Gene amplification is frequently present in human tumors, although specific target genes relevant to many amplified loci remain unidentified. An expression cloning assay enabled identification of a ...candidate oncogene derived from human chromosome 3p14.1. The cDNA retrieved from morphologically transformed cells contained the full-length protein coding region and detected an abundant transcript in the same cells. Sequence analysis revealed identity with the wild-type sequence of p44S10, a highly conserved subunit of the 26S proteasome that exhibits similarity to the Arabidopsis fus6/cop11 family of signaling molecules. p44S10 gene copy number and mRNA expression were increased in association with segmental 1.8 - 11-fold chromosomal gains in cutaneous malignant melanoma cell lines (5/13; 40%) and tumors (2/40; 5%), and in breast cancer MCF-7 cells. Likewise, malignant progression of human radial growth phase WM35 melanoma cells was associated with amplification and increased expression of endogenous p44S10, and increased expression of p44S10 was sufficient to induce proliferation of WM35 cells in vivo. The results demonstrate segmental copy number gains within chromosome 3p in cutaneous malignant melanoma and suggest that deregulation of a proteasome regulatory particle subunit may contribute to the malignant phenotype.
The unique biochemical properties of acetate kinase present a classic conundrum in the study of the mechanism of enzyme‐catalyzed phosphoryl transfer. Large, single crystals of acetate kinase from ...Methanosarcina thermophila were grown from a solution of ammonium sulfate in the presence of ATP. The crystals diffract to beyond 1.7 Å resolution. Analysis of X‐ray data from the crystals is consistent with a space group of C2 and unit cell dimensions a = 181 Å, b = 67 Å, c = 83 Å, β = 103°. Diffraction data have been collected from the crystals at 110 and 277 K. Data collected at 277 K extend to lower resolution, but are more reproducible. The orientation of a noncrystallographic twofold axis of symmetry has been determined. Based on an analysis of the predicted amino acid sequences of acetate kinase from several organisms, we hypothesize that acetate kinase is a member of the sugar kinase/actin/hsp70 structural family.
GTPase protocols Manser, Ed; Leung, Thomas
2002, 2002., 2002-12-15, Letnik:
189
eBook, Book
Small GTPase binding proteins (GTPases) are an ancient group of proteins that play key roles in almost every aspect of cell biology, from cell proliferation to nuclear transport. In GTPase Protocols: ...The Ras Superfamily, Edward J. Manser and Thomas Leung have collected the key techniques currently in use to probe the function of these ubiquitous proteins both in vitro and in vivo. Presented in a format that ensures ready reproducibility by accomplished experimentalists who have refined the various methods in their laboratories, each technique includes step-by-step instructions, tips on avoiding pitfalls and troubleshooting, and ancillary notes explaining how to adapt each procedure in the event of problems. The methods cover the spectrum of core techniques required for the five major GTPase subfamilies (Ras, Rho, Rab, Arf, and Ran) and permit a diversity of applications ranging from structural studies on a GTPase to real time in vivo analysis. Timely and highly practical, GTPase Protocols: The Ras Superfamily illuminates the powerful techniques used by investigators today to study this special family of proteins that plays such important roles in human health and disease.
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