Molecular cloning of the murine adenosine deaminase gene from a genetically enriched source: identification and characterization of the promoter region.

A genomic library was prepared with DNA from a genetically enriched mouse cell line in which amplified copies of the adenosine deaminase (ADA) gene account for over 5% of the genome. Overlapping cosmid clones encompassing the entire ADA structural gene were isolated from this genomic library and used for subsequent structural and functional analyses. Nuclease protection and primer extension analyses served to identify the location of multiple transcription initiation sites at the 5' end of the structural gene. Promoter activity was found by functional analyses to reside within a 240-base-pair fragment which contains the transcription initiation sites. Sequences upstream of the transcription initiation sites are very G + C rich (77%) and include a 22 nucleotide stretch of deoxyguanylate residues and two potential Sp1 transcription factor-binding sites. Comparison of the mouse and human ADA gene promoters revealed the presence of several regions that are highly conserved with regard to both sequence content and location and may represent genetic elements which are involved in ADA gene expression.

Partial purification and characterization of mitotic factors from HeLa cells.

Extracts from mitotic HeLa cells, when injected into Xenopus laevis oocytes, exhibit maturation-promoting activity (MPA) as evidenced by the breakdown of the germinal vesicle and the condensation of chromosomes. In this study we have attempted to purify and characterize these mitotic factors. When 0.2 M NaCl-soluble extracts of mitotic HeLa cells were concentrated by ultrafiltration and subjected to affinity chromatography on hydroxylapatite followed by DNA-cellulose, the proteins with MPA eluted as a single peak and their specific activity was increased approx. 200-fold compared with crude extracts. The molecular weight of the mitotic factors was estimated to be 100 kD as determined by chromatography on Sephacryl S-200. SDS-PAGE of the partially-purified mitotic factors indicated the presence of several polypeptides ranging from 40-150 kD with a major band of about 50 kD. The majority of these polypeptides were found to be phosphoproteins as revealed by 32P-labeling and autoradiography. Very little or no phosphorylation was observed at the 50 kD band. Several of these polypeptides were reactive with mitosis-specific monoclonal antibodies, MPM-1 or MPM-2, as shown by immunoblots of these proteins but the major polypeptide band at 50 kD was not. Removal of the immunoreactive polypeptides by precipitation with these antibodies did not destroy the MPA. The MPA of the crude or the partially-purified mitotic factors was destroyed by injection of (but not pretreatment with) alkaline phosphatase within 45 min after injection of mitotic factors. These results are discussed in terms of a possible role of phosphorylation-dephosphorylation of non-histone proteins in the regulation of mitosis and meiosis.

Inactivation of mitotic factors by ultraviolet irradiation of HeLa cells in mitosis.

Extracts from mitotic HeLa cells, when injected into fully grown Xenopus laevis oocytes, exhibit maturation-promoting activity (MPA) indicated by germinal vesicle breakdown (GVBD) and chromosome condensation. Recently, we observed that the MPA of mitotic cell extracts is neutralized by the inhibitors of mitotic factors (IMF) in HeLa cells, which are activated at telophase and remain active throughout the G1 period. The activity of the IMF coincides with the process of chromosome decondensation, which begins at telophase and continues until the beginning of S phase, when chromatin reaches its most decondensed state. The objective of the present study was to investigate whether these two phenomena - chromosome decondensation and the activation of IMF - were related. The activity of IMF was measured in N2O-blocked mitotic HeLa cells, in which chromosome decondensation was induced by exposure to ultraviolet light, and subsequent incubation in medium containing inhibitors of DNA synthesis, hydroxyurea and arabinosylcytosine (araC). u.v. irradiation activated IMF was seen even at very high doses of X-irradiation. The IMF seemed to inactivate the mitotic factors directly by forming a complex that precipitated on heating at 60 degrees C for 15 min. Mg2+ or polyamines (i.e. spermine, spermidine, and putrescine), agents known to promote chromatin condensation partially restored the MPA of the u.v.-irradiated mitotic cell extracts. These results tend to support the conclusion that the IMF play a role in the decondensation of chromosomes.