Two cDNAs representing alleles of the nitrate reductase gene of potato (Solanum tuberosum L. cv. Desirée): sequence analysis, genomic organization and expression.

Two, different, full-length cDNAs, StNR2 and StNR3, of 3049 and 3066 nucleotides, respectively, were isolated from a Solanum tuberosum L. cv. Desirée leaf cDNA library by an RT-PCR approach. Conceptual translation of the longest open reading frame of each cDNA showed that they could encode a protein of 911 amino acids in each case with an M(r) of 102.6 and 102.5 kDa, respectively, and with 99.7% identity with each other. The cDNAs have a high degree of sequence similarity with all higher plant nitrate reductases (NRs) and possess structural characteristics expected of NADH-NR proteins, consistent with enzyme activity data. Southern analysis of genomic DNA suggested the presence of a single NR gene in the potato genome whilst studies using the mapping population F1840, and the full-length StNR2 cDNA as hybridization probe, identified a single NR locus within the potato genome that is located on chromosome XI. The two cDNAs identified here are probably derived from two transcribing alleles of this single gene. Distribution of total NR transcript and of NADH-NR activity, in different organs of compost-grown plants, depended on the level of nitrate supplied: at low nitrate level transcript and activity were detected only in leaf and stem tissue whilst at high nitrate level they could also be detected in root and stolon. An RT-PCR approach was used to differentiate between the transcripts derived from the two identified alleles and to show that the pattern of transcription of the two identified alleles of the potato nia gene, in the organs studied, is the same.

Redefining reductive sulfate assimilation in higher plants: a role for APS reductase, a new member of the thioredoxin superfamily?

The reaction steps leading from the intermediate adenosine 5'-phosphosulfate (APS) to sulfide within the higher plant reductive sulfate assimilation pathway are the subject of controversy. Two pathways have been proposed: a 'bound intermediate' pathway in which the sulfo group of APS is first transferred by APS sulfotransferase to a carrier molecule to form a bound sulfite intermediate and is then further reduced by thiosulfonate reductase to bound sulfide; and a 'free intermediate' pathway in which APS is further activated to 3'-phosphoadenosine 5'-phosphosulfate (PAPS) by APS kinase followed by reduction of the sulfo group to free sulfite by PAPS reductase. Sulfite is then reduced to free sulfide by sulfite reductase. Sulfide, either free or bound, is then incorporated into organic form (as cysteine) by the enzyme O-acetylserine (thiol) lyase. In order to better characterize the pathway we attempted to clone PAPS reductase cDNAs by functional complementation of an Escherichia coli cysH mutant to prototrophy. We found no evidence for PAPS reductase cDNAs but did identify cDNAs that encode a small family of novel, chloroplast-localized proteins with APS reductase activity that are new members of the thioredoxin superfamily. We show here that the thioredoxin domain of these proteins is functional. We speculate that rather than proceeding via either of the pathways proposed above, reductive sulfate assimilation proceeds via the reduction of APS to sulfite by APS reductase and the subsequent reduction of sulfite to sulfide by sulfite reductase. In this scheme the product of the APS kinase reaction, PAPS, is not a direct intermediate in the pathway but rather acts as a substrate for sulfotransferase action and perhaps as a store of activated sulfate that can be returned to the pathway as APS via phosphohydrolase action on PAPS. Interactions between enzyme isoforms within the chloroplast stroma may bring about substrate channeling of APS and contribute to the partitioning of APS between sulfotransferase reactions on the one hand and the synthesis of cysteine and related metabolites via the reductive sulfate assimilation pathway on the other.

Defining teaching hospitals' GME strategy in response to new financial and market challenges.

