Dimers generated from tetrameric phosphorylating glyceraldehyde-3-phosphate dehydrogenase from Bacillus stearothermophilus are inactive but exhibit cooperativity in NAD binding

Tetrameric phosphorylating glyceraldehyde-3-phosphate dehydrogenase (GAPDH) from Bacillus stearothermophilus has been described as a "dimer of dimers" with three nonequivalent interfaces, P-axis (between subunits O and P and between subunits Q and R), Q-axis (between subunits O and Q and between subunits P and R), and R-axis interface (between subunits O and R and between subunits P and Q). O-P dimers, the most stable and the easiest to generate, have been created by selective disruption of hydrogen bonds across the R- and Q-axis interfaces by site-directed mutagenesis. Asp-186 and Ser-48, and Glu-276 and Tyr-46, which are hydrogen bond partners across the R- and Q-axis interfaces, respectively, have been replaced with glycine residues. All mutated residues are highly conserved among GAPDHs from different species and are located in loops. Both double mutants D186G/E276G and Y46G/S48G were dimeric, while all single mutants remained tetrameric. As previously described [Clermont, S., Corbier, C., Mely, Y., Gerard, D., Wonacott, A., and Branlant, G. (1993) Biochemistry 32, 10178-10184], NAD binding to wild type GAPDH (wtGAPDH) was interpreted according to the induced-fit model and exhibited negative cooperativity. However, NAD binding to wtGAPDH can be adequately described in terms of two independent dimers with two interacting binding sites in each dimer. Single mutants D186G, E276G, and Y46G exhibited behavior in NAD binding similar to that of the wild type, while both dimeric mutants D186G/E276G and Y46G/S48G exhibited positive cooperativity in binding the coenzyme NAD. The fact that O-P dimer mutants retained cooperative behavior shows that (1) the P-axis interface is important in transmitting the information induced upon NAD binding inside the O-P dimer from one subunit to the other and (2) the S-loop of the R-axis-related subunit is not directly involved in cooperative binding of NAD in the O-P dimer. In both O-P dimer mutants, the absorption band of the binary enzyme-NAD complex had a highly decreased intensity compared to that of the wild type and, in addition, totally disappeared in the presence of G3P or 1,3-dPG. However, no enzymatic activity was detected, indicating that the formed ternary enzyme-NAD-G3P or -1, 3-dPG complex was not catalytically efficient. In the O-P dimers, the interaction with the S-loop of the R-axis-related subunit is disrupted, and therefore, the S-loop should be less structured. This resulted in increased accessibility of the active site to the solvent, particularly for the adenosine-binding site of NAD. Thus, together, this is likely to explain both the lowered affinity of the dimeric enzyme for NAD and the absence of activity.     

Misfolded forms of glyceraldehyde-3-phosphate dehydrogenase interact with GroEL and inhibit chaperonin-assisted folding of the wild-type enzyme

We studied the interaction of chaperonin GroEL with different misfolded forms of tetrameric phosphorylating glyceraldehyde-3-phosphate dehydrogenase (GAPDH): (1) GAPDH from rabbit muscles with all SH-groups modified by 5,5'-dithiobis(2-nitrobenzoate); (2) O-R-type dimers of mutant GAPDH from Bacillus stearothermophilus with amino acid substitutions Y283V, D282G, and Y283V/W84F, and (3) O-P-type dimers of mutant GAPDH from B. stearothermophilus with amino acid substitutions Y46G/S48G and Y46G/R52G. It was shown that chemically modified GAPDH and the O-R-type mutant dimers bound to GroEL with 1:1 stoichiometry and dissociation constants K(d) of 0.4 and 0.9 muM, respectively. A striking feature of the resulting complexes with GroEL was their stability in the presence of Mg-ATP. Chemically modified GAPDH and the O-R-type mutant dimers inhibited GroEL-assisted refolding of urea-denatured wild-type GAPDH from B. stearothermophilus but did not affect its spontaneous reactivation. In contrast to the O-R-dimers, the O-P-type mutant dimers neither bound nor affected GroEL-assisted refolding of the wild-type GAPDH. Thus, we suggest that interaction of GroEL with certain types of misfolded proteins can result in the formation of stable complexes and the impairment of chaperonin activity.     

