Proc. Natl. Acad. Sci. USA Vol. 84, pp. 8623-8627, December 1987 Medical Sciences Human deficiency associated with a : Thermolabile aldolase due to a single base mutation (DNA sequencing/expression vector/hereditary disease) HIROYUKI KISHI*t, TSUNEHIRO MUKAI*, AKIRA HIRONOt, HISAICHI Fundi, SHIRO MIWAt, AND KATSUJI HORI*§ *Department of Biochemistry, Saga Medical School, Nabeshima, Saga 840-01, Japan; and tDepartment of Internal Medicine, Institute of Medical Science, Tokyo University, Tokyo 108, Japan Communicated by Charles C. Richardson, July 20, 1987

ABSTRACT -1,6-bisphosphate aldolase A (fruc- hemolytic anemia, has been described (11, 12). Two cases tose-bisphosphate aldolase; EC 4.1.2.13) deficiency is an (one kindred) of three were found in Japan. In these cases autosomal recessive disorder associated with hereditary hemo- erythrocyte aldolase activity was very low and thermolabile, lytic anemia. To clarify the molecular mechanism of the suggesting that the mutation is on the structural gene as deficiency at the nucleotide level, we have cloned aldolase A opposed to being a mutation of gene regulation. cDNA from a patient's poly(A)+ RNA that was expressed in In the present paper we examine a case of erythrocyte cultured lymphoblastoid cells. Nucleotide analysis of the pa- aldolase deficiency and report that by comparing the nucle- tient's aldolase A cDNA showed a substitution of a single otide sequence ofthe patient's erythrocyte aldolase A cDNA nucleotide (adenine to guanine) at position 386 in a coding with that ofa normal control, an A-G transversion was found region. As a result, the 128th , , was to occur in the codon for the 128th amino acid, aspartic acid replaced with (GAT to GGT). Furthermore, change of (GAU). This results in the production of an having the second letter of the aspartic acid codon extinguished a Fok glycine (GGU) instead of aspartic acid. We also discuss the I restriction site (GGATG to GGGTG). Southern blot analysis characteristics of the altered enzyme expressed in Esche- of the genomic DNA showed the patient carried a homozygous richia coli. mutation inherited from his parents. When compared with normal human aldolase A, the patient's enzyme from eryth- AND rocytes and from cultured lymphoblastoid cells was found to be MATERIALS METHODS highly thermolabile, suggesting that this mutation causes a Materials. Reagents for measuring aldolase activity were functional defect of the enzyme. To further examine this obtained from Boehringer Mannheim. The cDNA synthesis possibility, the thermal stability ofaldolase A ofthe patient and system was from Amersham; the nitrocellulose filter was of a normal control, expressed in Escherichia coli using from Schleicher & Schuell; the nylon filter was from New expression plasmids, was determined. The results of E. coli England Nuclear; and [a-32P]dCTP (3704 Ci/mmol; 1 Ci = 37 expression of the mutated aldolase A enzyme confirmed the GBq) was from ICN. Restriction and the other thermolabile nature of the abnormal enzyme. The Asp-128 is enzymes were from Nippon Gene and Takara Shuzo. conserved in aldolase A, B, and C of eukaryotes, including an Cell Cultures. A lymphoblastoid cell line was established insect, DrosophUa, suggesting that the Asp-128 of the aldolase from a patient (Y.K.) with erythrocyte A protein is likely to be an amino acid residue with a crucial role (12) and from a normal volunteer by transforming the periph- in maintaining the correct spatial structure or in performing eral blood with Epstein-Barr virus. These cell the catalytic function of the enzyme. lines were used in these studies because aldolase expressed in cultured lymphoblastoid cells was confirmed to be aldolase Fructose-1,6-bisphosphate aldolase (fructose-bisphosphate A (data not shown), the same type of enzyme as that aldolase; EC 4.1.2.13) is a glycolytic enzyme that is com- expressed in erythrocytes (13). posed of three distinct isozymic forms, aldolases A, B, and Preparations of Aldolase A and Enzyme Assay. For elec- C (1). This enzyme has been studied extensively with respect trophoresis, aldolase was partially purified from erythrocytes to tissue distribution, changes during development, and as follows. Cells that were briefly washed to remove