Characterization of the diphtheria toxin-resistance system in Chinese hamster ovary cells.

Variations in two general classes of diphtheria toxin-resistant mutants which may be selected from Chinese hamster ovary (CH0-K1) cells and the conditions for their selection are described. The resistance of class I mutants can be overcome with increasing concentrations of toxin. Their entire complement of EF-2 is susceptible to ADP-ribosylation by toxin. Class I includes those strains in which resistance resides at the level of the plasma membrane. The resistance of class II, translational, mutants cannot be overcome by high concentrations of toxin, as all, or a portion, of their EF-2 is insensitive to the action of diphtheria toxin and Pseudomonas exotoxin A. Adjustment of the concentration of toxin used to select resistant mutants can be used to regulate the class of mutant recovered. Metabolic cooperation between cells does not affect recovery of either class I or class II mutants. Resistance is stable in class I strains, but class IIb strains, which possess 50% resistant and 50% sensitive EF-2, display a transient high level of resistance which is retained for varying lengths of time following exposure to toxin. Class IIa strains, which possess 100% resistant EF-2, grow normally in saturating concentrations of toxin, but class IIb strains grow at a reduced rate. Evidence is presented which suggests that the gene for EF-2 is functionally diploid in CHO-K1 cells.     

Binding and uptake of diphtheria toxin by toxin-resistant Chinese hamster ovary and mouse cells.

We investigated two phenotypically distinct types of diphtheria toxin-resistant mutants of Chinese hamster cells and compared their resistance with that of naturally resistant mouse cells. All are resistant due to a defect in the process of internalization and delivery of toxin to its target in the cytosol, elongation factor 2. By cell hybridization studies, analysis of cross-resistance, and determination of specific binding sites for 125I-labeled diphtheria toxin, we showed that these cell strains fall into two distinct complementation groups. The Dipr group encompasses Chinese hamster strains that are resistant only to diphtheria toxin, as well as mouse LM cells. These strains possess a normal complement of high-affinity binding sites for diphtheria toxin, but these receptors are unable to deliver active toxin fragment A to the cytosol. Cells of the DPVr group have a broader spectrum of resistance, including Pseudomonas exotoxin A and several enveloped viruses as well as diphtheria toxin. In these studies, which investigate the resistance of these cells to diphtheria toxin, we demonstrate that they possess a reduced number of specific binding sites for this toxin and behave, phenotypically, like cells treated with the proton ionophore monensin. Their resistance is related to a defect in a mechanism required for release of active toxin from the endocytic vesicle.     

Selection and characterization of cells resistant to diphtheria toxin and pseudomonas exotoxin A: presumptive translational mutants.

Two classes of diphtheria toxin-resistant variants were selected from Chinese hamster ovary (CHO-K1) cells: permeability variants, in which uptake of toxin was impaired, and a new class of cytoplasmic variants, which were cross-resistant to Pseudomonas exotoxin. EF-2 prepared from the cytoplasmic variants was resistant to ADP-ribosylation by either toxin. The evidence presented suggests that these are translational variants possessing a mutationally altered EF-2 gene product. These studies also confirmed that Pseudomonas toxin ADP-ribosylates EF-2 in toxin-sensitive intact cells, as well as in cell-free systems.     

Diphtheria toxin resistance in human lymphoblast lines

Stable mutants (Dip^r), highly resistant to diphtheria toxin have been selected from a sensitive human lymphoblast line. A second human lymphoblast line, HH-4 (and its derivative TK6-1) were found to be highly resistant to diphtheria toxin without any previous selection, suggesting the presence of the Dip^r allele in the human population. The resistance of protein synthesis in extracts of mutant cells to diphtheria toxin indicates that the genetic lesion in the resistant lines examined involved an alteration in the protein synthesis. In comparison to sensitive cells, the mutant cell extracts contained reduced (30–40%) levels of ADP-ribosylatable elongation factor-2 activity suggesting that the lesion presumably affects elongation factor-2 in such cells. The biochemical phenotype of these mutants appears similar to that of the Dip^rIIb class of mutants of Chinese hamster cells (4,6) which behave codominantly in hybrids.     

Characterization of the endogenous ADP-ribosylation of wild-type and mutant elongation factor 2 in eukaryotic cells.