Mechanism of dissociation of human apolipoprotein A-I from complexes with dimyristoylphosphatidylcholine as studied by guanidine hydrochloride denaturation

The reversibility of the binding of human apolipoprotein A-I (apo A-I) to phospholipid has been monitored through the influence of guanidine hydrochloride (Gdn-HCl) on the isothermal denaturation and renaturation of apo A-1/dimyristoylphosphatidylcholine (DMPC) complexes at 24 degree C. Denaturation was studied by incubating discoidal 1:100 and vesicular 1:500 mol/mol apo A-I/DMPC complexes with up to 7 M Gdn-HCl for up to 72 h. Unfolding of apo A-I molecules was observed from circular dichroism spectra while the distribution of protein between free and lipid-associated states was monitored by density gradient ultracentrifugation. The ability of apo A-I to combine with DMPC in the presence of Gdn-HCl at 24 degrees C was also investigated by similar procedures. In both the denaturation and renaturation of 1:100 and 1:500 complexes, the final values of the molar ellipticity and the ratio of free to bound apo A-I at various concentrations of Gdn-HCl are dependent on the initial state of the lipid and protein; apo A-I is more resistant to denaturation when Gdn-HCl is added to existing complexes than to a mixture of apo A-I and DMPC. There is an intermediate state in the denaturation pathway of apo A-I/DMPC complexes which is not present in the renaturation; the intermediate comprises partially unfold apo A-I molecules still associated with the complex by some of their apolar residues. Complete unfolding of the alpha helix and subsequent desorption of the apo A-I molecules from the lipid/water interface involve cooperative exposure of these apolar residues to the aqueous phase. The energy barrier associated with this desorption step makes the binding of apo A-I to DMPC a thermodynamically irreversible process. Consequently, binding constants of apo A-I and PC cannot be calculated simply from equilibrium thermodynamic treatments of the partitioning of protein between free and bound states. Apo A-I molecules do not exchange freely between the lipid-free and lipid-bound states, and extra work is required to drive protein molecules off the surface. The required increased in surface pressure can be achieved by a net mass transfer of protein to the surface; in vivo, increases in the surface pressure of lipoproteins by lipolysis can cause protein desorption.     

Circular permutation within the coenzyme binding domain of the tetrameric glyceraldehyde-3-phosphate dehydrogenase from Bacillus stearothermophilus.

A circularly permuted (cp) variant of the phosphorylating NAD-dependent glyceraldehyde-3-phosphate dehydrogenase (GAPDH) from Bacillus stearothermophilus has been constructed with N- and C-termini created within the coenzyme binding domain. The cp variant has a kcat value equal to 40% of the wild-type value, whereas Km and KD values for NAD show a threefold decrease compared to wild type. These results indicate that the folding process and the conformational changes that accompany NAD binding during the catalytic event occur efficiently in the permuted variant and that NAD binding is tighter. Reversible denaturation experiments show that the stability of the variant is only reduced by 0.7 kcal/mol compared to the wild-type enzyme. These experiments confirm and extend results obtained recently on other permuted proteins. For multimeric proteins, such as GAPDH, which harbor subunits with two structural domains, the natural location of the N- and C-termini is not a prerequisite for optimal folding and biological activity.     

High pressure effects on the activity of glycolytic enzymes

High hydrostatic pressure inhibits growth in most organisms; this may be explained by a deactivation of enzymes involved in essential metabolic pathways. In order to check this hypothesis the enzymic activity of rabbit muscle lactic dehydrogenase and yeast glyceraldehyde-3-phosphate dehydrogenase was investigated in the presence of the coenzyme and excess of substrate at pressures up to 2 kbar.Kinetic analysis of an initial phase of pressure induced activation and of a second phase of reversible deactivation shows that the two enzymes respond to high pressures in different ways leading to a volume of activation of ΔV³(LDH) = 0 ± 1 cm³ mol⁻¹ and ΔV³(GAPDH) = 60 ± 4 cm³ mol⁻¹, respectively. Comparing the lower limits of pressure deaclivation, LDH is found to be more stable towards pressure than GAPDH. At p ≈ 2 kbar total deactivation of both enzymes is observed. A concentration dependent lag of GAPDH reactivation proves dissociation to participate in the process of deactivation, while the effects for LDH are explicable on the basis of reversible denaturation alone.     

