Effective methods are needed to identify and isolate those genes that are differentially expressed in various cells or under altered conditions. This report describes a method to separate and clone individual messenger RNAs (mRNAs) by means of the polymerase chain reaction. The key element is to use a set of oligonucleotide primers, one being anchored to the polyadenylate tail of a subset of mRNAs, the other being short and arbitrary in sequence so that it anneals at different positions relative to the first primer. The mRNA subpopulations defined by these primer pairs were amplified after reverse transcription and resolved on a DNA sequencing gel. When multiple primer sets were used, reproducible patterns of amplified complementary DNA fragments were obtained that showed strong dependence on sequence specificity of either primer.

Cells prepare for S phase during the G1 phase of the cell cycle. Cell biological methods have provided knowledge of cycle kinetics and of substages of G1 that are determined by extracellular signals. Through the use of biochemical and molecular biological techniques to study effects of growth factors, oncogenes, and inhibitors, intracellular events during G1 that lead to DNA synthesis are rapidly being discovered. Many cells in vivo are in a quiescent state (G0), with unduplicated DNA. Cells can be activated to reenter the cycle during G1. Similarly, cells in culture can be shifted between G0 and G1. These switches in and out of G1 are the main determinants of post-embryonic cell proliferation rate and are defectively controlled in cancer cells.

This paper provides evidence that normal animal cells possess a unique regulatory mechanism to shift them between proliferative and quiescent states. Cells cease to increase in number under a diversity of suboptimal nutritional conditions, whereas a uniformity of metabolic changes follows these nutritional shifts. Evidence is given here that cells are put into the same quiescent state by each of these diverse blocks to proliferation and that cells escape at the same point in GI of the cell cycle when nutrition is restored. The name restriction point is proposed for the specific time in the cell cycle at which this critical release event occurs.The restriction point control is proposed to permit normal cells to retain viability by a shift to a minimal metabolism upon differentiation in vivo and in vitro when conditions are suboptimal for growth. differentiation in vivo, or under nutritional deprivation to utilize this switching mechanism to achieve quiescence. A fundamental difference between normal and malignant cells may be that the malignant cells have lost their R-point control. As the consequences of this loss, although malignant cells' growth is less restricted, they would have reduced survival ability under adverse conditions. A preliminary report of this work has been presented elsewhere (7). Approximately 3 X 103 cells in 1 ml of medium were placed into each well of a Linbro tray and were allowed to grow into a loose network during 2-3 days. The cells remained attached to the bottoms of the wells throughout the experiments. Media were changed by aspiration and rinsing with phosphate-buffered saline (pH 7.2). After the cells had been allowed to incorporate [3H]thymidine (Amersham) added to 0.05 ,M and 0.1 juCi/ml, the wells were rinsed with 2 ml of phosphatebuffered saline and then with 1 ml of 5% trichloroacetic acid.The cells were dissolved in 0.8 ml of 2% Na2CO3 in 0.1 M NaOH. A 0.6-ml portion of this solution was added to 7 ml of Triton-toluene scintillator to which 0.2 ml of 50% trichloroacetic acid was added to neutralize the alkali. The samples were counted for 3H in a Nuclear Chicago Scintillation Spectrometer. Autoradiography was done with Kodak AR-10 stripping film. RESULTSThe cell cycle has been divided into four major phases (9) (G., S, G2, and M) but the sequence of essential events within GI, S, and G2 is just beginning to be investigated. A cell cannot be positioned within G1 because there are virtually no biochemical events established that can be used as landmarks.Our purpose in this paper is to compare the relative positions between M and S ("within G1") of a number of different quiescent cell populations, in order to ascertain whether or not they are at the same point. In the simplest approach, complete medium was added to quiescent cells and the transit time to the beginning of DNA synthesis was determined. For reasons to be summarized below, the results of these experiments were not fully adequate to locate the cells' positions.1286

Differential display has been developed as a tool to detect and characterize altered gene expression in eukaryotic cells. The basic principle is to systematically amplify messenger RNAs and then distribute their 3' termini on a denaturing polyacrylamide gel. Here we provide methodological details and examine in depth the specificity, sensitivity and reproducibility of the method. We show that the number of anchored oligo-dT primers can be reduced from twelve to four that are degenerate at the penultimate base from the 3' end. We also demonstrate that using optimized conditions described here, multiple RNA samples from related cells can be displayed simultaneously. Therefore process-specific rather than cell-specific genes could be more accurately identified. These results enable further streamlining of the technique and make it readily applicable to a broad spectrum of biological systems.