However, when quantitation is important, as when comparing gene expression levels in a series of tissue samples, simple RT–PCR1 may give distorted results because of the potential for introducing large differences in the amount of final reaction product during the exponential amplification or reverse transcription steps (1). To alleviate this problem, investigators have used internal standards, which when added to the reaction tube can be used to normalize for intertube variations in amplification efficiency (2, 3). The choice of an internal standard is not simple. Requirements include that the standard be amplified with the same efficiency as the target gene, that the primers be of similar composition, and that the products of the standard and target gene be easily distinguished and quantitated. Some approaches that have been used include use of genes on the same chromosome as the target gene. This should give reasonably equal amplification efficiency; however, because the primer sequences for the target and standard gene are different, significant differences in efficiency may occur. Another approach is to synthesize a sequence identical to the target gene, but with a larger product size, or an internal restriction site not present in the target gene. This has the advantage of using exactly the same primer sequences for both target and standard genes. The main disadvantage of this method is the difficulty in preparation of new standards for every gene to be studied. Furthermore, the standard is not part of a larger sequence (such as an mRNA), and this size difference may contribute to errors. We have developed and used a simple method for accurate quantitation of human DNA and RNA by PCR called the homologous internal standard (HIS) method. This method uses homologous genes from rodents as internal standards for quantitation of human genes or their messages. The standards use exactly the same primers as the target genes, no synthesis is necessary, and target and standard products are easily distinguished and quantitated using restriction digestion. The strategy behind the HIS technique is to locate in a rodent species a sequence of the gene cDNA that shares total homology with the human cDNA in two 20-bp regions separated by a sequence of 80 to 200 bp with less than perfect homology. In order to work therefore, the gene must have been cloned and se1

Abbreviations used: RT-PCR, reverse transcription–polymerase chain reaction; HIS, homologous internal standard.

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quenced from at least one rodent species as well as the human. In practice the identification of such sequences is quite simple, and usually requires an hour or two of gene library searching. Once the human and homologous sequences have been identified, a matching program such as BESTFIT or FASTA can be used to align the two sequences. A printout of such an alignment can be quickly and easily visually scanned to detect small regions of perfect homology separated by larger regions of less perfect homology. For most genes with human– nonhuman homologies of 75–90%, several alternative possible regions within a cDNA sequence become rapidly evident. If the homology is less than 65–70%, this may be more difficult. For genes with less than 60% homology between human and nonhuman homologs, the HIS method is not likely to be useful. Primer sequences (with perfect homology between human and nonhuman genes) of 20 bp are chosen to flank the amplicon region. Once primer sequences have been identified, the intervening amplicon sequence for both human and nonhuman genes must be mapped for restriction enzyme sites in order to find a unique restriction site in the nonhuman (HIS) amplicon which is absent in the human amplicon. This site should be located as close to the center of the HIS amplicon as possible. Usually there are between two and four such sites to choose from, and the choice of enzyme can be made on the basis of cost, convenience, and experience. It is possible to choose sequences with the unique site in the human gene; however, we have found that better results are obtained when the cutting site is in the standard sequence. Once the appropriate sequences are identified, and a single set of primers is prepared from the perfectly homologous flanking regions, a test titration should be performed using mixtures of human

FIG. 2. CYP1A1 mRNA Titration. Band intensities from Fig. 1 were determined by liquid scintillation counting, and data were plotted vs ratio of human/HIS RNA added to each tube. Linear correlation coefficient R Å 0.978, P õ 0.004.