Anti-[ADP-ribosylated elongation factor 2 (EF-2)] antiserum has been used to immunoprecipitate the modified form of EF-2 from polyoma-virus-transformed baby hamster kidney (pyBHK) cells [Fendrick, J. L. & Iglewski, W. J. (1989) Proc. Natl Acad. Sci. USA 86, 554-557]. This antiserum also immunoprecipitates a 32P-labelled protein of similar size to EF-2 from a variety of primary and continuous cell lines derived from many species of animals. One of these cell lines, chinese hamster ovary CHO-K1 cells was further characterized. The time course of labelling of ADP-ribosylated EF-2 with [32P]orthophosphate was similar in pyBHK cells and in CHO-K1 cells. The kinetics of labelling were more rapid for cells cultured in 2% serum than 10% serum, with incorporation of 32P reaching a maximum at 6 h and 10 h, respectively. EF-2 mutants of pyBHK and CHO-K1 cells resistant to diphtheria-toxin-catalyzed ADP-ribosylation of EF-2 remain sensitive to cellular ADP-ribosylation of EF-2. The 32P-labelled moiety of ADP-ribosylated EF-2 was digested by snake venom phosphodiesterase and the product was identified as AMP. The same 32P-labelled tryptic peptide was modified by toxin in wild-type EF-2 and by the cellular transferase in mutant EF-2. When purified EF-2 from pyBHK cells was incubated with [carbonyl-14C]nicotinamide and diphtheria toxin fragment A, under conditions for reversal of the ADP-ribosylation reaction, [14C]NAD was generated. The results suggest that cellular ADP-ribosylated EF-2 exists in a variety of cell types, and the ribosylated product is identical to that produced by toxin ADP-ribosylation of EF-2, except in diphthamide mutant cells. Studies with the mutant cell lines indicate that the toxin and the cellular transferase, however, recognize different determinants at the ADP-ribose acceptor site in EF-2. The cellular transferase does not require the diphthamide modification of the histidine ring in the amino acid sequence of EF-2 for the transfer of ADP-ribose to the ring. Therefore, we would expect the cellular transferase active site to be similar to, but not identical to, the critical amino acids demonstrated in the active site of diphtheria toxin and Pseudomonas exotoxin A.     

Codominant translational mutants of Chinese hamster ovary cells selected with diphtheria toxin.

Diphtheria toxin-resistance markers in two translational mutants, CH-RE1.22c, possessing no toxin-sensitive EF-2 (class IIa), and CH-RE1.32, with 50% toxin-sensitive and 50% toxin-resistant EF-2 (class IIb), behaved codominantly in somatic cell hybrids. There was no complementation in hybrids formed between the two resistant mutants. The mutant parents and their hybrids, except those formed by fusion of CH-RE1.32 and wild-type cells, grew in the presence of toxin. To explain these results we suggest that CHO-K1 cells possess two functional copies of the gene for EF-2 and that CH-RE1.22c and CH-RE1.32 represent the homozygous (R/R) and heterozygous (R/S) states of resistance at the EF-2 gene locus. The failure of hybrids formed between CH-RE1.32 and wild-type cells to grow in toxin is a gene dosage effect. Codominant class IIa translational resistance is a selectable marker for the isolation of hybrids. It can be combined with a second, recessive, marker to provide a cell which is a "universal hybridizer" (10).     

Highly frequent single amino acid substitution in mammalian elongation factor 2 (EF-2) results in expression of resistance to EF-2-ADP-ribosylating toxins

Toxin-resistant polypeptide chain elongation factor 2 cDNA has been cloned from a mutant hamster cell line with only non-ADP-ribosylatable elongation factor 2. The mutation conferring resistance to diphtheria toxin and Pseudomonas aeruginosa exotoxin A is a G-to-A transition in the first nucleotide of codon 717. Codon 715 encodes a histidine residue that is modified post-translationally to diphthamide, which is the target amino acid for ADP-ribosylation by both toxins. Transfection of mouse L cells with a recombinant elongation factor 2 cDNA differing from the wild-type only by this G-to-A transition confers resistance to P. aeruginosa exotoxin A. The degrees of toxin-resistant protein synthesis of stable transfectants are dependent on the ratio of non-ADP-ribosylated elongation factor 2 to wild-type elongation factor 2, not the amount of non-ADP-ribosylated elongation factor 2. The mutation creates a new Mbo II restriction site in the elongation factor 2 gene. Several independently isolated diphtheria toxin-resistant Chinese hamster ovary cell lines show the same alteration in the Mbo II restriction pattern.     

