We have studied the regulation of expression of the asparagine synthetase (AS) gene in tsll cells, a mutant of BHK hamster cells which encodes a temperature-sensitive AS and therefore does not produce endogenous asparagine at 39.5°C. Incubation of tsll cells at the nonpermissive temperature drastically increases the level of AS mRNA, and the stimulation of AS mRNA expression is effectively suppressed by the addition of asparagine to the medium. We show here that regulation of AS gene expression involves cis-acting elements which are contained in the mRNA as well as in the 5' genomic region. When a plasmid containing the human AS cDNA under the control of the human AS promoter region was stably transfected into tsll cells, the expression of human AS RNAs was regulated as that of the endogenous hamster transcripts, indicating that this construct contained all cis elements necessary for regulation. Expression of the AS cDNA in tsll cells under the control of a constitutive foreign promoter was also regulated by the concentration of asparagine, and this regulation required translation. When we introduced by mutagenesis a number of stop codons in the AS cDNA, the mutant mRNAs with short open reading frames were expressed at low levels that were not increased by asparagine deprivation. Inhibition of protein and RNA synthesis also prevented down-regulation of AS mRNA levels by high concentrations of asparagine. In a parallel series of experiments, we showed that an AS DNA fragment including the promoter and first exon can also regulate RNA expression in response to asparagne concentration. Furthermore, similar increases in the levels of AS RNAs are produced not only by asparagine deprivation in tsll cells but also by deprivation of human and wild-type BHK cells of leucine, isoleucine, or glutamine. Thus, regulation of AS gene expression is a response to amino acid starvation through mechanisms which appear to involve both changes in RNA stability and changes in the rate of transcription initiation or elongation.
We have previously shown that asparagine synthetase (AS) mRNA expression can be dramatically upregulated by asparagine deprivation in tsll cells, mutants of BHK hamster cells which encode a temperaturesensitive AS. The expression of AS mRNA was also induced upon starvation for one of several essential amino acids in HeLa cells. We also showed that regulation of AS mRNA expression by amino acid concentration has both transcriptional and posttranscriptional components. Here we report the analysis of the elements in the human AS promoter region important for its basal activity and activation by amino acid starvation. Our results indicate that a DNA fragment spanning from nucleotides -164 to +44 of the AS promoter is sufficient for uninduced and induced gene expression. Mutations in a region located 15 to 30 bp downstream from the major transcription start site that shows good homology to a sequence in the first exon of c-fos implicated as a negative regulatory element resulted in a significant increase in basal gene expression but did not affect regulation. Interestingly, this region binds single-stranded-DNA-binding proteins that are specific for the AS coding strand. Mutations in either one of two putative binding sites for transcription factor Spl, in a region of approximately 60 bp where many minor RNA start sites are located, or at the major transcription start site decreased promoter activity, but significant induction by amino acid starvation was still observed. Strikingly, mutations centered around nucleotide -68 not only decreased the basal promoter activity but also abolished amino acid regulation. This DNA region contains the sequence 5'-CATGATG-3', which we call the amino acid response element (AARE), that can bind a factor(s) present in HeLa cells nuclear extracts that is not capable of binding to an AS promoter with mutations or deletions of the AARE. This finding is in line with the hypothesis that transcriptional activation of AS gene expression is mediated through the binding of a positive regulatory element. We did not detect changes in the level of binding of this factor to the AARE by using nuclear extracts from HeLa cells grown under starved conditions, suggesting that activation of this factor(s) results from posttranslational modification or complexing with other proteins that do not affect its DNAbinding properties.The signals regulating gene expression in prokaryotes and lower eukaryotes in response to nutrient variation are well known and characterized; it is well established that these organisms are quickly able to adjust to variations in nutrient supply and to intracellular amino acid levels by altering their patterns of gene expression. Much less is known about the response of higher eukaryotes to nutrient deprivation (9).We have previously shown (13,15)
