(Received for publication, January 27, 1995; and in revised form, July 31, 1995)
From the
Both diphtheria toxin and Pseudomonas exotoxin A inhibit eukaryotic protein synthesis by ADP-ribosylating diphthamide, a posttranslationally modified histidine residue present in the elongation factor 2 (EF-2) protein. Elongation factor 2 cannot be ADP-ribosylated by the toxins unless this histidine is modified. In this report we identify three new point mutations in toxin-resistant alleles of the Chinese hamster ovary cell elongation factor 2 gene. The mutations resulted in amino acid substitutions at positions 584 (serine to glycine), 714 (isoleucine to asparagine), and 719 (glycine to aspartic acid). All three amino acid substitutions prevented the biosynthesis of diphthamide. The amount by which the toxins reduced protein synthesis in each of these mutant cell strains suggested that all three mutations also either impaired the function of EF-2 or reduced its steady state level in the cytoplasm. Western blot analysis showed that equal amounts of EF-2 were present in each of the cell strains, indicating that the mutations impaired the catalytic function of EF-2.
Elongation factor 2 (EF-2) ()is responsible for the
GTP hydrolysis-dependent translocation of eukaryotic and
archaebacterial ribosomes during polypeptide synthesis(1) . Its
counterpart in eubacteria, mitochondria, and chloroplasts is elongation
factor G (EF-G). The primary distinction between EF-2 and EF-G is the
presence in EF-2 of a unique posttranslationally modified histidine
residue
2-[3-carboxyamido-3-(trimethylammonio)propyl]histidine which
has been characterized in detail and given the trivial name
diphthamide(2, 3) . Posttranslational modification of
histidine to diphthamide requires the participation of at least six
gene products in yeast(4) . Despite the evolutionary
conservation of this complex enzymatic pathway in all eukaryotes and
archaebacteria, the role of diphthamide in the cell remains enigmatic.
Both diphtheria toxin (DT) and Pseudomonas exotoxin A (PEA)
catalyze transfer of the ADP-ribose moiety of NAD to
N1 of the histidine imidazole ring of diphthamide(5) .
Toxin-resistant mutants lacking enzyme activities required for
diphthamide synthesis have been isolated and studied in detail in both
yeast and mammalian cells (6, 7, 8, 9) and show no phenotypic
alterations other than resistance to DT and PEA. Several mutations in
the EF-2 structural gene can also result in the production of
toxin-resistant
EF-2(10, 11, 12, 13) . Mutant cell
lines with mutations in the EF-2 gene can be distinguished from those
with mutations in genes encoding enzymes required for diphthamide
synthesis by somatic cell dominance hybridization and complementation
analyses, dose-response assays with DT and PEA, and in vitro ADP-ribosylation of EF-2 by
DT(6, 8, 14, 15, 16) .
In the present study we screened toxin-resistant mutant strains derived from CHO-K1 cells to identify EF-2 gene mutations. We used restriction fragment length polymorphism (RFLP) analysis and DNA sequencing to characterize point mutations in the EF-2 gene that are responsible for toxin resistance. We determined if these mutations disrupted the biosynthesis of diphthamide. Expression of cloned mutant and wild-type EF-2 in wild-type CHO-K1 cells confirmed that each point mutation was sufficient to account for the toxin-resistant phenotype.
RFLP analysis
for an MboII-site indicative of a mutation in codon 717.
Genomic DNA was harvested from 4 10
cells. A
polymerase chain reaction (PCR) was used to amplify exon 11. Primers
were 11F = 5`-cctgggtgttccttctc-3` (bases 4812-4828;
numbered as in GenBank(TM) (31) entry J03200(26) )
and 11R = 5`-gggtgaatgggcgccacacaggcagg-3` (complimentary to
bases 5128-5103), generating a 316-base pair product. The PCR
reactions contained 0.75 µM concentration of each primer,
PCR mix (200 µM of each dNTP, 3 mM
MgCl
, 50 mM KCl, 10 mM Tris-HCl, pH 8.3,
100 µg/ml bovine serum albumin, 0.05% Triton X-100), 0.1 µg of
genomic DNA template and 1 unit of Taq polymerase (Life
Technologies, Inc.) in a 100-µl reaction volume, overlaid with 50
µl of sterile mineral oil. Reaction mixtures were cycled between 94
°C for 30 s, and 72 °C for 90 s, for a total of 25 cycles,
followed by a 7-min incubation at 72 °C and storage at 4 °C.
