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Allantoin uptake in both growing and resting cultures of Saccharomyces cerevisiae occurs by a low-Km (ca. 15 micrometer) transport system that uses energy that is likely generated in the cytoplasm. This conclusion was based on the observation that transport did not occur in the absence of glucose or the presence of dinitrophenol, carbonyl cyanide-m-chloro-phenyl hydrazine, fluoride, or arsenate ions. Normal uptake was observed, however, in the presence of cyanide. The rate of accumulation was maximal at pH 5.2. In contrast to the urea transport system, allantoin uptake appeared to be unidirectional. Preloaded, radioactive allantoin was not lost from cells suspended in allantoin-free buffer and did not exchange with exogenously added, nonradioactive allantoin. Treatment of preloaded cells with nystatin, however, released the accumulated radioactivity. Allantoin accumulated within cells was isolated and shown to be chemically unaltered.

Arginase, the enzyme responsible for arginine degradation in Saccharomyces cerevisiae, is an inducible protein whose inhibition of ornithine carbamoyl-transferase has been studied extensively. Mutant strains defective in the normal regulation of arginase production have also been isolated. However, in spite of these studies, the macromolecular biosynthetic events involved in production of arginase remain obscure. We have, therefore, studied the requirements of arginase induction. We observed that: (i) 4 min elapsed between the addition of inducer (homoarginine) and the appearance of arginase activity at 30 degrees C; (ii) induction required ribonucleic acid synthesis and a functional rna1 gene product; and (iii) production of arginase-specific synthetic capacity occurred in the absence of protein synthesis but could be expressed only when protein synthesis was not inhibited. Termination of induction by inducer removal, addition of the ribonucleic acid synthesis inhibitor lomofungin, or resuspension of a culture of organisms containing temperature-sensitive rna1 gene products in a medium at 35 degrees C resulted in loss of ability for continued arginase synthesis with half-lives of 5.5, 3.8, and 4.5 min, respectively. These and other recently published data suggest that a variety of inducible or repressible proteins responding rapidly to the environment may be derived from labile synthetic capacities, whereas constitutively produced proteins needed continuously throughout the cell cycle may be derived from synthetic capacities that are significantly more stable.

Addition of urea to an uninduced culture of Saccharomyces at 22 C results in appearance of allophanate hydrolase activity after a lag of 12 min. We have previously demonstrated that both ribonucleic acid (RNA) and protein synthesis are needed for this induction to occur. To elucidate the time intervals occupied by known processes involved in induction, temperature-sensitive mutants defective in messenger RNA transport from nucleus to cytoplasm (rna1) and in protein synthesis initiation (prt1) were employed along with an RNA polymerase inhibitor in experiments that measure cumulative synthetic capacity to produce allophanate hydrolase. These measurements identify the time within the lag period at which each of the above processes is completed. We observed that RNA synthesis, rna1 gene product function, and protein synthesis initiation are completed at 1 to 1.5, 4, and 9 to 10 min, respectively.

Experiments characterizing the urea active transport system in Saccharomyces cerevisiae indicate that (i) formamide and acetamide are strong competitive inhibitors of urea accumulation, (ii) uptake is maximal at pH 3.3 and is 80% inhibited at pH 6.0, and (iii) adenosine 5'-triphosphate generated by glycolysis in conjunction with formation of an ion gradient is likely the driving force behind urea transport. Mutant strains were isolated that are unable to accumulate urea at external concentrations of 0.25 mM. These strains also exhibit a depressed growth rate on 10 mM urea, indicating existence of a relationship between the active transport and facilitated diffusion modes of urea uptake.

Urea transport in Saccharomyces cerevisiae occurs by two pathways. The first mode of uptake is via an active transport system which: (i) has an apparent Km value of 14 muM, (ii) is absolutely dependent upon energy metabolism, (iii) requires pre-growth of the cultures in the presence of oxaluric acid, gratuitous inducer of the allantoin degradative enzymes, and (iv) is sensitive to nitrogen repression. The second mode of uptake which occurs at external urea concentrations in excess of 0.5 mM is via either passive or facilitated diffusion.