ADP-ribosyltransferase from beef liver which ADP-ribosylates elongation factor-2

Fragment A of diphtheria toxin and Pseudomonas toxin A intoxicate cells by ADP-ribosylating the diphthamide residue of elongation factor-2 (EF-2) resulting in an inhibition of protein synthesis [1-3]. A cellular enzyme from polyoma virus transformed baby hamster kidney (pyBHK) cells ADP-ribosylates EF-2 in an identical manner [4]. Here we describe a similar cellular enzyme from beef liver which transfers [adenosine-14C]ADP-ribose from NAD to EF-2. The 14C-label can be removed from the EF-2 by snake venom phosphodiesterase as a soluble product which comigrates with AMP on TLC plates, indicating the 14C-label is present on EF-2 as monomeric units of ADP-ribose. Furthermore, the forward transferase reaction catalyzed by the beef liver ADP-ribosyltransferase is reversible by excess diphtheria toxin fragment A, with the formation of 14C-labeled NAD, indicating that both transferases ADP-ribosylate the same site on the diphthamide residue of EF-2. Thus, beef liver and pyBHK mono(ADP-ribosyl)transferases both modify the diphthamide residue of EF-2, in a manner identical to diphtheria toxin fragment A and Pseudomonas toxin A. These results suggest the cellular enzyme is probably ubiquitous among eukaryotic cells.     

Complete nucleotide sequence and characterization of the 5'-flanking region of mammalian elongation factor 2 gene

The hamster elongation factor 2 gene was isolated from genomic libraries of diphtheria toxin- and Pseudomonas aeruginosa exotoxin A-resistant cells containing non-ADP-ribosylatable elongation factor 2, and its structure was determined by a combination of restriction endonuclease mapping and DNA sequence analysis. The entire gene is about 6 kilobases long and has 13 exons. Almost all the introns are about 90-200 bases long, except the first and third, which are about 1 kilobase and 400 bases long, respectively. The first exon is processed just after the initiation codon for translation. The promoter of this gene was also characterized. As this gene contains the mutation conferring resistance to diphtheria toxin and P. aeruginosa exotoxin A, introduction of this gene into mammalian cells results in expression of toxin resistance. Using this characteristic, gene expression by deletion mutants of the 5'-flanking region were examined, and results showed that about 60 base pairs upstream of the TATA sequence were most efficient for expression of the elongation factor 2 gene.     

Biosynthesis of diphthamide in Saccharomyces cerevisiae. Partial purification and characterization of a specific S-adenosylmethionine:elongation factor 2 methyltransferase

The inactivation of elongation factor 2 (EF-2) by diphtheria toxin requires the presence of a post-translationally modified histidine residue in EF-2. This residue, diphthamide, has the structure 2-[3-carboxyamido-3-(trimethylammonio)propyl]histidine. The present work was undertaken to study the pathway of diphthamide biosynthesis using diphtheria toxin-resistant yeast mutants (Chen. J.-Y., Bodley, J. W., and Livingston, D. M. (1985) Mol. Cell. Biol. 5, 3357-3360) which are defective in diphthamide formation. We demonstrate here that one of these mutants (dph5) contains a toxin-resistant form of EF-2 which can be converted in vitro to a toxin-sensitive form through the action of an enzyme present in other yeast strains. Both this toxin-resistant EF-2 and its modifying enzyme have been partially purified and evidence is presented that the modifying enzyme is a specific S-adenosylmethionine:EF-2 methyltransferase. In vitro complementation to diphtheria toxin sensitivity required S-adenosylmethionine, and when partially purified components were incubated with [methyl-3H]S-adenosylmethionine, label was incorporated specifically into EF-2. Hydrolysis of labeled EF-2 yielded diphthine (the unamidated form of diphthamide) and a single chromatographically separable labeling intermediate. We conclude that the S-adenosylmethionine:EF-2 methyltransferase adds at least the last two of the three methyl groups present in diphthine and that this modification is sufficient to create diphtheria toxin sensitivity. Evidence is also presented for the existence of an ATP-dependent amidating enzyme which catalyzes the final step in the biosynthesis of diphthamide in EF-2.     