The PCR product generated from a wild-type allele contains no MboII recognition sequence. The product derived from an allele
containing a G to A transition in codon 717 contains one MboII
site which results in cleavage into two fragments of 180 and 136 base
pairs. Ten µl of each PCR mixture were digested with MboII
and resolved on a 5% polyacrylamide gel, stained with ethidium bromide,
and photographed under UV light. The validity of this method was
confirmed by Southern blots of MboII-digested genomic DNA
performed as described in(11) .
Figure 1: Plasmids used in this study. Cloning strategy showing restriction maps of PCR products and plasmids. The plasmids were constructed as described under ``Experimental Procedures.''
Figure 2:
Intact cell assay for inhibition of
protein synthesis by DT. Incorporation of S-labeled amino
acids into trichloroacetic acid-precipitable protein during a 1-h pulse
after 24-h incubation in toxin is plotted as percentage of toxin-free
control.
, CHO-K1;
, RE1.22;
, RE1.22c;
,
RPE.10;
, R1.41;
, K1W1.P17;
, RPE.11a. Each data
point represents the average of three separate vials of cells. The
values for
S incorporation in the toxin-free controls
ranged from 22,775 ± 247 cpm for RE1.22 to 19,484 ± 422
cpm for RPE.11a in this assay.
Dose-response curves were generated for each mutant cell
strain using an intact cell assay(8) . With this assay, in
which incorporation of radiolabeled amino acids into protein is
measured after exposing cells to a range of concentrations of DT or
PEA, it is possible to classify toxin-resistant mutant strains into
three general categories: (i) class I mutants are defective in some
aspect of the entry of toxins into the cytosol, and protein synthesis
is completely inhibited by high concentrations of toxin; (ii) class IIa
mutants either lack enzymes needed to modify histidine 715 to
diphthamide (MOD) or have mutations in both copies of
the elongation factor 2 gene, and protein synthesis is unaffected by
any concentration of toxin; (iii) class IIb mutants have mutations in
one of the two copies of the elongation factor 2 gene, and protein
synthesis is typically reduced to 47-56% of control, by high
concentrations of toxin(8, 14, 22) . In this
study, 12 mutant cell strains (Table 1) produced typical class
IIb dose-response curves as illustrated in Fig. 2. Incorporation
of amino acids into protein in the presence of high doses of DT was
reduced to between 47.5 and 56% of toxin-free controls. In the case of
the mutant cell strains RPE.11a, R1.41, and K1W1.P17, protein synthesis
was reduced well below 50%, but was never reduced to less than 15% ( Table 1and Fig. 2). Wild-type CHO-K1, and class IIa
RE1.22c cell strains, were included as controls. Thus, the mutant
strains which are the focus of this study were confirmed to be class
IIb mutants.
Each class IIb mutant was screened for the presence of
an MboII restriction endonuclease recognition sequence (GAAGA)
indicative of a G to A transition in codon 717 of the EF-2 gene (GAGGA
GAAGA)(11, 13) . This mutation, which was
originally reported in four out of four independent isolates of
mutagenized CHO-K1 cells selected for toxin resistance(13) ,
prevents the first step in the biosynthesis of
diphthamide(11) . Fig. 3presents results from a
representative PCR RFLP analysis. Ten of the 13 class IIb mutant cell
strains analyzed possessed an MboII site indicative of a G to
A transition in codon 717, and three did not. Wild-type CHO-K1,
heterozygous RE1.22 and homozygous RE1.22c (11) were included
as controls. The three strains which did not possess an MboII
site, were the same three that showed significantly less than 50%
protein synthesis in the intact cell assay. This suggested either that
their toxin-resistant EF-2 was functionally impaired or that it was
less stable than wild-type EF-2.
Figure 3: MboII restriction digest of exon 11 PCR products. A 316-base pair region, which included exon 11 of the EF-2 gene, was amplified from genomic DNA harvested from wild-type and mutant strains of CHO-K1 cells as described under ``Experimental Procedures.'' The PCR products were digested with MboII, separated on a 5% nondenaturing polyacrylamide gel, and stained with ethidium bromide.