Induced expression of the allantoin (DAL) catabolic genes in Saccharomyces cerevisiae has been suggested to be mediated by interaction of three different types of promoter elements. First is an inducer-independent upstream activation sequence, UASNTR, whose operation is sensitive to nitrogen catabolite repression. The GLN3 product is required for UASNTR-mediated transcriptional activation. This site consists of two separated elements, each of which has a GATAA sequence at its core. Response of the DAL genes to inducer is mediated by a second type of cis-acting element, DAL UIS. The DAL82 and DAL81 genes are required for response to inducer; DAL82 protein is the UIS-binding protein. When only the UASNTR and UIS elements are present, DAL gene expression occurs at high levels in the absence of inducer. We, therefore, hypothesized that a third element, an upstream repressor sequence (URS) mediates maintenance of DAL gene expression at a low level when inducer is absent. Since the DAL and UGA genes are overexpressed and largely inducer independent in dal80 deletion mutants, we have suggested DAL80 protein negatively regulates a wide spectrum of nitrogen-catabolic gene expression, likely in conjunction with a URS element. Here we show that DAL80 protein binds to DAL3 and UGA4 upstream DNA sequences, designated URSGATA, consisting of two GATAA-containing sites separated by at least 15 bp. The preferred orientation of the sites is tail to tail, but reasonable binding activity is also observed with a head-to-tail configuration. URSGATA elements contain the sequence GATAA at their core and hence share sequence homology with UASNTR elements.

Regulated expression of the inducible arginase (CAR1) gene of Saccharomyces cerevisiae has been shown to require three upstream activation sequences (UASs) and an upstream repression sequence, URS1. Two of the UAS elements, UASC1 and UASC2, operate in an inducer-independent manner, while the third, UASI, is inducer dependent. UASC1 and UASC2 were previously shown to contain ABF-1 binding sites that were required for normal transcription. In this work, we demonstrate that UASC1 and UASC2 also contain two and three sites, respectively, that are able to bind RAP1 protein. RAP1 binding to these sites, however, is significantly weaker than that to sites in TEF2 and HMRE. The effects of mutating the sites individually or in combination suggest that at least three of them, two in UASC1 and one in UASC2, probably participate in CAR1 expression.

Expression of allantoin pathway genes is subject to induction and nitrogen catabolite repression. Two classes of cis-dominant mutations (DUR80 and DUR1,2-Oh) result in overproduction of DUR1,2 mRNA. In DUR80 mutants, DUR1,2 expression remained inducible, nitrogen catabolite repression sensitive, and unresponsive to cell ploidy, i.e., overproduction was superimposed on normal gene regulation. DUR1,2-Oh mutations, in contrast, generated a pattern of DUR1,2 expression similar to that often reported when a Ty element inserts upstream of a gene, the ROAM phenotype. We analyzed four independent DUR80 and DUR1,2-Oh alleles. The DUR1,2-Oh mutation was, as expected, a Ty insertion at -445 3' of the native DUR1,2 upstream activation sequences (UASs). All three DUR80 alleles were also Ty insertions between -644 and -653 immediately 5' of the native DUR1,2 USASs. We suggest that the difference in DUR1,2-Oh and DUR80 phenotypes depends on whether the native cis-acting elements and transcription factors associated with them can operate. If they can, enhancement of normally regulated DUR1,2 expression is observed. This is a novel phenotype for Ty insertions. If the native DUR1,2 cis-acting elements are not present, the case when Ty insertion occurs 3' of them, a ROAM phenotype is generated. Nitrogen-regulated upstream activation sequence (UASNTR)-homologous sequences present in the Ty delta elements rather than cis-acting elements required for Ty transcription are the most likely candidates to serve as the cis-acting elements mediating the DUR80 phenotype.

