In Saccharomyces cerevisiae, Expression of Arginine Catabolic Genes CAR1 and CAR2 in Response to Exogenous Nitrogen Availability Is Mediated by the Ume6 (CargRI)-Sin3 (CargRII)-Rpd3 (CargRIII) Complex

ABSTRACT The products of three genes named CARGRI,CARGRII, and CARGRIII were shown to repress the expression of CAR1 and CAR2 genes, involved in arginine catabolism. CARGRI is identical toUME6 and encodes a regulator of early meiotic genes. In this work we identify CARGRII as SIN3 andCARGRIII as RPD3. The associated gene products are components of a high-molecular-weight complex with histone deacetylase activity and are recruited by Ume6 to promoters containing a URS1 sequence. Sap30, another component of this complex, is also required to repress CAR1 expression. This histone deacetylase complex prevents the synthesis of the two arginine catabolic enzymes, arginase (CAR1) and ornithine transaminase (CAR2), as long as exogenous nitrogen is available. Upon nitrogen depletion, repression at URS1 is released and Ume6 interacts with ArgRI and ArgRII, two proteins involved in arginine-dependent activation of CAR1 and CAR2, leading to high levels of the two catabolic enzymes despite a low cytosolic arginine pool. Our data also show that the deletion of theUME6 gene impairs cell growth more strongly than the deletion of the SIN3 or RPD3 gene, especially in the Σ1278b background.

Synergistic Operation of the CAR2(Ornithine Transaminase) Promoter Elements in Saccharomyces cerevisiae

ABSTRACT Dal82p binds to the UIS ALL sites of allophanate-induced genes of the allantoin-degradative pathway and functions synergistically with the GATA family Gln3p and Gat1p transcriptional activators that are responsible for nitrogen catabolite repression-sensitive gene expression. CAR2, which encodes the arginine-degradative enzyme ornithine transaminase, is not nitrogen catabolite repression sensitive, but its expression can be modestly induced by the allantoin pathway inducer. The dominant activators ofCAR2 transcription have been thought to be the ArgR and Mcm1 factors, which mediate arginine-dependent induction. These observations prompted us to investigate the structure of theCAR2 promoter with the objectives of determining whether other transcription factors were required for CAR2expression and, if so, of ascertaining their relative contributions toCAR2’s expression and control. We show that Rap1p binds upstream of CAR2 and plays a central role in its induced expression irrespective of whether the inducer is arginine or the allantoin pathway inducer analogue oxalurate (OXLU). Our data also explain the early report that ornithine transaminase production is induced when cells are grown with urea. OXLU induction derives from the Dal82p binding site, which is immediately downstream of the Rap1p site, and Dal82p functions synergistically with Rap1p. This synergism is unlike all other known instances of Dal82p synergism, namely, that with the GATA family transcription activators Gln3p and Gat1p, which occurs only in the presence of an inducer. The observations reported suggest that CAR2 gene expression results from strong constitutive transcriptional activation mediated by Rap1p and Dal82p being balanced by the down regulation of an equally strong transcriptional repressor, Ume6p. This balance is then tipped in the direction of expression by the presence of the inducer. The formal structure of theCAR2 promoter and its operation closely follow the model proposed for CAR1.

Transgenic mice over-producing putrescine in their tissues do not convert the diamine into higher polyamines

We recently described a transgenic mouse line over-expressing the human ornithine decarboxylase gene virtually in all tissues. Despite strikingly elevated tissue putrescine concentrations, no or minimal changes were found in the levels of the higher polyamines spermidine and spermine. We have now extended these studies by further increasing tissue putrescine with the aid of 5-fluoromethylornithine, a specific inhibitor of ornithine transaminase and hence the catabolism of L-ornithine. As a result of the treatment with the latter drug, the concentration of putrescine was further increased by a factor of 2-3 without any changes in the concentrations of spermidine and spermine. In the testis of transgenic mice treated with 5-fluoromethylornithine, the concentration of putrescine was nearly 60 times that in non-transgenic untreated animals, yet the concentration of spermidine was only 1.5-fold higher. A similar small increase in brain spermidine was accompanied by a 40-fold elevation in the concentration of putrescine. The apparent blockade between putrescine and spermidine was in all likelihood not attributable to an inhibition of S-adenosylmethionine decarboxylase, the rate-controlling enzyme in the biosynthesis of spermidine and spermine. Our results are more compatible with the view that in non-dividing adult tissues putrescine is sequestered through some unknown mechanisms in a way that makes it unavailable for the synthesis of the higher polyamines.

MATING-TYPE EFFECT ON CIS MUTATIONS LEADING TO CONSTITUTIVITY OF ORNITHINE TRANSAMINASE IN DIPLOID CELLS OF SACCHAROMYCES CEREVISIAE

ABSTRACT Cis-acting regulatory mutations have been isolated that affect L-ornithine transaminase (OTAse), an enzyme catalyzing the second step of arginine breakdown in yeast. These mutations lead to constitutive synthesis of OTAse at various levels. Two different types of mutations have been recovered, both of which are tightly linked to the structural gene (cargB) for this enzyme. One type behaves as a classical operator-constitutive mutation similar to the cargB+O-—l mutation previously described (DUBOIS et al. 1978) .—The second type is peculiar in two respects : the higher level of constitutive OTAse synthesis and the expression of constitutivity in diploid cells. These mutations are designated curgB+Oh. They behave as usual operator-constitutive mutations in diploid strains homozygous for mating type (a/a or α/α), but the constitutivity is strongly reduced in a/α diploid cells.