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  • The mutation in Caucasian people of European heritage is qui


    The Δ32 mutation in Caucasian people of European heritage is quite common but extremely rare in Asian and African countries (Huang et al., 1996). We recently reported the presence of this mutation from India (Husain et al., 1998a) and established the complete inheritance pattern (Husain et al., 1998b). This T0901317 results in the formation of severely truncated protein which results in the loss of both the signal transduction and HIV coreceptor function. Even with this kind of homozygous deletion, people were found to be leading an healthy life. The major reason for this is the redundancy of chemokine receptors that can use overlapping ligands. It is, for this reason, that we had earlier described the function of a hammer-head ribozyme and a DNA-enzyme against the CCR5 gene and showed its intracellular efficacy (Goila and Banerjea, 1998). These general purpose RNA cleaving DNA-enzymes were described by Santoro and Joyce (1997) that were functional under simulated physiological conditions and two kinds of catalytic motifs, 10–23 and 8–17, were identified. We have earlier reported extremely efficient cleavage of HIV-1 envelope RNA by a DNA-enzyme and demonstrated its ability to interfere functionally with the expression of HIV-1 envelope gene (Dash et al., 1998).
    Materials and methods
    Discussion This is the first report of a catalytic DNA or RNA cleaving DNA-enzyme that cleaved the CXCR-4 gene efficiently in a sequence specific manner. This is an important coreceptor for X4 viruses, the appearance of which usually coincides with the later stages of the disease. The cleavage obtained was specific as evident from the expected cleavage pattern that was observed. Ability of the CXCR-4 DNA-enzyme to specifically cleave the target RNA was tested using two kinds of substrates. In the first case we synthesized a CXCR-4-CCR5 fusion gene by using a 60 nucleotide long DNA fragment that was generated by using recombinant DNA techniques and cloned into an expression vector so that labeled transcripts could be generated by in vitro transcription. In the second case, the CXCR-4 encoding DNA, was linearized in its coding region using BamH1 enzyme which could direct the formation of ∼500 nucleotide long transcript. The CXCR-4-DNA-enzyme cleaved both the transcripts with equal efficiency. Also the kinetics of cleavage was very similar to the one earlier described (Santoro and Joyce, 1997, Dash et al., 1998). With the aim to design DNA-enzymes that could simultaneously cleave the CXCR-4 and CCR5 transcripts, a di-DNA-enzyme was constructed by placing them in tandem. This di-DNA-enzyme possessed the ability to cleave both the targets, CXCR-4 and CCR5, in a sequence-specific manner. We had earlier reported the construction of DNA-enzyme-916 that was directed against CCR5 gene and we also showed its ability to act intracellularly (Goila and Banerjea 1998) on the full-length authentic CCR5 transcript. Thus, the di-DNA-enzyme that has been constructed, possessed the ability to cleave authentic CXCR-4 and CCR5 target RNAs. It is also remarkable that the di-DNA-enzyme retained the cleavage specificity of the mono-DNA-enzymes.
    Introduction The large yellow croaker Larimichthys crocea is an economically important marine fish species in China that is largely endemic to coastal waters of the eastern and southern parts of the country. Unfortunately, wild stocks have severely suffered from overfishing in the last few decades. Although artificial mariculture in China has made advances in croaker rearing since 1985, aquacultured croakers are likely to have reduced genetic diversity and disease resistance. Moreover, aquacultured croakers are extremely vulnerable to various marine pathogens, in particular, Vibrio anguillarum, a common pathogenic bacterial species. Outbreaks of infectious bacterial diseases and decreased disease resistance [1] in maricultured croaker species have markedly affected the fishery industry.