The histidine residue of codon 715 is essential for function of elongation factor 2

Several mutant cDNAs of elongation factor 2 (EF-2) were constructed by site-directed mutagenesis and their products expressed in mouse cells were investigated. Amino acid substitution for the histidine residue of codon 715, which is modified post-translationally to diphthamide, resulted in non-functional EF-2 and this substitution did not render EF-2 resistant to Pseudomonas aeruginosa exotoxin A, which inactivates EF-2 transferring ADP-ribose to the diphthamide residue. These non-functional EF-2s with replacements of the histidine-715 residue showed various extents of inhibition of protein synthesis by competing with functional EF-2 in vivo. These results suggest that histidine-715 is essential for the translocase activity of EF-2 and that the region around diphthamide functions in recognition of, and/or binding to ribosomes. Substitution of proline for the alanine-713 residue and substitution of glutamine for the glycine-717 residue converted EF-2 to partially toxin-resistant forms. Two-dimensional gel analysis with fragment A of diphtheria toxin of these toxin-resistant EF-2s revealed that their ADP-ribosylations by toxin were much less than that of wild-type EF-2.     

Diphtheria toxin-resistant mutants of Saccharomyces cerevisiae.

We developed a selection procedure based on the observation that diphtheria toxin kills spheroplasts of Saccharomyces cerevisiae (Murakami et al., Mol. Cell. Biol. 2:588-592, 1982); this procedure yielded mutants resistant to the in vitro action of the toxin. Spheroplasts of mutagenized S. cerevisiae were transformed in the presence of diphtheria toxin, and the transformed survivors were screened in vitro for toxin-resistant elongation factor 2. Thirty-one haploid ADP ribosylation-negative mutants comprising five complementation groups were obtained by this procedure. The mutants grew normally and were stable to prolonged storage. Heterozygous diploids produced by mating wild-type sensitive cells with the mutants revealed that in each case the resistant phenotype was recessive to the sensitive phenotype. Sporulation of these diploids yielded tetrads in which the resistant phenotype segregated as a single Mendelian character. From these observations, we concluded that these mutants are defective in the enzymatic steps responsible for the posttranslational modification of elongation factor 2 which is necessary for recognition by diphtheria toxin.     

Targeted introduction of a diphtheria toxin-resistant point mutation into the chromosomal EF-2 locus by in vivo homologous recombination.

We have introduced a specific point mutation into the gene for chromosomal elongation factor 2 (EF-2) in Chinese hamster ovary cells (CHO-K1) by in vivo homologous recombination. To obtain a selectable construct for gene-targeting, we modified a diphtheria toxin-resistant mutant EF-2 gene (Gly717 to Arg) by deleting its promoter and first exon so that homologous recombinants could be distinguished from randomly integrated transformants, and also by inserting a second positive selection marker, the neomycin-resistance gene, into the 3'-flanking region to increase the selective accuracy. More than 80% of the clones surviving after selection for resistance to both the toxin and neomycin were the desired homologous recombinants in which the wild-type, toxin-sensitive EF-2 gene was replaced by the modified gene giving resistance to both the toxin and neomycin. This result shows that the specific point mutation was co-introduced with a second selective marker into an endogenous chromosomal gene and that the modified gene was expressed.     

Autoradiographic detection of mutation to exotoxin-A resistance in mouse fibroblasts treated with ethyl methanesulfonate, X-rays and ultraviolet light

P. aeruginosa exotoxin-A (PE) blocks protein synthesis in mammalian cells by inactivating elongation factor 2 (EF-2). Toxin-resistant mutant cells can be detected autoradiographically, in cultures grown on microscope coverslips in the presence of PE, and then exposed to [3H]leucine. The frequency of PE-resistant cells detected by the autoradiographic assay in non-mutagenized cells of the established mouse cell line LTKA is 9.7 +/- 0.6 X 10(-5). Upon treatment with ethyl methanesulfonate (EMS), X-rays or ultraviolet (UV) light it increases in a dose-dependent fashion. The mutational nature of the resistance detected by the assay is indicated by its clonal inheritance, and by the dose-dependent increase in the frequency of resistant cells after mutagenesis. On the basis of the high frequency of PE-resistant cells detected by the autoradiographic assay, and their cross-resistance to diphtheria toxin (DT), we suggest that the PE-resistant mutants detected by the autoradiographic assay are of class II, i.e., they are altered in the structural gene for EF-2. The autoradiographic assay for PE resistance is similar to that for DT resistance, but is applicable also to mouse cells, which are naturally resistant to DT. Being independent of colony formation, the autoradiographic assay for PE resistance can be used with non-dividing cells, either in vitro or in vivo.     

