Cloning and analysis of a constitutive heat shock (cognate) protein 70 gene inducible by L-glutamine.

An intronless gene encoding a protein of 652 amino acid residues with an M(r) of 71,266, showing between 79% and 59% identity in nucleotide sequence with heat shock protein 70 (HSP 70) genes of Bremia lactucae (a parasitic Oomycete of lettuce) and a wide range of organisms that include humans, was isolated from the nonparasitic Oomycete Achlya klebsiana. While the gene appears to be constitutively expressed, L-glutamine augmented its expression particularly under conditions of nutritional stress. L-Glutamine enhanced the transcription of a 2.4-kilobase poly(A)+ RNA simultaneously in the same way as it elevated the cellular level of the HSP 70-like protein. A polyclonal antibody (affinity-purified) raised in rabbit against the purified monomeric (M(r) 120,000) form of an NAD-specific glutamate dehydrogenase (Yang, B., and LéJohn, H.B. (1994) J. Biol. Chem. 269, 4506-4512) immunoprecipitated the HSP 70-like protein, and it was used to study the kinetics of induction of this stress-related protein and the effect of proteinase inhibitors on its metabolism. By using as probes four partial length cDNA clones, nine overlapping DNA fragments of the organism's genome carrying the HSP 70-like protein gene were isolated from a genomic library. The nucleotide sequence of the gene, including its boundaries, was determined by using these genomic clones. The 5'-untranslated boundary of the gene displayed the classical nucleotide arrangement of heat shock elements as well as CCAAT and TATA box motifs. Within the coding region are the typical conserved amino acid heat shock protein signatures 1 and 2 at the predicted locations. By primer extension and S1 nuclease protection mapping system, we estimated that the gene is probably transcribed into a message of 2.2 kilobases.

NADP(+)-activable, NAD(+)-specific glutamate dehydrogenase. Purification and immunological analysis.

An NAD(+)-specific glutamate dehydrogenase (NAD-GDH) that is inducible by L-glutamine was isolated from Achlya klebsiana and purified to electrophoretic homogeneity. The enzyme is only partially active in vitro unless NADP+ (an activator) is present in both its oxidative deamination and reductive amination reactions. This type of enzyme was reported (LéJohn, H.B. (1971) Nature 231, 164-168) to be widespread among the amorphous group of algae-related fungi classified as Oomycota. The enzyme retained its dependence on NADP+ at all stages of its purification. NADP+ decreased the Km of substrates 3-fold and increased the Vmax 4-fold. M(r) of the undernatured enzyme was 480,000, and, denatured, only a single subunit of M(r) 120,000 was seen. A polyclonal antibody raised in rabbit against purified enzyme subunit excised from SDS-polyacrylamide gel electrophoresis gels immunoprecipitated the M(r) 120,000 polypeptide, the undenatured enzyme, and a physically distinct polypeptide of M(r) 74,000. The antibody, purified against the M(r) 120,000 enzyme subunit as anchored antigen on Sepharose, still immunoprecipitated the M(r) 74,000 polypeptide. The M(r) 74,000 polypeptide was found to be a subunit of a M(r) 220,000 native protein.

Molecular characterization of an NAD-specific glutamate dehydrogenase gene inducible by L-glutamine. Antisense gene pair arrangement with L-glutamine-inducible heat shock 70-like protein gene.

The gene for an NAD-specific glutamate dehydrogenase (NAD-GDH) that is allosterically activated by NADP+ (non-substrate) was cloned, and its physical structure and nucleotide sequence was determined. The gene consists of 9 introns and 10 exons; the 10th and largest exon, which is 1863 nucleotides long, is at the 3'-end of the gene. The shortest exon of 33 base pairs is the first and is located at the 5'-end of the gene. The large exon is in perfect register along the complementary strand with a heat shock 70 (HSP)-like protein gene. The NAD-GDH gene is inducible with L-glutamine, just as the HSP 70-like protein gene (LéJohn, H.B., Cameron, L.E., Yang, B., MacBeath, G., Barker, D.S., and Williams, S.A. (1994) J. Biol. Chem. 269, 4513-4522). The phenomenon of anti-parallel coupling of two genes is named antisense gene pair. By Northern and Western blotting techniques, we obtained indirect evidence that the gene is expressed in vivo. The gene encodes a protein of M(r) 118,740 which consists of 1063 amino acid residues. The 5' and 3' borders of the gene display typical but unproven promoter motifs of CCAAT, TATAAT, and AAATAAAA polyadenylation signal bounded by a pyrimidine-rich transcription termination-type format. Restriction endonuclease site mapping of all the genomic clones isolated that carry most or all of the gene, and of the genome itself, gave hybridization patterns that are consistent with the interpretation that the organism, Achlya klebsiana, has only one form of the gene. 3'-End-labeling of a 5.2-kb XbaI DNA fragment (carrying the antisense gene pair) that was then asymmetrically cleaved to produce two single 3'-end-labeled pieces that were used as probes on L-glutamine-induced cell poly(A)+ RNA, showed that the end-labeled DNA equivalent to the HSP 70-like protein mRNA hybridized to a 3.4-kb transcript and the end-labeled DNA equivalent to the NAD-GDH mRNA hybridized to a 2.4-kb transcript.

