Serial microanalysis of renal transcriptomes.

Large-scale gene expression studies can now be routinely performed on macroamounts of cells, but it is unclear to which extent current methods are valuable for analyzing complex tissues. In the present study, we used the method of serial analysis of gene expression (SAGE) for quantitative mRNA profiling in the mouse kidney. We first performed SAGE at the whole-kidney level by sequencing 12,000 mRNA tags. Most abundant tags corresponded to transcripts widely distributed or enriched in the predominant kidney epithelial cells (proximal tubular cells), whereas transcripts specific for minor cell types were barely evidenced. To better explore such cells, we set up a SAGE adaptation for downsized extracts, enabling a 1, 000-fold reduction of the amount of starting material. The potential of this approach was evaluated by studying gene expression in microdissected kidney tubules (50,000 cells). Specific gene expression profiles were obtained, and known markers (e.g., uromodulin in the thick ascending limb of Henle's loop and aquaporin-2 in the collecting duct) were found appropriately enriched. In addition, several enriched tags had no databank match, suggesting that they correspond to unknown or poorly characterized transcripts with specific tissue distribution. It is concluded that SAGE adaptation for downsized extracts makes possible large-scale quantitative gene expression measurements in small biological samples and will help to study the tissue expression and function of genes not evidenced with other high-throughput methods.

Rpb4p is necessary for RNA polymerase II activity at high temperature.

Rpb4p and Rpb7p are two subunits of the yeast RNA polymerase II, which form a subcomplex that can dissociate from the enzyme in vitro. Whereas RPB7 is essential, RPB4 is dispensable for cellular viability. However, the rpb4 null mutant is heat-sensitive, and it has been suggested that Rpb4p is an essential component for cellular stress response. To examine this hypothesis, we used two-dimensional gel electrophoresis to analyze the protein expression pattern of the rpb4 null mutant in response to heat shock, oxidative stress, osmotic stress, and in the post-diauxic phase. We show that this mutant is not impaired in stress induced transcriptional activation: the absence of heat shock response of the mutant is due to a general defect in RNA polymerase II activity at high temperature. Under this condition, Rpb4p is necessary to maintain the polymerase activity in vivo. The heat growth defect of the rpb4 null mutant can be partially suppressed by overexpression of RPB7, suggesting that Rpb4p maintains or stabilizes Rpb7p in the RNA polymerase. We also demonstrate that rpb4 null mutant is an appropriate tool to analyze the involvement of transcriptional events in the survival and adaptation to heat shock or other stresses.

Altered balance between matrix gelatinases (MMP-2 and MMP-9) and their tissue inhibitors in human dilated cardiomyopathy: potential role of MMP-9 in myosin-heavy chain degradation.

BACKGROUND: End-stage of human dilated cardiomyopathy (DCM) is characterized by myocyte loss and fibrosis, and associated with ventricular dilatation and reduced cardiac function. Matrix metalloproteinases (MMPs) and their natural tissue inhibitors (TIMPs) have been involved in the myocardial remodeling. AIMS: To evaluate the potential role of matrix gelatinases (MMP-2 and MMP-9) in DCM, the balance between gelatinases and TIMPs and the gelatinase localization were investigated in left free wall ventricles from six normal donors and six patients with DCM at the transplantation time. METHODS: TIMP-(1, 2, 3 and 4) mRNAs were analyzed by quantitative reverse transcription-polymerase chain reaction (RT-PCR). TIMP-1 and -2 protein content was assessed by ELISA. MMP-2 and MMP-9 expression were examined by zymography and immunological techniques. RESULTS: All TIMPs were down-regulated in DCM hearts, especially TIMP-1 (reduced by 80%). Gel zymography revealed similar activity of MMP-2 and MMP-9 in both tissues. By in situ zymography and immunohistochemistry, active and immunoreactive gelatinases were pericardiomyocyte in control hearts and intracardiomyocyte in DCM hearts. Intracellular MMPs were associated with sarcomeric structure in DCM. To estimate a putative role of these gelatinases, several sarcomeric contractile proteins were digested in vitro by purified active MMP-9. Only myosin-heavy chain was cleaved in vitro giving 180-, 120-, 80- and 20-kDa proteolytic fragments. In vivo, two major myosin-heavy chain proteolytic fragments (80 and 20 kDa) were detected by specific monoclonal antibody against myosin-heavy chain in DCM left ventricular homogenates, only. CONCLUSIONS: Taken together, these data highly suggest that MMP-2 and MMP-9 may be involved in the disorganization of the contractile apparatus in DCM hearts.

