Noncomplementing Mutants Research Articles

Hydroxylamine-induced purple mutants (ad-3) in Neurospora crassa. II. Identification of genetic alteration at the molecular level.

There is evidence that most hydroxylamine (HA)-induced mutants in Neurospora arise from a GC-to-AT base-pair transition. Evidence supporting this hypothesis was obtained by a study of the revertibility of HA-induced mutants after treatment with specific chemical mutagens. Hydroxylamine-induced ad-3 mutants of Neurospora crassa were tested for revertibility after treatment with nitrous acid (NA), ethyl methanesulfonate (EMS), O-methyl-hydroxylamine (OMHA), and 2-methoxy-6-chloro-9- [3-(ethyl-2-chloraethyl) amino propyl-amino] acridine dihydrochloride (ICR-170). Mutants not reverting after treatment with these mutagens were tested for revertibility after treatment with N-methyl-N'-nitro-N-nitroso-guanidine (MNNG). An analysis was made of mutants induced in a 2-component heterokaryon. Of this material 78.2% reverted by an AT-to-GC transition; 10% by a GC-to-AT transition, 4.5% by base-pair insertion or deletion; and 7.3% reverted spontaneously but not after treatment with any of the 5 mutagens. Thus, HA produces mainly GC-to-AT transitions when the treatment is carried out under conditions promoting this specificity. This means that HA has a mutagenic specificity in higher organisms similar to that observed in phage and transforming DNA from B. subtilis. HA can be used as an analytical tool for mutagenesis in higher organisms to characterize the mutagenic activity of agents with an unknown mutation mechanism. HA can also be used to produce mutants with AT at the mutant site. The spectra of genetic alterations among the ad-3B mutants with nonpolarized or polarized complementation patterns and among noncomplementing mutants do not differ from each other, because the majority of HA-induced mutants result from the same genetic alteration.

Mutation discovery in the mouse using genetically guided array capture and resequencing

Forward genetics (phenotype-driven approaches) remain the primary source for allelic variants in the mouse. Unfortunately, the gap between observable phenotype and causative genotype limits the widespread use of spontaneous and induced mouse mutants. As alternatives to traditional positional cloning and mutation detection approaches, sequence capture and next-generation sequencing technologies can be used to rapidly sequence subsets of the genome. Application of these technologies to mutation detection efforts in the mouse has the potential to significantly reduce the time and resources required for mutation identification by abrogating the need for high-resolution genetic mapping, long-range PCR, and sequencing of individual PCR amplimers. As proof of principle, we used array-based sequence capture and pyrosequencing to sequence an allelic series from the classically defined Kit locus (approximately 200 kb) from each of five noncomplementing Kit mutants (one known allele and four unknown alleles) and have successfully identified and validated a nonsynonymous coding mutation for each allele. These data represent the first documentation and validation that these new technologies can be used to efficiently discover causative mutations. Importantly, these data also provide a specific methodological foundation for the development of large-scale mutation detection efforts in the laboratory mouse.

Effects of charged cluster mutations on the function of herpes simplex virus type 1 UL34 protein.

Herpes simplex virus type 1 (HSV-1) is a DNA virus that acquires an envelope by budding into the inner nuclear membrane of an infected cell. Recombinant HSV-1 lacking the U(L)34 gene cannot undergo this event. U(L)34 and U(L)31, another viral protein, colocalize in an infected cell and are necessary and sufficient to target both proteins to the inner nuclear envelope. In order to define and characterize sequences of U(L)34 that are necessary for primary envelopment to occur, a library of 19 U(L)34 charged cluster mutants and a truncation mutant lacking the putative transmembrane domain (DeltaTM) were generated. Mutants in this library were analyzed in a complementation assay for their ability to function in the production of infectious virus. Seven of the mutants failed to complement a U(L)34-null virus. The remainder of the mutants complemented at or near wild-type U(L)34 levels. Failure of a mutant protein to function might be the result of incorrect subcellular localization. To address this possibility, confocal microscopy was used to determine the localization of the U(L)34 protein in charged cluster mutants and DeltaTM. In transfection-infection experiments, all of the functional U(L)34 mutants and four of the six noncomplementing mutants localized to the inner nuclear envelope in a manner indistinguishable from that of wild-type U(L)34. All of the noncomplementing U(L)34 mutants mediated proper localization of U(L)31. Charged clusters critical for U(L)34 function are dispersed throughout the protein sequence and do not correlate well with highly conserved regions of the protein. These data suggest that U(L)34 has at least one function in addition to mediating proper localization of U(L)31 in infected cells and provide further support for the role of U(L)34 in mediating proper localization of U(L)31 in infected cells.