We have determined the nucleotide sequence of the yeast CAR1 gene along with its 5' and 3' flanking sequences. The structural portion of this gene encodes a protein of 333 amino acid residues with a calculated Mr value of 35,616. The transcripts produced from this gene are modestly heterogeneous at their 5' and 3' termini. Most 5' termini map to a position 42 to 49 base pairs upstream from the ATG at the beginning of the open reading frame. The 3' termini map to a position 108 to 127 base pairs downstream of the amber codon which terminates the open reading frame. A variety of potentially significant sequences were identified in the regions flanking the structural portion of the gene.

Saturation mutagenesis of the UASNTR element responsible for GLN3-dependent, nitrogen catabolite repression-sensitive transcriptional activation of allantoin pathway genes in yeast cells identified the dodecanucleotide sequence 5'-TTNCTGATAAGG-3' as the minimum required for UAS activity. There was significant flexibility in mutant sequences capable of supporting UAS activity, which correlates well with the high variation in UASNTR homologous sequences reported to be upstream of the DAL and DUR genes. Three of nine UASNTR-like sequences 5' of the DAL5 gene supported high-level transcriptional activation. The others, which contained nonpermissive substitutions, were not active.

The DUR3 gene, which encodes a component required for active transport of urea in Saccharomyces cerevisiae, has been isolated, and its sequence has been determined. The deduced DUR3 protein profile possesses alternating hydrophobic and hydrophilic regions characteristics of integral membrane proteins. Strong negative complementation observed during genetic analysis of the DUR3 locus suggests that the DUR3 product may polymerize to carry out its physiological function. Expression of DUR3 is regulated in a manner similar to that of other genes in the allantoin pathway. High-level expression is inducer dependent, requiring functional DAL81 and DAL82 genes. Maintenance of DUR3 mRNA at uninduced, nonrepressed basal levels requires the negatively acting DAL80 gene product. DUR3 expression is highly sensitive to nitrogen catabolite repression and also has a partial requirement for the GLN3 product.

We demonstrate that expression of the UGA1, CAN1, GAP1, PUT1, PUT2, PUT4, and DAL4 genes is sensitive to nitrogen catabolite repression. The expression of all these genes, with the exception of UGA1 and PUT2, also required a functional GLN3 protein. In addition, GLN3 protein was required for expression of the DAL1, DAL2, DAL7, GDH1, and GDH2 genes. The UGA1, CAN1, GAP1, and DAL4 genes markedly increased their expression when the DAL80 locus, encoding a negative regulatory element, was disrupted. Expression of the GDH1, PUT1, PUT2, and PUT4 genes also responded to DAL80 disruption, but much more modestly. Expression of GLN1 and GDH2 exhibited parallel responses to the provision of asparagine and glutamine as nitrogen sources but did not follow the regulatory responses noted above for the nitrogen catabolic genes such as DAL5. Steady-state mRNA levels of both genes did not significantly decrease when glutamine was provided as nitrogen source but were lowered by the provision of asparagine. They also did not respond to disruption of DAL80.

Arginase (CAR1) gene expression in Saccharomyces cerevisiae is induced by arginine. The 5' regulatory region of CAR1 contains four separable regulatory elements--two inducer-independent upstream activation sequences (UASs) (UASC1 and UASC2), an inducer-dependent UAS (UASI), and an upstream repression sequence (URS1) which negatively regulates CAR1 and many other yeast genes. Here we demonstrate that three homologous DNA sequences originally reported to be present in the inducer-responsive UASI are in fact three exchangeable elements (UASI-A, UASI-B, and UASI-C). Although two of these elements, either the same or different ones, are required for transcriptional activation to occur, all three are required for maximal levels of induction. The elements operate in all orientations relative to one another and to the TATA sequence. All three UASI elements bind protein(s); protein binding does not require arginine or overproduction of any of the putative arginine pathway regulatory proteins. The UASI-protein complex was also observed even when extracts were derived from arg80/argRI or arg81/argRII deletion mutants. Similar sequences situated upstream of ARG5,6 and ARG3 and reported to negatively regulate their expression are able to functionally substitute for the CAR1 UASI elements and mediate reporter gene expression.