The H2O2 stimulon in Saccharomyces cerevisiae.

The changes in gene expression underlying the yeast adaptive stress response to H2O2 were analyzed by comparative two-dimensional gel electrophoresis of total cell proteins. The synthesis of at least 115 proteins is stimulated by H2O2, whereas 52 other proteins are repressed by this treatment. We have identified 71 of the stimulated and 44 of the repressed targets. The kinetics and dose-response parameters of the H2O2 genomic response were also analyzed. Identification of these proteins and their mapping into specific cellular processes give a distinct picture of the way in which yeast cells adapt to oxidative stress. As expected, H2O2-responsive targets include an important number of heat shock proteins and proteins with reactive oxygen intermediate scavenging activities. Exposure to H2O2 also results in a slowdown of protein biosynthetic processes and a stimulation of protein degradation pathways. Finally, the most remarkable result inferred from this study is the resetting of carbohydrate metabolism minutes after the exposure to H2O2. Carbohydrate fluxes are redirected to the regeneration of NADPH at the expense of glycolysis. This study represents the first genome-wide characterization of a H2O2-inducible stimulon in a eukaryote.

Angiotensin II potentiates vasopressin-dependent cAMP accumulation in CHO transfected cells. Mechanisms of cross-talk between AT1A and V2 receptors.

The V2 vasopressin and the AT1A angiotensin II receptors are respectively coupled to the adenylyl cyclase and the phosphoinositide pathways. The cross-talk between these two receptors and their transduction pathways were investigated in CHO cells transfected with cDNA of both AT1A and V2 receptors. In these cells, angiotensin II induced an increase in intracellular calcium, and vasopressin a rise in intracellular cAMP accumulation. The simultaneous addition of angiotensin II and vasopressin potentiated the production of cAMP by the V2 receptor. This potentiation was dose-dependent and, at a concentration of 10(-7) M angiotensin II, the accumulation of cAMP was 4-fold greater than that induced by 10(-7) M vasopressin alone. Such cross-talk occurred in the presence and absence of cyclic nucleotide phosphodiesterase inhibitors, indicating that inhibition of phosphodiesterase activity was not the principal cause of potentiation. This was confirmed by the absence of calcium-inhibitable isoforms of phosphodiesterases in CHO cells. The addition of angiotensin II to forskolin, which stimulates the adenylyl cyclase, did not modify the production of cAMP. Phorbol 12-myristate 13-acetate (PMA), an activator of protein kinase C (PKC), partially mimicked, and staurosporine, an inhibitor of PKC, partially inhibited the effect of angiotensin II on vasopressin. Chelation of intracellular calcium with BAPTA-AM markedly reduced the potentiation of V2 receptor by angiotensin II. However, increase in intracellular calcium with thapsigargin did not modify the cAMP accumulation induced by vasopressin. It was concluded that, in CHO cells, activation of the AT1A receptor by angiotensin II potentiates the V2 receptor through activation of protein kinase C in the presence of intracellular calcium at a step located between the receptor and the adenylyl cyclase.

Divergent regulation of 92-kDa gelatinase and TIMP-1 by HBECs in response to IL-1beta and TNF-alpha.

In this study, we addressed the question of whether human bronchial epithelial cells (HBECs) contribute to the regulation of 92-kDa gelatinase activity by secreting tissue inhibitor of metalloproteinase (TIMP)-1. We investigated expression of 92-kDa gelatinase and TIMP-1 in response to lipopolysaccharide (LPS) and to the proinflammatory cytokines interleukin (IL)-1beta and tumor necrosis factor (TNF)-alpha. Confluent HBECs from explants were cultured in plastic dishes coated with type I and III collagen. We demonstrated that TIMP-1 was expressed at both the protein and mRNA levels by primary cultures of HBECs. Gelatin zymography of HBEC-conditioned media showed that exposure of HBECs to LPS, IL-1beta, or TNF-alpha induced a twofold increase in the latent form of 92-kDa gelatinase production, as well as its activation. Also, quantitative reverse transcriptase (RT)-polymerase chain reaction (PCR) demonstrated a twofold increase in the 92-kDa mRNA level in response to both cytokines. In contrast, TIMP-1 production evaluated by immunoblotting was unchanged in the presence of LPS and IL-1beta and was clearly decreased in the presence of TNF-alpha. Quantitative RT-PCR demonstrated that TIMP-1 mRNA levels remained unchanged in response to LPS or IL-1beta but decreased by 70% in the presence of TNF-alpha. All of these results strongly suggest that the control mechanisms regulating the expression of 92-kDa gelatinase and TIMP-1 by HBECs in response to inflammatory stimuli are divergent and result in an imbalance between 92-kDa gelatinase and TIMP-1 in favor of the metalloproteinase. Such an imbalance may contribute significantly to acute airway inflammation.