FIG. 1. CYP1A1 mRNA titration gel. Varying amounts (shown in nanograms on the figure) of human RNA from MCF-7 breast cancer cells were mixed with 1 ng mouse CYP1A1 plasmid (ATCC No. 63006). RT–PCR products were digested with BstNI and run on 8% urea acrylamide gel in TBE buffer. The gel was exposed to X-ray film for 2 h. The top band is the human amplicon (255 bp), and the bottom band (164 bp) is the digested mouse amplicon.

and the appropriate rodent DNA or RNA at different ratios. For accurate quantitation we have used primers endlabeled with radioactive 32P. Relative levels of amplicons can be determined by liquid scintillation counting after the human and HIS bands are cut from the gel. Densitometric scanning, binding to specific antibodies (4–6), chemiluminescence (7, 8), fluorescence (9), or other quantitation techniques may also be used after the restriction digestion step. Titration experiments illustrated in Figs. 1 and 2 are critical to determine the optimum conditions of the assay for each gene. Figure 1 shows a representative RT–PCR gel with human and mouse (as the HIS) bands for CYP1A1. The lower band is the mouse amplicon after digestion with BstNI. Figure 2 presents the titration curve of the ratio of human to HIS band intensity determined as a function of the ratio of human to HIS RNA. We have performed the HIS technique on samples of human peripheral blood lymphocytes, frozen for over 1 year at 0207C, in order to assess the level of expression of the CYP1A1 gene; small samples of cells obtained by a simple nasal wash to determine the level of metallothionein gene expression; and DNA obtained by extraction from histology slides to quantitate the degree of c-myc gene amplification in human breast cancer. The HIS method for quantitative PCR and RT–PCR is applicable to the analysis of any human gene, as long as a suitable rodent homolog has been cloned and sequenced. Examples of other genes for which the method may be used are listed in Table 1. For each gene, a search of GenBank revealed several possible alternative HIS sequences. We used the BESTFIT program to align human and homologous sequences, and then DNA STRIDER to produce restriction maps of the amplicon regions. The choice of primers and amplicons was made by visual examination of the homology alignment. Clearly, the number of genes for which the

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Restriction Enzymes, Amplicons, and Fragment Sizes Used in the HIS RT–PCR Method for Nine Representative Genes HIS species

Gene

% Homology

Cathepsin D

Rat

75

Heme oxygenase

Mouse

78

p53

Mouse

79

PDGF

Mouse

87

PKC

Rat

90

TGF b receptor

Rat

82

c-myc Metallothionein CYP1A1

Rat Hamster Mouse

87

Enzymea BglII, HinfI Tth111 II, Bsu36 I HphI, EcoRII NcoI, MseI Cfr10 I, BbvI NspbII, BsteII BsiHkI HgiAI BstNI

method may be used is quite large, and the examples in the table are only for illustration. Given the nature of the PCR reaction, accurate quantitation is not possible without the use of an internal standard which is amplified in the same tube and with the same conditions as the target sequence. Because amplification efficiencies may differ significantly as a function of primer sequence, it has become apparent that the use of different primers for the target and the standard may introduce new problems in the quantitation of the assay. Many laboratories have addressed the problem of doing quantitative PCR by the use of internal standards which are as similar as possible to the target sequence. Most of these have relied on synthetic standards (4, 5, 8, 10–14) often produced by sitespecific mutagenesis (15, 16) to be able to use the identical primer sequences for amplification of both target and standard genes. Other approaches have been taken in order to satisfy the requirement for single primer sets such as the use of deletion mutants (6, 7, 17), the use of a mammalian gene homolog to quantitate expression of a viral gene (18), or other creative strategies (19, 20). While these methods are certainly useful, they require considerable laboratory effort for production of a standard for each gene to be studied. One group developed a standard for use with a series of genes (14); however, usually such an approach must be followed for one gene at a time. Furthermore, in order to separate standard from target amplicons by gel electrophoresis after PCR, fairly large differences in size are necessary which may introduce new possibilities for error (21). The HIS method offers several advantages over other methods to achieve reliable quantification of PCR of either DNA or RNA. It is much simpler and faster to determine the proper primer sequences by computer

