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We have obtained a cDNA clone encoding most of human X-linked 3-phosphoglycerate kinase (PGK; ATP:3-phospho-D-glycerate 1-phosphotransferase, EC 2.7.2.3). Total mRNA was prepared from human adenocarcinoma-derived cell line LS174T and used for cDNA preparation. Double-stranded cDNA was inserted, after tailing with oligo(dC), into the plasmid vector pBR327 and cloned in Escherichia coli K-12. Transformants were screened by colony hybridization with a mixture of 32P-labeled oligodeoxyribonucleotides. A pool of hexadecamers complementary to all 32 possible sequences encoding amino acids 291-296 of X-linked PGK was used for the initial screen. One clone among 2,500 gave a strong positive signal. Plasmid DNA from this clone was purified and characterized by hybridization first to the hexadecamer probe mixture and then to an undecamer probe consisting ofa mixture offour sequences. The cloned fragment hybridizes preferentially to DNA from human cells with five X chromosomes. DNA sequence analysis has established that the 1.2-kilobase-pair fragment encodes PGK from amino acid 121 through the COOH terminus.

Mycoplasmas were examined on the surfaces of tissue culture cells prepared for transmission and scanning electron microscopy. The pleomorphic bodies seen were proved to be mycoplasmas by the use of thin sections, passage of the infection from one cell line to another, and by autoradiography with [3H]thymidine both on the sections and the replicas. The mycoplasmas were not always evenly distributed over the cell's surface; their arrangement seemed to correlate with the activity or morphology of the cell. The use of replicas and scanning electron microscopy in routine examination of cultures for mycoplasma contamination is discussed.A number of surveys (9,11,15,27) indicate that many cell cultures, particularly when grown in the presence of antibiotics (10,15), are contaminated with mycoplasmas, and undetected infections have been an important source of artifact in many experiments (27).There are established techniques for the detection and identification of mycoplasmas (3,9,14,15,27,29). Many, however, are time consuming and complex. We have now found that very simple electron microscopic techniques such as cell-surface replicas (8,21,22) can provide a rapid and sensitive assay for the presence of mycoplasmas in tissue cultures. If available, scanning electron microscopy (5) can also be used to good advantage. This paper describes the morphology of mycoplasmas as observed in replicas and in preparations for the scanning electron microscope (SEM), presents an electron microscope autoradiographic technique for identification of these organisms in replicas, and discusses preliminary data on the interactions of culture cells with mycoplasmas. MATERIALS AND METHODSWe have examined cells from a number of different types (BHK, 3T3, Cl-1-D, LA9, L929, LD, HeLa, ME180, Chimp, CV-1, and AGMK), often obtaining the same cell line from a number of different sources. These cells were cultured on glass coverslips in the absence of antibiotics. * They were fixed before reaching confluency in 2.5% glutaraldehyde-0.5% osmium tetroxide in 0.1 M cacodylate buffer pH 7.4 at 0C (12). After dehydration in a graded series of ethanols, they were dried from Freon in a critical-point bomb (6). If lower-quality replicas can be tolerated (see Discussion) one can also dry the Abbreviation: SEM, scanning electron microscope. * Mycoplasma contamination can be detected in the presence of antibiotics. However, we have found that cells may need to be grown without antibiotics for several weeks before the mycoplasmas are present in more than marginally detectable numbers.samples from amyl acetate (21). For examination in the scanning electron microscope (ETEC; ETEC Corp., Hayward, Ca.), coverslips were rotary coated with gold. For transmission electron microscopy, samples were shadowed at a 450 angle with platinum-palladium (80:20) and at a 900 angle with carbon; the replicas were floated off the glass with hydrofluoric acid; and the cells were digested away with Clorox (21). The replicas were then rinsed in water, picked up on grids, and e...

Folded chromosomes were isolated from Mycoplasma hyorhinis. When examined by electron microscopy, these molecules show variability of loop size, number of loops, total contour length and degree of twisting of the DNA. Sedimentation velocity was unaltered after treatment with RNase, proteinase K, SDS, temperatures up to 65 degrees C and NaCl concentrations from 0.1 M to 4 M.

A putative HeLa cell culture line was discovered to be contaminated with mouse cells by examination of agarose gel profiles of restriction endonuclease digests of mitochondrial DNA. The contamination was confirmed by karyotypic analysis, and by observation of the mouse satellite band in an analytical buoyant density centrifugation of total cellular DNA. Restriction endonuclease analysis of mitochondrial DNA is suggested as a useful method for monitoring the species of cells in culture.