Expression of matrix metalloproteinase gelatinases A and B by cultured epithelial cells from human bronchial explants.

To investigate the role of human bronchial epithelial cells (HBECs) in the maintenance and remodeling of the extracellular matrix, we evaluated the expression by HBECs of 72- and 92-kDa gelatinases under basal conditions and after exposure to bacterial lipopolysaccharides (LPS). Confluent HBECs from explants were cultured in plastic dishes coated with type I and III collagens. Gelatin zymography of HBEC-conditioned media showed constitutive major 92-kDa and minor 72-kDa gelatinases recognized by specific human antibodies and totally inhibited by the metalloproteinase inhibitor EDTA. The identification of the two matrix metalloproteinases was confirmed by quantitative reverse transcription-polymerase chain reaction. Identical patterns of gelatinase expression were observed with repetitive primary cultures issued from the same explants. Zymography showed that exposure of HBECs to LPS induced 2- and 20-fold increases in 92-kDa gelatinase production and activation, respectively, as well as a smaller increase in activated 68-kDa gelatinase. With [3H]gelatin substrate, elevated metallogelatinolytic activity (138 microgram of hydrolyzed gelatin/48 h/10(6) cells) was also observed, whereas no activity was detected in the absence of LPS. A human epithelial cell line (16HBE14o-) exhibited the same basal profile of gelatinase activity, but this profile remained unchanged after exposure to LPS. Quantitative reverse transcription-polymerase chain reaction demonstrated only minimal changes in 92-kDa mRNA levels in response to LPS, but the half-life of 92-kDa gelatinase mRNA was increased with exposure to LPS. In contrast, concomitant slight increases in 72-kDa gelatinase protein and mRNA were found, suggesting that the control mechanisms regulating the expression of 92- and 72-kDa gelatinases by HBECs in response to LPS are divergent. All these data allowed us to propose that HBECs may be actively involved in the physiological and physiopathological remodeling of the airway basement membrane.

Gene RPA43 in Saccharomyces cerevisiae encodes an essential subunit of RNA polymerase I.

Yeast RNA polymerase I contains 14 distinct polypeptides, including A43, a component of about 43 kDa. The corresponding gene, RPA43, encodes a 326-amino acid polypeptide matching the peptidic sequence of two tryptic fragments isolated from A43. Gene inactivation leads to a lethal phenotype that is rescued by a plasmid containing the 35S ribosomal RNA gene fused to the GAL7 promoter, which allows the synthesis of 35S rRNA by RNA polymerase II in the presence of galactose. A screening for mutants rescued by the presence of GAL7-35SrDNA identified a nonsense rpa43 allele truncating the protein at amino acid position 217. [3H]Uridine pulse labeling showed that this mutation abolishes 35S rRNA synthesis without significant effects on the synthesis of 5 S RNA and tRNAs. These properties establish that A43 is an essential component of RNA polymerase I. This highly hydrophilic phosphoprotein has a strongly acidic carboxyl-terminal domain, and shows no homology to entries in current sequence data banks, including all the genetically identified components of the other two yeast RNA polymerases. RPA43 mapped next to RPA190, encoding the largest subunit of polymerase I. These genes are divergently transcribed and may thus share upstream regulatory elements ensuring their co-regulation.

A second nitrogen permease regulator in Saccharomyces cerevisiae.

We describe a Saccharomyces cerevisiae mutant affected in its urea and proline transport capacities, and a gene coding for a protein complementing this mutation. This protein is not membrane-embedded and contains two PEST sequences, often found in regulatory factors. The mRNA is not down-regulated under nitrogen catabolite repression, and is induced by urea and proline. In the mutant, the PUT4 mRNA encoding the proline permease is not affected, whereas the DUR3 mRNA, involved in urea active transport, is strongly increased. Our data suggest that this protein is a post-transcriptional regulator of nitrogen permeases.

Distribution of mRNA encoding the FA-CHIP water channel in amphibian tissues: effects of salt adaptation.

