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Identification of Human RNA Transcripts Among Heterogeneous Proc. Nat. Acad. Sci. USA Vol. 72, No. 5, pp. 1868-1872, May 1975 Identification of Human RNA Transcripts among Heterogeneous Nuclear RNA from Man-Mouse Somatic Cell Hybrids (human X chromosome/repetitive DNA sequences/DNA- RNA hybridization) R. A. DI CIOCCIO*, R. VOSS*, M. KRIM*, K. H. GRZESCHIKt, AND M. SINISCALCO* * Sloan-Xettering Institute for Cancer Research, New York, N.Y. 10021 and t Institute for Human Genetics, University of Mflnster, Munster, Germany Communicated by James E. Darnell, Jr., February 14, 1975 ABSTRACT In a man-mouse hybrid line from our X-linked in man (7). Consequently, when phosphoribosyl- cell library, the only cytological detectable portion of the cells are fused with phosphoribo- human genome is the X chromosome, and the only genetic transferase-negative mouse markers regularly expressed are coded by genes known to syltransferase-positive human cells, the resulting hybrid clones be X-linked. A component of the heterogeneous nuclear grown in HAT medium may lose every part of the human RNA of these cells was found to be complementary to genome but the X chromosome, or at least the portion of it repetitive human DNA sequences by means of RNA-DNA bearing the locus for the human enzyme (8, 9). hybridization on nitrocellulose filters. The same pro- cedure also permitted the identification of hybrid cell The identification of human RNA transcripts in man- DNA sequences that are complementary to human hetero- mouse somatic cell hybrids of the type described above is geneous nuclear RNA. This experimental approach, based on RNA- DNA hybridization. Molecular hybrids coupled with hybridization studies in situ, is expected to formed by annealing RNA from A9/HRBC2 cells with human yield critical data on the distribution and the specificity to allow detection for human RNA syn- of the repetitive DNA sequences present in the human DNA are expected genome and to provide a new tool for cytological mapping thesized in A9/HRBC2 cells. Likewise, molecular hybrids ob- of human chromosomes. tained by annealing RNA from human cells with DNA from A9/HRBC2 should detect the human DNA carried by this Highly unstable somatic cell hybrids offer a unique op- hybrid cell line. To be meaningful, the levels of molecular portunity for basic experimentation of molecular biology at hybridization obtained in such experiments should be con- the level of single human chromosomes. The Sendai virus- stantly higher than those between the RNA of the parental mediated fusion between somatic cells of human and rodent mouse cells with human DNA and vice versa, since these origin usually yields interspecific somatic cell hybrids which measure the amount of interspecific crosshybridization. undergo the progressive loss of the human genome (1-5). Soeiro and Darnell (10) have demonstrated that hybridization When the rodent parental cell line is deficient in a metabolic between L-cell hnRNA and HeLa cell DNA and vice versa is function that is essential for survival in a given selective negligible. For this reason, hnRNA was chosen as experi- medium, these types of somatic cell hybrids evolve rapidly mental material. While this work was in progress, Coon et al. towards a reduced karyotype where the only component of (11) found that complementary RNA of human mitochondrial the human genome retained is the chromosome (or chromo- or nuclear DNA does not significantly hybridize with rodent some portion) carrying the human gene that codes for the DNA and vice versa. essential metabolic function. The present report describes the rationale and the method- MATERIALS AND METHODS ologies devised for the identification of human RNA tran- The A9/HRBC2 hybrid line was derived from the man- scripts among the heterogeneous nuclear RNA (hnRNA) mouse hybrid line established by Miller et al. (8) through the isolated from a man-mouse somatic cell hybrid line (A9/ fusion of hypoxanthine phosphoribosyltransferase-positive HRBC2) that has lost almost entirely the parental human human male diploid cells (HRBC2) with mouse A9-cells genome with the exception of the human X chromosome. that lack this enzyme (12) as well as adenine phosphoribosyl- The selective system that has permitted the isolation of this transferase (EC 2.4.2.7; AMP:pyrophosphate phosphoribo- hybrid cell line was devised by Szybalski and Szybalska (6) syltransferase) (13). The original hybrid line had been prop- to select against mammalian cells deficient in hypoxanthine agated in the HAT-selective medium for about 1 year, phosphoribosyltransferase (EC 2.4.2.8; IMP: pyrophosphate before being stored in liquid nitrogen. During that period it phosphoribosyltransferase). This