Accumulation of intracellular allantoin and allantoate is mediated by two distinct active transport systems in Saccharomyces cerevisiae. Allantoin transport (DAL4 gene) is inducible, while allantoate uptake is constitutive (it occurs at full levels in the absence of any allantoate-related compounds from the culture medium). Both systems appear to be sensitive to nitrogen catabolite repression, feedback inhibition, and trans-inhibition. Mutants (dal5) that lack allantoate transport have been isolated. These strains also exhibit a 60% loss of allantoin transport capability. Conversely, dal4 mutants previously described are unable to transport allantoin and exhibit a 50% loss of allantoate transport. We interpret the pleiotropic behavior of the dal4 and dal5 mutations as deriving from a functional interaction between elements of the two transport systems.

Expression of the Saccharomyces cerevisiae arginase (CAR1) gene is regulated by induction and nitrogen catabolite repression (NCR). Arginine was demonstrated to be the native inducer. CAR1 sensitivity to NCR has long been accepted to be accomplished through a negative control mechanism, and cis-acting sites for it have been hypothesized. In search of this negatively acting site, we discovered that CAR1 sensitivity to NCR derives from regulated inducer (arginine) exclusion. The route of catabolic entry of arginine into the cell, the general amino acid permease (GAP1), is sensitive to NCR. However, CAR1 expression in the presence of sufficient intracellular arginine is NCR insensitive.

Expression of the DAL2, DAL4, DAL7, DUR1,2, and DUR3 genes in Saccharomyces cerevisiae is induced by the presence of allophanate, the last intermediate of the allantoin degradative pathway. Analysis of the DAL7 5'-flanking region identified an element, designated the DAL upstream induction sequence (DAL UIS), required for response to inducer. The operation of this cis-acting element requires functional DAL81 and DAL82 gene products. We determined the DAL UIS structure by using saturation mutagenesis. A specific dodecanucleotide sequence is the minimum required for response of reporter gene transcription to inducer. There are two copies of the sequence in the 5'-flanking region of the DAL7 gene. There are one or more copies of the sequence upstream of each allantoin pathway gene that responds to inducer. The sequence is also found 5' of the allophanate-inducible CAR2 gene as well. No such sequences were detected upstream of allantoin pathway genes that do not respond to the presence of inducer. We also demonstrated that the presence of a UIS element adjacent to the nitrogen-regulated upstream activation sequence significantly enhances its operation.

DNA fragments previously shown to be required for expression of the CAR1 (arginase) gene in Saccharomyces cerevisiae and to support transcriptional activation of a reporter gene in a heterologous expression vector were shown to bind purified regulatory protein ABF-1. Two ABF-1 sites were identified in the CAR1 upstream region, one to which ABF-1 protein bound with high affinity and a second to which it bound much less avidly. The higher-affinity ABF-1 binding site upstream of CAR1 was an effective competitor of the HMRE, ARS1 B domain, and COR2-GFI binding sequences for protein binding. Point mutations in the CAR1 high-affinity ABF-1 binding site resulted in a 12-fold loss of transcriptional activation of a reporter gene compared with the wild-type CAR1 DNA fragment. These data are consistent with the suggestion that ABF-1 protein is one of the transcription factors involved in expression of the CAR1 gene.

Several highly inducible enzyme activities are required for the degradation of allantoin in Saccharomyces cerevisiae. Induction of these pathway enzymes has been shown to be regulated at transcription, and response to inducer is lost in dal81 and dal82/durM mutants. The similar phenotypes generated by dal81 and dal82 mutations prompted the question of whether they were allelic. We demonstrated that the DAL81 and DAL82 loci are distinct, unlinked genes situated on chromosomes IX and XIV. DAL82 gene expression did not respond to induction by the allantoin pathway inducer or to nitrogen catabolite repression. Expression was also not significantly affected by mutation of the dal80 locus. From the nucleotide sequence of the DAL82 gene, we deduced that it encodes a protein with a mass of 29,079 Da that may possess the structural motifs expected of a regulatory protein. This protein was shown to be required for the function mediated by the cis-acting upstream induction sequence situated in the 5'-flanking regions of the inducible allantoin pathway genes.