A water channel, the frog aquaporin-CHIP (FA-CHIP) was recently cloned from Rana esculenta urinary bladder. The 28.9 kDa encoded protein shows 78.8%, 77.4%, 42.4% and 35.6% identity with rat CHIP28, human CHIP28, rat WCH-CD and gamma-TIP, other members of the new transmembrane water channel family (Aquaporin-CHIP). We have now studied membranes from different frog (R. esculenta) organs employing semiquantitative PCR using FA-CHIP specific primers and an internal standard to quantify the PCR products. The FA-CHIP mRNA was abundantly expressed in the frog urinary bladder, skin, lung and gall bladder, while a lower expression was detected in the colon, liver and oviduct. FA-CHIP mRNA was not detected in the frog kidney, erythrocytes and brain but its expression was observed in the toad (Bufo arenarum) urinary bladder and skin, showing that FA-CHIP is probably a general amphibian water channel. Salt acclimation is known to increase the water permeability of frog and toad epithelia. We have now observed that salt acclimation for 1, 3, 4 or 5 days markedly increased skin and urinary bladder FA-CHIP mRNA expression. It is generally accepted that water permeability is controlled in these tissues by the rate of water channel transfer from subapical vesicles (aggrephores) to the apical membrane. Our results indicate that water permeability is also regulated at the level of the FA-CHIP transcription.

Molecular analysis of vasopressin receptors in the rat nephron. Evidence for alternative splicing of the V2 receptor.

Expression and regulation of vasopressin V2 and V1a receptors were studied at the mRNA level in the rat kidney. Two V2 mRNA variants were identified and shown to arise from a single gene by alternative splicing using one donor and two different acceptor sites. The long (V2L) form encodes the adenylyl cyclase-coupled receptor. The short (V2S) form lacks the nucleotide sequence encoding the putative seventh transmembrane domain and undergoes a frame shift in its 3'end coding region; it is inactive on the cyclase pathway in transfected cells. Measurement of mRNAs, carried out by quantitative reverse transcription-polymerase chain reaction (RT-PCR) on microdissected nephrons, demonstrated that neither V2L, V2S nor V1A mRNAs are expressed in glomeruli and proximal tubules (< 100 mRNA copies/glomerulus or mm of tubular length), whereas they are present in the ascending limb of Henle's loop and in the collecting tubule. The V2L mRNA, which is always predominant in these structures, is expressed throughout the collecting tubule at 10 times higher levels (30,000 copies/mm) than in the thin and thick ascending limbs. The ratio of the V2S over V2L mRNA is constant (15%) in all nephron segments; hence high V2S levels are only observed in the collecting tubule. The V1A mRNA is slightly expressed in the thin ascending limb, absent in the thick ascending limb and reaches its maximum in the cortical collecting duct (4,000 copies/mm), before gradually decreasing to undetectable levels in the terminal collecting duct. Finally, in vivo administration of a vasopressin V2 agonist decreased by 50% V2L and V2S mRNAs, but did not alter the V1A mRNA level. We conclude that this study provides the quantitation, on a molar basis, of vasopressin receptor mRNAs in kidney tubules and demonstrates the occurrence of two V2 mRNA spliced variants which are similarly down-regulated.

Sequence and functional expression of an amphibian water channel, FA-CHIP: a new member of the MIP family.

A new member of the family of water channel proteins (aquaporin-CHIP) related to the major intrinsic protein (MIP) family is described. The cDNA coding for this amphibian CHIP was cloned from frog (Rana esculenta) urinary bladder, a model for the kidney collecting duct, using a RT-PCR cloning strategy. The encoded protein, designated FA-CHIP (frog aquaporin-CHIP), shows 77.4%, 42.4% and 35.6% identity with the three proteins now referred to as the aquaporins of the MIP family, i.e., human CHIP28, WCH-CD and gamma-TIP, respectively. Xenopus leavis injected with FA-CHIP cRNA exhibited a marked increase of the osmotic water permeability.

Predominant expression of beta 1-adrenergic receptor in the thick ascending limb of rat kidney. Absolute mRNA quantitation by reverse transcription and polymerase chain reaction.

