Effect of tryptic or peptic digestion or mechanical isolation on the extraction of proteins, antigens, and ribonucleic acids from Sarcocystis muris bradyzoites.
Various methods have been used for the isolation of bradyzoites of Sarcocystis species. Using Sarcocystis muris as a model, the effect of 3 methods, trypsin digestion, pepsin digestion, and mechanical isolation, on the subsequent extraction of S. muris proteins, antigens, and RNA was examined. Although some quantitative differences were found among the proteins, antigens, and RNA extracted after the 3 isolation methods, qualitative differences were not evident. The overall isoelectric focusing protein profile showed approximately 30 bands in the pH range of 3-9 and was dominated by 5 bands with pI values of approximately 5.3, 6.3, 6.8, 7.0, and 7.4. In experimentally infected mice, antibodies were recorded from 35 days postinoculation (PI) until the end of examination (130 days PI). The S. muris RNA appeared to consist of at least 10 subunits in the range of 200-4,200 nucleotides. A Toxoplasma gondii DNA fragment specific for small subunit ribosomal RNA (rRNA) hybridized mainly to the S. muris 1,900-nucleotide subunit and a range of smaller subunits, and a probe specific for large subunit rRNA hybridized mainly to the 4,200-nucleotide subunit and a range of smaller subunits. All 3 methods of bradyzoite isolation gave good yields of intact messenger RNAs that showed similar in vitro translation curves.
Tenter
A M
AM
Department of Clinical Microbiology, Flinders University School of Medicine, Flinders Medical Centre, South Australia.
Johnson
M R
MR
Johnson
A M
AM
D000818:Animals; D000953:Antigens, Protozoan / Q000032:analysis*; D004797:Enzyme-Linked Immunosorbent Assay; D005455:Fluorescent Antibody Technique; D007525:Isoelectric Focusing; D051379:Mice; D009693:Nucleic Acid Hybridization; D010434:Pepsin A; D015800:Protozoan Proteins / Q000032:analysis*; D015347:RNA Probes; D016053:RNA, Protozoan / Q000032:analysis*; D012522:Sarcocystis / Q000032:analysis / Q000235:genetics / Q000276:immunology / Q000302:isolation & purification*; D013047:Specific Pathogen-Free Organisms; D014357:Trypsin
D000953:Antigens, Protozoan; D015800:Protozoan Proteins; D015347:RNA Probes; D016053:RNA, Protozoan; D014357:Trypsin; D010434:Pepsin A