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Clones for human prothymosin a have been identified in cDNA libraries from staphylococcal enterotoxin A-stimulated normal human lymphocytes and from simian virus 40-transformed fibroblasts. The 1198-base-pair fibroblast clone has been sequenced. The encoded protein is highly acidic (54 residues out of 111) and shares >90% sequence homology with rat prothymosin a. The peptide "hormone" thymosin al appears at positions 2-29 of the prothymosin a amino acid sequence. There is no N-terminal signal peptide.

Human lymphocyte lysates prepared by detergent treatment of intact, normal resting cells contain ribonucleases that are insensitive to many inhibitors commonly used with eucaryotic cells. Phenol-extracted ribonucleic acid (RNA) obtained directly from unfractionated cytoplasm is sometimes degraded, but after fractionation of the cytoplasmic material by sucrose density gradient centrifugation, the polyadenylated RNA, in particular, is inevitably destroyed. An extensive survey of ribonuclease inhibitors, undertaken as a consequence, indicated that the complexes formed between the oxovanadium ion and the four ribonucleosides were unique in their ability to suppress lymphocyte nuclease activity. It proved possible to isolate intact ribosomal RNA and polyadenylated messenger RNA from lymphocyte cytoplasm fractionated on sucrose gradients when 2.5 mM each of the four ribonucleosidevanadyl complexes was used throughout the procedure. The data showed that the size distribution of poly(A)-bearing RNA remained unchanged, with a peak at ~16 S under denaturing conditions, regardless of whether the mRNA was originally associated with polysomes or was nonpolysome bound. The cytoplasmic RNAs were completely free of contamination by either intact nuclear RNA or nuclear fragments. Furthermore, exogenous globin mRNA mixed with lymphocytes and reisolated together with endogenous cytoplasmic polyadenylated RNA was fully translatable only when ribonucleoside-vanadyl complexes were employed during the preparation. The use of this inhibitor should therefore be considered for all tissues in which ribonucleases impede isolation of intact RNA. e isolation of intact RNA from most animal cells relies on the use of exogenous ribonuclease inhibitors. Many substances including diethyl pyrocarbonate, polyvinyl sulfate, heparin, bentonite, macaloid, an assortment of ribonucleotides, sodium dodecyl sulfate, and proteinase K have been employed

The function of prothymosin a has been investigated by using four different antisense oligodeoxyribonucleotides directed at selected regions of its mRNA. In every case, when synchronized human myeloma cells were released from stationary phase by incubation in fresh medium containing antisense oligomers, cell division was prevented or inhibited; sense oligomers and random antisense oligomers had no effect. A detailed analysis of synchronized cell populations indicated that sense-treated and untreated cells divided m17 hr after growth initiation, whereas cells incubated with antisense oligomer 183, a 16-mer targeted 5 bases downstream of the initiation codon, entered mitosis approximately one cell division late. The failure to divide correlated directly with a deficit in prothymosin a and with the continued presence of intact intracellular antisense oligomers over a period ofat least 24 hr. Because antisense oligomers had no effect either on the timing of the induction of prothymosin a mRNA upon growth stimulation or on mRNA levels seen throughout the cell cycle, we concluded that antisense DNA caused specific hybrid arrest of translation. Our data suggest that prothymosin a is required for cell division. However, there is no evidence that prothymosin a directly regulates mitosis.The function of prothymosin a is debatable. Evidence from this and other laboratories suggests that the protein is neither a precursor of thymosin a,, a putative thymic hormone, nor a secreted thymic hormone itself (1-3). Instead, several observations support a role in cell proliferation: (i) Prothymosin a mRNA and protein are present in virtually all mammalian tissues, and a homologous protein has been detected in yeast (1,(4)(5)(6)(7)(8); these findings are consistent with an activity essential to most cells. (ii) The amounts of prothymosin a and its mRNA are roughly proportional to the proliferative activity of the tissue from which they are isolated (1,4,7). (iii) Prothymosin a mRNA is induced in normal human lymphocytes and in serum-starved NIH 3T3 cells upon growth stimulation with mitogens or serum, respectively (1).Using COS cells transfected with the human prothymosin a gene, we have recently shown that prothymosin a is a nuclear protein. We have also found that chimeric proteins composed of all or part of prothymosin a fused with (3-galactosidase are targeted to the nucleus only when the basic amino acids at the carboxyl terminus of prothymosin a are included (9). The presence of a nuclear localization signal indicates a function carried out, at least in part, in the nucleus; the precise nature of that function is unknown.In the present study, we have directly evaluated the relationship between prothymosin a and cell division. Using antisense oligomers to inhibit accumulation ofprothymosin a in a synchronized population of myeloma cells, we have established that cells deficient in prothymosin a cannot divide, that the inhibition is reversible, and that degradation of intracellular antisense oligomers accompanies resumpti...

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