Beta 1- and beta 2-adrenergic receptor (beta-ARs) expression in the thick ascending limb of rat kidney was studied at the level of mRNA and receptor coupling to adenylyl cyclase. Absolute quantitation of beta 1- and beta 2-AR mRNAs in microdissected nephron segments was performed with an assay based on reverse transcription and polymerase chain reaction, using in vitro transcribed mutant RNAs as internal standards. In the cortical thick ascending limb (CTAL), the number of mRNA molecules/mm of tubular length was 2,806 +/- 328 (n = 12) for beta 1-AR and 159 +/- 26 for beta 2-AR (P < 0.01). Lower levels were obtained in the medullary thick ascending, beta 1-AR mRNA still being predominant. The pharmacological properties of beta-ARS was also studied in the CTAL. Cyclic AMP accumulation was stimulated by beta-agonist with a rank order of potency of isoproterenol > norepinephrine > epinephrine. This observation, and the higher efficiency of a beta 1 than of a beta 2 antagonist to inhibit isoproterenol-induced cAMP accumulation, establish the typical beta 1-AR sensitivity of the CTAL. No detectable contribution of atypical or beta 3-ARs to adenylyl cyclase stimulation could be found. In conclusion, this study, which shows markedly different levels of beta 1- and beta 2-AR mRNAS in the CTAL, provides a molecular basis for the predominant expression of the beta 1 receptor subtype in this nephron segment.

RPC53 encodes a subunit of Saccharomyces cerevisiae RNA polymerase C (III) whose inactivation leads to a predominantly G1 arrest.

RPC53 is shown to be an essential gene encoding the C53 subunit specifically associated with yeast RNA polymerase C (III). Temperature-sensitive rpc53 mutants were generated and showed a rapid inhibition of tRNA synthesis after transfer to the restrictive temperature. Unexpectedly, the rpc53 mutants preferentially arrested their cell division in the G1 phase as large, round, unbudded cells. The RPC53 DNA sequence is predicted to code for a hydrophilic M(r)-46,916 protein enriched in charged amino acid residues. The carboxy-terminal 136 amino acids of C53 are significantly similar (25% identical amino acid residues) to the same region of the human BN51 protein. The BN51 cDNA was originally isolated by its ability to complement a temperature-sensitive hamster cell mutant that undergoes a G1 cell division arrest, as is true for the rpc53 mutants.

Characterization and mutagenesis of the gene encoding the A49 subunit of RNA polymerase A in Saccharomyces cerevisiae.

The gene encoding the 49-kDa subunit of RNA polymerase A in Saccharomyces cerevisiae has been identified by formation of a hybrid enzyme between the S. cerevisiae A49 subunit and Saccharomyces douglasii subunits based on a polymorphism existing between the subunits of RNA polymerase A in these two species. The sequence of the gene reveals a basic protein with an unusually high lysine content, which may account for the affinity for DNA shown by the subunit. No appreciable homology with any polymerase subunits, enzymes, or transcription factors is found. Complete deletion of the single-copy RPA49 gene leads to viable but slowly growing colonies. Insertion of the HIS3 gene halfway into the RPA49 coding region results in synthesis of a truncated A49 subunit that is incorporated into the polymerase. The truncated and wild-type subunits compete equally for assembly in the heterozygous diploid, although the wild type is phenotypically dominant.

The MAT locus revisited within a 9.8 kb fragment of chromosome III containing BUD5 and two new open reading frames.

This paper reports the DNA sequence of a segment of 9.8 kb of the chromosome III. The sequenced DNA contains the MAT alpha locus. The new sequence of the MAT alpha locus differs from the previously reported sequence by six modifications in the W segment. We have found the same modifications in the HML locus. The corrected sequence contains, in HML, an open reading frame (ORF) of 190 codons which ends at the border between the W segment and the flanking DNA. In the MAT locus, this ORF extends in the flanking DNA up to 538 codons. This ORF corresponds to a gene independently identified as BUD5 (Chant et al., 1991). This gene presents homologies with the exchange factors SDC25 and CDC25. A large ORF of 1399 codons is found on the opposite side of MAT alpha (toward the telomere). This ORF corresponds to a new gene YCR724. Next to this gene is a small ORF, YCR725, of 127 codons. The localization of this fragment on chromosome III, originally supposed to be distal from the MAT locus based on genetic distance, illustrates variation in recombination frequency along the chromosome and suggests the existence of hot spots of recombination between MAT and the THR4 locus.

ABF1 binding sites in yeast RNA polymerase genes.