We show that mutation at the GLN3 locus results in decreased steady-state levels of DAL7, DUR1,2, CAR1, and URA3 mRNAs derived from cultures grown in the presence of inducer. Basal levels of these RNA species, however, were not significantly affected by a gln3 mutation. The GLN3 product appears to affect gene expression in two ways. The pleiotropic requirement of GLN3 for induced gene expression probably derives from the need of the GLN3 product for inducer uptake into the cell and its loss in gln3 mutants. We also demonstrate that transcriptional activation, mediated by the DAL5 and DAL7 upstream activation sequences, requires a functional GLN3 gene product. This observation identified transcriptional activation as the most likely point of GLN3 participation in the expression of allantoin system genes.

We demonstrate that the DAL5 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 DAL5 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.

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 DAL5 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 expected characteristics of an integral membrane protein, which correlate well with DAL5 gene function. S1 protection fragments generated by DAL5 transcripts exhibited high heterogeneity over a 30-base-pair range. This pattern of fragments was not affected by growth conditions of the cells or the conditions of the assay.

We show that the allantoin transport system of Saccharomyces cerevisiae responds to two induction systems, one mediated by allophanate or its analog oxalurate and the other mediated by allantoin or its analog hydantoin acetate. The effects of the two inducers were additive in strain M85. Like other allantoin pathway genes, oxalurate-mediated induction of allantoin transport required a functional DAL81 gene product. Hydantoin acetate-mediated induction of the system, on the other hand, occurred normally in dal81 mutants. This suggests that induction was not only mediated by two separate inducers, but also involved different regulatory proteins. Induction is probably a transcriptionally regulated process, because addition of hydantoin acetate or oxalurate to the culture medium increased the steady-state levels of mRNA encoded by a gene required for allantoin transport (DAL4).

Our previous work has shown that both induction, after addition of inducer, and loss of ability to produce allophanate hydrolase, after removal of inducer, proceed more rapidly than expected from the reported half-life of messenger ribonucleic acid in Saccharomyces cerevisiae. As a basis of rectifying these observations, we have characterized induction and repression of allophanate hydrolase synthesis and find that: (i) induction of the hydrolase begins immediately upon addition of inducer, (ii) once induction has been initiated removal of inducer does not result in immediate loss of synthetic capacity, (iii) induction of the capacity to produce hydrolase can occur in the absence of protein synthesis, (iv) the half-life of hydrolase synthetic capacity increases if protein synthesis is inhibited, (v) allophanate hydrolase itself is not degraded upon removal of inducer, and (vi) induction and repression of allophanate hydrolase synthetic capacity likely occurs at the level of transcription.

In contrast to the previously held notion that nitrogen catabolite repression is primarily responsible for the ability of yeast cells to use good nitrogen sources in preference to poor ones, we demonstrate that this ability is probably the result of other control mechanisms, such as metabolite compartmentation. We suggest that nitrogen repression is functionally a long-term adaptation to changes in the nutritional environment of yeast cells.

We have previously shown that allophanate acts as an inducer for five structural genes whose products participate in the degradation of allantoin by Saccharomyces cerevisiae. This observation led us to hypothesize that these genes might be controlled in common and to test the hypothesis by searching for mutants unable to induce production of the allantoin-degrading enzymes. Such mutants have been found. These strains grew poorly when provided with any of the allantoin pathway intermediates, but used other nitrogen sources normally. The mutations carried in these strains were recessive to wild-type alleles and complemented mutations in all known loci associated with the allantoin pathway. The locus containing the most thoroughly studied mutation (dal81-1) was not fund to be tightly linked to any of the allantoin pathway structural genes. The low basal levels of allantoin pathway enzymes observed in Dal81- strains remained the same whether or not the inducer was present in the growth medium. However, the levels of enzyme increased moderately when mutants were grown on poor nitrogen sources. From these observations, we conclude that dal81 mutant strains possess a defect in the induction of enzyme synthesis; enzyme production due to relief of nitrogen catabolite repression, however, appears normal. The observed epistatic relationships of mutations in the DAL80 and DAL81 loci suggest that their products may possess a reasonable degree of functional independence.