After determining that DT-resistant cell strains RPE.11a, R1.41, and K1W1.P17 did not have a G to A transition in codon 717, we identified the mutations in their EF-2 alleles responsible for resistance to DT. Because the histidine precursor of diphthamide is encoded by exon 11 of the EF-2 gene, we first sequenced exons 11-13 from each of the mutants. PCR amplification of this region of genomic DNA using primers which hybridize to the previously published cDNA sequence (25) proved problematic. The PCR product produced by such primers always produced a product that was shorter in length than would be predicted for the gene, but identical in length to the product predicted for cDNA. Pretreatment of the template genomic DNA with RNase did not eliminate this artifact. The shorter product is most likely derived from an EF-2 pseudogene, based on partial sequencing of the artifact band (data not shown). Use of primers complementary to sequences in intron 10 and exon 13 enabled us to successfully amplify the correct region of the EF-2 gene. The intron 10-exon 13 PCR products from RPE.11a, R1.41, and K1W1.P17 were ligated into M13mp18 and M13mp19 and single-stranded DNA from several clones of each was harvested and sequenced. Roughly half of the R1.41 clones contained a T to A transversion in the second position of codon 714, resulting in a change from isoleucine to asparagine in the predicted protein, the other half were derived from the wild-type allele. Roughly half of the clones derived from K1W1.P17 contained a G to A transition in the second position of codon 719, resulting in a glycine to aspartic acid substitution in the predicted protein. No mutation was found in the intron 10-exon 13 clones derived from RPE.11a. We then cloned full-length cDNA from RPE.11a, in order to avoid sequencing introns. RPE.11a cDNA contained an A to G transition in the first position of codon 584, resulting in a serine to glycine substitution in the predicted protein.
Having demonstrated that RPE.11a, R1.41, and K1W1.P17 each possessed a unique mutation resulting in an amino acid change in EF-2, we analyzed the EF-2 protein from each cell strain to determine the effect of these mutations on the posttranslational biosynthesis of diphthamide. The posttranslational modification of histidine 715 to diphthamide proceeds in several steps and four intermediate forms have been identified. The intermediates are 2-(3-carboxy-3-aminopropyl)histidine and the unamidated 1-, 2-, and 3-methylammonio forms of diphthamide. Each form can be resolved chromatographically, all eluting before histidine with the buffer system used(7, 16) .
Amino acid analysis of EF-2
tryptic peptides, that contained the histidine 715 residue, was carried
out for each mutant. Peptides from ADP-ribosylated and nonribosylated
EF-2 from CHO-K1 cells were included as controls. Because each mutant
cell strain is heterozygous at the EF-2 locus and produces both
toxin-sensitive and toxin-resistant EF-2, the EF-2 in cellular extracts
was first ADP-ribosylated to completion with an excess of
NAD and DT to ADP-ribosylate all of the wild-type
EF-2. This enabled us to determine by chromatographic analysis if the
product of the mutant allele possessed diphthamide that could not be
ADP-ribosylated by toxin. Amino acid analysis revealed that only
ribosyl-diphthamide and histidine were recovered from the EF-2
extracted from each mutant. No intermediate forms in the diphthamide
biosynthetic pathway were detected (Fig. 4). A small but
significant amount of ADP-ribosylated diphthamide was detected in
extracts of CHO-K1 cells that had not been exposed to toxin. This is in
agreement with other studies, which have shown that a small percentage
of EF-2 in normal cells is ADP-ribosylated by an endogenous
enzyme(23) , apparently in a cell cycle-dependent
manner(24) .
Figure 4:
Amino acid analysis of
His-715/diphthamide. Elution profiles of
[H]histidine (A-E)- or
[
C]NAD
(F)-labeled
enzymatic digests of EF-2 tryptic peptide. A, CHO-K1. B, CHO-K1 ADP-ribosylated with DT adn NAD
. C, R1.41 ADP-ribosylated with DT and NAD
. D, RPE.11a ADP-ribosylated with DT and NAD+. E,
K1W1.P17 ADP-ribosylated with DT and NAD
. F,
CHO-K1 ADP-ribosylated with DT and
[
C]NAD
. The ADP group was
removed from ADP-ribosylated diphthamide by phosphodiesterase during
the enzymatic digestion of the tryptic peptide, leaving
ribosyl-diphthamide.