Site-specific mutations of FtsZ--effects on GTPase and in vitro assembly.

FtsZ, the major cytoskeletal protein in bacterial cytokinesis, assembles in vitro into protofilaments, which can further associate into sheets, bundles or tubes. We have constructed 16 site-directed mutants of E. coli ftsZ, and tested them for GTP hydrolysis and assembly in vitro, and for their ability to complement the temperature sensitive ftsZ84 mutation in E. coli. The mutants were grouped into three classes. Benign mutants, which mapped mostly to the front and back surface of the protofilament, were able to complement ftsZ84 in vivo and showed normal assembly in vitro. GTP contact mutations had less than 10% of wild type GTPase activity. They could all assemble in vitro, and several of these mutants could complement ftsZ84. A third, and newly discovered, class of mutations mapped to the sides of the protofilaments. These lateral mutants had mostly normal GTPase and assembly in vitro, but none of them complemented ftsZ84. The non-complementing mutants showed greatly reduced expression from the pBS58 vector, suggesting possible dominant negative effects. Several mutants with greatly reduced GTPase could still complement ftsZ84, suggesting that the high level of GTPase observed in vitro is not essential for in vivo function. All of the lateral mutants failed to complement ftsZ84, which suggests that these surfaces of the protofilaments are important for function in cell division. These lateral surfaces may mediate association of FtsZ protofilaments into pairs or small sheets, although their structure is apparently different from the sheets assembled in DEAE dextran or calcium.

Protein interaction maps for complete genomes based on gene fusion events.

A large-scale effort to measure, detect and analyse protein-protein interactions using experimental methods is under way. These include biochemistry such as co-immunoprecipitation or crosslinking, molecular biology such as the two-hybrid system or phage display, and genetics such as unlinked noncomplementing mutant detection. Using the two-hybrid system, an international effort to analyse the complete yeast genome is in progress. Evidently, all these approaches are tedious, labour intensive and inaccurate. From a computational perspective, the question is how can we predict that two proteins interact from structure or sequence alone. Here we present a method that identifies gene-fusion events in complete genomes, solely based on sequence comparison. Because there must be selective pressure for certain genes to be fused over the course of evolution, we are able to predict functional associations of proteins. We show that 215 genes or proteins in the complete genomes of Escherichia coli, Haemophilus influenzae and Methanococcus jannaschii are involved in 64 unique fusion events. The approach is general, and can be applied even to genes of unknown function.

Mutations in the dim-1 gene of Neurospora crassa reduce the level of DNA methylation.

Mutants that show reduced DNA methylation were identified in a mutant screen based on the assumptions that (i) the nucleoside analog 5-azacytidine (5-azaC) promotes the formation of potentially lethal DNA-methyltransferase adducts; (ii) reduction in DNA methyltransferase will decrease the sensitivity of cells to 5-azaC; and (iii) this potential selective advantage will be enhanced in mutants that are deficient in the repair of 5-azaC-induced DNA damage. Of fifteen potential repair mutants screened for sensitivity to 5-azaC, five (mus-9, mus-10, mus-11, mus-18, and uvs-3) showed moderately increased sensitivity and two (mus-20, mei-3) showed highly increased sensitivity. A mus-20 mutation was used to isolate three non-complementing methylation mutants. The mutations, named dim-1 (defective in methylation), reduced female fertility, reduced methylation by 40-50%, and altered patterns of methylation. In wild-type strains hypomethylation per se fails to alter methylation specificity. We demonstrate a growth-phase-dependent change in methylation patterns, detectable only in hypomethylated DNA from dim+ cultures. This may represent a growth-phase-dependent change in the relative amounts of distinct species of methyltransferase, one of which may be encoded by the dim-1 gene.

