1.Functional Characterization Of Human Dopamine Responsive Gene-1, DRG-1 2.Functional Complementation Between Human Gene LASS2 And Its Yeast Homologue LAG1

Human dopamine responsive gene-1 (DRG-1) was cloned in 2002. Its expression level can be up-regulated in the rat astrocytes treated with dopamine. However, its functions are not clear up to now. In this study, DRG-1 was identified to be a conserved gene in metazoans and share NF-κB binding sites with its homologues in their promoters. DRG-1 was highly expressed in human liver, kidney, pancreas and spleen. In yeast two-hybrid screen, DRG-1 was shown to bind with p75^NTR-associated cell death executor (NADE). The interaction was further verified both by co-immunoprecipitation assay in vitro and GST pull down assay in vivo. Furthermore, DRG-1 and NADE were co-localized in the cytoplasm of 293 and PC12 cells. The regions responsible for interaction were subsequently mapped to the 1-150 amino acid of DRG-1 at N-terminal and 59-111 amino acid of NADE at C-terminal. Noticeably, stable expression of DRG-1 in 293 cells could promote cell proliferation in MTTassay and the promotion was suppressed by overexpression of NADE. The findings provide explanation for the mechanism of the upreglation of DRG-1 expression by dopamine stimulation. And the results also suggest that DRG-1 may contribute to the dopamine receptor-induced cell growth, which is negatively regulated by NADE.LAG1(Longevity Assurance Gene 1) is the first ageing-related gene cloned in Saccharomyces cerevisiae. LAG1 and its yeast homolgue LAC1 encode the catalytic subunits of the ceramide synthase. Double deletion of LAG1 and LAC1 leads to the slow growth defect or lethality under certain genetic background. Some LAG1 homologues can rescue the defect of lag1 △ lac1 △ strain by functional complementation.LASS2 containing conserved TLC domain and specific HOX domain is a human homologue of Lag1p. In this study, shuffling test and tetrads analysis were carried out to examine the complementation between Lag1p and LASS2 or its fragment containing TLC domain but excluding HOX domain (LASS2 △ HOX). Behind either intact LAG1 or strong ADH1 promoter, LASS2 and LASS2△HOX couldn't rescue the slow growth defect of double mutant. The results indicated that LASS2 or LASS2△HOX couldn't functionally complement Lag1p.