The authors present an overview of current graduate medical education (GME) issues, particularly the financial challenges to teaching hospitals resulting from the Balanced Budget and Tax Payer Relief Acts of 1997 and other recent market-driven factors. They describe in detail the nature of Medicare GME payments before and after the 1997 legislation, with specific examples, and explain the negative financial impact of the legislation and aspects of the legislation that are designed to alleviate that impact. Other factors influencing GME program size and composition are also discussed, including oversupplies or shortages of physicians, the concern that teaching hospitals are using public funds to train international medical graduates, changing training requirements, etc. The authors also describe a recent consulting assignment during which they assisted a major teaching hospital to develop a GME strategy that was responsive to the organization's mission and patients and that took into account future GME financing challenges. Detailed explanations are given of how the consultants analyzed the hospital's GME programs and finances, developed and ranked key institution-specific program criteria (strategic, organizational and operational, and financial), and, in consultation with all key stakeholders, formulated a GME strategy specific to the institution's needs. The authors conclude by cautioning that each institution's GME strategy will be different, but that it is important for institutions to develop such strategies to better face future challenges.

Cysteine biosynthesis in higher plants: a new member of the Arabidopsis thaliana serine acetyltransferase small gene-family obtained by functional complementation of an Escherichia coli cysteine auxotroph.

A cDNA clone, Sat-52, encoding a novel isoform of serine acetyltransferase (EC 2.3.1.30) was isolated by functional complementation of an Escherichia coli cysE mutant defective in serine acetyltransferase. The 1158 base pair clone contains a full-length open reading frame encoding a deduced protein of 312 amino acids with an M(r) of 32.77 kDa. Northern analysis revealed a single transcript of ca 1.19 kb that did not increase in abundance under sulfate limitation. Genomic Southern hybridization suggests the presence of a single copy of the Sat-52 gene.

Three members of a novel small gene-family from Arabidopsis thaliana able to complement functionally an Escherichia coli mutant defective in PAPS reductase activity encode proteins with a thioredoxin-like domain and "APS reductase" activity.

Three different cDNAs, Prh-19, Prh-26, and Prh-43 [3'-phosphoadenosine-5'-phosphosulfate (PAPS) reductase homolog], have been isolated by complementation of an Escherichia coli cysH mutant, defective in PAPS reductase activity, to prototrophy with an Arabidopsis thaliana cDNA library in the expression vector lambda YES. Sequence analysis of the cDNAs revealed continuous open reading frames encoding polypeptides of 465, 458, and 453 amino acids, with calculated molecular masses of 51.3, 50.5, and 50.4 kDa, respectively, that have strong homology with fungal, yeast and bacterial PAPS reductases. However, unlike microbial PAPS reductases, each PRH protein has an N-terminal extension, characteristic of a plastid transit peptide, and a C-terminal extension that has amino acid and deduced three-dimensional homology to thioredoxin proteins. Adenosine 5'-phosphosulfate (APS) was shown to be a much more efficient substrate than PAPS when the activity of the PRH proteins was tested by their ability to convert 35S-labeled substrate to acid-volatile 35S-sulfite. We speculate that the thioredoxin-like domain is involved in catalytic function, and that the PRH proteins may function as novel "APS reductase" enzymes. Southern hybridization analysis showed the presence of a small multigene family in the Arabidopsis genome. RNA blot hybridization with gene-specific probes revealed for each gene the presence of a transcript of approximately 1.85 kb in leaves, stems, and roots that increased on sulfate starvation. To our knowledge, this is the first report of the cloning and characterization of plant genes that encode proteins with APS reductase activity and supports the suggestion that APS can be utilized directly, without activation to PAPS, as an intermediary substrate in reductive sulfate assimilation.

Cloning and characterization of an Arabidopsis thaliana cDNA clone encoding an organellar isoform of serine acetyltransferase.

We have cloned an Arabidopsis thaliana cDNA encoding serine acetyltransferase (EC 2.3.1.30) by functional complementation of the Escherichia coli cysE mutant JM15. The cDNA clone Sat-1 conferred serine acetyltransferase activity (with apparent Km for the two substrates acetyl CoA and L-serine of 0.043 and 3.47 mmol/dm3 respectively) on the cysE mutant. The 1515 bp full-length cDNA encodes a deduced protein of 391 amino acids which includes a putative chloroplastic targeting presequence. Northern analysis revealed a single message of 1.5 kb, while Southern hybridisation suggests a small multigene family of related sequences.

