Expression profile and down-regulation of argininosuccinate synthetase in hepatocellular carcinoma in a transgenic mouse model.

BACKGROUND: Argininosuccinate synthetase (ASS) participates in urea and nitric oxide production and is a rate-limiting enzyme in arginine biosynthesis. Regulation of ASS expression appears complex and dynamic. In addition to transcriptional regulation, a novel post-transcriptional regulation affecting nuclear precursor RNA stability has been reported. Moreover, many cancers, including hepatocellular carcinoma (HCC), have been found not to express ASS mRNA; therefore, they are auxotrophic for arginine. To study when and where ASS is expressed and whether post-transcriptional regulation is undermined in particular temporal and spatial expression and in pathological events such as HCC, we set up a transgenic mouse system with modified BAC (bacterial artificial chromosome) carrying the human ASS gene tagged with an EGFP reporter. RESULTS: We established and characterized the transgenic mouse models based on the use of two BAC-based EGFP reporter cassettes: a transcription reporter and a transcription/post-transcription coupled reporter. Using such a transgenic mouse system, EGFP fluorescence pattern in E14.5 embryo was examined. Profiles of fluorescence and that of Ass RNA in in situ hybridization were found to be in good agreement in general, yet our system has the advantages of sensitivity and direct fluorescence visualization. By comparing expression patterns between mice carrying the transcription reporter and those carrying the transcription/post-transcription couple reporter, a post-transcriptional up-regulation of ASS was found around the ventricular zone/subventricular zone of E14.5 embryonic brain. In the EGFP fluorescence pattern and mRNA level in adult tissues, tissue-specific regulation was found to be mainly controlled at transcriptional initiation. Furthermore, strong EGFP expression was found in brain regions of olfactory bulb, septum, habenular nucleus and choroid plexus of the young transgenic mice. On the other hand, in crossing to hepatitis B virus X protein (HBx)-transgenic mice, the Tg (ASS-EGFP, HBx) double transgenic mice developed HCC in which ASS expression was down-regulated, as in clinical samples. CONCLUSIONS: The BAC transgenic mouse model described is a valuable tool for studying ASS gene expression. Moreover, this mouse model is a close reproduction of clinical behavior of ASS in HCC and is useful in testing arginine-depleting agents and for studies of the role of ASS in tumorigenesis.

A transgenic approach to study argininosuccinate synthetase gene expression.

BACKGROUND: Argininosuccinate synthetase (ASS) participates in urea, nitric oxide and arginine production. Besides transcriptional regulation, a post-transcriptional regulation affecting nuclear precursor RNA stability has been reported. To study whether such post-transcriptional regulation underlines particular temporal and spatial ASS expression, and to investigate how human ASS gene behaves in a mouse background, a transgenic mouse system using a modified bacterial artificial chromosome carrying the human ASS gene tagged with EGFP was employed. RESULTS: Two lines of ASS-EGFP transgenic mice were generated: one with EGFP under transcriptional control similar to that of the endogenous ASS gene, another with EGFP under both transcriptional and post-transcriptional regulation as that of the endogenous ASS mRNA. EGFP expression in the liver, the organ for urea production, and in the intestine and kidney that are responsible for arginine biosynthesis, was examined. Organs taken from embryos E14.5 stage to young adult were examined under a fluorescence microscope either directly or after cryosectioning. The levels of EGFP and endogenous mouse Ass mRNAs were also quantified by S1 nuclease mapping. EGFP fluorescence and EGFP mRNA levels in both the liver and kidney were found to increase progressively from embryonic stage toward birth. In contrast, EGFP expression in the intestine was higher in neonates and started to decline at about 3 weeks after birth. Comparison between the EGFP profiles of the two transgenic lines indicated the developmental and tissue-specific regulation was mainly controlled at the transcriptional level. The ASS transgene was of human origin. EGFP expression in the liver followed essentially the mouse Ass pattern as evidenced by zonation distribution of fluorescence and the level of EGFP mRNA at birth. However, in the small intestine, Ass mRNA level declined sharply at 3 week of age, and yet substantial EGFP mRNA was still detectable at this stage. Thus, the time course of EGFP expression in the transgenic mice resembled that of the human ASS gene. CONCLUSIONS: We demonstrate that the transgenic mouse system reported here has the merit of sensitivity and direct visualization advantage, and is ideal for annotating temporal and spatial expression profiles and the regulation mode of the ASS gene.

