A procedure for making simultaneous determinations of the relative levels of gene transcripts in tissues or cells.

In this paper, we describe a method for making simultaneous determinations of the relative levels of selected resident gene transcripts in biological samples. The procedure consists of immobilizing a battery of cloned genes on a nitrocellulose or nylon filter and hybridizing the filter with radiolabeled cDNA synthesized from mRNA extracted from tissue or cell lines. The intensity of the autoradiographic signals obtained from the various genes on the filter is interpreted to be roughly proportional to the relative numbers of transcripts of the genes present in the original mRNA population. The reliability of the method was established by detecting the expression of c-N-ras and c-myc in human promyelocytic HL-60 cells, which are known to express these two oncogenes. The accuracy of the technique was further established by using a conventional method to hybridize filter-bound, cytoplasmic RNA with labeled probes synthesized from plasmid inserts of genes identified by the screening procedure. The utility of the procedure was demonstrated by our ability to simultaneously examine the relative levels of expression of 21 oncogenes in a radiation-induced canine lung carcinoma.

The subcellular distribution of 32P-labelled phospholipids, 32P-labelled ribonucleic acid and 125I-labelled odoprotein in pig thyroid slices. Effect in vitro of thyrotrophic hormone and dibutyryl-3',5'-(cyclic)-adeosine onophosphate.

1. The incorporation in vitro of [(32)P]phosphate into phospholipids and RNA and of [(125)I]iodide into protein-bound iodine by pig thyroid slices incubated for up to 6hr. was studied. The subcellular distribution of the labelled products formed after incubation with radioactive precursor in the nuclear, mitochondrial, smooth-microsomal, rough-microsomal and cell-sap fractions was also studied. 2. Pig thyroid slices actively took up [(32)P]phosphate from the medium during 6hr. of incubation; the rate of incorporation of (32)P into phospholipids was two to five times that into RNA. 3. The uptake of [(125)I]iodide by the slices from the medium was rapid for 4hr. of incubation, 6-10% of the label being incorporated into iodoprotein. 4. Much of the (32)P-labelled phospholipid accumulated in mitochondria and microsomes, whereas the nuclear fraction contained most of the (32)P-labelled RNA. After 2hr. of incubation most of the (32)P-labelled cytoplasmic RNA accumulated in the rough-microsomal fraction. The major site of localization of proteinbound (125)I was the smooth-microsomal fraction, and gradually increasing amounts appeared in the soluble cytoplasm fraction, suggesting a vectorial discharge of [(125)I]iodoprotein (presumably thyroglobulin) from smooth vesicles into the colloid. 5. The addition of 0.1-0.4 unit of thyrotrophic hormone/ml. of incubation medium markedly enhanced the accumulation of (32)P-labelled phospholipids in the microsomal fractions and to a much smaller extent that of (32)P-labelled RNA without any increase in the total uptake of the label. Almost simultaneously the hormone increased the uptake of [(125)I]iodide by the slices and enhanced the accumulation of protein-bound (125)I in the smooth-microsomal fraction. 6. As a function of time of incubation, thyrotrophic hormone had a biphasic effect on [(125)I]iodide uptake and protein-bound (125)I formation, the stimulatory effect being reversed after 4hr. of incubation. 7. 6-N-2'-O-Dibutyryl-3',5'-(cyclic)-AMP, but not 3',5'-(cyclic)-AMP or 5'-AMP, mimicked the action of thyrotrophic hormone on iodine uptake as well as on iodination of protein. On the other hand, the mimicry by 6-N-2'-O-dibutyryl-3',5'-(cyclic)-AMP of the stimulatory effect of thyrotrophic hormone on the formation of labelled thyroid phospholipids and RNA was only an apparent one resulting from an enhanced uptake of [(32)P]phosphate. 8. It is concluded that thyrotrophic hormone causes a co-ordinated increase in the formation or accumulation of phospholipids, RNA and iodoprotein associated with the endoplasmic reticulum, and that 6-N-2'-O-dibutyryl-3',5'-(cyclic)-AMP mimics the more rapid effects of thyrotrophic hormone on transport and metabolic functions of thyroid cells, but does not influence their slower biosynthetic responses to the hormone.