Thermal unfolding used as a probe to characterize the intra- and intersubunit stabilizing interactions in phosphorylating D-glyceraldehyde-3-phosphate dehydrogenase from Bacillus stearothermophilus

Tetrameric phosphorylating glyceraldehyde-3-phosphate dehydrogenase (GAPDH) from Bacillus stearothermophilus can be described as a dimer of dimers with three nonequivalent interfaces. To investigate the contribution of intra- and intersubunit interactions to GAPDH thermostability, 10 residues located either at the cofactor domain (amino acids 1-148 and 313-333) or at the catalytic domain (amino acids 149-312) were mutated and the thermal unfolding of the mutants was studied by differential scanning calorimetry in the absence and presence of saturating concentrations of NAD. Disruptions of intrasubunit interactions lead to a drastic decrease in thermostability of the N313T, Y283V, and W310F mutants. Moreover, for the N313T mutant, a weakening of cooperative interactions between the catalytic and the cofactor domains and an inefficient binding of NAD are observed. This is likely the consequences of modification or loss of the hydrogen bonding network associating N313 and residues 236-238 and N313 and the nicotinamide carboxyamide of NAD, respectively. For the residues Y283 and W310, which are involved in stacking hydrophobic interactions, mutating both positions does not affect the efficiency of NAD binding. This shows that the factors involved in the thermostability of the tetrameric apo GAPDH are then different from those induced by NAD binding. Disruption of intersubunit hydrogen bonds between the catalytic domain and the NAD-binding domain of a neighboring subunit also leads to a significant destabilization of the apo tetrameric form as observed for the D282G mutant. Moreover, no efficient binding of NAD is observed. Both results are likely the consequence of a loss of hydrogen bonds across the P-axis and the Q-axis between D282 and R197 and between D282 and R52, respectively. Similar results, i.e., a destabilizing effect and inefficient NAD binding, are observed with the T34Q/T39S/L43Q mutant in which steric hindrance is introduced at the S-loop of the R-axis-related subunit via mutations at the adenosine subsite. The dimeric form of the D282G mutant exhibits a single partial heat absorption peak, whereas the Y46G/R52G mutant which exists only as a dimer shows two peaks. Taking into account the recent small-angle X-ray scattering studies which suggested that the dimeric form of the D282G mutant and of the dimeric Y46G/R52G mutant are of the O-R and O-P types, respectively (Vachette, unpublished results), we propose that the presence of one or two peaks in thermal unfolding of dimers is a signature of the dimer type.     

Ribosomal proteins and DNA-binding protein II from the extreme thermophile Bacillus caldolyticus

Crystallographic studies, presently on ribosomal and DNA-binding proteins from the moderate thermophile Bacillus stearothermophilus, can be expected to benefit from the use of even more stable proteins from extreme thermophiles. Bacillus caldolyticus, which is able to grow in the temperature range of 70-80 degrees C, appears to be a suitable candidate. We have compared the two bacilli using two criteria: the two-dimensional gel patterns of ribosomal proteins and the properties of DNA-binding protein II. The latter protein is ubiquitous in the eubacterial kingdom and can be purified in large quantities. B. caldolyticus can be grown at 75 degrees C in continuous culture with a generation time of 45-60 min. The yield of ribosomes compares favorably with that of B. stearothermophilus. The gel patterns of the ribosomal proteins are very similar but several differences, in particular among the 50S proteins, are observed. The N-terminal amino-acid sequence of the DNA-binding protein differs in 3 positions (out of 39) from B. stearothermophilus and the protein shows an increased resistance to thermal denaturation. Tetragonal and monoclinic crystals of DNA-binding protein II have been obtained which are suitable for X-ray studies and the diffraction patterns of the two crystal forms are shown.     

Different Thermostability of Skeletal Muscle Glyceraldehyde-3-phosphate Dehydrogenase from Hibernating and Euthermic Jerboa （Jaculus orientalis）

D Glyceraldehyde 3 phosphatedehydrogenase(GAPDH ,EC 1.2 .1.12 )isakeyenzymeoftheglycolyticpathwaythatispresentinthecytosolofallorganismssofarstudied[1] .TheglycolyticGAPDHhasbeenremarkablyconservedduringevolution ,havingahomotetramericstructurewithsubunitsof 35 - 37kD[1] .GAPDHhasbeenisolatedfromavarietyofspecies[2 ] ,includingmesophilic ,moderatelythermophilicandhyperthermophilicmicroorganisms[3 ] .Theseenzymes ,whichdifferinthermalstability ,havebeenshowntobehighlysimilarinaminoacidse…     

Studies on the role of polyamines associated with the ribosomes from Bacillus stearothermophilus

