Cytogcnet. Cell Genet. 22: 228-231 (1978)
Assignment of the gene for phosphoribosyl pyrophosphate amidotransferase to the p te r-q 2 1 region of human chromosome4 W. Stanley and E.H.Y. Cnu Department of Human Genetics, University of Michigan Medical School, Ann Arbor, Mich.
Supported by NSF grant GB 34302.
Downloaded by: Univ. of California Santa Barbara 128.111.121.42 - 2/25/2018 4:53:06 PM
Pliosphoribosylpyrophosphate (PRPP) amidotransferase (PRPPATF, E.C. 2.4.2.14) catalyzes the formation of 5-phosphoribosyl-l-amine, the first intermediate of the de novo purine biosynthetic pathway. An aux otrophic mutant (Pur-1) exhibiting a nutritional requirement for exogenous purines was isolated from the Chinese hamster V79 cell line.1This mutant clone is deficient in the activity of PRPP amidotransferase.2’3 Interspecific cell hybrids formed by fusion of Pur-1 cells with those from an established human lymphoblastoid line (UM-61) were isolated in purine-deficient medium. Most hybrid clones exhibited extensive breakage of human chromosomes. One clone, designated 22C, contained three intact human chromosomes, 4, 5, and 16. Cells from clone 22C were plated at low density in purine-supplemented medium, and 11 colonies were isolated and tested for growth in purine-deficient medium. Eight of these subclones were purine independent and three were purine dependent. Karyotype analysis of six of the eight purine-independent subclones indicated the presence of human chromosomes 4, 5, and 16, while those subclones requiring exogenous purines had lost all three of these human chromo somes. One of these subclones, designated 22C-35, was plated at low density, and 60 colonies were isolated and analyzed. Cell lines established from two such colonies, designated 22C-35-35 and 22C-35-65, contained cells retaining only human chromosomes 4 and 5. C reagan et a!.1 have shown that the gene that confers sensitivity to diphtheria toxin (DTS) was located on human chromosome 5. We found that Chinese hamster V79 cells were approximately 10 times more resistant
Stanley, C hu
Somatic cell hybrids/Enzym e loci
229
to the cytotoxic action of DT than were hybrid clones containing human chromosome 5 (fig. 1). Using diphtheria toxin as a selective agent, a purine-independent colony, designated 9T, was isolated from cell line 22C-35-65, which was less sensitive to this toxin. Ail cells of line 9T contained one intact human chromosome 4, but in different cells a small metacentric chromosome and one or two small telocentrics might also be present. In addition, structural rearrangements involving a humanhamster translocation and/or break at the centromere region of one of the hamster acrocentric chromosomes were seen in certain cells in the population. To obtain cell lines uniform for their chromosomal comple ment, line 9T was further subcloned. The important karyotypic charac teristics of two purine-independent lines (9T-B and 9T-K) and one purinedependent line (9T-2-1) derived from diphtheria toxin-resistant cell line 9T are illustrated in fig. 2. The telocentric chromosome present in cells of 9T-K and 9T-B is most likely the short arm of a hamster acrocentric
Downloaded by: Univ. of California Santa Barbara 128.111.121.42 - 2/25/2018 4:53:06 PM
Fig. 1. Cytotoxic effect of diphtheria toxin on hybrid cells containing human chro mosome 5: (A) Chinese hamster V79 cells, control; (B) hybrid cells, control; (C) V79 cells, 3 pM diphtheria toxin; (D) hybrid cells, 3 pM diphtheria toxin.
230
Stanley. C hu
% a V
#
Somatic cell hybrids/Enzyme loci
* • •» *
*
* i
C
A
B
I
CD
Assignment of the gene for phosphoribosyl pyrophosphate amidotransferase to the p te r-q 2 1 region of human chromosome4 W. Stanley and E.H.Y. Cnu Department of Human Genetics, University of Michigan Medical School, Ann Arbor, Mich.
Supported by NSF grant GB 34302.
Downloaded by: Univ. of California Santa Barbara 128.111.121.42 - 2/25/2018 4:53:06 PM
Pliosphoribosylpyrophosphate (PRPP) amidotransferase (PRPPATF, E.C. 2.4.2.14) catalyzes the formation of 5-phosphoribosyl-l-amine, the first intermediate of the de novo purine biosynthetic pathway. An aux otrophic mutant (Pur-1) exhibiting a nutritional requirement for exogenous purines was isolated from the Chinese hamster V79 cell line.1This mutant clone is deficient in the activity of PRPP amidotransferase.2’3 Interspecific cell hybrids formed by fusion of Pur-1 cells with those from an established human lymphoblastoid line (UM-61) were isolated in purine-deficient medium. Most hybrid clones exhibited extensive breakage of human chromosomes. One clone, designated 22C, contained three intact human chromosomes, 4, 5, and 16. Cells from clone 22C were plated at low density in purine-supplemented medium, and 11 colonies were isolated and tested for growth in purine-deficient medium. Eight of these subclones were purine independent and three were purine dependent. Karyotype analysis of six of the eight purine-independent subclones indicated the presence of human chromosomes 4, 5, and 16, while those subclones requiring exogenous purines had lost all three of these human chromo somes. One of these subclones, designated 22C-35, was plated at low density, and 60 colonies were isolated and analyzed. Cell lines established from two such colonies, designated 22C-35-35 and 22C-35-65, contained cells retaining only human chromosomes 4 and 5. C reagan et a!.1 have shown that the gene that confers sensitivity to diphtheria toxin (DTS) was located on human chromosome 5. We found that Chinese hamster V79 cells were approximately 10 times more resistant
Stanley, C hu
Somatic cell hybrids/Enzym e loci
229
to the cytotoxic action of DT than were hybrid clones containing human chromosome 5 (fig. 1). Using diphtheria toxin as a selective agent, a purine-independent colony, designated 9T, was isolated from cell line 22C-35-65, which was less sensitive to this toxin. Ail cells of line 9T contained one intact human chromosome 4, but in different cells a small metacentric chromosome and one or two small telocentrics might also be present. In addition, structural rearrangements involving a humanhamster translocation and/or break at the centromere region of one of the hamster acrocentric chromosomes were seen in certain cells in the population. To obtain cell lines uniform for their chromosomal comple ment, line 9T was further subcloned. The important karyotypic charac teristics of two purine-independent lines (9T-B and 9T-K) and one purinedependent line (9T-2-1) derived from diphtheria toxin-resistant cell line 9T are illustrated in fig. 2. The telocentric chromosome present in cells of 9T-K and 9T-B is most likely the short arm of a hamster acrocentric
Downloaded by: Univ. of California Santa Barbara 128.111.121.42 - 2/25/2018 4:53:06 PM
Fig. 1. Cytotoxic effect of diphtheria toxin on hybrid cells containing human chro mosome 5: (A) Chinese hamster V79 cells, control; (B) hybrid cells, control; (C) V79 cells, 3 pM diphtheria toxin; (D) hybrid cells, 3 pM diphtheria toxin.
230
Stanley. C hu
% a V
#
Somatic cell hybrids/Enzyme loci
* • •» *
*
* i
C
A
B
I
CD