The Nir1 locus in barley is tightly linked to the nitrite reductase apoprotein gene Nii.

pBNiR1, a cDNA clone encoding part of the barley nitrite reductase apoprotein, was isolated from a barley (cv. Maris Mink) leaf cDNA library using the 1.85 kb insert of the maize nitrite reductase cDNA clone pCIB808 as a heterologous probe. The cDNA insert of pBNiR1 is 503 bp in length. The nucleotide coding sequence could be aligned with the 3' end of other higher plant nitrite reductase apoprotein cDNA sequences but diverges in the 3' untranslated region. The whole-plant barley mutant STA3999, previously isolated from the cultivar Tweed, accumulates nitrite after nitrate treatment in the light, has very much lowered levels of nitrite reductase activity and lacks detectable nitrite reductase cross-reacting material due to a recessive mutation in a single nuclear gene which we have designated Nir1. STA3999 has the characteristics expected of a nitrite reductase apoprotein gene mutant. Here we have used pB-NiR1 in RFLP analysis to determine whether the mutation carried by STA3999 is linked to the nitrite reductase apoprotein gene locus Nii. An RFLP was identified between the wild-type barley cultivars Tweed (major hybridising band of 11.5 kb) and Golden Promise (major hybridising band of 7.5 kb) when DraI-digested DNA was probed with the insert from the partial barley nitrite reductase cDNA clone, pBNiR1. DraI-digested DNA from the mutant STA3999 also exhibited a major hybridising band of 11.5 kb after hybridisation with the insert from pBNiR1.(ABSTRACT TRUNCATED AT 250 WORDS)

nir1, a conditional-lethal mutation in barley causing a defect in nitrite reduction.

Eleven green individuals were isolated when 95000 M2 plants of barley (Hordeum vulgare L.), mutagenised with azide in the M1, were screened for nitrite accumulation in their leaves after nitrate treatment in the light. The selected plants were maintained in aerated liquid culture solution containing glutamine as sole nitrogen source. Not all plants survived to flowering and some others that did were not fertile. One of the selected plants, STA3999, from the cultivar Tweed could be crossed to the wild-type cultivar and analysis of the F2 progeny showed that leaf nitrite accumulation was due to a recessive mutation in a single nuclear gene, which has been designated Nir1. The homozygous nir1 mutant could be maintained to flowering in liquid culture with either glutamine or ammonium as sole nitrogen source, but died within 14 days after transfer to compost. The nitrite reductase cross-reacting material seen in nitrate-treated wild-type plants could not be detected in either the leaf or the root of the homozygous nir1 mutant. Nitrite reductase activity, measured with dithionite-reduced methyl viologen as electron donor, of the nitrate-treated homozygous nir1 mutant was much reduced but NADH-nitrate reductase activity was elevated compared to wild-type plants. We conclude that the Nir1 locus determines the formation of nitrite reductase apoprotein in both the leaf and root of barley and speculate that it represents either the nitrite reductase apoprotein gene locus or, less likely, a regulatory locus whose product is required for the synthesis of nitrite reductase, but not nitrate reductase.(ABSTRACT TRUNCATED AT 250 WORDS)

Leucine: tRNA Ligase from Cultured Cells of Nicotiana tabacum var. Xanthi: Evidence for de Novo Synthesis and for Loss of Functional Enzyme Molecules.