Because the tryptic peptide contains one other
histidine residue in addition to histidine 715, and the procedure for
preparing the tryptic peptide does not eliminate all other
histidine-containing peptides, a large histidine peak was always
detected. Neither the size of the ribosyl-diphthamide peak, nor the
ratio of histidine to ribosyl-diphthamide can be used to calculate the
amount of EF-2 present in cell extracts. Thus, these analyses did not
prove that the mutant EF-2 contained only an unmodified histidine 715.
The possibility remained that the mutant EF-2 contained diphthamide
which had been ADP-ribosylated, but was still able to function in
protein synthesis. Analysis of the amount of
[C]ADP-ribose incorporated into EF-2 by DT in
cell extracts from each of the mutants eliminated this possibility.
Mutant cell strains RPE.11a, K1W1.P17, and R1.41 each possessed half as
much ADP-ribosylatable EF-2 per mg of cellular protein as did CHO-K1 (Fig. 5).
Figure 5:
ADP-ribosylatable EF-2. Cytosolic extracts
prepared from CHO-K1 cells and mutant strains were ADP-ribosylated with
[C]NAD
and DT as described
under ``Experimental Procedures.'' Results are plotted as a
percentage of the ADP-ribosylated EF-2 in CHO-K1 cells (114 pmol/mg of
extract protein). Each bar represents the average of three
separate determinations, the error bars indicate the maximum
value obtained.
In cell strains RPE.11a, R1.41, and K1W1.P17, protein synthesis was reduced to 20.8, 27.3, and 24% of control, respectively, by high concentrations of diphtheria toxin (Table 1). In order to address the issue of whether the mutant, toxin-resistant EF-2 produced from the mutant allele in cell strains RPE.11a, R1.41, and K1W1.P17 was functionally impaired, or was less stable, we performed quantitative Western blots to assess the total amount of EF-2 present in each of these strains (Fig. 6). There was no significant difference in the total amount of EF-2 detected in these cell strains, compared with CHO-K1 and RE1.22, indicating that the mutant EF-2 is not degraded more rapidly or produced in lower quantity than the wild-type EF-2. We therefore conclude that these mutations reduce the catalytic efficiency of the EF-2 protein.
Figure 6:
Immunodetection of EF-2 by Western
blotting. Cytosolic cell extracts were prepared from cells, and the
protein concentration in each extract was determined. Five, 10, 15, and
20 µg of protein from each extract was assayed. Extract from CHO-K1 (lane 1, ) was compared with extracts from RE1.22 (lane 2,
), RPE.11a (lane 3,
), R1.41 (lane 4,
) and K1W1.P17 (lane 5,
).
The radioactivity in each band was quantified using a Bio-Rad GS-250
molecular imager and is expressed in pixel density units per band after
a 12-h exposure to the phosphor screen. Data points for the 15
µg/lane sample are the average of two separate gel
loadings.
In order to prove that the point mutations we identified were responsible for the DTX-resistant phenotype of these cell strains, we constructed EF-2 cDNA expression vectors which differed from each other only by these single point mutations by domain swapping, as described under ``Experimental Procedures'' (Fig. 1). We first sequenced a clone of RE1.22c to ascertain that no reverse transcription or PCR misincorporations were present. The sequence of the clone derived from cell strain RE1.22c was identical to the sequence with GenBank(TM) accession number M13708 (25) with two exceptions: (i) position 1400 in M13708 is A, whereas it is C in RE1.22c and also C in the genomic clone with accession number J03200. The authors of J03200 (26) found a C at this position in the genomic DNA sequence and speculated that the cDNA sequence in M13708 might have had an error at this position. Our sequence data indicate that the C is correct, and the A in M13708 is an error. (ii) In the first position of codon 717, we found a G to A transition, predicted by the presence of an MboII site in exon 11.
