BACKGROUND: The aim of this study was to perform a comprehensive evaluation of the occurrence of DNA in human prostasomes. METHODS: Prostasomes were purified from seminal fluid (seminal prostasomes) and from PC-3-cells (PC-3 cell prostasomes). DNA extracted from both sources of prostasomes was visualized on agarose gels. Further, the DNA was cloned and sequenced (13 clones from seminal prostasomal DNA and 16 clones from PC-3 cell prostasomal DNA) and identified by alignment in the BLAST-nucleotide search database. In order to decide if the DNA was internally or externally located in/on prostasomes, prostasomes were treated with nuclease (DNase) and A(260) was measured before and after treatment. Additionally, flow cytometric studies were performed with membrane permeable and membrane impermeable DNA stains. RESULTS: We identified human chromosomal DNA in purified prostasomes from both sources and treatment with DNase demonstrated that the prostasome-shielded DNA was protected from enzyme attack. Membrane-permeable DNA stain raised the fluorescence contrary to membrane-impermeable stain. Clearly discernible nucleic acid of prostasomes was separated on 1% agarose gel yielding DNA fragments of about 13 kbp and below with a marked band at about 1 kbp. Cloning and sequencing of 13 fragments from seminal prostasomes and 16 from PC-3 cell prostasomes revealed a chromosomal origin of the DNA. In purified seminal prostasomes, 4 out of 13 DNA clones featured gene sequences (31%). The corresponding figure for PC3-derived prostasomes was 4 out of 16 clones featuring gene sequences (25%). CONCLUSION: Human prostasomes contain chromosomal DNA. Both nuclease treatment and differential DNA stainings indicated an inside location of the prostasomal DNA. Our findings suggest a DNA-delivery function of prostasomes to sperm cells. 2009 Wiley-Liss, Inc.
BACKGROUND: The aim of this study was to perform a comprehensive evaluation of the occurrence of DNA in human prostasomes. METHODS: Prostasomes were purified from seminal fluid (seminal prostasomes) and from PC-3-cells (PC-3 cell prostasomes). DNA extracted from both sources of prostasomes was visualized on agarose gels. Further, the DNA was cloned and sequenced (13 clones from seminal prostasomal DNA and 16 clones from PC-3 cell prostasomal DNA) and identified by alignment in the BLAST-nucleotide search database. In order to decide if the DNA was internally or externally located in/on prostasomes, prostasomes were treated with nuclease (DNase) and A(260) was measured before and after treatment. Additionally, flow cytometric studies were performed with membrane permeable and membrane impermeable DNA stains. RESULTS: We identified human chromosomal DNA in purified prostasomes from both sources and treatment with DNase demonstrated that the prostasome-shielded DNA was protected from enzyme attack. Membrane-permeable DNA stain raised the fluorescence contrary to membrane-impermeable stain. Clearly discernible nucleic acid of prostasomes was separated on 1% agarose gel yielding DNA fragments of about 13 kbp and below with a marked band at about 1 kbp. Cloning and sequencing of 13 fragments from seminal prostasomes and 16 from PC-3 cell prostasomes revealed a chromosomal origin of the DNA. In purified seminal prostasomes, 4 out of 13 DNA clones featured gene sequences (31%). The corresponding figure for PC3-derived prostasomes was 4 out of 16 clones featuring gene sequences (25%). CONCLUSION:Human prostasomes contain chromosomal DNA. Both nuclease treatment and differential DNA stainings indicated an inside location of the prostasomal DNA. Our findings suggest a DNA-delivery function of prostasomes to sperm cells. 2009 Wiley-Liss, Inc.
Authors: Javier Conde-Vancells; Esperanza Gonzalez; Shelly C Lu; Jose M Mato; Juan M Falcon-Perez Journal: Expert Opin Drug Metab Toxicol Date: 2010-05 Impact factor: 4.481