In HeLa cells deprived of valine, histidine, or methionine initiation of protein synthesis decreases rapidly and disaggregation of polyribosomes also occurs. The mechanism of inhibition does not seem to involve the supply of RNA in the cell, and thus it differs from the initiation of inhibition at elevated temperatures. Polyribosomes rapidly form again if the missing amino acid is restored, even in the presence of actinomycin D.Studies of HeLa cells deprived of single essential amino acids -have shown that protein synthesis continues in this situation at a reduced rate determined by the rate of endogenous protein turnover (1). Such cells are in a condition of suspended growth and offer a unique opportunity for the study of metabolic regulation. It has previously been established that the rate of synthesis of ribosomal precursor RNA is reduced in these cells (2, 3), and maturation into complete ribosomal subunits is restricted (3), though synthesis of ribosomal proteins is uninterrupted (4).In several recent studies (5, 6), it was found that ascites tumor cells deprived of a complete mixture of essential -amino acids had extensive disaggregation of polyribosomes into inactive monoribosomes. The disaggregation was reversed if the set of essential amino acids was restored. However, since all of the essential amino acids were withheld or supplied at the same time, it is uncertain whether the apparent -regulation of protein synthesis that was observed involved the availability of amino acids per se, or amino acids in the role of donors of amide or methyl groups.We have found that HeLa cells, deprived of any one of several essential amino acids, promptly exhibit a marked inhibition of translational initiation. This inhibition and its -reversal have been studied and compared to previous demonstrations of control of translational initiation in cells undergoing mitosis (7) and in cells exposed to elevated temperatures (8). MATERIALS AND METHODS Cell cultureSuspension cultures of HeLa cells were maintained in Eagle's medium with 5% calf serum, as described (9, 10), at a con-* To whom reprint requests should be sent. Reprints will be available only outside of North America. Amino-acid Depriation, Cell Labeling, and Fractionation. Cells were collected by centrifugation and rinsed in Eagle's medium without valine, histidine, or methionine, but with leucine at a concentration of 0.02 mM (one-twentieth of the normal concentration), and 5% dialyzed calf serum. They were then resuspended in a medium of the same composition at a concentration of 3 X 106 cells/ml. Cells acting as growing controls were resuspended in medium to which the missing amino acid had been added to its normal concentration. All cultures were incubated at 370C, except where noted Suspension cultures deprived of an amino acid for specified times, or growing control cultures, were incubated for 45 min, and then labeled for various times with ['4C]leucine (0.25 pCi/ml, 12.5 Ci/mol final specific activity). 8-ml samples were then rapidly cooled and fract...

The mRNA for type II collagen was purified from embryonic chick sternum or from purified sternal chondrocytes with guanidine thiocyanate as the extractant. Double-stranded cDNAs to procollagen mRNAs from sternum were synthesized and dC-tailed. After annealing with PstI-cleaved, dG-tailed pBR322, this DNA was used to transform Escherichia coli X1776. Transformed colonies were screened by colony hybridization to type I and II collagen cDNAs. Clones that preferentially hybridized to type II cDNA were characterized further. Four such cDNA clones, pCgII-2, 3, 10 and 12, with inserts of 400, 320, 260 and 750 bp, have been identified as type II collagen cDNA clones by several criteria, including their preference for hybridizing with type II rather than type I collagen mRNAs in hybrid-selected translation experiments.

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