the buffy (1). It seems, therefore, that this enzyme is coat were disrupted and the lysates were passed through useful for CM-Sephadex C-50 and fractionated by ammonium sulfate to studying molecular mechanisms ofgene expression remove hemoglobin. Aldolase activity was determined by and also for understanding the evolution of the gene. In two methods: activity staining (zymogram) and spectroscop- addition, elucidation of the molecular mechanism of aldolase ic methods. Electrophoresis on cellulose polyacetate strips deficiency is helpful for understanding the regulation of and staining for aldolase activity were carried out as de- aldolase expression. scribed by Susor et al. (14). Aldolase activity in the eryth- Genes for aldolases A and B have been cloned and rocyte lysates was determined spectrophotometrically as characterized in rat (2-4), human (5-7), and other animals (8, described (15). The human aldolase activity expressed in E. 9). These results now permit us to study in detail the coli was determined by the two methods described above in molecular basis of hereditary diseases caused by human the presence of 5 mM EDTA to inhibit E. coli aldolase aldolase deficiency. In inherited deficiency ofaldolase in man activity (16). To determine thermal stability of aldolase, cell there is a fructose intolerance, which is due to a aldolase lysates were incubated for 30 min at various temperatures in B deficiency (10). Recently, another type of clinical entity, the presence of proteinase inhibitors-leupeptin, pepstatin, erythrocyte aldolase deficiency associated with hereditary Abbreviation: PhMeSO2F, phenylmethylsulfonyl fluoride. The publication costs of this article were defrayed in part by page charge tPresent address: Medical Institute of Bioregulation, Kyushu Uni- payment. This article must therefore be hereby marked "advertisement" versity, Fukuoka 812, Japan. in accordance with 18 U.S.C. §1734 solely to indicate this fact. §To whom reprint requests should be addressed. 8623 Downloaded by guest on September 25, 2021 8624 Medical Sciences: Kishi et al. Proc. Natl. Acad. Sci. USA 84 (1987) N-a-tosyllysine chloromethyl ketone, and phenylmethylsul- the isozymic form of the aldolase, the enzyme was subjected fonyl fluoride (PhMeSO2F)-and then assayed for the re- to zymography using the crude lysates of erythrocytes or maining activity at 30'C. The remaining activity in the heated lymphoblastoid cell lines from the patient and a healthy sample is expressed as a percentage of the activity in the volunteer along with an authentic human aldolase A prepa- nontreated sample. ration. Aldolases in the normal control erythrocytes and the Construction and Isolation of cDNA Clones and DNA Se- lymphoblastoid cell line migrated toward the anode along quencing. Poly(A)+ RNA was prepared from the lymphoblas- with the authentic enzyme (data not shown), indicating that toid cell line. Construction of a double-stranded cDNA and the enzymes expressed in these human cells are of type A. its ligation to XgtlO DNA was carried out according to Gubler Aldolase A activities in erythrocytes ofthe patient (Y.K.) and and Hoffman (17) and Huynh et al. (18), respectively. The a normal control were 0.12 and 2.99 units/g of Hb, respec- cDNA fragments cleaved with restriction enzymes were tively, supporting previous observation (12). In the lympho- subcloned in pUC13 plasmid. DNA sequencing was per- blastoid cell line of the patient and the normal control the formed by the modified method ofdideoxy chain-termination aldolase A activities were 53.2 x 103 and 154.8 x 103 units/g (19). of protein, respectively. These results indicate that the Construction of Patient Aldolase A Expression Plasmids and aldolase activity in the patient's lymphoblastoid cells is also Their Expressions in E. coli. pHAA47, an original E. coli significantly lower than that of normal lymphoblastoid cells. expression plasmid of human aldolase A, was constructed To examine the kinetics ofthe enzyme activity in the cell lines from pIN-I1, an E. coli expression vector containing lpp of the patient and the normal control, the thermal stability of promoter and lacUV5 promoter-operator (20), pUC13 DNA, the aldolase was measured in cell lysates that were incubated and