Cloning and analysis of beta-tubulin gene from a protoctist.

We have isolated and characterized by restriction endonuclease mapping, transcription pattern, and DNA sequencing a beta-tubulin gene from the coenocytic freshwater protoctist, Achlya klebsiana. The gene is intronless and has a single open reading frame that encodes a 444-amino acid residue polypeptide of Mr 49,856. The protein shows a high degree of homology to other beta-tubulins, 85% identity to human beta-tubulin and 89% identity to beta-tubulin of the sporozoan (also a protoctist) Plasmodium falciparum. Fungal beta-tubulins are among the least identical to A. klebsiana beta-tubulin. Through Southern blot hybridization analysis, we determined that there is just one form of beta-tubulin gene in A. klebsiana. Transcription of the gene was studied during sporogenesis. Following induction of sporogenesis, the level of the mRNA increased markedly at 2 h and declined in the next 2 h when mitosis, cytokinesis, and spore development occurred. At the same time, beta-tubulin content increased about 6-fold in the cells. Sporulation in A. klebsiana is not inhibited by antimitotic drugs such as benomyl, colcemid, and colchicine. Benomyl resistance in Neurospora crassa and Aspergillus nidulans has been genetically and molecularly linked to single amino acid substitutions at positions 167 and 165, respectively. The change from phenylalanine to tyrosine conferring benomyl resistance to N. crassa is seen in A. klebsiana, but the valine substitution for alanine in A. nidulans is marked by cysteine replacement in A. klebsiana. The amino acid found at position 165 is not conserved in various beta-tubulins, but phenylalanine at position 167 is extremely conserved.

Structure and expression of fungal calmodulin gene.

I report on the isolation, structural analysis, and in vivo expression patterns of a fungal calmodulin gene. The gene is intronless and encodes a protein of 148 amino acid residues that is 92% homologous with vertebrate calmodulins. Through S1 nuclease transcript mapping, it was determined that the cloned gene (a) is transcribed in vivo, (b) has a 5'-untranslated region of about 400 nucleotides, and (c) has a 3'-untranslated end of about 300 nucleotides. Southern blot hybridization analysis of the genomic DNA and the cloned gene provide evidence for the existence of only one type of calmodulin gene in the organism. The amino acid sequence deduced from the DNA sequence shows that Achlya klebsiana calmodulin has amino acid substitutions that are a mix of those seen in calmodulins from invertebrates such as Drosophila and trypanosome when compared to mammalian calmodulins. Not surprisingly, it has less resemblance to calmodulins from Saccharomyces and Dictyostelium.

Involvement of protease in L-glutamine control of nucleic acid and polyphosphate metabolism in cells transformed by 9,10-dimethyl-1,2-benzanthracene, SV40 and H-ras oncogene.

Mammalian cells transformed with either 9,10-dimethyl-1,2-benzanthracene, SV40 or H-ras oncogene dramatically changed their ability to synthesize DNA and RNA and metabolize polyphosphate when L-glutamine was withdrawn from the growth medium or when heat shocked (growth at 42 degrees C). Untransformed, DNA and RNA synthesis decreased by 50-80% when glutamine was withdrawn, but polyphosphate accumulated whether or not glutamine was supplied. Heat shock did not alter this response. Transformed isogenic cells responded differently; at 37 degrees C, they decreased their synthesis of DNA and RNA if starved for glutamine, whereas at 42 degrees C, synthesis was optimal without glutamine. Transformed cells accumulated polyphosphate at 37 degrees C when starved for glutamine, but at 42 degrees C, no polyphosphate accumulated. This apparent non-dependence on glutamine by transformed cells when heat shocked was found to be due to the production of glutamine from serum proteins through induction of a protease(s).

