DAL5 is a constitutively expressed allantoin system gene whose product is required for allantoate transport. Its simple pattern of expression prompted us to use this gene for identifying the element(s) that mediates transcriptional activation of allantoin system genes. Deletion analysis of the DAL5 5'-flanking sequences resulted in identification of two small regions required for DAL5 expression. Analysis of these two regions with synthetic oligonucleotides localized the sequences supporting transcriptional activation to two DNA fragments of 10 to 12 base pairs, each containing one copy of the pentanucleotide 5'-GATAA-3'. The 5'-flanking region of DAL5 contained eight copies of this sequence. Synthetic constructions containing single copies of these fragments were unable to support transcriptional activation, while those containing two or more copies supported high-level activation. The 5'-GATAA-3' sequence was also found beneath the footprint of a DNA-binding protein. These observations are consistent with the suggestion that DNA fragments containing the sequence 5'-GATAA-3' play an important role in DAL5 gene expression, probably representing a portion of the binding site for a transcriptional activation factor.

This report describes the isolation of the genes encoding allantoicase (DAL2) and ureidoglycolate hydrolase (DAL3), which are components of the large DAL gene cluster on the right arm of chromosome IX of Saccharomyces cerevisiae. During this work a new gene (DAL7) was identified and found to be regulated in the manner expected for an allantoin pathway gene. Its expression was (i) induced by allophanate, (ii) sensitive to nitrogen catabolite repression, and (iii) responsive to mutation of the DAL80 and DAL8J loci, which have previously been shown to regulate the allantoin degradation system. Hybridization probes generated from these cloned genes were used to analyze expression of the allantoin pathway genes in wild-type and mutant cells grown under a variety of physiological conditions. When comparison was possible, the patterns of mRNA and enzyme levels observed in various strains and physiological conditions were very similar, suggesting that the system is predominantly regulated at the level of gene expression. Although all of the genes seem to be controlled by a common mechanism, their detailed patterns of expression were, at the same time, highly individual and diverse.

We demonstrate that the DALS gene, encoding a necessary component of the allantoate transport system, is constitutively expressed in Saccharomyces cerevisiae. Its relatively high basal level of expression did not increase further upon addition of allantoin pathway intermediates. However, steady-state DALS mRNA levels dropped precipitously when a repressive nitrogen source was provided. These control characteristics of DAL5 expression make this gene a good model with which to unravel the mechanism of nitrogen catabolite repression. Its particular advantage relative to other potentially useful genes derives from its lack of control by induction and hence the complicating effects of inducer exclusion.

The DUR1,2 gene from Saccharomyces cerevisiae has been isolated on recombinant plasmids along with all DNA between the DURI,2 and MET8 loci. DUR1,2 was found to encode a 5.7-kilobase transcript, which is consistent with our earlier suggestion that the DURi and DUR2 loci are two domains of a single multifunctional gene. Steady-state levels of the DUR1,2 transcript responded to induction and nitrogen catabolite repression in the same way as urea amidolyase activity. da)81 mutants (grown with inducer) contained barely detectable amounts of DURI,2 RNA, whereas dal80 mutants (grown without inducer) contained the same amount as a wild-type induced culture. These observations support our earlier hypothesis that DUR1,2 is transcriptionally regulated, with control being mediated by the DAL80 and DAL8I gene products. We cloned the DUR1,2_Oh mutation and found it to be a Ty insertion near sequences required for complementation of durl,2 mutations.The ROAM phenotype of the DUR1,2_Oh mutation is sharply different from that of cis-dominant, DUR80 mutations, which enhance DUR1,2 expression but do not affect the normal control pattern of the gene. There is evidence that DUR80 mutations may also be Ty insertions, which generate phenotypes that are different from those in DUR1,2-Oh mutations.An appreciation for the molecular mechanisms involved in control and integration of procaryotic metabolic pathways has been gained by studying regulons with widely differing physiological functions (28, 31). Similar information is now beginning to accumulate for eucaryotic systems (42). Nitrogen catabolic systems are particularly useful for such investigations, because most are subject to multiple layers of regulation. Genes encoding the allantoin-degradative system in Saccharomyces cerevisiae, for example, respond to both induction and nitrogen catabolite repression (15,25,38 its 3-min half-life after the addition of asparagine to the medium (12). In contrast, enzyme activity is not lost after the addition of a repressive nitrogen source, but continued enzyme synthesis ceases (12). The lack of enzyme induction does not result from inducer exclusion, because daI80 mutants, which do not require the presence of an inducer for enzyme production, are similarly devoid of allantoindegrading enzymes when grown in glucose-asparagine medium (8).Earlier kinetic studies are consistent with the hypothesis that both induction and nitrogen catabolite repression are exerted at gene expression (1-4, 16, 24-26

We determined the nucleotide sequence of the DAL5 gene, which encodes a component of the allantoate transport system. Translation of the sequence revealed that the DALS gene product is highly hydrophobic. It possesses an alternating motif of hydrophilic sequences that can potentially be folded into alpha-helices and hydrophobic sequences that can potentially be folded into beta-pleated sheets. These are the expected characteristics of an integral membrane protein, which correlate well with DALS gene function. Si protection fragments generated by DAL5 transcripts exhibited high heterogeneity over a 30-base-pair range. This pattern of fragnments was not affected by growth conditions of the celis or the conditions of the assay.Allantoin degradation in Saccharomyces cerevisiae is accomplished by the products of an exquisitely regulated set of genes. Four of the genes are highly inducible (DAL4, DAL7, DURI,2, and DUR3), two are partially inducible (DAL] and DAL2), and the remaining two (DAL3 and DALS) are expressed constitutively (8,9,12,17,22,26,28) (i.e., their expression fails to respond to the addition of allantoinpathway-related metabolites to the culture medium). Allophanate and oxalurate (OXLU) have been shown to serve as the native and gratuitous inducers, respectively (24). Transcripts from all eight genes are markedly overproduced in dal80 mutants (4, 12, 22, 28; R. Buckholz and T. G. Cooper, unpublished observations). The highly pleiotropic phenotype of dat80 mutants suggests that all eight pathway genes are regulated in parallel and therefore likely contain recognizable sequences associated with this control in their 5'-flanking regions. The broad range of inducibility, however, is less easy to explain on the molecular level. The important question here is, how does one account for constitutive, partially inducible, and highly inducible expression observed for various allantoin system genes? In our view, the most effective rmeans of addressing this question is to begin with a gene that exhibits the simplest pattern of metabolic regulation and to attempt to explain its activation and regulation. Once a simple case is understood in detail, we will be better able to understand the pathway genes that exhibit more complicated patterns of regulation.The l)AL5 gene, which encodes a required component of the allantoate transport system, is one of two allantoin pathway genes that are expressed constitutively (5, 22). Its availability in cloned form (22) and the simplicity of its physiological regulation make it a good candidate for use in identifying sequences responsible for allantoin system activation.A second reason for determining the structure of this gene relates to its function. The required participation of its product in allantoate transport suggests that it potentially encodes either a ligand-binding protein secreted into the periplasm or an integral memnbrane protein which participates in some unknown manner in the translocation of ligand across the membrane. Some insight into these possibilities may be...