Response of cultured mammalian cells to the exotoxins of Pseudomonas aeruginosa and Corynebacterium diphtheriae: differential cytotoxicity.

The sensitivities of 21 mammalian cell lines to the exotoxins of Pseudomonas aeruginosa and Corynebacterium diphtheriae were measured. Each line exhibited 1-4 log differences in sensitivities to the two toxins. No species-specific sensitivities were noted for Pseudomonas exotoxin while diphtheria exotoxin was most potent in cells of monkey origin, followed by human and hamster cells. Rat- and mouse-derived cell lines were very insensitive to diphtheria exotoxin. The rates of cellular intoxication by both toxins exhibited apparent first-order kinetics and were indistinguishable from one another when equipotent doses were used. Our preparation of diphtheria exotoxin appeared to have a slightly higher ADP-ribosylating efficiency than did Pseudomonas toxin. However, neither toxin exhibited cell line-specific differences in ribosylating efficiencies which could have explained the wide range in potencies for intact cells. Our results suggest that there are significant differences in the mechanisms of cellular intoxication by Pseudomonas and diphtheria exotoxins and that these differences probably exist in the attachment or internalization stages of toxin action.     

Codominant and recessive 9-beta-D-arabinofuranosyladenine-resistant mutations of baby hamster cells

9-beta-D-Arabinosyladenine (araA)-resistant mutants of baby hamster kidney (BHK) cells can be classified into 3 classes. In order to gain a better understanding of the mechanism(s) of resistance and the biochemical basis of cytotoxicity of various purine nucleosides, cell hybrids of the mutant and wild-type cells were made and analyzed. The class I araA-resistant, adenosine-kinase-deficient (AK-) allele was shown to be recessive to the wild-type araA-sensitive (AK+) gene. The class II mutant allele, which encodes an altered ribonucleoside diphosphate reductase, was shown to be codominant. The class III mutants show multiple phenotypes, araAr/dAdor/adenosine sensitive (Ados) and alteration in AK activity. The araA- and dAdo-resistant alleles of araS10d, ara-16c, and ara-19a in class III mutant/wild-type hybrid cells are all recessive to the wild-type allele, consistent with a common mechanism of resistance. In contrast the Ados allele of ara-S10d is dominant while those of ara-16c and ara-19a are recessive. The difference may be a reflection of two distinct mechanisms of enhanced Ado sensitivity or, alternatively, it suggests that the sensitivity of the hybrids to Ado is highly dependent on the level of AK activity.     

The frequency of mutants in human fibroblasts UV-irradiated at various times during S-phase suggests that genes for thioguanine- and diphtheria toxin-resistance are replicated early

Human cells deficient in rate of excision repair of DNA damage induced by UV-radiation, i.e., xeroderma pigmentosum (XP) cells, are much more sensitive to the mutagenic effect of UV than are cells from normal persons. The lower frequency of mutants in the latter cells has been attributed to the fact that, unlike XP cells, they excise most of the potentially mutagenic lesions before these can be converted into mutations. If semi-conservative DNA synthesis on a template still containing unexcised lesions is responsible for introducing mutations and if replication of the gene of interest, e.g., hypoxanthine (guanine)phosphoribosyltransferase (HPRT) for thioguanine resistance or the elongation factor 2 (EF-2) for diphtheria toxin resistance, occurs at a particular time during S-phase, it should be possible to shorten the time available for such repair by synchronizing cells and irradiating them just as the gene is to be replicated. The predicted result would be a much higher frequency of mutants at one part in the S-phase than at other times. To test this, cells were synchronized using the alpha-polymerase inhibitor aphidicolin, which blocks cells at the G1/S border. Autoradiography, cytofluorimetry, and incorporation of tritiated thymidine studies showed that DNA synthesis started immediately after release from aphidicolin and was completed in 8-10 h. Cells irradiated with 6 J/m2 at various times post-release were assayed for survival and mutations. The frequency of thioguanine- or diphtheria toxin-resistant cells in the population was highest in cells irradiated during the first fifth of the S-phase, i.e., 0-1.5 h post-release. It was significantly lower in cells irradiated at later times. In contrast, UV-induced cytotoxicity showed no significant time dependence during S-phase. These data suggest that the HPRT and EF-2 genes are replicated early in S-phase.     