normal human aldolase A cDNA pHAAL116-3 (6), as for 30 min at various temperatures as described above (see will be described elsewhere (I. Takahashi, Y. Takasaki, M. Materials and Methods). Fig. 1 shows that the aldolase of Sakakibara, T.M., and K.H., unpublished data). An E. coli erythrocytes and lymphoblastoid cells of the normal control expression plasmid carrying the patient aldolase A cDNA retained almost all of its original activity after incubation at fragment was constructed by substituting a restriction frag- 55TC, whereas the patient's aldolase in these cells was almost ment ofthe patient aldolase A cDNA for the same restriction completely lost under the same conditions. These results fragment of aldolase A cDNA in pHAA47 as follows. First, indicate that the aldolase A expressed in the patient's cells is pHAA47 was digested with Acc I to remove a 312-base-pair much less stable than that in normal cells and also that (bp)-long Acc I DNA fragment that was cleaved at the Acc I aldolase A in the lymphoblastoid cell lines has the same sites located at the 325th and 637th positions from the ATG defect as in the erythrocytes. The temperature sensitivity of initiation codon. Then, the same Acc I fragment from the the patient's aldolase A suggested to us that the mutation patient aldolase A cDNAs (pHAdA 5-2) that contained the probably occurred in the coding region ofthe aldolase A gene substituted base at the 386th position from an initiation codon and that sequence analysis of the gene may be informative. (see Results) was ligated to the large fragment of E. coli A Single Base Mutation Associated with Disappearance of a expression plasmid pHAA47 at the Acc I sites in the sense Restriction Site and an Amino Acid Substitution. cDNA clones direction and transfected into E. coli strain JM83 (see Fig. 4A, were constructed from poly(A)+ RNA of the patient's lym- pHAdA524 and pHAdA526). The Acc I fragment of phoblastoid cell line using XgtlO as a vector and were pHAdA526 was again replaced with that of normal human screened using a rat aldolase A cDNA fragment as a probe aldolase A cDNA to construct pHAA471 and pHAA473. The (21). Ten clones positive for aldolase A were obtained from tranisformants thus obtained were cultured overnight at 370C, 20,000 plaques of cDNA library. One of the clones that suspended in buffer (25 mM Tris HCI, pH 7.5/1 mM EDTA/1 appeared to carry the entire length ofcDNA (pHAdA5-2) was mM PhMeSO2F), and sonicated to prepare cell lysates. The completely sequenced. The nucleotide sequence in the cod- lysates were then used for enzyme assays as described ing region of pHAdA5-2 was the same as that of normal above. aldolase A cDNA (6) except for one nucleotide. In the patient's cDNA clone, the 386th base from the ATG start RESULTS codon, adenine, was replaced with guanine (Fig. 2). As a result, the 128th amino acid, aspartic acid (GAT), was Thermolabile Aldolase Activity in a Patient with Hemolytic replaced with glycine (GGT). Anemia. Two cases (one kindred) of erythrocyte aldolase There are four Fok I recognition sites (GGATG) in normal deficiency associated with hereditary hemolytic anemia have human aldolase A cDNA that produce internal fragments of been reported in a Japanese family (12). The activity of 33, 120, and 144 bp. One of the recognition sequences is aldolase in the patient's erythrocytes was shown to be about located at the site including the 128th aspartic acid codon 5% ofthat of normal and was thermolabile (12). To determine (GAT). Therefore, this single base change that occurs in the A B

11

FIG. 1. Effect of temperature 4- on aldolase activity in the pa- tient's erythrocytes (A) or a lym- 50 phoblastoid cell line (B). Extracts from erythrocytes or the lympho- blastoid cell line were incubated at the indicated temperature for 30 min and cooled on ice and then the aldolase activity was measured. o 1.10 and A, activity of the extract from 40 45 50 55 a normal control; a, activity ofthe Incubation Temperature (*C) extract from the patient. Downloaded by guest on September 25, 2021 Medical Sciences: Kishi et A Proc. Natl. Acad. Sci. USA 84 (1987) 8625