Homologous expression and purification of mutants of an essential protein by reverse epitope-tagging.

Purification of mutant enzymes is a prime requirement of biophysical and biochemical studies. Our investigations on the essential Escherichia coli enzyme glutaminyl-tRNA synthetase demand mutant enzymes free of any wild-type protein contamination. However, as it is not possible to express noncomplementing mutant enzymes in an E. coli glnS-deletion strain, we developed a novel strategy to address these problems. Instead of following the common tactic of epitope-tagging the mutant protein of interest on an extrachromosomal genetic element, we fused a reporter epitope to the 5' end of the chromosomal glnS-gene copy: this is referred to as 'reverse epitope-tagging.' The corresponding strain, E. coli HAPPY101, displays a normal phenotype, and glutaminyl-tRNA synthetase is exclusively present as an epitope-tagged form in cell-free extracts. Here we report the use of E. coli HAPPY101 to express and purify a number of mutant glutaminyl-tRNA synthetases independently of their enzymatic activity. In this process, epitope-tagged wild-type protein is readily separated from mutant enzymes by conventional chromatographic methods. In addition, the absence of wild-type can be monitored by immunodetection using a monoclonal antibody specific for the epitope. The strategy described here for expression and purification of an essential enzyme is not restricted to glutaminyl-tRNA synthetase and should be applicable to any essential enzyme that retains sufficient activity to sustain growth following reverse epitope-tagging.

Protein-protein interactions: methods for detection and analysis.

The function and activity of a protein are often modulated by other proteins with which it interacts. This review is intended as a practical guide to the analysis of such protein-protein interactions. We discuss biochemical methods such as protein affinity chromatography, affinity blotting, coimmunoprecipitation, and cross-linking; molecular biological methods such as protein probing, the two-hybrid system, and phage display: and genetic methods such as the isolation of extragenic suppressors, synthetic mutants, and unlinked noncomplementing mutants. We next describe how binding affinities can be evaluated by techniques including protein affinity chromatography, sedimentation, gel filtration, fluorescence methods, solid-phase sampling of equilibrium solutions, and surface plasmon resonance. Finally, three examples of well-characterized domains involved in multiple protein-protein interactions are examined. The emphasis of the discussion is on variations in the approaches, concerns in evaluating the results, and advantages and disadvantages of the techniques.

Protein-protein interactions: methods for detection and analysis.

The function and activity of a protein are often modulated by other proteins with which it interacts. This review is intended as a practical guide to the analysis of such protein-protein interactions. We discuss biochemical methods such as protein affinity chromatography, affinity blotting, coimmunoprecipitation, and cross-linking; molecular biological methods such as protein probing, the two-hybrid system, and phage display: and genetic methods such as the isolation of extragenic suppressors, synthetic mutants, and unlinked noncomplementing mutants. We next describe how binding affinities can be evaluated by techniques including protein affinity chromatography, sedimentation, gel filtration, fluorescence methods, solid-phase sampling of equilibrium solutions, and surface plasmon resonance. Finally, three examples of well-characterized domains involved in multiple protein-protein interactions are examined. The emphasis of the discussion is on variations in the approaches, concerns in evaluating the results, and advantages and disadvantages of the techniques.

Possible involvement of the yeast POLIII DNA polymerase in induced gene conversion

In Saccharomyces cerevisiae, three different DNA polymerase complexes, POLI, POLII and POLIII, are known to be involved in DNA replication. The catalytic subunit of POLIII is encoded by the essential CDC2 gene. The existence of different thermosensitive noncomplementing mutants of CDC2 offers the possibility of using a genetic approach to investigate the involvement of POLIII in induced gene conversion. When cdc2 heteroallelic cells were irradiated and incubated under restrictive conditions, almost no induction of thermoresistant cells could be detected, suggesting an essential role for POLIII in mitotic gene conversion events.