Leucine:tRNA ligase was assayed in extracts from cultured tobacco (Nicotiana tabacum) XD cells by measuring the initial rate of aminoacylation of transfer RNA with l-[4,5-(3)H]leucine. Transfer RNA was purified from tobacco XD cells after the method of Vanderhoef et al. (Phytochemistry 9: 2291-2304). The buoyant density of leucine:tRNA ligase from cells grown for 100 generations in 2.5 mm [(15)N]nitrate and 30% deuterium oxide was 1.3397. After transfer of cells into light medium (2.5 mm [(14)N]nitrate and 100% H(2)O) the ligase activity increased and the buoyant density decreased with time to 1.3174 at 72 hours after transfer. It was concluded that leucine:tRNA ligase molecules were synthesized de novo from light amino acids during the period of activity increase. The width at half-peak height of the enzyme distribution profiles following isopycnic equilibrium centrifugation in caesium chloride remained constant at all times after transfer into light medium providing evidence for the loss of preexisting functional ligase molecules. It was concluded that during the period of activity increase the cellular level of enzyme activity was determined by a balance between de novo synthesis and the loss of functional enzyme molecules due to either inactivation or degradation.

Structural and functional relationships of enzyme activities induced by nitrate in barley.

1. Nitrate induces the development of NADH-nitrate reductase (EC 1.6.6.1), FMNH(2)-nitrate reductase and NADH-cytochrome c reductase activities in barley shoots. 2. Sucrose-density-gradient analysis shows one band of NADH-nitrate reductase (8S), one band of FMNH(2)-nitrate reductase activity (8S) and three bands of NADH-cytochrome c reductase activity (bottom layer, 8S and 3.7S). Both 8S and 3.7S NADH-cytochrome c reductase activities are inducible by nitrate, but the induction of the 8S band is much more marked. 3. The 8S NADH-cytochrome c reductase band co-sediments with both NADH-nitrate reductase activity and FMNH(2)-nitrate reductase activity. Nitrite reductase activity (4.6S) did not coincide with the activity of either the 8S or the 3.7S NADH-cytochrome c reductase. 4. FMNH(2)-nitrate reductase activity is more stable (t((1/2)) 12.5min) than either NADH-nitrate reductase activity (t((1/2)) 0.5min) or total NADH-cytochrome c reductase activity (t((1/2)) 1.5min) at 45 degrees C. 5. NADH-cytochrome c reductase and NADH-nitrate reductase activities are more sensitive to p-chloromercuribenzoate than is FMNH(2)-nitrate reductase activity. 6. Tungstate prevents the formation of NADH-nitrate reductase and FMNH(2)-nitrate reductase activities, but it causes superinduction of NADH-cytochrome c reductase activity. Molybdate overcomes the effects of tungstate. 7. The same three bands (bottom layer, 8S and 3.7S) of NADH-cytochrome c reductase activity are observed irrespective of whether induction is carried out in the presence or absence of tungstate, but only the activities in the 8S and 3.7S bands are increased. 8. The results support the idea that NADH-nitrate reductase, FMNH(2)-nitrate reductase and NADH-cytochrome c reductase are activities of the same enzyme complex, and that in the presence of tungstate the 8S enzyme complex is formed but is functional only with respect to NADH-cytochrome c reductase activity.

Correlated changes of some enzyme activities and cofactor and substrate contents of pea cotyledon tissue during germination.

1. The activities of six enzymes (hexokinase, phosphoglucose isomerase, phosphofructokinase, aldolase, glucose 6-phosphate dehydrogenase and amylase) in extracts of pea cotyledons were determined. The activities during the first 10 days after germination showed individual and characteristic changes that indicate a specific control of both synthesis and destruction of enzymes. 2. Tissue contents of glucose, inorganic phosphate, glucose 6-phosphate, fructose 6-phosphate, ATP, ADP, AMP, NAD and NADP were also determined, and a correlation is reported between the substrate concentrations at day 1 and the subsequent enzymic activity. 3. The initial NAD(+)/NADH ratio value of 1 changed to about 3 by day 4; the NADP content was lower and changes in the oxidation state were less striking. The ratio of ATP to ADP and AMP remained virtually constant.