CHO-K1 cells were transfected with wild-type and toxin-resistant EF-2 cDNA in the pCEP4 expression vector. The expression constructs were constructed by replacing the 3` end of the fully sequenced RE1.22c cDNA clone with the 3` end from wild-type or mutant clones which had been sequenced (Fig. 1). The region swapped into each construct was then sequenced again. Twenty-four hours after transfection, 600 units/ml of hygromycin B was added. Within 7 days untransfected control cells exposed to hygromycin had died, and transfected cells had formed colonies of 20-60 cells. Response to DT was then measured in an intact cell assay. Cells transfected with mutant EF-2 showed significant levels of incorporation of labeled amino acids into protein after exposure to high concentrations of DT, while those transfected with wild-type EF-2 were not significantly different from untransfected controls (Table 2). The results of these assays appear to agree very well with results previously published for mouse L cells transiently transfected with mutant EF-2 cDNA in a plasmid utilizing the SV40 early promoter(12, 13) . However, it is not possible to make a direct comparison, because toxin-free controls were not included in the previous studies.
Although our results were in agreement with those previously published for expression of cloned EF-2 cDNA in mammalian cells, the level of protein synthesis obtained in high concentrations of toxin was lower than expected. In an attempt to achieve higher levels of expression than previously published(12, 13) , we used pCEP4, an expression vector with the cytomegalovirus enhancer/promoter, which has been reported to be severalfold more active than the SV40 promoter/enhancer (27) and we selected for hygromycin resistance, rather than assaying the whole population of transiently transfected cells. We had hoped that the recombinant EF-2 would be stably expressed at high levels. Our results indicate that in a population of cells transformed to hygromycin resistance, the level of EF-2 protein produced from the plasmid is less than 10% of that produced from the two functional EF-2 alleles in the CHO-K1 genome (Table 2). Transfections with the pMSG vector constructs, containing the murine mammary tumor virus promoter produced similar results (data not shown).
In order to select cells which stably expressed high levels of toxin-resistant EF-2, we exposed transfected populations, which had grown in hygromycin-containing medium for a period of 2 weeks, to PEA at a concentration of 100 ng/ml for 48 h. This exposure to PEA resulted in cessation of cell division or cell death in roughly 80-90% of the cells. The remaining cells grew into discrete colonies of varying sizes. One week after exposure to toxin, several individual colonies were picked and grown in toxin-free medium for 2 months. Intact cell assays were performed on these clonal lines, and their resistance to toxin was highly variable (Table 3). The resistance correlated with the size of the colonies from which each clone was picked.
We have characterized 13 mutant strains derived from CHO-K1 cells that are resistant to diphtheria toxin and Pseudomonas exotoxin A due to mutations in the elongation factor 2 gene. Ten of the strains have a G to A transition in the first position of codon 717 of the EF-2 gene, resulting in a glycine to arginine substitution in the protein. Three others have unique mutations: (i) a G to A transition in the second position of codon 719, resulting in a glycine to aspartic acid substitution in the predicted protein; (ii) a T to A transversion in the second position of codon 714, resulting in an isoleucine to asparagine change in the protein; (iii) an A to G transition in the first position of codon 584, resulting in a serine to glycine change in the protein. The mutant strains contained one-half as much ADP-ribosylatable EF-2 as parental cells and equal amounts of total EF-2. Only ribosyldiphthamide, but not free diphthamide or intermediate forms of diphthamide, was detected following ADP-ribosylation of the EF-2 in vitro. All four mutations prevent the addition of the backbone of methionine from S-adenosyl-methionine to the imidazole ring of histidine 715 in the posttranslational synthesis of diphthamide(2, 3) .
Although it is tempting to label codon 717 of the EF-2 gene as a mutational ``hot spot'' (13) due to the high proportion of mutations to toxin resistance which occurred in this codon, our results indicate that this may be an artifact of the method of selection. While mutations at sites other than the first position of codon 717 result in EF-2 that is resistant to ADP-ribosylation, they also may result in a rate of protein synthesis in the presence of toxin that is well below 50% of control. These cell strains grow much more slowly, if at all, in the presence of DT or PEA. Previous studies, using somatic cell fusions between wild-type and class IIb mutants, showed that the resulting hybrids could not survive in high levels of toxin, although they synthesized roughly 25% as much protein in the presence of toxin as they did in toxin-free medium(14) . Thus, these mutant cell strains may be overlooked when picking clones of toxin-resistant cells selected in toxin. Other investigators, using site-directed mutagenesis of the Saccharomyces cerevisiae EF-2 gene, have also recently identified new toxin-resistance mutations, all of which result in an increased doubling time, compared with cells expressing only wild-type EF-2(10, 28) . In these studies Kimata and Kohno (28) recovered nine amino acid substitutions at codon 699 (equivalent to codon 715 in CHO-K1 EF-2) that were not recovered by Phan et al.(10) and postulated that the difference in growth rates led to this differential recovery(28) .