X-Ray-induced specific-locus mutations in the ad-3 region of two-component heterokaryons of Neurospora crassa. VIII. Dose-dependence of the overall spectrum

There is considerable controversy in the literature concerning the nature of X-ray-induced specific-locus mutations in various experimental organisms. To investigate this problem in Neurospora crassa a series of experiments (Webber and de Serres, 1965) was performed to study the induction-kinetics of X-ray-induced mutation in the adenine-3 ( ad-3) region of a two-component heterokaryon (H-12). Subsequent genetic analyses (de Serres, 1989a,b,c, 1990a), on a series of 832 mutants recovered in these experiments, have shown that 3 different classes of ad-3 mutants were recovered, namely gene/point mutations, multilocus deletions and multiple-site mutations. Complementation studies with a series of genetic markers that define 21 genetic loci in the ad-3 and immediately adjacent genetic regions have shown that ad-3 mutants classified as multilocus deletions result from the inactivation of a series of loci in the ad-3 and immediately adjacent regions of Linkage Group I, whereas multiple-locus mutations result from combinations of gene/point mutations and multilocus deletions. Analysis of the induction kinetics of these 3 different classes, after completion of the genetic characterization of all mutants (de Serres, 1990b) demonstrated that gene/point mutations increase linearly with X-ray dose, whereas multilocus deletions and multiple-site mutations increase as the square of X-ray dose. Further analysis of allelic complementation among the gene/point mutations at the ad-3B locus (de Serres, 1990c), demonstrated that the spectrum of complementation patterns was dose-dependent: complementing mutants with nonpolarized patterns decreased and noncomplementing mutants increased with increasing X-ray dose. There was little or no change with dose in the frequency of mutants with polarized patterns. In the present report, data from studies published previously have been utilized, along with additional data from the original X-ray experiments (12-5, 12-6, 12-7, and 12-10; see Webber and de Serres, 1965) to develop composite complementation maps of the X-ray-induced specific-locus mutations in the ad-3 and immediately adjacent regions as a function of X-ray dose. This analysis of the overall spectrum of X-ray-induced specific-locus mutations in the ad-3 region demonstrated marked dose-dependence and provides an explanation for the discrepancies in the literature with regard to specific-locus studies in different experimental organisms.

X-ray-induced specific-locus mutations in the ad-3 region of two-component heterokaryons of Neurospora crassa: VII. Genetic lesions resulting in gene/point mutations at the ad-3B locus have different dose-response relationships

Genetic characterization of X-ray-induced ad-3 mutants, induced in a two-component heterokaryon (H-12) of Neurospora crassa, has been performed to determine genotype, patterns of allelic complementation, and leakiness, anf to distinguish gene/point mutations from multilocus deletions and multiple locus mutations (de Serres, 1989c, 1990a). The arrau of genotypes in the classes and subclasses in the spectrum of ad-3 mutants induced by a mutagenic agent constitute its mutagenicity profile; for X-rays this profile consists of 29 different genotypes. In the present paper, the data on gene/point mutations induced at the ad-3B locus (designated ad-3B R mutations) have been tabulated as a function of X-ray dose to determine the dose-dependent relationships of complementing and noncomplementing mutants. This analysis has shown that the overall percentages of mutants showing allelic complementation and the percentages of complementing mutants with nonpolarized patterns (both leaky and nonleaky) and noncomplementing mutants were dose-dependent; the former class decreased with increasing X-ray dose, and the latter class increased with increasing X-ray dose. The percentages of complementing mutants with polarized patterns were X-ray dose-independent. In addition, an unexpectedly high frequency of mutants with nonpolarized complementation patterns are discontinuous and probably result from multiple-site mutations.

