Jihua Yao

DAXX interacts with phage ΦC31 integrase and inhibits recombination

Phage ΦC31 integrase has potential as a means of inserting therapeutic genes into specific sites in the human genome. However, the possible interactions between ΦC31 integrase and cellular proteins have never been investigated. Using pLexA-ΦC31 integrase as bait, we screened a pB42AD-human fetal brain cDNA library for potential interacting cellular proteins. Among 61 positives isolated from 106 independent clones, 51 contained DAXX C-terminal fragments. The strong interaction between DAXX and ΦC31 was further confirmed by co-immunoprecipitation. Deletion analysis revealed that the fas-binding domain of DAXX is also the region for ΦC31 binding. Hybridization between a ΦC31 integrase peptide array and an HEK293 cell extract revealed that a tetramer, 451RFGK454, in the C-terminus of ΦC31 is responsible for the interaction with DAXX. This tetramer is also necessary for ΦC31 integrase activity as removal of this tetramer resulted in a complete loss of integrase activity. Co-expression of DAXX with ΦC31 integrase in a HEK293-derived ΦC31 integrase activity reporter cell line significantly reduced the ΦC31-mediated recombination rate. Knocking down DAXX with a DAXX-specific duplex RNA resulted in increased recombination efficiency. Therefore, endogenous DAXX may interact with ΦC31 causing a mild inhibition in the integration efficiency. This is the first time that ΦC31 was shown to interact with an important cellular protein and the potential effect of this interaction should be further studied.

Molecular cloning and expression of two closely related GTP-binding proteins from zebrafish.

Developmentally regulated GTP-binding proteins (DRGs) are a subclass of GTP-binding proteins that have been discovered recently. Here we report two zebrafish DRG cDNA clones closely related to human and mouse DRG genes. The two DRG sequences showed a high degree of similarity (55% identity, 72% similarity) at the amino acids level. Whole mount in situ hybridization revealed expression of zebrafish DRGs maternally, following the onset of zygotic transcription at the mid-blastula transition (MBT) and throughout embryonic. The expression of these two genes in different tissues follows a similar pattern, suggesting that they may serve a similar function.

Efficient expression of human factor IX cDNA in liver mediated by hydrodynamics-based plasmid administration

Hydrodynamics-based administration via tail vein was used to deliver naked plasmid with human factor IX (hFIX) cDNA in 2.2 mL Ringer’s solution into mice within 7 s. The peak level of expression of hFIX was 2921 ng/mL in mouse plasma. The hFIX cDNA expression increased with increasing the amount of plasmid DNA injected. The peak level of gene expression declined after repeated injection of plasmid (1459 ng/mL). The hFIX cDNA was detected in various organs, but the highest level of gene expression appeared in liver. Transaminase levels and liver histological results showed that rapid intravenous plasmid injection into mice induced transient focal acute liver damage, which was rapidly repaired within 3–10 d. These results suggested that high-level expression of hFIX cDNA can be achieved by hydrodynamics-based plasmid transfer and this method is now further used for gene therapy and gene function study in our lab.

Cloning and expression of translation elongation factor 2 (EF-2) in zebrafish.

We have identified a developmentally regulated gene translation elongation factor 2 (EF-2) in zebrafish (GenBank Accession No. AAQ91234). Analysis of DNA sequence of zebrafish EF-2 shows that the 2826 bp cDNA spans an open reading frame from nucleotide 55 to 2631 and encodes a protein of 858 amino acids. It shares an identity of 92, 93, 93, 92, 79 and 80% in amino acid sequence to human, mouse, Chinese hamster, Gallus gullus, C. elegans and Drosophila EF-2, respectively. Zebrafish EF-2 protein has 16 conserved domains, GTP-binding domain is found in the NH2 terminus, and the ADP-ribosylation domain locates at the COOH terminus. Whole mount in situ hybridization on zebrafish embryos shows that the transcripts of EF-2 gene are detected during the early development of zebrafish embryo and constantly change from 5-somite stage to protruding-mouth stage. It expresses strongly throughout envelope at 5-somite stage. Then the stained cells concentrate strongly in the eyes, brain and muscle tissue. From prim-25 stage the stained cells only appear strongly in the lens and the anterior portion of the cerebellum.