Allantoate uptake appears to be mediated by an energy-dependent active transport system with an apparent Michaelis constant of about 50 microM. Cells were able to accumulate allantoate to greater than 3,000 times the extracellular concentration. The rate of accumulation was maximum at pH 5.7 to 5.8. The energy source for allantoate uptake is probably different from that for uptake of the other allantoin pathway intermediates. The latter systems are inhibited by arsenate, fluoride, dinitrophenol, and carboxyl cyanide-m-chlorophenyl hydrazone, whereas allantoate accumulation was sensitive to only dinitrophenol and carboxyl cyanide-m-chlorophenyl hydrazone. Efflux of preloaded allanotate did not occur at detectable levels. However, exchange of intra- and extracellular allantoate was found to occur very slowly. The latter two characteristics are shared with the allantoin uptake system and may result from the sequestering of intracellular allantoate within the cell vacuole. During the course of these studies, we found that, contrary to earlier reports, the reaction catalyzed by allantoinase is freely reversible.

Oxalurate, the gratuitous inducer of the allantoin degradative enzymes, was taken into the cell by an energy-dependent active transport system with an apparent Km of 1.2 mM. Efflux of previously accumulated oxalurate was rapid, with a half-life of about 2 min. The oxalurate uptake system appears to be both constitutively produced and insensitive to nitrogen catabolite repression. The latter observations suggest that failure of oxalurate to bring about induction of allophanate hydrolase in cultures growing under repressive conditions does not result from inducer exclusion, but rather from repression of dur1,2 gene expression.

Saccharomyces cerevisiae responds to deperivation of nutrients by arresting cell division at the unbudded G1 stage. Cells situated outside of G1 at the time of deperivation complete the cell cycle before arresting. This prompted an investigation of the source of nutrients used by these cells to complete division and the mechanisms controlling their availability. We found a close correlation between accumulation of unbudded cells and loss of previously formed allophanate hydrolase activity after nutrient starvation. These losses were not specific to the allantoin, system since they have been observed for a number of other enzymes and also when cellular protein levels were monitored with [3H]leucine. Loss of hydrolase activity was also observed when protein synthesis was inhibited either by addition of inhibitors or loss of the prtl gene product. We found that onset of nutrient starvation brought about release of large quantities of arginine and allantoin normally sequestered in the cell vacuole. Treatment of a cells with alpha-factor resulted in both the release of allantoin and arginine from the cell vacuole and the onset of intracellular protein degradation. These effects were not observed when either alpha cells or a/alpha diploid strains were treated with alpha-factor. These data suggest that release of vacuolar constitutents and protein turnover may be regulated by the G1 arrest signal.

Uninduced cultures of Saccharomyces cerevisiae exhibit high basal levels of allantoinase, allantoicase, and ureidoglycolate hydrolase, the enzymes responsible for degrading allantoin to urea. As a result, these activities increase only 4- to 8-fold upon induction, whereas the urea-degrading enzymes, urea carboxylase and allophanate hydrolase, have very low basal levels and routinely increase 30-fold on induction. Differences in the inducibility of these five enzymes were somewhat surprising because they are all part of the same pathway and have the same inducer, allophanate. Our current studies reconcile these observations. S. cerevisiae normally contained up to 1 mM allantoin sequestered in a cellular organelle, most likely the vacuole. Separation of the large amounts of allantoin and the enzymes that degrade it provide the cell with an efficient nitrogen reserve. On starvation, sequestered allantoin likely becomes accessible to these degradative enzymes. Because they are already present at high levels, the fact that their inducer is considerably removed from the input allantoin is of little consequence. This suggests that at times metabolite compartmentation may play an equal role with enzyme induction in the regulation of allantoin metabolism. Metabolism of arginine, another sequestered metabolite, must be controlled both by induction of arginase and compartmentation because arginine serves both as a reserve nitrogen source and a precursor of protein synthesis. The latter function precludes the existence of high basal levels of arginase.