a 0.3-kb fragment should disappear. As shown in Fig. 3B, the A- 0.3-kb fragment (labeled d in Fig. 3) completely disappeared GATCAT C7 'GAT C_ and, instead, a 1-kb fragment (labeled a' in Fig. 3) appeared 129 GLy in the patient's DNA, whereas DNA from a normal lympho- blastoid cell line and genomic DNA cloned by us (T.M., H. Yatsuki, K. Joh, Y. Arai, and K.H., unpublished data) gave 128 Asp a 0.3-kb fragment (Fig. 3B). The leukocyte DNA from the patient also gave essentially the same result (data not shown). Thermal Stability ofthe Patient's Aldolase A Expressed in E. 127 Lou coli. To examine whether the adenine to guanine nucleotide substitution in genomic DNA resulted in the production of 126 Gty thermolabile aldolase A in the patient's erythrocytes, aldolase A cDNAs, prepared from normal control and pa- tient's lymphoblastoid cell lines, were inserted into an E. coli FIG. 2. Comparison of the nucleotide sequence of the patient's expression vector that contained an lpp and lacUVS promot- and normal aldolase A. The nucleotide sequence was determined er-operator (20). The expression vector was then transfected from a cloned cDNA. The amino acid sequence shown was deduced into E. coli strain JM83. The human aldolases, encoded for by from the nucleotide sequence as described (6). A' represents a part normal and patient cDNAs and expressed in E. coli, were of the sequence of normal aldolase A cDNA. A- represents the then used for a comparison of enzyme thermal stability (Fig. corresponding sequence of the patient's aldolase A cDNA. The 4). Normal aldolase A retained about 70% of its activity after substituted nucleotides are circled. The arrowhead shows the sub- heat treatment at 500C (pHAA47), whereas the patient's stituted guanine. aldolase A entirely lost its activity even at 40TC (pHAdA524 and pHAdA526). Thermal stability ofthe aldolases expressed patient's cDNA should be accompanied by the disappearance in E. coli was quite similar to that of the enzymes from of this site and, therefore, results in the disappearance of one erythrocytes and lymphoblastoid cells, although the enzymes of the Fok I fragments. In fact, the 120-bp Fok I fragment synthesized in E. coli were less stable. disappeared in the patient's cDNA (data not shown), indi- When the Acc I fragment of pHAdA526 was substituted cating that there was a base change in the GGATG of a Fok with the corresponding Acc I fragment of pHAA47, a normal I site. aldolase A cDNA, the enzyme encoded for by the reconsti- To verify that the base change in the patient's aldolase A tuted plasmid (pHAA471 and pHAA473) displayed the ther- cDNA was a consequence of the nucleotide substitution in mal stability of the enzyme encoded for by the pHAA47. the patient's genomic DNA, high molecular weight DNA These results indicated that a single base change of adenine (extracted from the patient's lymphoblastoid cell line) was to guanine at the 386th position was responsible for heat with Fok on transferred to lability of the enzyme but that the remaining portion of the digested I, separated agarose gel, DNA a nylon filter, and probed with the Fok I-Sau3A fragment of expression plasmid was normal. the normal human aldolase A cDNA (6) (Fig. 3A). If the Fok I site of the genomic DNA (corresponding to that of cDNA) DISCUSSION was lost, then a 1-kilobase (kb) fragment should appear and Three cases (two kindreds) of aldolase deficiency associated with congenital nonspherocytic hemolytic anemia have been A B described (11, 12). The present study presents an analysis of 1 2 3 one ofthese cases that demonstrated the aldolase A defect at the nucleotide level. The Probe(cDNA) nucleotide sequence analysis of the a - patient's aldolase A cDNA showed a substitution ofthe 386th 0* I":.., base (adenine by guanine) in the coding region and the J": ., .