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Humanb amplicon

HISc amplicon

353

348

343

347

391

391

256

255

206

206

127

130

125 130 255

125 130 256

HIS fragmentsd 202 232 169 203 188 196 162 179 94 130 62 76 65 75 164

{ { { { { { { { { { { { { { {

146 116 172 138 203 195 93 76 112 76 68 54 60 55 92

searching and matching than to synthesize specific standards for each gene to be investigated. Once the primer sequences, restriction enzymes and homologous species have been identified, the source of the HIS can be any cell line or tissue from the appropriate species. The competitive titration methods or other techniques could also be used with the HIS approach to provide more precise values for the quantity of RNA in a particular sample. REFERENCES 1. Foley, K. P., Leoneard, M. W., and Engel, J. D. (1993) Trends Genet. 11, 380–385. 2. Duchmann, R., Strober, W., and James, S. P. (1993) DNA Cell Biol. 12, 217–225. 3. Vanden Heuvel, J. P., Tyson, F. L., and Bell, D. A. (1993) BioTechniques 14, 395–398. 4. Tsuruta, H., Matsui, S., Oka, K., Namba, T., Shinngu, M., and Nakamura, M. J. (1995) Immunol. Methods 180, 259–64. 5. Berndt, C., Bebenroth, M., Oehlschlegel, K., Hiepe, F., and Schossler, W. (1995) Anal. Biochem. 225, 252–257. 6. Ravaggi, A., Zonaro, A., Mazza, C., Albertini, A., and Cariani, E. (1995) J. Clin. Microbiol. 33, 265–269. 7. Boivin, G., Olson, C. A., Quirk, M. R., St-Cyr, S. M., and Jordan, M. C. (1995) J. Virol. Methods 51, 329–342. 8. Karet, F. E., Charnock-Jones, D. S., Harrison-Woolrych, M. L., O’Reilly, G., Davenport, A. P., and Smith, S. K. (1994) Anal. Biochem. 220, 384–390. 9. Jesson-Eller, K., Picozza, E., and Crivello, J. F. (1994) BioTechniques 17, 962–973. 10. Thiery, R., Boutin, P., Arnauld, C., and Jestin, A. (1995) BioTechniques 18, 212–213. 11. Riedy, M. C., Timm, E. A., Jr., and Stewart, C. C. (1995) BioTechniques 18, 70–74. 12. Hamalainen, E. R., Keppainen, R., Kuivaniemi, H., Tromp, G., Vaheri, A., Pihlajaniemi, T., and Kivirikko, K. I. (1995) J. Biol. Chem. 270, 21590–21593.

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13. Jia, G. Q., and Gutierrez-Ramos, J. C. (1995) Eur. J. Immunol. 25, 2096–2100. 14. Sestini, R., Orlando, C., Zentilin, L., Lami, D., Gelmini, S., Pinzani, P., Giacca, M., and Pazzagli, M. (1995) Clin. Chem. 41, 826–832. 15. Kinoshita, M., Shin, S., and Aono, T. (1993) Genet. Anal. Tech. Appl. 10, 116–121. 16. Guyon, T., Levasseur, P., Truffault, F., Cottin, C., Gaud, C., and Berrih-Aknin, S. R. (1994) J. Clin. Invest. 94, 16–24. 17. Wagner, J., Drab, M., Gehlen, F., Langheinrich, M., Volk, S., Ganten, D., and Ritz, E. (1994) Kidney Int. 46, 1542–1545. 18. Schang, L. M., and Osorio, F. A. (1994) J. Virol. Methods 50, 269–280. 19. Zenilman, M. E., Graham, W., Tanner, K., and Shuldiner, A. R. (1995) Anal. Biochem. 224, 339–346. 20. Huang, SK., Yi, M., Palmer, E., and Marsh, D. G. (1995) J. Immunol. 154, 6157–6162. 21. Repp, R., Borkhardt, A., Gossen, R., Kreuder, J., Hammermann, J., and Lampert, (1995) BioTechniques 19, 84–90.

