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Home , Chromosome, Chromosome 2

The Is Located on 2 JAMES V. TRICOLI, GRAEME I. BELL, AND THOMAS B. SHOWS

Since the physiologic effects of glucagon play an important SUMMARY DNA samples prepared from a panel of human-mouse role in diabetes, such genetic information may eventually hybrids with different numbers and combinations lead to a better understanding of the regulation of this hor- of human were examined for the pres- mone during the hyperglycemia associated with this dis- ence of the human preproglucagon gene by hybridiza- ease. tion with a cloned segment of the human gene. The cell hybrids formed between cultured human and segregation of the human glucagon gene specific DNA mouse cells were used to map the human gene coding for fragment and human in these cell hy- preproglucagon.2'4 These hybrids contain mouse chromo- brids indicated that the preproglucagon gene (desig- somes but retain only a partial and random complement of nated GCG) is on chromosome 2 in . DIABETES human chromosomes. DNA from a panel of human-mouse 33:200-202, February 1984. hybrids whose human chromosome composi- tion was known was analyzed for the presence or absence of the human preproglucagon gene by hybridization with a complementary DNA encoding Syrian hamster cloned segment of the human gene. This study indicated preproglucagon messenger RNA as well as the that the human preproglucagon gene (designated GCG) is human gene was isolated and characterized.1 on chromosome 2. These studies indicated that mammalian gluca- goAn is derived from a 179-180 amino acid precursor (the human has one less amino acid at its carboxyl ter- MATERIALS AND METHODS minus than the hamster protein). The precursor, in addition, Cell hybrids. The somatic cell hybrids were constructed and contains the sequences of two polypeptides of 37 and 35 maintained as previously described.25-10 Clones were se- amino acids that are bound by a pair of basic amino acids. lected and analyzed for characteristic human mark- The two polypeptides are related to glucagon and are des- ers whose had been assigned to a particular chro- ignated glucagon-like peptides (GLP) 1 and 2, respectively. mosome.211 In addition, on the same passage the human Although GLP-1 and -2 have not been previously isolated chromosomes were identified by Giemsa-trypsin staining8 from the pancreas or other tissues, their with glu- and DNA was isolated for use in filter hybridization.12 cagon and other members of the glucagon-secretion family Identification of the human glucagon gene. Five micro- suggests that they have unique, although presently unde- grams of DNA from each cell line was digested to completion termined, physiologic properties. It is suggested that a series with the restriction endonuclease EcoRI. The resulting DNA of duplications of the coding regions or exons and subse- fragments were separated by electrophoresis in a 0.8% aga- quent produced glucagon and the two glucagon- rose gel and then transferred to a nitrocellulose filter (see like peptides. The assignment of the preproglucagon gene ref. 13). The filters were hybridized as described previously14 to a human chromosome would be important in determining with a 32P-labeled 2134 EcoRI fragment from its relationship and close linkage to other genetic markers. \hglucagon1,1 which contained a region, exon 2, coding for preproglucagon 11-65 and part of another exon corre- sponding to preproglucagon 66-99, as well as flanking in- From the Department of Human , Roswell Park Memorial Institute, tervening sequences or . This probe hybridized to a New York State Department of Health, Buffalo, New York 14263 (J.V.T., TBS); and Chiron Corporation, Emeryville, California (G.I.B.). 2.0-kilobase pair (kb) human EcoRI fragment, as expected, Received for publication 18 November 1983. and to a 7.8-kb mouse EcoRI fragment.

200 DIABETES, VOL. 33, FEBRUARY 1984 TABLE 1 Human chromosomes determined by karyotyping

Chromosomes ° n kh Cell hybrids GCG band 1 2 3 4 5 6 7 I3 9 10 11 12 13 14 15 16 17 18 19 20 21 22 X