L-glutamine alteration of gene expression, not of polyphosphate and calcium metabolism, is a key event in arresting fungal sporulation.

Vegetatively growing cells of the coenocytic freshwater mould Achlya developed asexual sporangia and sporulated within 6 h of postransfer to a nutrient-free (starvation) medium. Sporangial development was arrested by the addition of L-glutamine to starving cells. During starvation (minus glutamine), three polyphosphate substances accumulated intracellularly, ATP was rapidly depleted, and a protein of molecular weight 42 000 (presumed to be actin) was actively synthesized, whereas synthesis of the most abundant detergent-soluble protein of molecular weight 83 000 (p83) ceased. In the presence of glutamine, starving cells used up the polyphosphates faster than they were formed. ATP depletion was delayed, cell calcium (Ca) exited rapidly, and synthesis of actin diminished while p83 synthesis continued unabated. Several pyrimidine analogues, including 5-diazouracil (which inhibited pyridimide nucleotide biosynthesis), and inorganic phosphate prevented Ca exit from glutamine-supplemented starving cells. The pyrimidine analogues delayed but did not inhibit sporangial development; however, they did not overcome glutamine suppression of sporangial development. Vegetatively growing and starving cells displayed significantly different protein synthesis patterns (monitored by polyacrylamide gel electrophoresis) but, when glutamine was added, it changed the protein synthesis pattern of starving cells to a form typical of vegetatively growing cells. Glutamine withdrawal reversed the effect and the cells differentiated. Pyrimidine analogues and inorganic phosphate did not alter the protein synthesis patterns of starving cells in the presence and absence of glutamine. The conclusion is that glutamine inhibition of sporangial development may be linked to its ability to subvert starving cell metabolism by making it vegetative like.

Recharacterization of fungal dinucleoside polyphosphate (HS3).

Three polyphosphorylated dinucleosides given the pseudonyms of HS3, HS2, and HS1 that were erroneously described as diguanosine polyphosphates (LéJohn, H. B., Cameron, L. E., McNaughton, D. R. & Klassen, G. R. (1975) Biochem, Biophys, Res, Commun. 66, 460-467) have been repurified and partially recharacterized. They have proved to be extremely complex molecules; chemical (HCl and KOH hydrolysis), physical (ultraviolet-light spectral analysis and ion-exchange chromatography), and enzymic (nucleotide pyrophosphatase and bacterial alkaline phosphatase hydrolysis) studies showed that (i) all three HS compounds are uracil rich and (ii) only HS3 contains a purine nucleoside and glutamate. The partial structure of HS3 was deciphered as a moiety of ADP--sugar X--glutamate (the mode of attachment of glutamate is obscure) that is covalently linked to another moiety composed of UDP, mannitol, and four phosphates. Sugar X had chromatographic characteristics of ribitol, but the chromatographic isolate also contained a ninhydrin-sensitive entity presumed to be an amino group. Sugar X, THEREFore, may be an amino sugar polyol. Only the general chemical compositions of HS2 and HS1 were determined. Each contained two uridines and HS2 had 10 phosphates whereas HS1 had 12.

Glucose transport in Achlya: characterization and possible regulatory aspects.

The freshwater fungus Achlya transported D-(+)glucose (glucose) and 2-deoxy-D-glucose (deoxyglucose) by an energy-related system. Their transport4 was inhibited by uncouplers of metabolic energy such as 2,4-dinitrophenol, cyanide, azide, and carbonylcyanide-p-chlorophenylhydrazone. Besides inhibiting each other, glucose and deoxyglucose transport was inhibited by D-(+)galactose, D-(+)mannose, and D-(+)xylose. Many other sugars tested failed to inhibit glucose transport implying a certain degree of specificity. Glucose transport was pH (optimum at 6.5) and temperature (optimum at 30-40 degrees C) dependent. Glucose transport was also inhibited by citrate, N6-substituted adenines (cytokinins), and iodine. None of these agents penetrated the cell membrane within the brief (1-3-min) period in which glucose transport was measured. In every case, transport was inhibited within 10 s (the shortest time in which measurements could be made). When cells were osmotically shocked to release a cell-wall membrane phosphorylated proteoglycan (PPG), they became incapable of transporting glucose for several hours until new PPG material was reisolable from the membrane by osmotic-shock treatment. The osmotically shocked cells could not transport glucose or deoxyglucose. No glucose-binding protein was detected in the shock fluid. Practically all of the glucose transported within 1-2 min was recovered as glucose-6-phosphate. No other phosphorylated sugar was detected suggesting that glucose may be phosphorylated in transport. Related studies have shown that citrate removed calcium bound by PPG; N6-substituted adenines were bound by PPG while three polyphosphorylated dinucleosides, HS3, HS2, and HS1, were displaced from it. Iodine formed stable complexes with the HS compounds. All of these agents inhibited glucose transport without entering the cell. It is therefore possible that HS compounds, calcium and PPG may be involved in maintaining the cell membrane in proper form for glucose transport.