A scan of the GenBank(TM) nucleotide sequence data base release 84.0 (31) shows that the EF-2 or EF-G gene or cDNA has been sequenced from 27 species, including that from eukaryotic mitochondria and chloroplast organelles. A multiple sequence alignment of the protein translations of these sequences allowed us to identify many regions that are conserved in all 27 sequences (data available in computer-readable format upon request). Fig. 7is a multiple sequence alignment of the two regions of EF-2/EF-G in which we found mutations. We chose sequences from five eukaryotes, two archaebacteria, and two eubacteria as representative. The mutation in codon 584, identified in cell strain RPE.11a, lies at the carboxyl-terminal end of one of the conserved regions of the protein (Fig. 7B). This mutation is of particular interest, because it is located far from the modified histidine. The three-dimensional structure of EF-G from Thermus thermophilus has recently been determined by crystallography(29, 30) . The structure shows that amino acid 584 is located close to amino acid 715, on the surface of domain 4 of the protein. Thus, in the biosynthesis of diphthamide one or both of the enzymes (16) required for the addition of the backbone of methionine to the imidazole ring of histidine 715 in EF-2 may interact directly with amino acid 584. Alternatively, alteration of amino acid 584 could possibly disrupt the native conformation of domain 4 and prevent the enzymes(s) from modifying histidine 715.
Figure 7: Multiple sequence alignment of EF-2/EF-G. EF-2 and EF-G protein sequences were obtained from GenBank(TM) and aligned using the University of Wisconsin Genetics Computer Group PILEUP program. Five eukaryotic sequences (Cricetulus griseus, U17362; Homo sapiens, Z11692; Drosophila melanogaster, X15805; Saccharomyces cerevisiae, M59369) are aligned with two archaebacterial (Methanococcus vannielii, X12384; Thermoplasma acidophilum, X56840) and two eubacterial (Micrococcus luteus, M17788; Escherichia coli, X00415) sequences. Fig. 6A shows the regions in which we report mutations. Amino acids which are conserved between all nine species are highlighted in black, and amino acids which are conserved between members of two of the three different ``domains'' (Eucarya, Archaea, Bacteria) (37) are highlighted in gray. Bold amino acids above the alignment indicate the mutations discussed in this study. Bold amino acids below the alignment indicate the mutations discussed in this study. Bold amino acids below the alignment indicate the E. coli EF-G mutations described in(32) . The histidine residue which is posttranslationally modified in eukaryotes and archaebacteria is indicated with an asterisk and highlighted in bold. Fig. 6B is a schematic representation of domains conserved between all nine of the proteins. The University of Wisconsin Genetics Computer Group PLOTSIMILARITY program was used with the Dayhoff table (38) to score similar as well as identical amino acids. Regions with a similarity score above 1.0 are indicated with thick boxes. Gray boxes indicate domains conserved among other GTP-binding proteins.
The mutation in codon 584 results in EF-2, which apparently functions less well in protein synthesis than wild-type EF-2, as indicated by levels of protein synthesis that are 20% of control, while expressing equal amounts of wild-type and total EF-2 ( Fig. 5and Fig. 6), when cell strain RPE.11a is grown in the presence of DT ( Fig. 2and Table 1). Two mutations that result in kanamycin resistance have been identified in the E. coli fusA gene that encodes EF-G(32) . Both of the fusA mutations result in colonies that exhibit very slow growth at 42 °C. These mutations are both located within eight amino acids of the mutation we identified in cell strain RPE.11a (Fig. 7). The similar effects of mutations in this region of both EF-2 and EF-G suggest a common conserved functional role for this domain of the protein.