is achieved by carrying out had lost almost the entire human genome, with the exception cell growth in the so-called HAT selective medium, containing of the human X chromosome and all or some of its markers a potent inhibitor of purine and pyrimidine synthesis (A for (8, 9). The present clonal derivative originated from the few aminopterin) together with preformed nitrogen bases (H for cells that recovered in HAT medium after 4 years of storage. hypoxanthine and T for thymidine). Cell survival in this The HeLa-S3 cell line (14) was used as a source of human selective system depends upon the ability of the cell to use the hnRNA. This line is known to have from 43 to 69 chromo- preformed bases as a salvage pathway for nucleic acid syn- somes with two or more doses of each human chromosome (15) thesis. This ability is provided by normal activity of the phos- and exhibited normal activity for all X-linked and autosomal phoribosyltransferase, whose structural gene is known to be enzyme markers mentioned in Table 1. All cells wvere grown in monolayer cultures in 1585 cm2 Abbreviations: hnRNA, heterogeneous nuclear RNA; HAT roller bottles. HeLa and A9 cells were constantly propagated medium, hypoxanthine/aminopterin/thymidine medium. in minimum essential medium, and A9/HRBC2 cells in HAT 1868 Downloaded by guest on September 29, 2021 Proc. Nat. Acad. Sci. USA 72 (1975) Human RNA in Man-Mouse Cell Hybrids 1869 medium (16), both supplemented with the standard additions TABLE 1. Characterization of enzymatic markers of sera and antibiotics reported in full elsewhere4I All cell in A9/HRBC2 cells stocks were checked for mycoplasma with Levine's method A B C (17) and frozen. Each experiment was conducted with a freshly thawed vial of mycoplasma-free frozen cells. Con- G6PD EC 1.1.1.49 X M ( hy ( H fluent monolayers of cells were labeled for 1 hr with 2 mCi of HGPRT EC 2.4.2.8 X - H [3H]uridine (27 Ci/mmol) in 25 ml of medium. The cells were PGK EC2.7.2.3 X M H chilled to 40 and harvested, and their nuclei were isolated NAD-MDH-1 EC1.1.1.37 2 M >>> hy >>> H (18, 19). Nuclear RNA was extracted (10, 19) and hnRNA, IDH-1 EC1.1.1.42 2 M >>> hy >>> H with a sedimentation constant greater than 45 S, was isolated (A) List of human enzyme markers (with trivial abbreviations (10). DNA was extracted (20) from isolated nuclei of cultured and EC numbers) retained by A9/HRBC2 hybrid cell. (B) cells (18, 19) and of human placenta (21). Minor modifica- Chromosomes carrying relevant loci in man. (C) Relative in- tions of these classical nucleic acid isolation procedures were tensity of mouse (M), human (H), and heteropolymeric (hy) applied and are described elsewhere.T RNA* DNA hybridiza- enzymatic bands in the electrophoretic pattern of A9/HRBC2 tion on nitrocellulose filters (22) was carried out as described cell lysates. The following additional 23 enzyme markers, identi- by Soeiro and Darnell (10). Background binding to blank fying 13 of the 22 human autosomes, were found to be regularly filters under these experimental conditions ranges from 1 to absent: AK2(EC 2.7.4.3); PGM-1(EC 2.7.5.1); Pep C(EC 3% of the radioactive RNA hybridized to DNA-bearing 3.4.3.-); PPH(EC 4.2.1.11); Me-l(EC 1.1.1.40); IPO-B(EC filters. 1.6.4.3); sGOT-1(EC 2.6.1.1); HK(EC 2.7.1.1); LDH-A(EC Multistep hybridization experiments were conducted as 1.1.1.27); LDH-B(EC 1.1.1.27); Pep B(EC 3.4.3.-); NP(EC described by and Szybalski (23) with the following 2.4.2.1); PK-3(EC 2.7.1.40); MPI(EC 5.3.1.8); APRT(EC Bovre 2.4.2.7); TK(EC 2.7.1.21); Pep A(EC 3.4.3.-); GPI(EC 5.3.1.9); modifications. The first step or preparative hybridization was ADA(EC 3.5.4.4); IPO-A(EC 1.6.4.3); ,-Gluc(EC 3.2.1.31); conducted as described by Soeiro and Darnell (10). After and Pep-D(EC 3.4.3.-). For details concerning these enzyme radioactivity determination, the filter was removed from the markers, their nomenclature, and the chromosomal assignment scintillation fluid, air-dried, and treated with iodoacetate of relevant human genes, see ref. 32. (23). Then 0.75 ml of 0.30 M NaCl/0.030 M sodium citrate (pH 7) containing 0.1% sodium dodecyl sulfate was added to phoresis. This procedure ensures that conclusions drawn from the vial containing the filter, and the solution with the filter enzyme and chromosomal studies can be confidently con- was heated in a boiling-water bath for 10 min. The solution sidered pertinent to the same population of A9/HRBC2 cells was immediately chilled to 40, and the filter was carefully re- used in the experiments of molecular hybridization, despite moved and dried for radioactivity measurements to determine possible variations of the hybrid genome known to occur the efficiency of elution.
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