-It b'- 0 *0 " resultant replacement of aspartic acid, the 128th amino acid, C- W.W-' 40 by glycine. Using a system expressing human aldolase A in 7 910a1112 E. coli, the characteristic of temperature sensitivity of the aldolase A was reproduced in E. coli (Fig. 4). We clearly a' d a d. demonstrated that glycine instead ofaspartic acid at the 128th d- ~ ~ ~ : position caused the aldolase A to be thermolabile. Although there are many instances of inherited diseases in which the mutation of nucleotide sequence is identified (22), FIG. 3. Southern blot analysis ofaldolase A gene in chromosomal it is generally difficult to assess whether, if more than one DNA. (A) Fok I map of the relevant region of the human genomic mutation exists in a sequence, a particular mutation only DNA (shown by open and closed circles) (T.M., H. Yatsuki, K. Joh, a Y. Arai, and K.H., unpublished results). The closed circle under represents DNA polymorphism or causes a functional exon 8 corresponds to the Fok I site not present in the patient's defect. However, our use of the E. coli expression vector in cDNA. (B) High molecular weight DNA was extracted from lym- this study has proved to be very useful in surveying for phoblastoid cell lines. DNA was digested with Fok I, fractionated on mutations in coding sequences of genes that may influence a 2% agarose gel, transferred to a nylon filter, and hybridized with the function of proteins. In addition, this method enables us a 32P-labeled Fok I-Sau3A cDNA fragment as indicated. a, a', and to purify proteins from E. coli extract that contains the d correspond to the DNA fragments denoted by the same letters in product encoded by E. coli expression plasmid and further to the Fok I map (A); b and c correspond to bands derived from an characterize the proteins biochemically. aldolase A (data not shown). DNA was obtained from a Our study proves that the patient's aldolase A gene carries normal lymphoblastoid cell line (10 ,ug, lane 1), the patient's a homozygous mutation in the coding region of the genome lymphoblastoid cell line (10 ,ug, lane 2), and the cloned human since in Southern at the relevant aldolase A gene, pHAAR10 (95 ng, lane 3). We observe only a faint blotting analysis cleavage "a" band in lanes 1 and 2 because a smaller amount of the gene Fok I site did not occur in the patient's genomic DNA. The sequence was applied as compared with that in lane 3 (<0.001 with patient's parents carry the same mutation and are heterozy- respect to the aldolase A gene sequence) and, in addition, because gous for the mutant gene since they are phenotypically the smaller hybridizing region was shared with the probe. normal and have aldolase activities in erythrocytes that are Downloaded by guest on September 25, 2021 8626 Medical Sciences: Kishi et al. Proc. Natl. Acad. Sci. USA 84 (1987) A

LpptLac G /Ampr _--a- pHAdA526 1 pHAdA524 FIG. 4. Effect of temperature on aldolase A activity expressed in E. coli. (A) Construction of plasmid DNA containing normal or the patient's cDNA. Aldolase A cDNA was inserted into the B expression vector (pHAA47); then the Acc I fragment was re- placed with that of the patient's cDNA (pHAdA524, pHAdA526). Finally, the Acc I fragment was replaced with that of a normal control (pHAA471, pHA473). Ampr, ampicillin resistant. (B) Aldolase activity after treatment at the indicated temperatures. E. coli strain JM83 transformed with 0J' the above plasmid DNA was cul- +o tured overnight at 370C. The ex- Noo tracts of the transformants were incubated for 30 min at varying temperatures and the aldolase ac- tivities in the extracts were mea- sured. The full activities were 7.76 nmol/min for pHAA47 (o), 11.74 nmol/min for pHAA471 (A), 9.41 nmol/min for pHAA473 (o), 3.62 40 45 50 55 nmol/min for pHAdA524 (e), and Incubation Temperature (°C) 7.50 nmol/min for pHAdA526 (A). intermediate between normal and affected levels (12). These our colleagues for helpful discussions and comments, and Dr. Mark observations, together with the evidence that the aldolase A Bogart for reviewing the manuscript. This investigation was sup- gene (ALDOA) is on 16 (23), support the idea ported in part by Special Project Research Grant 60127010 (Inborn that this hereditary disorder has an autosomal recessive mode Errors of ) from the Ministry of Education, Science and of inheritance. Culture of Japan. Vertebrate aldolases have three isozymic forms: A, B, and 1. Horecker, B. L., Tsolas, 0. & Lai, C. Y. (1972) in The Enzyme, C. The amino acid sequences ofaldolase A and are ed. Boyer, P. D. (Academic, New York), Vol. 7, pp. 213-258. highly conserved (24). The 128th amino acid, aspartic acid, 2. Tsutsumi, K., Mukai, T., Tsutsumi, R., Hidaka, S., Arai, Y., found in normal human aldolase A is conserved in all aldolase Hori, K. & Ishikawa, K. (1985) J. Mol. Biol. 