Inhibition of mammalian ribonucleotide reductase by a dinucleotide produced in eucaryotic cells.

HS3, a highly phosphorylated dinucleoside originally purified from the fungus Achlya, has been isolated from Chinese hamster ovary cells undergoing glutamine starvation. The HS3 compounds obtained from the fungal and mammalian sources exhibited similar physical and chemical properties. This unusual dinucleotide may be an important regulator of eucaryotic ribonucleoside diphosphate reductase activity; for 50 micrometer HS3, isolated from either mammalian or fungal cells, significantly inhibited CDP reduction in Achlya or hamster cell preparations, but only marginally affected the activity of the enzyme from E. coli. Studies with HS3 isolated from Achlya and partially purified mammalian ribonucleotide reductase indicated that the compound noncompetitively inhibited the reduction of varying concentrations of the substrates CDP, ADP and GDP with Ki values of 23 micrometer, 14 micron and 16 micron respectively. These inhibitor concentrations are well below the estimated intracellular levels of HS3 in glutamine starved cells and suggest that HS3 inhibition of ribonucleotide reduction may be responsible for the rapid inhibition of DNA synthesis seen under these culture conditions.

Amino acid transport in a water-mould: the possible regulatory roles of calcium and N6-(delta2-isopentenyl)adenine.

Transport of amino acids in the water-mould Achlya is an energy-dependent process. Based on competition kinetics and studies involving the influence of pH and temperature on the initial transport rates, it was concluded that the 20 amino acids (L-isomers) commonly found in proteins were transported by more than one, possibly nine, uptake systems. This is similar to the pattern elucidated for some bacteria but unlike those uncovered for all fungi studied to date. The nine different systems elucidated are: (i) methionine, (ii) cysteine. (iii) proline, (iv) serine-threonine, (v) aspartic and glutamic acids, (vi) glutamine and asparagine, (vii) glycine and alanine, (viii) histidine, lysine, and arginine, and (ix) phenylalanine-tyrosine-tryptophan and leucine-isoleucine-valine as two overlapping groups. Transport of all of these amino acids was inhibited by azide, cyanide, and its derivatives and 2,4-dinitrophenol. These agents normally interfere with metabolism at the level of the electron transport chain and oxidative phosphorylation. Osmotic shock treatment of the cells released, into the shock fluid, a glycopeptide that binds calcium as well as tryptophan but no other amino acid. The shocked cells are incapable of concentrating amino acids, but remain viable and reacquire this capacity when the glycopeptide is resynthesized.

A rapid and sensitive auxin binding system for detecting N6-substituted adenines, and some urea and thiourea derivatives, that show cytokinin activity in cell division tests.

A selective, sensitive and rapid (2 min or less) method for detecting compounds with potential for cytokinin activity is described. The method does not measure cytokinesis; instead, it determines the ability of cytokinin-active agents to (i) activate the intake of either L-tryptophan or indoleacetic acid by germinated spores of the water-mould Achlya, while inhibiting the energy-dependent transport of all L-amino acids usually found in proteins; (ii) inhibit the energy-dependent transport of nucleosides and sugars by the same organism. The compounds with cytokinin activity generally activate auxin (tryptophan) intake at 10(-8) M or greater and inhibit at 10(-6) M or greater. The most effective activating compounds were N6-(delta2-isopentenyl)adenine, N6-benzyladenine. N6-furfuryladenine, and N6-(trans-hydroxy-3-methyl-but-2-enyl)adenine. These compounds are classed generally as cytokinins in plant growth studies. A cell membrane - localized glycopeptide of molecular weight 6000 was isolated from this organism and shown to be the site at which cytokinins, auxin, and tryptophan bind. An earlier study had also established that calcium ions bind to this entity as well. Tryptophan binding to the glycopeptide was enhanced by cytokinins, suggesting that this may be the way in which whole cells display enhanced tryptophan binding in the bioassay. On the other hand, calcium binding was antagonized by cytokinin. The results suggest that this may be an important experimental system for use in studying one possible way in which cytokinin may regulate plant growth.