We have used gel retardation and DNase protection assays to investigate the trans-acting factors involved in the regulation of yeast RNA polymerase genes RPC160 and RPC40. The same binding component was found to interact with the promoter of the two genes, at a short distance (100-150 base pairs) from the transcription start sites. From its size, its DNA-binding specificity and its immunological properties, this factor appears to correspond to the autonomous replication sequences and silencer-binding factor ABF1/SBF-B. The interaction of ABF1 with the polymerase upstream box sequence was characterized using gel DNA-binding assay. The factor binds with high affinity to the polymerase upstream box sequence (Kapp = 5.10(-10) M). A mutational analysis showed that nine base pairs belonging to two separated attachment sites are involved in factor binding. The consensus sequence RTCRYB(N)4ACG was derived from the present binding studies. These data provide an experimental basis for evaluating the efficiency of known or potential ABF1 sites and for comparing several factors with ABF1-like binding properties.

Contacts between the factor TUF and RPG sequences.

The yeast TUF factor binds specifically to RPG-like sequences involved in multiple functions at enhancers, silencers, and telomeres. We have characterized the interaction of TUF with its optimal binding sequence, rpg-1 (1-ACACCCATACATTT-14), using a gel DNA-binding assay in combination with methylation protection and mutagenesis experiments. As many as 10 base pairs appear to be engaged in factor binding. Analysis of a collection of 30 different RPG mutants demonstrated the importance of 8 base pairs at position 2, 3, 4, 5, 6, 7, 10, and 12 and the critical role of the central GC pair at position 5. Methylation protection data on four different natural sites confirmed a close contact at positions 4, 5, 6, and 10 and suggested additional contacts at base pairs 8, 12, and 13. The derived consensus sequence was RCAAYCCRYNCAYY. A quantitative band shift analysis was used to determine the equilibrium dissociation constant for the complex of TUF and its optimal binding site rpg-1. The specific dissociation constant (K8) was found to be 1.3 x 10(-11) M. The comparison of the K8 value with the dissociation constant obtained for nonspecific DNA sites (Kn8 = 8.7 x 10(-6) M) shows the high binding selectivity of TUF for its specific RPG target.

Conditional expression of RPA190, the gene encoding the largest subunit of yeast RNA polymerase I: effects of decreased rRNA synthesis on ribosomal protein synthesis.

The synthesis of ribosomal proteins (r proteins) under the conditions of greatly reduced RNA synthesis were studied by using a strain of the yeast Saccharomyces cerevisiae in which the production of the largest subunit (RPA190) of RNA polymerase I was controlled by the galactose promoter. Although growth on galactose medium was normal, the strain was unable to sustain growth when shifted to glucose medium. This growth defect was shown to be due to a preferential decrease in RNA synthesis caused by deprivation of RNA polymerase I. Under these conditions, the accumulation of r proteins decreased to match the rRNA synthesis rate. When proteins were pulse-labeled for short periods, no or only a weak decrease was observed in the differential synthesis rate of several r proteins (L5, L39, L29 and/or L28, L27 and/or S21) relative to those of control cells synthesizing RPA190 from the normal promoter. Degradation of these r proteins synthesized in excess was observed during subsequent chase periods. Analysis of the amounts of mRNAs for L3 and L29 and their locations in polysomes also suggested that the synthesis of these proteins relative to other cellular proteins were comparable to those observed in control cells. However, Northern analysis of several r-protein mRNAs revealed that the unspliced precursor mRNA for r-protein L32 accumulated when rRNA synthesis rates were decreased. This result supports the feedback regulation model in which excess L32 protein inhibits the splicing of its own precursor mRNA, as proposed by previous workers (M. D. Dabeva, M. A. Post-Beittenmiller, and J. R. Warner, Proc. Natl. Acad. Sci. USA 83:5854-5857, 1986).

Isolation, characterization, and inactivation of the APA1 gene encoding yeast diadenosine 5',5'''-P1,P4-tetraphosphate phosphorylase.

The gene encoding diadenosine 5',5'''-P1,P4-tetraphosphate (Ap4A) phosphorylase from yeast was isolated from a lambda gt11 library. The DNA sequence of the coding region was determined, and more than 90% of the deduced amino acid sequence was confirmed by peptide sequencing. The Ap4A phosphorylase gene (APA1) is unique in the yeast genome. Disruption experiments with this gene, first, supported the conclusion that, in vivo, Ap4A phosphorylase catabolizes the Ap4N nucleotides (where N is A, C, G, or U) and second, revealed the occurrence of a second Ap4A phosphorylase activity in yeast cells. Finally, evidence is provided that the APA1 gene product is responsible for most of the ADP sulfurylase activity in yeast extracts.