The human tsJl gene was isolated on the basis of its ability to complement the mutation of the BHK cell cycle tsil mutant, which is blocked in GI at the nonpermissive temperature. This gene has now been identified as the structural gene for asparagine synthetase (AS) on the bases of sequence homology and the ability of exogenous asparagine to bypass the tsil block. The tsll (AS) mRNA has a size of about 2 kilobases and is induced in mid-GI phase in human, mouse, and hamster cell lines. We have studied the organization and regulation of expression of the tsil gene. The human tsil gene consists of 13 exons (the first two noncoding) interspersed in a region of about 21 kilobases of DNA. Transient expression assays using the bacterial chloramphenicol acetyltransferase reporter gene identified two separate promoters: one (ts]l P1) contained in a 280-base-pair region upstream of the first exon and the other (tsil P2) contained in the first intron. tsil P1 produced about sixfold more chloramphenicol acetyltransferase activity than did tsil P2 and had features of the promoters of housekeeping genes: high G+C content, multiple transcription start sites, absence of a TATA box, and presence of putative Spl binding sites. ts]l P2 contained a TATA sequence and other elements characteristic of a promoter, but so far we have no evidence of its physiological utilization. The tsll gene was overexpressed in tsil cells exposed to the nonpermissive temperature. Addition of asparagine to the culture medium led to a drastic decrease in mRNA levels and prevented Gl induction in serum-stimulated cells, which indicated that expression of the AS gene is regulated by a mechanism of end product inhibition.Molecular analysis of growth regulation in mammalian cells has been carried out by several approaches, including characterization of the mode of action of growth factors and their receptors (21) and isolation of genes whose expression is specific for growing or growth-arrested cells (1, 10, 25, 36). Another approach is represented by the study of genes mutated in temperature-sensitive (ts) cell cycle mutants, which are blocked in specific phases of the cell cycle at the nonpermissive temperature (16,17,37). We previously reported the isolation and characterization of the human gene complementing the mutation of the GI cell cycle mutant, ts]l, isolated in our laboratory from the BHK-21 Syrian hamster cell line (16). The tsll mRNA is expressed in all human, mouse, and hamster cell lines tested and could encode a protein of 550 amino acids. Its expression is induced in mid-GI phase.We have identified the ts/l-encoded protein as asparagine synthetase (AS) on the bases of sequence homology and the ability of exogenous asparagine to bypass the tslI block. Thus, tsll cells appear to encode a temperature-sensitive AS, and the lack of asparagine leads to a block in mid-Gl phase by a mechanism not yet understood. We also report here the characterization of the genomic structure of the tsl l gene and a study of the regulation of its expression. The ...
While the cytoplasmic phase of the hepadnavirus replication cycle is well understood, very little is known about the nuclear phase. In contrast to retroviruses, proviral integration is not required for hepadnavirus replication; however, some of the viral DNAs in the nucleus are diverted into an integration pathway. Under certain conditions these integrations function as carcinogenic agents. In order to study the integration process, we have utilized LMH-D2 cells, which replicate wild-type duck hepatitis B virus (DHBV), to develop the first protocol to detect and characterize integrations of DHBV originating from episomal viral DNAs. Contrary to expectations, our results showed that stable new integrations are readily detectable in subclones of LMH-D2 cells. Complete characterization of one integration revealed a single-genome-length integrant with the structure of double-stranded linear (DSL) DHBV DNAs which are produced by in situ priming during viral replication. The integration contained a terminal redundancy of 6 bp from the r region of the virus DNA minus strand as well as a direct repeat of 70 bp of cellular DNA. On the basis of the structure of the integrant and the cellular DNA target site, we propose a molecular model for the integration mechanism that has some similarities to that of retroviruses. Identification of DSL hepadnavirus DNA integration suggests the possibility that modified DSL viral DNAs may be the precursors to a class of simple, unrearranged hepadnavirus integrations.
ts11 is a temperature-sensitive (ts) mutant isolated from the BHK-21 Syrian hamster cell line that is blocked in the G1 phase of the cell cycle at the non-permissive temperature (39.5 degrees C). We previously showed that the human gene encoding asparagine synthetase (AS) transformed ts11 cells to a ts+ phenotype and that ts11 cells were auxotrophic for asparagine at 39.5 degrees C. We show here that ts11 cells exhibit a ts phenotype for AS activity, and that the ts11 AS was much heat-labile than the wt enzyme. We have isolated AS cDNAs from wt BHK and ts11 cells and found that wt, but not ts11 AS cDNAs were capable of transformation. The deduced amino acid sequence of Syrian hamster AS showed 95% identity to the human protein as well as the same number of residues. The inability of the ts11 AS cDNAs to transform was due to a single base change, a C to T transition, that would result in the substitution of leucine with phenylalanine at a residue located in the C-terminal fourth of the enzyme. Thus the ts11 mutation identifies a mutated, thermolabile AS.
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