181, 153-160. so far examined, including human, rat, and rabbit 3. Mukai, T., Joh, K., Arai, Y., Yatsuki, H. & Hori, K. (1986) J. isozymes Biol. Chem. 261, 3347-3354. A, human, rat, and chicken B, and rat and mouse C (25) and 4. Joh, K., Arai, Y., Mukai, T. & Hori, K. (1986) J. Mol. Biol. even in Drosophila aldolase (26). One of the reasons that the 190, 401-410. substitution of aspartic acid by glycine causes the thermola- 5. Rottmann, W. H., Tolan, D. R. & Penhoet, E. E. (1984) Proc. bility of the enzyme may be due to the loss of a negative Natl. Acad. Sci. USA 81, 2738-2742. charge that is indispensable for retaining its conformational 6. Sakakibara, M., Mukai, T. & Hori, K. (1985) Biochem. stability, especially at high temperature. It is thus possible Biophys. Res. Commun. 131, 413-420. that the aspartic acid fulfills an important role in the function 7. Sakakibara, M., Mukai, T., Yatsuki, H. & Hori, K. (1985) of aldolase either by maintaining its structural stability or as Nucleic Acids Res. 13, 5055-5069. a regulatory site. 8. Tolan, D. R., Amsden, A. B., Putney, S. D., Urdea, M. S. & Penhoet, E. E. (1984) J. Biol. Chem. 259, 1127-1131. The patient's aldolase activity in erythrocytes was as low 9. Burgess, D. G. & Penhoet, E. E. (1985) J. Biol. Chem. 260, as about 5% of that of the normal control and was 4604-4614. thermolabile. In the patient's lymphoblastoid cell line the 10. Schapira, F., Schapira, G. & Dreyfus, J. C. (1961) Enzymol. activity was about 30% of that of the normal control and also Biol. Clin. 1, 170-175. was thermolabile. We cannot explain at present why the 11. Beutler, E., Scott, S., Bishop, A., Margolis, N., Matsumoto, F. remaining aldolase activities in lymphoblastoid cell line are & Kuhl, W. (1973) Trans. Assoc. Am. Physicians 76, 154-166. higher than those in erythrocytes. Since the level of aldolase 12. Miwa, S., Fujii, H., Tani, K., Takahashi, K., Takegawa, S., A mRNA in the patient's lymphoblastoid cell line is usually Fujinami, N., Sakurai, M., Kubo, M., Tanimoto, Y., Kato, T. higher than that in the normal control (data not shown), it is & Matsumoto, N. (1981) Am. J. Hematol. 11, 425-437. 13. Yeltman, D. R. & Harris, B. G. (1977) Biochim. Biophys. Acta rather likely that large quantities of aldolase mRNA in the 484, 188-198. cultured cells partially compensate for its low activity, 14. Susor, W. A., Penhoet, E. E. & Rutter, W. J. (1975) Methods although we cannot eliminate other possibilities. Further Enzymol. 61, 66-73. studies are necessary to answer this question, including 15. Beutler, E., Blume, K. G., Kaplan, J. C., Lohr, G. W., Ramot, determination of the relative amount of aldolase mRNA in B. & Valentine, W. N. (1977) Br. J. Haematol. 35, 331-340. or the 16. Rutter, W. J. (1964) Fed. Proc. Fed. Am. Soc. Exp. Biol. 23, reticulocytes lymphocytes of patient. 1248-1257. We are grateful to Dr. M. Inouye of the State University of New 17. Gubler, U. & Hoffman, B. J. (1983) Gene 25, 263-269. York at Stony Brook for providing E. coli expression vector pIN-Ill, 18. Huynh, T. V., Young, R. A. & Davis, R. W. (1985) in DNA Downloaded by guest on September 25, 2021 Medical Sciences: Kishi et al. Proc. Nadl. Acad. Sci. USA 84 (1987) 8627

Cloning, ed. Glover, D. M. (IRL, Oxford), Vol. 1, pp. 49-78. Joh, K., Mukai, T. & Hori, K. (1987) Hum. Genet. 76, 20-26. 19. Hattori, M. & Sakaki, Y. (1986) Anal. Biochem. 152, 232-238. 24. Hori, K., Mukai, T., Joh, K., Arai, Y., Sakakibara, M. & 20. Masui, Y., Coleman, J. & Inouye, M. (1983) in Experimental Yatsuki, H. (1987) in Isozymes: Current Topics in Biological Manipulation ofGene Expression, ed. Inouye, M. (Academic, and Medical Research, eds. Rattszzi, M. C., Scandalios, J. G. New York), pp. 15-30. & Whitt, G. S. (ARL, New York), Vol. 14, pp. 153-175. 21. Joh, K., Mukai, T., Yatsuki, H. & Hori, K. (1985) Gene 39, 25. Paolella, G., Buono, P., Mancini, F. P., Izzo, P. & Salvatore, 17-24. F. (1986) Eur. J. Biochem. 156, 229-235. 22. Cooper, D. N. & Schmidtke, J. (1986) Hum. Genet. 73, 1-11. 26. Malek, A. A., Suter, F. X., Frank, G. & Brenner-Holzach, 0. 23. Kukita, A., Yoshida, M. C., Fukushige, S., Sakakibara, M., (1985) Biochem. Biophys. Res. Commun. 126, 199-205. Downloaded by guest on September 25, 2021