1. Spermine and spermidine were the main polyamines detectable in Bacillus stearothermophilus. 2. When grown at 65 degrees B. stearothermophilus contained lower concentrations of polyamines per mg. of RNA than when grown at 45 degrees or at 55 degrees . 3. Ribosomes isolated from B. stearothermophilus in 0.01m-tris-hydrochloric acid buffer (pH7.4)-0.01m-magnesium chloride contained sufficient polyamines to neutralize between 4% and 9% of their RNA phosphorus. 4. Removal of polyamines from the ribosomes by dialysis against m-potassium chloride did not appreciably alter the hypochromicity or thermal denaturation profiles of the ribosomes when measured in 0.01m-tris-hydrochloric acid buffer (pH7.4)-0.01m-magnesium chloride, though it did cause a loss of ribosome particles sedimenting at greater than 78s. 5. When ribosomes were dialysed against acridine orange solutions acridine orange bound to the ribosomes and did not displace spermine, but when a mixture of ribosomal RNA and spermine was dialysed against acridine orange the acridine orange displaced the spermine. It is concluded that polyamines in the ribosomes are less accessible for displacement by acridine orange than when polyamines are bound to ribosomal RNA.     

Cloning and characterization of the gene for a methanol-utilising alcohol dehydrogenase from Bacillus stearothermophilus

The cloning and characterization of the alcohol dehydrogenase (ADH) gene (adh) from Bacillus stearothermophilus strain DSM2334, an obligate aerobe, are described. The clone directed the synthesis of ADH as judged on Western blots, activity gels and tetrazolium plates. It specified an enzyme that oxidised methanol as well as ethanol. The enzyme was found to be encoded by a single gene in B. stearothermophilus which did not cross-hybridize to adh clones from Escherichia coli, yeast or maize. The cloned gene was expressed in E. coli but activity was not detected in Bacillus subtilis, despite stable maintenance of the recombinant plasmid in this host. The gene is catabolite-repressed in DSM2334.     

Cation-induced thermostability of yeast and Escherichia coli pyrophosphatases

Inorganic pyrophosphatases (PPiases) from both yeast and Escherichia coli were found to be stable against heat denaturation in the presence of Mg2+, as previously observed with the enzymes from thermophilic bacteria. No loss of activity was observed after 1 h of incubation at 50 degrees C and pHs between 6 and 9 in the yeast enzyme, and at 60 degrees C and pHs between 7.2 and 9.2 in the E. coli enzyme. Such an induced thermostability of the E. coli enzyme was detected when Mn2+, Co2+, Ca2+, Cd2+, and Zn2+ were added in place of Mg2+. On the other hand, the degree of induced thermostability of the yeast enzyme was dependent upon the divalent cations used, and Ni2+ and Cu2+ accelerated the heat inactivation. On adding the divalent cations, the difference spectra of the E. coli enzyme always showed negative peaks in the ultraviolet region, but those of the yeast enzyme changed again depending upon the divalent cations. The circular dichroism spectra in the near ultraviolet region of both enzymes greatly differed from each other, but both were not affected so much by adding the divalent cations unlike the thermophilic enzymes from Bacillus stearothermophilus and thermophilic bacterium PS-3. Yeast and E. coli PPiases did not cross-link with the anti-immunoglobulin G's from the thermophilic enzymes, but the thermophilic enzymes did with each other's antisera. The results in the present study indicated that the conformation of PPiase, in which the aromatic amino acid residues were buried in the interior of the protein molecule, was very important for the thermostability and also that the protein structures of PPiases from B. stearothermophilus and thermophilic bacterium PS-3 were very similar to each other, but were very different from those of the mesophilic enzymes.     

Oestradiol Up-regulates Oestrogen Receptor, Cyclophilin, and Glyceraldehyde Phosphate Dehydrogenase mRNA Concentrations in Endometrium, but Down-regulates Them in Liver

