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To test the hypothesis that prothymosin and parathymosin contain amino acid sequences that cause them to be targeted to the cell nucleus, expression vectors were constructed containing a simian virus 40 promoter and cDNAs that would code for chimeric proteins composed of truncated human growth hormone (hGH) linked to the NH2 terminus of prothymosin or parathymosin. The truncated hGH lacked the signal peptide sequence required for its secretion. After transfection of these constructs into HeLa S3 cells, which do not normally synthesize hGH, the use of indirect immunofluorescence staining to follow the localization of the hGH chimeras demonstrated that both prothymosin and parathymosin caused targeting to the cell nucleus. Controls with a construct coding for native hGH only, and one coding for the truncated hGH lacking the signal peptide, revealed secretion into culture medium and staining in the endoplasmic reticulum and Golgi apparatus in the first case, and diffuse staining throughout the cytoplasm in the second. The results provide direct evidence, with proteins synthesized in situ, for the presence of nuclear localization signals in both prothymosin and parathymosin.Interest in proteins of thymic origin was stimulated by the observation that a crude preparation from calf thymus, designated thymosin fraction 5, showed immunomodulatory properties in a number of in vitro test systems (reviewed in ref. 1). Many of these properties were attributed to a single peptide purified from this fraction, named thymosin a, (2), which was proposed to function as a "thymic hormone" influencing lymphocyte maturation (3). Based on evidence suggesting that this thymosin a, might be a proteolytic artifact that arose during the preparation ofthymosin fraction 5 (4, 5), a search was initiated for the native protein or polypeptide precursor. A procedure designed to minimize endogenous proteolysis led to the isolation of two homologous polypeptides, named prothymosin (6) and parathymosin (7), the first of which contained the 28-amino acid sequence of thymosin a, at its NH2 terminus. The proposed role of thymosin a, as a thymic hormone was brought into question when the putative precursor, prothymosin, and the mRNA coding for this polypeptide were shown to be present in all mammalian tissues examined (8,9). In addition, the cDNAs coding for both human (10, 11) and rat (12) prothymosins were found to lack sequences coding for the signal peptides expected in secretory proteins. An extracellular function was also rendered unlikely by the observation that the mRNA for prothymosin was localized exclusively on free polysomes (13).The experimental findings relating to parathymosin have closely mimicked those for prothymosin, with quantitative differences in tissue polypeptide and mRNA content (7,9,12,14).More recent studies have suggested a relationship between prothymosin and cell growth. Increased prothymosin mRNA in response to a variety of stimulators of mammalian cell division has been reported (11), and elevated mRNA was also ...

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Differential display (DD) is a powerful molecular tool that allows the identification and subsequent isolation of transcripts differentially expressed between biological samples, for example, between undifferentiated and differentiated cells, between different tissues or in one tissue at different stages of development. However, significantly high rates of apparent false positives have been reported using this technique. We suggest that the vast majority of false positives do not represent the originally selected transcript, but instead result from the re-amplification of cDNA species that co-migrate with the cDNA of interest in DD gels. Here we describe the use of a procedure to resolve co-migrating cDNAs and to purify the candidate of interest before cloning. The use of this modified technique resolves downstream problems encountered during DD experiments.

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