To study gene mutations in different organs and tissues of an experimental animal, we produced transgenic mice harboring bacteriophage lambda shuttle vectors integrated in the genome in a head-to-tail arrangement. As a target for mutagenesis, the selectable bacterial lacZ gene was cloned in the vector. The integrated vectors were rescued from total genomic DNA with high efficiency by in vitro packaging and propagation of the phages in a LacZ- strain of Escherichia coli C. The background mutation frequencies in brain and liver DNA appeared to be low, as was indicated by the absence of colorless plaques among 138,816 and 168,160 phage isolated from brain and liver DNA, respectively. Treatment of adult female transgenic mice with N-ethyl-N-nitrosourea resulted in a dose-dependent increase of the frequency of mutated vectors isolated from brain DNA, up to 7.4 x 10(-5) at 250 mg of the alkylating agent per kilogram of body weight. At this dose, in liver DNA of the same mice, mutation frequencies were approximately 3 x 10(-5). DNA sequence analysis of four mutant vectors isolated from brain DNA indicated predominantly G.C----A.T transitions. These results demonstrate the value of this transgenic mouse model in studying gene mutations in vivo. In addition to its use in fundamental research, the system could be used as a sensitive, organ-specific, short-term mutagenicity assay.
We report the identification of a Cryptobia genomic DNA gene predicted to encode a hydrophobic protein containing a zinc metalloproteinase motif, HEXXH, and hence named it a major surface proteinase 1-like (MSP-1). The MSP-1 gene was identified using universal genome walking. Southern blot analysis revealed it to be a multicopy gene. Fragments of DNA encoding a segment homologous to the HEXXH motif of MSP-1 are widely found in bacteria, yeast, parasitic protozoans, plants, and animals including humans. These results suggest that the MSP-1 may have highly conserved functions, such as in intracellular proteolysis.
Cysteine protease is a metabolic enzyme, whereas metalloprotease is the virulent factor in cryptobiosis caused by Cryptobia salmositica. Recombinant DNA vaccines were produced with the insertion of either the metalloprotease or cysteine protease gene of C. salmositica into plasmid vectors (pEGFP-N). As expected, fishes (Oncorhynchus mykiss and Salmo salar) injected intramuscularly with the metalloprotease-DNA (MP-DNA) vaccine (50 microg/fish) were consistently more anemic (lower packed cell volume, PCV) than controls (injected only with the plasmid) at 3-5 weeks post-inoculation. Also, there were no difference in PCV between fish injected with the cysteine-DNA plasmids and the controls. In addition, agglutinating antibodies against Cryptobia were detected only in the blood of MP-DNA-vaccinated fish at 5-7 weeks post-vaccination and not in cysteine-DNA plasmids and the control groups. MP-DNA-vaccinated fish when challenged with the pathogen had consistently lower parasitemia, delayed peak parasitemia, and faster recovery compared with the controls. All fish vaccinated with attenuated strain were protected when challenged with the pathogen; this positive control group confirmed that the two vaccines operate through different mechanisms.
The present study describes the identification of a cathepsin L-like cysteine proteinase gene (CYS) from the hemoflagellate Cryptobia salmositica. Genomic DNA sequence of cysteine proteinase was obtained by genome walking using degenerate primers. Specific primers were designed to amplify the cDNA of cysteine proteinase from mRNA by rapid amplification of cDNA ends-PCR. The open reading frame of CYS is 1,329 bp, with 443 deduced amino acids. Based on the sequence analysis, cysteine proteinase of C. salmositica is similar to the cathepsin L-like cysteine proteinase of kinetoplastid parasites such as Leishmania spp. and Trypanosoma spp. The identification of CYS proteinase gene could help to design cysteine proteinase specific inhibitors. Further studies are required to characterize the complete genomic organization of the cysteine proteinase.
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