Given that the mutations we have studied interfere with the biosynthesis of diphthamide, the location of mutations near histidine 715 is less surprising than the mutation in codon 584. A combination of multiple sequence alignments with the recently published three-dimensional x-ray crystallographic structure of EF-G(29, 30) shows that the diphthamide residue occurs in EF-2 at a position equivalent to the tip of domain four of the EF-G protein. Amino acids in this domain which are conserved between eukaryotic EF-2 and prokaryotic EF-G are primarily located on the surface of the three-dimensional structure. The multiple sequence alignments show that diphthamide occurs within a region of 100 amino acids near the carboxyl-terminal end of EF-2 that, like the GTP-binding domain near the amino-terminal end of the protein, is highly conserved (Fig. 7B). The conservation of amino acid sequences in the region surrounding the site of diphthamide indicates that this region is important to the function of EF-2.
The results in Table 3demonstrate that CHO-K1 cells can survive exposure to toxin even when protein synthesis is inhibited to less than 10% of control. Previous studies on cell-cell hybrids showed that cells could not survive in saturating doses (0.5 µg/ml) of DT if their level of protein synthesis was less than 25% of control(14) . These results have important implications regarding strategies for recovering new mutations in the EF-2 gene. A brief exposure to toxin, followed by selecting clones that grow at different rates, may allow recovery of a wider variety of mutations than is possible by selecting clones which grow when continuously exposed to toxin. With continuous exposure to toxin, colonies of class IIa mutants and class IIb mutants with mutations in codon 717 may overgrow the plate before other mutants can form colonies large enough to pick. Many of the clones of stable transfectants expressing EF-2 with a Gly to Arg mutation in codon 717 synthesized protein in the presence of toxin at a rate near 50% of toxin-free control (Table 3). It is unlikely that this is due to allelic replacement of one of the two chromosomal copies of the EF-2 gene via homologous recombination, because homologous recombination has previously been shown to be an extremely rare event(33, 34) .
Modified forms of DT and PEA are
being tested for use in cancer therapy. These toxins are used because
of their extreme toxicity to mammalian cells, and the ability to link
the toxic domain to different ligand-binding domains, such as
immunoglobulins specific for a certain cell type, either by chemical
cross-linking or by genetic engineering (35) . As with any
chemotherapy, a major concern is the development of a toxin-resistant
subset of target cells that escape the chemotherapy. The frequency of
occurrence of class II toxin-resistant mutants varies widely from one
cell line to another. Class II toxin-resistant cells arise at a
frequency of less than 5.6 10
in recently
cloned CHO-K1 cells(14) , but occur at a very high frequency
(approximately 1
10
) in Vero monkey kidney
cells. (
)Within the class II mutants, the ratio of EF-2 gene
mutations to mutations in genes responsible for the posttranslational
modification of His-715 to diphthamide is also variable. In CHO-K1
cells, both MOD
and EF-2 mutants have been isolated.
In S. cerevisiae only MOD
mutants have been
described, but this is due to the selection criteria used(36) ,
not an indication of a low rate of mutation in the EF-2 genes. In most
cultured mammalian cell lines, only class IIb mutants have been
isolated.
Cancer cells are often found to be defective in
one or more DNA damage and repair pathways, and this leads to the
possibility that the frequency of occurrence of DT and PEA-resistant
cells within a population of tumor cells will be highly variable from
tumor to tumor. More studies with human tumor cell lines will be
necessary to confirm this.
Despite extensive study, the role of diphthamide in eukaryotes is unknown. Also unknown is the extent to which EF-2 participates in the regulation of protein synthesis and the role that regulation of protein synthesis plays in growth, development, and cell cycle regulation. In this report we identify several new mutations which help map critical regions of the EF-2 protein and provide insight into modifications of selection procedures which will help to identify additional mutations that can be used to address these important issues. The mapping of bacterial EF-G mutations and eukaryotic EF-2 mutations to homologous regions of the protein sequence provides evidence that studies of the mechanism of ribosomal translocation in either system will be applicable to the other. The role of diphthamide, however, must be studied in archaebacterial or eukaryotic cells. Our expression studies demonstrate that transfected cell strains expressing different levels of toxin-resistant EF-2 can be produced. The strains should prove valuable in studies to determine the role EF-2 plays in regulating protein synthesis.
The nucleotide sequence(s) reported in this paper has been submitted to the GenBank(TM)/EMBL Data Bank with accession number(s) U17362[GenBank].