WIL-1 WIL-2 WIL-5 1 i I II I I WIL-6 + III 7 1 I WIL-7 WIL-8X _1 _l_ l_ _J_ I WIL-13 i WIL-14 + WIL-15 + REW-7 + + + REW-10 + + - REW-11 - + REW-15 + + - XER-7 + i XER-9 - + — + XER-11 I + - + + JWR-22H I + + JWR-26C - + + + 1 + + NSL-9 + + + + REX-26 + + - + XSC-7 + + TSL-2 - + - 1 + + ICL-15 - + - + + XTR-22 + + XTR-3BSAGB + _ ATR-13 % Discordancy 39 0 30 26 22 22 26 39 35 22 22 43 30 26 30 57 52 22 30 39 48 52 39 Segregation of the human preproglucagon gene (GCG) in 26 human-mouse somatic cell hybrids. Positive or negative symbols indicate the presence or absence of the indicated human chromosome and chromosome-specific isozyme markers. The presence or absence of the 2.0-kb GCG band as assayed by Southern filter hybridization is indicated. % Discordancy is a measure of the percentage of hybrids in which the probe does not segregate with the indicated chromosome.

RESULTS mouse band found in all the cell . Thus, the Chromosomal mapping of the glucagon gene. The human human glucagon gene, GCG, is located on chromosome 2. gene was mapped by correlating the presence of specific human chromosomes in human-mouse somatic cell hybrids with the human glucagon gene sequences in the hybrid DISCUSSION DNA. The human chromosomes in these hybrid cells were Preproglucagon is synthesized in the pancreas, intestine, identified through a combination of karyotyping, Giemsa- and possibly the ; tissue-specific posttranslational trypsin banding, and analysis of human specific gene mark- processing generates different peptides in the pancreas and ers.28" The human glucagon gene was found to be present intestine.1 Although the physiologic properties of glucagon or absent as determined by hybridization with phg 14-4 have been extensively studied and its role in the mainte- together with human chromosome 2 and human IDH1 (iso- nance of normal glucose levels is well established, the phys- citrate dehydrogenase, soluble), the enzyme marker for hu- iologic functions of the larger glucagon-containing peptides man chromosome 2, in an analysis of 26 hybrids (Table 1). found in the intestine or glucagon-like peptides, GLP-1 and No other chromosomes or chromosome-specific isozyme -2, whose presence is predicted from the sequence of pre- markers cosegregated with GCG. Examples of human glu- , remain to be determined. Understanding the cagon {GCG) gene positive and negative cell hybrid DNAs function of the other peptides derived from preproglucagon are shown in Figure 1. The phg 14-4 glucagon probe hy- may indicate their role, if any, in human . There has bridized to both mouse and human DNAs yielding bands of been only one published case of congenital isolated glu- -7.8 kb (Figure 1A, lanes 2 and 3) and ~2.0 kb (Figure 1A, cagon deficiency15 since, unlike deficiency, the ab- lanes 1 and 4), respectively. The human-mouse hybrid cell sence of glucagon is lethal. However, recent evidence1617 DNA (Figure 1B, lanes 1, 2, 4, 6, 8, 9, and 10) is positive indicates that in diabetes it is the hyperglycemia resulting for the glucagon gene and for human chromosome 2. They from hyperglucagonemia that is the most damaging aspect contain a single human band at 2.0 kb. Lanes 3, 5, 7, and of the disease. The elucidation of the structure of prepro- 11 show hybrid DNAs that are negative for both the glucagon glucagon and its gene, and now the assignment of the gene gene and human chromosome 2. They contain only the to human chromosome 2, will facilitate physiologic and ge-

DIABETES, VOL 33, FEBRUARY 1984 201 HUMAN GLUCAGON GENE IS LOCATED ON CHROMOSOME 2 A.

M

H

12 1 2 3456 7 89 10 11

FIGURE 1. Hybridization of human giucagon gene probe to EcoRI digests of human, mouse, and human-mouse hybrid cell DNAs. (A) Hybridiza- tion to human DNA isolated from HeLa and WI-38 cells, lanes 1 and 4, respectively; or mouse DNA isolated from LMTK~ and RAG cells, lanes 2 and 3, respectively. H indicates the 2.0-kb human EcoRI fragment that hybridizes, and M the 7.8-kb mouse EcoRI fragment. (B) Hybridization to human-mouse hybrid cell DNAs from cells containing (lanes 1, 2, 4, 6, 8, 9, and 10) or lacking (lanes 3, 5, 7, and 11) human chromosome 2 and the preproglucagon gene.

