ABSTRACT
The goal of this study was to determine whether it will be feasible to study the expression of a large, human gene, such as the BCL2 proto-oncogene, by DNA transfection. The BCL2 proto-oncogene is 230 kb in size and is deregulated in tumor cells by translocation into the immunoglobulin heavy-chain locus. Yeast artificial chromosomes (YACs) containing the human BCL2 gene were altered by homologous recombination in Saccharomyces cerevisiae to yield replicas of the normal and translocated alleles. Constructions containing either allele and ranging in size from 360 to 800 kb were integrated stably into a mouse tumor line. Fifty-eight percent of the clones contained a copy of the entire YAC insert. Over 50% of these clones expressed appropriate levels of human BCL2 RNA and protein. These studies suggested that the expression of large human genes and their pathologic rearrangements can be studied by transfection techniques employing YACs propagated in S. cerevisiae.
Subject(s)
Proto-Oncogene Proteins/genetics , Animals , Base Sequence , Chromosomes, Fungal , Cloning, Molecular , DNA Mutational Analysis , Genes , Genetic Vectors , Humans , In Situ Hybridization , Mice , Molecular Sequence Data , Oligodeoxyribonucleotides/chemistry , Proto-Oncogene Mas , Proto-Oncogene Proteins c-bcl-2 , Saccharomyces cerevisiae , Structure-Activity Relationship , Transfection , Translocation, GeneticABSTRACT
Five DNase I-hypersensitive regions were associated with the Saccharomyces cerevisiae galactose gene cluster during both galactose induction and glucose repression of transcription. Four hypersensitive regions were located in areas flanking the GAL cluster genes, and one site occurred within GAL10. A DNase I-hypersensitive region located between the 5' ends of divergently transcribed GAL10 and GAL1 contained sequences essential for the transcription of both genes.
Subject(s)
DNA, Fungal/genetics , Galactose/pharmacology , Genes, Fungal , Genes, Regulator , Saccharomyces cerevisiae/genetics , Transcription, Genetic/drug effects , Base Sequence , DNA, Fungal/metabolism , Deoxyribonuclease I/metabolismABSTRACT
We examined the DNA of Saccharomyces cerevisiae by both HpaII-MspI restriction enzyme digestion and high-performance liquid chromatography analysis for the possible presence of 5-methylcytosine. Both of these methods failed to detect cytosine methylation within this yeast DNA; i.e., there is less than 1 5-methylcytosine per 3,100 to 6,000 cytosine residues.
Subject(s)
Cytosine/analogs & derivatives , DNA, Fungal/isolation & purification , Saccharomyces cerevisiae/analysis , 5-Methylcytosine , Chromatography, High Pressure Liquid/methods , Cytosine/analysisSubject(s)
Liver/metabolism , Taurine/biosynthesis , Animals , Cats , Cystine , Female , Male , Rats , Sulfinic Acids/metabolismABSTRACT
In vitro, addition of taurine to liver homogenates increases the proportion of cholic acid conjugated with taurine. In the present study, the relation between hepatic taurine concentration and the proportion of infused sodium cholate conjugated with taurine was studied in the whole organ. The isolated perfused liver was studied to eliminate possible transfer of taurine to or from the large extrahepatic poosl present in vivo. During cholate infusion, the proportion of taurocholate excreted in bile decreased, and the proportion of glycocholate increased in a complementary fashion. Infusion of taurine with cholate prevented these changes. Hepatic taurine concentration, calculated from measured hepatic taurine concentrations before and at the end of cholate infusion, fell. Fall in proportion of total bile acid excreted as taurocholate was most rapid at low hepatic taurine concentrations between about 1.4 and 0.65 mumol/g liver. Hepatic taurine concentrations is a major determinant of the proportion of bile acid conjugated with taurine.