The mutation spectrum of the phenylalanine hydroxylase (PAH) gene and associated haplotypes reveal ethnic heterogeneity in the Taiwanese population.

Phenylalanine hydroxylase (PAH) deficiency is responsible for most cases of phenylketonuria (PKU). In this study of the PAH mutation spectrum in the Taiwanese population, 139 alleles were identified including 34 different mutations. The V190G, Q267R and F392I mutations are first reported in this study. The most common mutations, R241C, R408Q and Ex6-96A>G, account for 23.2%, 12.0% and 9.2%, of the mutant alleles, respectively. Haplotype analysis shows that R241C and Ex6-96A>G are exclusively associated with haplotype 4.3 to suggest founder effects. On the other hand, R408Q is found on two distinct haplotypes suggesting recurrent mutations. The spectrum of PAH mutations in Taiwan shows various links to those of other Asian regions, yet remarkable differences exist. Notably, R408Q, E286K and -4173_-407del, accounting for 21% of all mutant alleles in Taiwan, are very rare or are undetected among PKU cohorts of other Asian regions to suggest local founder effects. Moreover, the low homozygosity value of 0.092 hints at a high degree of ethnic heterogeneity within the Taiwanese population. Our study of PAH mutation spectrum and the associated haplotypes is useful for subsequent study on the origin and migration pattern via Taiwan, an island at the historical crossroad of migration of ancient populations.

Modulation of formation of the 3'-end of the human argininosuccinate synthetase mRNA by GT-repeat polymorphism.

Microsatellites are abundant in the human genome and may acquire context-dependent functions. A highly polymorphic GT microsatellite is located downstream of the poly(A) signal of the human argininosuccinate synthetase (ASS1) gene. The ASS1 participates in urea and nitric oxide production and is a rate-limiting enzyme in arginine biosynthesis. To examine possible involvement of the GT microsatellite in ASS1 mRNA 3'-end formation, ASS1 minigene constructs were used in transient transfection for assessment of poly(A) site usage by S1 nuclease mapping. Synthesis of the major human ASS1 mRNA is found to be controlled by two consecutive non-canonical poly(A) signals, UAUAAA and AUUAAA, located 7 nucleotides apart where a U-rich sequence and the GU microsatellite serve as their respective downstream GU/U-rich elements. Moreover, AUUAAA utilization is affected by the GU-repeat number possibly leading to differential regulation of ASS1 polyadenylation in individuals with different repeat numbers. Interestingly, the less efficient UAUAAA motif is noted to be the major ASS1 poly(A) signal possibly as a result of an indispensable downstream U-rich element and restricted utilization of the AUUAAA motif by the presence of extended GU-repeats. The UAUAAA motif and the GT microsatellite are conserved only in primates whereas AUUAAA motif is present in all mammals analyzed. The suboptimal UAUAAA motif and the utilization of the polymorphic GT microsatellite as polyadenylation signal of the ASS1 gene may be used as a strategy in primates to modulate ASS1 level in response to interactions of genetic and environmental factors.

Simultaneous assessment of the effects of exonic mutations on RNA splicing and protein functions.

To simultaneously assess the effects of exonic mutations on RNA splicing and protein functions, we report here an intron-inclusive cDNA (Intinc) expression system. As a test model, twenty-four mutations in exon 9 of the phenylalanine hydroxylase (PAH) gene were examined in an Intinc expression plasmid composed of the PAH cDNA with the exon 9 flanked by its authentic introns. When the PAH enzyme activities from the Intinc plasmid-transfected cells were compared to those of a standard cDNA expression system, five mutations resulted in significant relative differences in PAH activities attributed to altered exon 9-inclusive mRNA levels. Two of the mutations affected exon recognition probably through splice site modifications and the remaining three affected experimentally verified exon splicing enhancer (ESE) motifs. The Intinc expression system allows not only a better link between mutation genotype to disease phenotype but also contributes to further understanding of molecular mechanisms of deleterious effects of mutations.