Mutation induction by okadaic acid, a protein phosphatase inhibitor, in CHL cells, but not in S. typhimurium.

Okadaic acid (OA) is a specific and strong inhibitor of protein phosphatase 1 and 2A present in eukaryotes, and a potent promoter of carcinogenesis in mouse skin. In this study, we examined the mutagenicity of OA. OA did not induce mutations in S. typhimurium TA100 and TA98, with or without a microsomal metabolic activation system. However, it was strongly mutagenic to Chinese hamster lung (CHL) cells without a microsomal activation system, as shown using diphtheria toxin (DT) resistance (DT^r) as a selective marker. Treatment of CHL cells with OA at 17.5 ng/ml induced 164 DT^r mutants per 10⁶ survivors. A plot of the mutation frequency against the OA concentration gave a concave curve, and the mutant frequency was calculated to be 5500/10⁶ survivors/μg, with OA in the dose range of 10–15 ng/ml. This value was about 680 times that of ethyl methanesulfonate (EMS), and comparable to that of 2-amino-N⁶-hydroxyadenine, one of the strongest knowon mutgens. Elongation factor 2 (EF-2) obtained from 4 DT^r clones was not ADP-ribosylated by DT fragment A. PCR-direct sequencing revealed that the hot spot of EF-2 for EMS mutagenesis in CHO-K1 cells, the first letter of codon 717, was not a t spot for OA mutagenesis in CHL cells.     

An in situ assay for induced diphtheria-toxin-resistant mutants of diploid human fibroblasts

The sensitivity of diploid human fibroblasts to the cytotoxic effects of diphtheria toxin (DT) depended on the cell growth status. Exponentially growing cells treated with 10^?3-1 lethal flocculating units (LF) of DT/ml for 4 days survived with a frequency of 4 × 10^?4. However, the DT-resistant phenotype of colonies isolated under these conditions was not stable. When the growth of the cells had been arrested by confluence or deprivation of serum growth factors prior to treatment with DT (4 days, 10^?3-0.6 LF/ml), the survival decreased to 2 × 10^?6 and the resistance of isolated colonies was stable. An in situ assay for induced DT-resistant mutants was developed in order to avoid problems associated with the possible reduced viability of the mutants relative to that of wild-type cells. A reproducible and linear dose response was obtained for the induction of DT-resistant mutants by ethylnitrosourea. The mutants were induced with high frequency by this compound (e.g., 10^?3 mutants/viable cell at a 37% survival dose); complete expression of the mutant phenotype occurred after 6 generations of growth under nonselective conditions. Isolated mutant colonies showed stable resistance to DT and were cross-resistant to Pseudomonas aeruginosa exotoxin A.     

Structure and expression of elongation factor 2 gene during development of Dictyostelium discoideum

A cDNA library constructed from poly(A)+ RNA isolated from Dictyostelium discoideum cells at 12 h of development was screened with the hamster elongation factor 2 (EF-2) cDNA. Several different cDNA clones which hybridized were isolated after a second screening. A cDNA clone representing the 5'-end of the mRNA was obtained by primer extension. By comparing the amino acid sequence deduced from the nucleotide sequences of these clones with that of hamster EF-2, we found enough homology between them to conclude that the isolated clones were complementary to the mRNA of D. discoideum EF-2. The N terminus which is the GTP-binding domain and the C-terminal half where it interacts with a ribosome showed a high degree of homology. The amino acid sequence of the carboxyl half includes that it contain a site of ADP-ribosylation by diphtheria toxin. From the Northern blotting analysis, the size of the mRNA was estimated to be 2.6 kilobases. The expression of the mRNA was high in vegetative cells, became maximal at the aggregation stage, and decreased thereafter through development. Upon differentiation of prespore and prestalk cells, the mRNA was highly enriched in the former over the latter. ADP-ribosylation assay of EF-2 protein by diphtheria toxin showed nearly the same developmental changes for the protein as the mRNA. However, prestalk cells were found to contain the same amount of the protein as prespore cells. The Southern blot analyses indicated that the gene encoding EF-2 is unique.