Oestradiol regulates reproductive physiology and cardiovascular health in women. In the endometrium of ovariectomized ewes, previous work demonstrated that a single dose of oestradiol (50 μg) up-regulates oestrogen receptor- (ER) and progesterone receptor (PR) gene expression within 24 h. Here we compared responses to different doses of oestradiol and different dosing regimens in two diverse tissues: endometrium and liver. ER, c-fos, cyclophilin and glyceraldehyde phosphate dehydrogenase (GAPDH) mRNA concentrations were analyzed on replicate RNA slot blots in both tissues, while PR and apolipoprotein AI (apo AI) mRNA concentrations were only analyzed in endometrium or liver, respectively. Along with ER mRNA, oestradiol strongly up-regulated GAPDH and cyclophilin mRNA concentrations in endometrium. In liver, however, oestradiol down-regulated them, along with apo AI mRNA. Responses to different doses and dose regimens, including repeated 50 μg doses, were similar to those evoked by a single 50 μg dose of oestradiol. Thus, oestradiol appears to have all-or-none effects which include up-regulation of ER, cyclophilin and GAPDH gene expression in endometrium and down-regulation of ER, apo AI, cyclophilin and GAPDH gene expression in liver. These results illustrate the sharp contrast between two mammalian tissues in their responses to physiological levels of oestradiol.     

Antibodies specific to modified glyceraldehyde-3-phosphate dehydrogenase induce inactivation of the native enzyme and change its conformation

The antibodies specific to an inactive glyceraldehyde-3-phosphate dehydrogenase (GAPDH) from Bacillus stearothermophilus prepared by the treatment of the tetrameric holoenzyme with glutaraldehyde were obtained. They were purified from the pool of polyclonal rabbit antibodies to GAPDH with the use of immobilized GAPDH cross-linked by glutaraldehyde as an affinity sorbent. Such antibodies were capable of interacting with the native enzyme, inducing its time-dependent inactivation; the effect was different with the apo- and holoenzyme forms. Differential scanning calorimetry of the purified [GAPDH].[antibody] complex revealed a large shift of the temperature corresponding to the maximal heat capacity of the holoenzyme towards the lower temperature. Again, the effect appeared to be different with the apoenzyme. Together, the results are consistent with the hypothesis that a specific antibody is able to exercise a certain strain on the target protein, altering its conformation toward the structure of the species which served to select the antibody. The possibility of preparing selective enzyme inhibitors based on the antibodies specific to inactive enzyme conformations is considered.     

Alpha-glucosidase inhibitory activity of Syzygium cumini (Linn.) Skeels seed kernel in vitro and in Goto-Kakizaki (GK) rats

Syzygium cumini seed kernel extracts were evaluated for the inhibition of alpha-glucosidase from mammalian (rat intestine), bacterial (Bacillus stearothermophilus), and yeast (Saccharomyces cerevisiae, baker's yeast). In vitro studies using the mammalian alpha-glucosidase from rat intestine showed the extracts to be more effective in inhibiting maltase when compared to the acarbose control. Since acarbose is inactive against both the bacterial and the yeast enzymes, the extracts were compared to 1-deoxynojirimycin. We found all extracts to be more potent against alpha-glucosidase derived from B. stearothermophilus than that against the enzymes from either baker's yeast or rat intestine. In an in vivo study using Goto-Kakizaki (GK) rats, the acetone extract was found to be a potent inhibitor of alpha-glucosidase hydrolysis of maltose when compared to untreated control animals. Therefore, these results point to the inhibition of alpha-glucosidase as a possible mechanism by which this herb acts as an anti-diabetic agent.     

Distinct metal dependence for catalytic and structural functions in the L-arabinose isomerases from the mesophilic Bacillus halodurans and the thermophilic Geobacillus stearothermophilus

L-Arabinose isomerase (AI) catalyzes the isomerization of L-arabinose to L-ribulose. It can also convert d-galactose to d-tagatose at elevated temperatures in the presence of divalent metal ions. The araA genes, encoding AI, from the mesophilic bacterium Bacillus halodurans and the thermophilic Geobacillus stearothermophilus were cloned and overexpressed in Escherichia coli, and the recombinant enzymes were purified to homogeneity. The purified enzymes are homotetramers with a molecular mass of 232 kDa and close amino acid sequence identity (67%). However, they exhibit quite different temperature dependence and metal requirements. B. halodurans AI has maximal activity at 50 degrees C under the assay conditions used and is not dependent on divalent metal ions. Its apparent K(m) values are 36 mM for L-arabinose and 167 mM for d-galactose, and the catalytic efficiencies (k(cat)/K(m)) of the enzyme were 51.4 mM(-1)min(-1) (L-arabinose) and 0.4 mM(-1)min(-1) (d-galactose). Unlike B. halodurans AI, G. stearothermophilus AI has maximal activity at 65-70 degrees C, and is strongly activated by Mn(2+). It also has a much higher catalytic efficiency of 4.3 mM(-1)min(-1) for d-galactose and 32.5 mM(-1)min(-1)for L-arabinose, with apparent K(m) values of 117 and 63 mM, respectively. Irreversible thermal denaturation experiments using circular dichroism (CD) spectroscopy showed that the apparent melting temperature of B. halodurans AI (T(m)=65-67 degrees C) was unaffected by the presence of metal ions, whereas EDTA-treated G. stearothermophilus AI had a lower T(m) (72 degrees C) than the holoenzyme (78 degrees C). CD studies of both enzymes demonstrated that metal-mediated significant conformational changes were found in holo G. stearothermophilus AI, and there is an active tertiary structure for G. stearothermophilus AI at elevated temperatures for its catalytic activity. This is in marked contrast to the mesophilic B. halodurans AI where cofactor coordination is not necessary for proper protein folding. The metal dependence of G. stearothermophilus AI seems to be correlated with their catalytic and structural functions. We therefore propose that the metal ion requirement of the thermophilic G. stearothermophilus AI reflects the need to adopt the correct substrate-binding conformation and the structural stability at elevated temperatures.     

