Several established clonal strains of rat pituitary cells which produce growth hormone in culture have been shown to secrete a second protein hormone, prolactin . Prolactin was measured immunologically in culture medium and within cells by complement fixation . Rates of prolactin production varied from 6 .6 to 12 µg/mg cell protein per 24 hr in four different cell strains . In these cultures ratios of production of prolactin to growth hormone varied from I .0 to 4 .1 . A fifth clonal strain produced growth hormone but no detectable prolactin . Intracellular prolactin was equivalent to the amount secreted into medium in a period of about 1-2 hr . Both cycloheximide and puromycin suppressed prolactin production by at least 94%. Hydrocortisone (3 X 10 -1 M), which stimulated the production of growth hormone 4-to 8-fold in most of the cell strains, reduced the rate of prolactin production to less than 25 % of that in control cultures . Conversely, addition of simple acid extracts of several tissues, including hypothalamus, to the medium of all strains increased the rate of production of prolactin six to nine times and decreased growth hormone production by about 50% . We conclude that multifunctional rat pituitary cells in culture show unusual promise for further studies of the control of expression of organ-specific activities in mammalian cells.

Thyroid or glucocorticoid hormone increases the synthesis of growth hormone (GH) by clonal lines of rat pituitary tumor cells. To investigate whether these increases arise from increased accumulation of GH-specific RNA sequences in the cytoplasm and nuclei of these cells, we adapted two existing procedures so that a 32P-labeled hybrid plasmid containing a cDNA sequence could be used to quantitate relative concentrations ofthe corresponding mRNA. One method (RNA gel blot hybridization) used electrophoresis of RNA, transfer to nitrocellulose paper, and hybridization to 32P-labeled plasmid. The other (RNA dot hybridization) used covalent attachment of RNA to activated cellulose paper squares and hybridization to 32P-labeled plasmid. As probe, we used a hybrid plasmid (pBR322-GHl) which we show by restriction analysis to contain a DNA sequence coding for rat GH. The results were comparable from both techniques and showed that incubation of GH3 cells with a thyroid hormone (triiodothyronine), a glucocorticoid hormone (dexamethasone), or both hormones caused an increase of cytoplasmic pre-GH mRNA sequences of about 4-, 22-, and 13-fold, respectively. Results obtained with the RNA gel blot hybridization method showed that hormonal stimulation leads to the induction of a single 1.0-kilobase species of pre-GH mRNA in the cytoplasm and of 2.7-and 1.0-kilobase species of GH-specific RNA in the nucleus.GH-cells are related lines of rat pituitary tumor cells that produce the protein hormones growth hormone (GH) and prolactin (1,2). Glucocorticoids increase GH production by . The use of hypothyroid serum in the growth medium later led to the demonstration that thyroid hormones also increase GH production by these cells (4). Since then, GH-cells have been used extensively to study the mechanisms of action of glucocorticoid and thyroid hormones (5-7).Recent experiments using in vitro mRNA translation systems have shown that increases in GH synthesis in response to these hormones are accompanied by increases in translatable cytoplasmic pre-GH (pGH) mRNA (8-11). However, the intracellular events leading to increased GH synthesis by GH-cells upon exposure to a glucocorticoid or a thyroid hormone remain unclear. For example, it is not known whether the hormoneinduced increases in translatable cytoplasmic pGH mRNA arise from an increase in cytoplasmic pGH mRNA sequences or from some cellular alteration in the structure of pGH mRNA that increases its translational efficiency. Furthermore, the recent demonstration of a probable nuclear precursor of pGH mRNA (12) raises the possibility that hormone action on GH-cells could be exerted at the level of processing of this precursor into mature cytoplasmic pGH mRNA.To investigate these questions, we have adapted the gel blot hybridization technique of Thomas (13) and the dot hybridization procedure of Kafatos et al. (14) so that a 32P-labeled GH cDNA plasmid [pBR322-GH1 (15)] can be used as probe to detect and quantitate GH-specific RNA sequences. We report here that exposure of G...

The construction, identification, and use of a recombinant DNA clone containing a growth hormone structural gene sequence is described. A cDNA copy of partially purified pregrowth hormone mRNA from cultured rat pituitary tumor (GC) cells was employed in the construction of a hybrid plasmid, designated pBR322-GH1. The cloned DNA sequence was positively identified by a hybridization-translation procedure which should be applicable to any cloned structural gene sequence. This procedure involved hybridization of cytoplasmic poly(A)-containing RNA from GC cells to the cloned DNA immobilized on nitrocellulose filters, followed by elution of the hybridized RNA and translation in a mRNA-depleted rabbit reticulocyte lysate system. Physical and immunological criteria were employed to show that the translation products were enriched for pregrowth hormone. Hybridization to excess plasmid DNA of [3H]uridine-labeled, size fractionated GC cell cytoplasmic RNA was used to show that all growth hormone-specific RNA sequences are the same size as functional pregrowth hormone mRNA.

Previous determinations of the chromosomal location of unique genes have required that the chromosomes of interest be fractionated, either by species-specific chromosome loss from interspecies hybrids, or by physical fractionation procedures. We An understanding ofthe mechanisms involved in the expression and regulation of any eukaryotic genes will ultimately require a determination of the location of its genetic elements within the genome of the corresponding organism. A first step in this determination is the localization ofthe gene to a particular chromosome and, if possible, to a particular subchromosomal region.Early gene localization techniques involved the use ofeither pedigree analysis or somatic cell genetics. More recently, hybridization in situ has been employed to localize reiterated genes in various organisms (see ref. 1 and references therein) and some unique genes in species containing polytene chromosomes (2). By contrast, the use of molecular hybridization procedures to localize unique genes in organisms without polytene chromosomes has depended on prior fractionation of the chromosomes under investigation. This has been achieved either by constructing interspecies (human-mouse) cell hybrids that preferentially lose human chromosomes (3, 4) or by physical fractionation of chromosomes (5, 6). A more direct in situ hybridization technique, in which preformed networks tailed with 5-bromodeoxyuridine are used to detect hybrids between chromosomes and a polyadenylylated RNA probe, has been employed successfully to localize an integrated retrovirus on chicken chromosomes (7). However, the extension of this method to localize other genes would require the use of probe RNA preparations that are as highly purified as retroviral RNA.For most mRNA species, this is a goal that would be quite difficult to achieve.We report here the development of a method for employing a hybrid plasmid containing a cDNA sequence to determine by hybridization in situ the chromosomal location ofa unique gene. To demonstrate that this method yields the correct result, we have used it to localize the gene that codes for human a-globin, whose chromosomal location has previously been determined through the use of somatic cell hybrids plus molecular hybridization (8, 9). EXPERIMENTAL PROCEDURES Precoating of Slides. Slides are incubated for a minimum of 5 hr in lOx Denhardt's solution (lx is 0.02% each of bovine serum albumin, Ficoll, and polyvinylpyrrolidone) (10) at 60C to reduce binding ofthe probe to the glass slide (11). The slides are then washed profusely in double-distilled water and air dried; they can be stored for several months after preparation.Preparation of Chromosomes. Penta X (69,XXXXX), a human lymphoblastoid cell line, was kindly supplied by M. Siniscalco of this institute. Metaphase chromosomes are prepared as described (12). The slides are either used for hybridization in situ within a day or stored with desiccant at -70'C.Preparation of 125I-Labeled dCTP. 125I-Labeled dCTP (specific activity 300-100...