netic studies of the role of this important hormone in normal 7Owerbach, D., Bell, G. I., Rutter, W. J., Brown, J. A., and Shows, and disease states. T. B.: The insulin gene is located on the short arm of in humans. Diabetes 1981; 30:267-70. 8 Shows, T B., Brown, J. A., Haley, L. L, Byers, M. G., Eddy, R. L, Cooper, E. S., and Goggin, A. P.: Assignment of the p-glucuronidase structural ACKNOWLEDGMENTS gene to the pter—>q22 region of in man. Cytogenet. Cell We are indebted to L. Haley, M. Byers, R. Eddy, and M. Genet. 1978; 21:99-104. 9 Henry for excellent technical assistance and to C. Young for Lalley, P. A., Brown, J. A., and Shows, T B.: Assignment of the hex- osaminidase B gene to and its segregation after diphtheria manuscript preparation. toxin selection employing an X;5 translocation; and the linkage of hexos- This work was supported by NIH grants GM 20454 and aminidase A, mannosephosphate isomerase, and pyruvate kinase (M2) genes using man-rodent cell hybrids. Cytogenet. Cell Genet. 1976; 16:188-91. HD 05196. 10 Naylor, S. L, Elliott, R. W., Brown, J. A., and Shows, T B.: Mapping of aminoacylase-1 and p-galactosidase-A to homologous regions of human and mouse suggests location of additional REFERENCES genes. Am. J. Human Genet. 1982; 34:235-44. 1 Bell, G. I., Sanchez-Pescador, R., Laybourn, P. J., and Najarian, 11 Shows, T. B., Sakaguchi, A. Y, and Naylor, S. L: Mapping the human R. C: Exon duplication and divergence in the human preproglucagon gene. , cloned genes, DNA polymorphisms, and inherited disease. In Ad- Nature 1983; 304:368-71. vances in . Vol. 12. Harris, H., and Hirschhorn, K., Eds. New 2 Shows, T. B.: Genetics of human-mouse somatic cell hybrids: linkage York and London, Plenum Press, 1982:341-452. 12 of human genes for lactate dehydrogenase-A and esterase-A4. Proc. Natl. Naylor, S. L, Sakaguchi, A. Y, Shows, T B., Law, M. L, Goeddel, Acad. Sci. USA 1972; 69:348-52. D. V., and Gray, P. W.: Human immune interferon gene is located on chro- 3 Shows, T. B., and Brown, J. A.: Human X-linked genes regionally mosome 12. J. Exp. Med. 1983; 157:1020-27. mapped utilizing X- translocations and somatic cell hybrids. Proc. 13 Southern, E. M.: Detection of specific sequences among DNA frag- Natl. Acad. Sci. USA 1975; 72:2125-29. ments separated by gel electrophoresis. J. Mol. Biol. 1975; 98:503-17. 4 Davidson, R., and Gerald, P. S.: Improved techniques for the induction 14 Naylor, S. L, Sakaguchi, A. Y, Shen, L.-P, Bell, G. I., Rutter, W. J., of mammalian cell hybridization by polyethylene glycol. Somatic Cell Genet. and Shows, T B.: Polymorphic human gene is located on chro- 1976; 2:165-76. mosome 3. Proc. Natl. Acad. Sci. USA 1983; 80:2686-89. 5 Lalley, P. A., Brown, J. A., Eddy, R. L, Haley, L. L, Byers, M. G., 15 Vidnes, J., and 0yasaeter, S.: Giucagon deficiency causing severe Goggin, A., and Shows, T. B.: Human p-glucuronidase: assignment of the neonatal hypoglycemia in a patient with normal insulin secretion. Pediatr. Res. structural gene to chromosome 7 using somatic cell hybrids. Biochem. Genet. 1977; 11:943-49. 1977; 15:367-82. 16 Unger, R. H., and Orci, L: Giucagon and the a cell, physiology and 6 Shows, T. B., and Brown, J. A.: Mapping chromosomes 1 and 2 , part 1. N. Engl. J. Med. 1981; 304:1518-24. employing a V2 translocation in somatic cell hybrids. Cytogenet. Cell Genet. 17 Unger, R. H., and Orci, L: Giucagon and the a cell, physiology and 1975; 14:251-55. pathophysiology, part 2. N. Engl. J. Med. 1981; 304:1575-80.

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