Allantoin uptake in Saccharomyces cerevisiae is mediated by an energy-dependent, low-Km, active transport system. However, there is at present little information concerning its regulation. In view of this, we investigated the control of alloantoin transport and found that it was regulated quite differently from the other pathway components. Preincubation of appropriate mutant cultures with purified allantoate (commercial preparations contain 17% allantoin), urea, or oxalurate did not significantly increase allantoin uptake. Preincubation with allantoin, however, resulted in a 10- to 15-fold increase in the rate of allantoin accumulation. Two allantoin analogs were also found to elicit dramatic increases in allantoin uptake. Hydantoin and hydantoin acetic acid were able to induce allantoin transport to 63 and 95% of the levels observed with allantoin. Neither of these compounds was able to serve as a sole nitrogen source for S. cerevisiae, and they may be non-metabolizable inducers of the allantoin permease. The rna1 gene product appeared to be required for allantoin permease induction, suggesting that control was exerted at the level of gene expression. In addition, we have shown that allantoin uptake is not unidirectional; efflux merely occurs at a very low rate. Allantoin uptake is also transinhibited by addition of certain amino acids to the culture medium, and several models concerning the operation of such inhibition were discussed.

Ornithine carbamoyltransferase synthesis is subject to two major regulatory systems in Saccharomyces cerevisiae. One system is specific for the arginine biosynthetic enzymes, whereas the other appears to be general, acting on a variety of other amino acid pathways as well. We observed that the synthetic capacity for continued ornithine carbamoyltransferase synthesis had the same short half-life (ca. 5 to 7 min) whether repression of enzyme production was brought about by action of the specific or general control system. We present evidence suggesting that both control systems regulate accumulation or ornithine carbamoyltransferase-specific synthetic capacity, rather than modulating its expression.

We have measured, by ribonucleic acid-deoxyribonucleic acid hybrid formation, the amounts of hut-specific ribonucleic acid contained in extracts of various mutant strains of Salmonella typhimurium. Our data are consistent with a model in which regulation of Hut enzyme production occurs at the level of transcription and support earlier genetic evidence indicating that all of the hut genes are transcribed in the clockwise direction on the S. typhimurium chromosome. These results also suggest that promoter sites of the two hut operons may differ in their ability to initiate transcription.

We have observed that transcription, involved in production of allophanate hydrolase, is completed 2.5 min after the addition of inducer at 15 degrees C. The rna1 gene product must be functional up unti 10 min; protein synthesis is initiated at 20 min and is terminated by 24 min. Two minutes later, active enzyme appears. The results confirm our earlier observations and eliminate any uncertainty that might have clouded identification of the time within the lag period that is occupied by ribonucleic acid synthesis.

The regulatory characteristics exhibited by ureidosuccinate transport in Saccharomyces cerevisiae led us to hypothesize that this biosynthetic intermediate was transported via the degradative allantoate transport system. The hypothesis was verified by the finding that neither dal5 nor urep1 mutant strains could transport allantoate or ureidosuccinate. Mutations in the two loci were tightly linked and failed to complement one another, suggesting that they were allelic. The use of a common transport system for accumulation of both biosynthetic and degradative metabolites explains the paradoxical characteristics observed for control of ureidosuccinate and allantoate transport.

Arginase-minus mutants of Saccharomyces cerevisiae were arrested in growth and accumulated at the unbudded G-1 stage of the cell cycle when starved for nitrogen. If, however, arginine was added to the culture medium at the time of starvation, growth ceased but the cells did not collect at the unbudded G-1 stage. We suggest that arginine addition prevented the cells from collecting at the G-1 stage by starving them for histidine and lysine, thereby inhibiting synthesis of proteins needed to complete the cell cycle.