A cDNA library of Sarcocystis muris cyst merozoites was screened using a digoxigenin-labeled probe. This probe was derived from a 504-bp polymerase-chain-reaction fragment representing part of a thiol proteinase. Several cDNA clones were isolated, one of which (PH08) consists of a nucleotide sequence of 1694 bp and encodes the complete prepropolypeptide of a cathepsin L-like proteinase. PH08 contains an open reading frame of 394 amino acid (aa) residues with a 46-residue signal sequence, which is followed by a 129-residue propeptide and 219 aa residues of the mature enzyme. Two potential glycosylation sites and a putative polyadenylation signal were also identified. The occurrence of the highly conserved interspersed ERFNIN aa motif, not found in cathepsin B-like proteinases, suggests the classification of the enzyme as a cathepsin L-like proteinase. Results worked out in this study will enable production of the recombinant thiol proteinase of S. muris cyst merozoites necessary for study of the substrate specificity as well as other biochemical parameters of this enzyme.
Two subgenomic libraries constructed from Sarcocystis muris total DNA were screened with a cDNA probe, specific for a 32-kDa protein associated with the dense granules. Two clones reacted positively and were isolated, gDG 32/1 and gDG 32/2. Genomic clone gDG32/1 and part of clone gDG 32/2 have been sequenced. The composite nucleotide sequence of these genomic clones comprises 4.34 kb. It contains a 5' region of 2.14 kb, a first coding region (222 bp, exon I), a noncoding region (608 bp, intron), a second coding region (660 bp, exon II), and a 3' region of 693 bp. The upstream region shows a eukaryotic promoter structure and a consensus sequence for the 5' and 3' splicing sites. Thus the open reading frame (ORF DG32) coding for the 32-kDa protein of the dense granules of S. muris cyst merozoites is interrupted by an intron. To our knowledge, dg32 is the first sarcosporidian mosaic gene to be characterized.
Parasites of the phylum Apicomplexa (Sporozoa) cause diseases such as malaria, toxoplasmosis, or intestinal coccidiosis. Invasive stages possess typical apical organelles such as dense granules that harbor a broad range of polypeptides that are believed to take part in the parasite-host cell interaction. In previous studies a 26-kDa polypeptide of dense granules from Sarcocystis muris cyst merozoites (bradyzoites) was characterized as a thiol (cysteine) proteinase. In this paper a method is demonstrated to amplify DNA fragments from genomic DNA of S. muris cyst merozoites by polymerase chain reaction, which probably code for the 26-kDa antigen.
Type 1 diabetes is an immune-mediated disease with pancreatic infiltration and subsequent beta cell destruction. In this study pancreatic exocrinopathy in non-obese diabetic mice (NOD) was identified using gene subtraction methods (SSH) and macroarray analysis. Female NOD mice were treated with cyclophosphamide for acceleration and synchronization of the disease process at 70 d of age and analysed 10 d later, before the onset of overt diabetes. Extraction of total RNA of pancreas was followed by subtraction using the SSH technique. Pools of cDNA were generated using total RNA from treated and untreated NOD mice. Subtraction of cDNA pools of cyclophosphamide treated mice from cDNA pools of untreated mice resulted in a cDNA library, from which 480 clones were randomly selected. The clones were hybridized against labelled cDNA-probes generated from cyclophosphamide-treated and control NOD mice. Fifty-three clones (11 %) revealed at least twofold differential gene expression after cyclophosphamide treatment. Three of the downregulated genes (amylase, carboxypeptidase and preprotrypsin) were selected for evaluation of macroarray data by quantitative real-time PCR. Analysis of real-time PCR data confirmed suppression of gene expression with highest fold change for amylase (4.68-fold) followed by carboxypeptidase (2.79-fold) and preprotrypsin (2.14-fold). These results lead to the conclusion that inflammation in this animal model of type 1 diabetes is not restricted to pancreatic islets and that subtraction followed by macroarray analysis is capable of identifying genes responsible associated with disease progression.
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