Direct inhibition of tombusvirus plus-strand RNA synthesis by a dominant negative mutant of a host metabolic enzyme, glyceraldehyde-3-phosphate dehydrogenase, in yeast and plants

The replication of plus-strand RNA viruses depends on many cellular factors. Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) is an abundant metabolic enzyme that is recruited to the replicase complex of Tomato bushy stunt virus (TBSV) and affects asymmetric viral RNA synthesis. To further our understanding on the role of GAPDH in TBSV replication, we used an in vitro TBSV replication assay based on recombinant p33 and p92(pol) viral replication proteins and cell-free yeast extract. We found that the addition of purified recombinant GAPDH to the cell extract prepared from GAPDH-depleted yeast results in increased plus-strand RNA synthesis and asymmetric production of viral RNAs. Our data also demonstrate that GAPDH interacts with p92(pol) viral replication protein, which may facilitate the recruitment of GAPDH into the viral replicase complex in the yeast model host. In addition, we have identified a dominant negative mutant of GAPDH, which inhibits RNA synthesis and RNA recruitment in vitro. Moreover, this mutant also exhibits strong suppression of tombusvirus accumulation in yeast and in virus-infected Nicotiana benthamiana. Overall, the obtained data support the model that the co-opted GAPDH plays a direct role in TBSV replication by stimulating plus-strand synthesis by the viral replicase.     

High-molecular-weight penicillin-binding proteins from membranes of bacilli

Mixtures of high-molecular-weight, cephalosporin-sensitive penicillin-binding proteins (PBPs) can be purified from Bacillus subtilis membranes by cephalosporin affinity chromatography (G. Kleppe and J. L. Strominger, J. Biol. Chem. 254:4856-4862, 1979). By appropriate modification of this technique, B. subtilis PBP 1 was purified to homogeneity, and a mixture of Bacillus stearothermophilus PBPs 1, 2, and 4 was isolated. [14C]penicillin-PBP complexes of high-molecular-weight PBPs purified from membranes of these two bacilli, after denaturation, were found to have chemical reactivities typical of the penicilloyl-serine derivative formed by D-alanine carboxypeptidase from B. stearothermophilus. Although enzymatic activity catalyzed by these and several other high-molecular-weight PBPs from gram-positive organisms has not been detected with cell wall-related substrates, a slow, enzymatic acylation of B. subtilis PBPs 1, 2ab, and 4 by [14C]-diacetyl-L-lysyl-D-alanyl-D-lactate was demonstrated. Further study is necessary to clarify the physiological relevance of the slow acylation by this analog of a natural cell wall biosynthetic intermediate.     

Transcriptional mapping of two yeast genes coding for glyceraldehyde 3-phosphate dehydrogenase isolated by sequence homology with the chicken gene

Homology between the coding regions of the chicken and yeast glyceraldehyde 3-phosphate dehydrogenase (GAPDH) genes was directly demonstrated by the hybridization of a cDNA clone coding for GAPDH in the chicken with EcoRI-digested yeast DNA. A yeast EcoRI fragment library in bacteriophage lambda was screened using the chicken cDNA plasmid as probe, and two recombinant phages were isolated, each one containing a different GAPDH gene. The initiation and termination sites for the GAPDH mRNA were localized for the two different GAPDH genes and compared to those of other yeast genes. Measurements of the relative mRNA levels for the two genes show that both genes are transcribed at about the same level when yeasts are grown on glucose media.