Identification of the Glycation Site of Lens gB-Crystallin by Fast Atom Bombardment Tandem Mass Spectrometry Jean B. Smith,* Stacy R. A. Hanson,* Ronald L. Cerny,* Hui-Ren Zhao,† and Edathara C. Abraham† *Department of Chemistry, University of Nebraska, Lincoln, Nebraska 68588-0304; and †Department of Biochemistry and Molecular Biology, Medical College of Georgia, Augusta, Georgia 30912-2100 Received June 14, 1996

Previous investigations of the glycation of lens gBcrystallin have produced conflicting results. In their investigation of [3H]NaBH4-treated crystallins from diabetic rats, Abraham et al. (1) found evidence that the N-terminus Gly-1, Lys-2, and Lys-163 of gB were all glycated. In contrast, Pennington et al. (2) examined [14C]fructosylated gB-crystallin that had been digested with trypsin and concluded that only Gly-1 was glycated. In this investigation, amino acid analysis of the major radioactive peptide, presumably Gly-Lys, had detected only Lys. They assumed that Gly was not detected because glycation blocked the N-terminus, preventing detection by amino acid analysis. In a third study, Casey et al. (3) determined the extent of glycation of gB-crystallin that had been altered by site-directed mutagenesis to replace Lys-2 with Thr. Because they found that glycation of the mutated form was significantly reduced, they concluded that Lys-2 is a major glycation site. On the other hand, the same study reported that amino acid analysis of both [14C]ANALYTICAL BIOCHEMISTRY ARTICLE NO.

glycosylated wild-type and mutated gB-crystallin indicated that Gly-1 was the major site of reaction. These apparently conflicting results could be rationalized if replacement of Lys-2 with Thr affected the rate of glycation, not only by preventing glycation at residue 2, but also because substitution of Thr for Lys could affect the rate of glycation at the N-terminus of Gly-1. Glycation of gB-crystallin is of interest because, as one of the structural proteins of the eye lens, it undergoes very little turnover and therefore has a lifetime of opportunity for posttranslational modification. A variety of evidence indicates that cataract develops when posttranslational modifications interfere with the proper close-packing of the lens crystallins (4) and that the early onset of cataract among diabetics may be due to nonenzymatic glycation of the e-amino NH2 groups of lysine residues or the amino termini of lens crystallins (5). Among the lens crystallins, only the highly homologous g-crystallins have a free N-terminus. The g-crystallins are a major constituent of the water-insoluble portion of the lens, which is thought to be a precursor of cataract (6). The uncertainty about the predominant glycation site of gB-crystallin is due to the close proximity of two of the possible sites, the N-terminus of Gly-1 and the e-amino group of Lys-2. We report the use of fast atom bombardment tandem mass spectrometry (FAB-MS/ MS) to unequivocally determine the site of glycation of gB-crystallin. Although tandem mass spectrometry has been used extensively to locate modification sites of proteins (7, 8), including lens proteins (9, 10) and Nand O-linked glycosylation sites of other proteins (11), this technique has not been used previously to locate sites of nonenzymatic glycation. In our investigation, the glycated protein was enzymatically digested, the glycated peptides were isolated by affinity chromatography, and the location of the modified site of the peptide was determined by examination of the fragmentation pattern of the peptide following analysis by FABMS/MS. Calf lenses were decapsulated and homogenized in 50 mM phosphate buffer, pH 7.4, containing 50 mM NaCl and the resulting homogenate was centrifuged at 10,000g for 30 min at 47C. The supernatant containing the water-soluble proteins was fractionated by size-exclusion chromatography (Sephacryl-S-300HR) into a-, b-, and g-crystallins. The g-crystallin fraction was pooled and dialyzed against a buffer of 0.02 M Tris– acetate, 1 mM EDTA, 0.1 mM DTT,1 pH 6.0. gB-Crystallin was isolated by cation-exchange HPLC using a 250 1 4.6-mm SynChropak CM300 column and a 50 1 4.6-mm guard column (Synchrom Inc., Lafayette, IN) according to the method of Siezen et al. (12) with minor modifications. The mobile phase consisted of buffer A 1

Abbreviations used: DTT, dithiothreitol; TFA, trifluoroacetic acid.

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