ABSTRACT
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 of a mixture of four 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.
Subject(s)
Phosphoglycerate Kinase/genetics , Sex Chromosomes , X Chromosome , Amino Acid Sequence , Base Sequence , Cloning, Molecular , DNA/genetics , Female , Humans , Nucleic Acid Hybridization , Oligodeoxyribonucleotides/geneticsABSTRACT
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.
Subject(s)
Chromosomes, Bacterial/ultrastructure , DNA, Bacterial , Mycoplasma/ultrastructure , Cell Fractionation , Centrifugation, Density Gradient , DNA, Bacterial/isolation & purification , Molecular Weight , Nucleic Acid ConformationABSTRACT
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.
Subject(s)
DNA Restriction Enzymes , DNA, Mitochondrial , Endonucleases , HeLa Cells/analysis , Animals , Cell Line , DNA, Mitochondrial/analysis , DNA, Neoplasm/analysis , Haemophilus influenzae/enzymology , Karyotyping , Mice , Molecular WeightABSTRACT
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 [(3)H]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.