Classical and molecular genetic analyses of his-3 mutants of Neurospora crassa: II. Southern blot analyses and molecular mechanisms of mutagenicity

Previous studies (Overton et al., Mutation Res., 1989) on specific revertibility of 81 his-3 mutants have shown a correlation between complementation pattern and presumed genetic alteration similar to that shown by ad-3B mutants. In the present study, restriction enzyme analyses were used to further characterize the genetic alterations in individual his-3 mutants. The restriction fragment banding patterns of the majority of mutants were identical with that shown by wild-type 74-OR23-1A and were consistent with expectations based on previous data suggesting that they resulted from single base-pair alterations (Overton et al., Mutation Res., 1989). His-3 mutants with altered banding patterns were only found among those with polarized complementation patterns or noncomplementing mutants. One of the mutants with a polarized complementation pattern, 1-189-83, and another noncomplementing mutant, 1-189-85, are associated with genetic alterations proximal to the his-3 locus. In one other mutant, 1-226-565 (with a polarized complementation pattern), an insertion of approx. 2 kb has occurred in the proximal region of the his-3 locus. Two other mutants, 1-155-270 and 1-155-276 (both noncomplementing), contained a large insertion of approx. 12.8 kb in the proximal region of the his-3 locus.

Molecular and classical genetic analyses of his-3 mutants of Neurospora crassa: I. Tests for allelic complementation and specific revertibility

A collection of 81 his-3 mutants of Neurospora crassa was analyzed in assays for allelic complementation and specific reverbility. In these studies, the linearity of the complementation map of the his-3 cistron (Webber, 1965) was confirmed and mutants were classified as complementing with non-polarized or polarized complementation patterens, or non-complementing. In the assays for spontaneous or induced revertibility, 89% (71/80) of the mutants reverted either spontaneously or after treatment with the chemical mutagens N-methyl- N′-nitro- N-nitrosoguanidine, ethyl methanesulfonate, 2-methoxy-6-chloro-9- (3-[ethyl-2-chloroethyl]aminopropylamino) acridine dihydrochloride, nitrous acid or hyrdoxylamine. The frequency of revertible mutants among the non-polarized complementing mutants was 96% (45/47), and 79% (15/19) for the polarized complementing and 79% (11/14) for the non-complementing mutants. The results of these classical genetic assays for allelic complementation and specific revertibility suggest a correlation between complementation pattern and presumptive genetic alterations at the molecular level among his-3 mutants similar to that found with ad-3B mutants induced by nitrous acid (Malling and de Serres, 1967), ethyl methanesulfonate (Malling and de Serres, 1968), or ultraviolet (Kilbey et al., 1971).

Identification of temperature-sensitive mutants of vaccinia virus that are defective in conversion of concatemeric replicative intermediates to the mature linear DNA genome.

Pulsed-field gel electrophoresis was used to screen temperature-sensitive mutants of vaccinia virus for the ability to convert replicated viral DNA into mature linear 185-kilobase hairpin-terminated genomes. Of 30 mutually noncomplementing mutants tested, 5 displayed a temperature-sensitive defect in the resolution of the telomere fusion configuration within concatemeric replicative intermediates, resulting in a failure to convert such intermediates to the linear monomeric genome. Adjacent genomic units in the concatemeric arrays generated in these mutants were arranged in both tandem and inverted orientations. The observation that four of the five mutants had a severe general defect in the synthesis of the late class of viral proteins suggests that at least one late protein is directly required to resolve the telomere fusion intermediate to hairpin termini. The identification of such telomere resolution proteins should be facilitated by genetic and molecular characterization of resolution-defective mutants, such as C63, in which late protein synthesis is not severely affected.

A complementation analysis by parasexual recombination of Candida albicans morphological mutants.

Benomyl treatment (at 100 micrograms ml-1) of Candida albicans 1001, and other strains derived from it, determined the appearance of morphological mutants similar to those derived from UV irradiation treatment. A permanent alteration in the morphogenesis of these mutant strains determined their inability to grow by budding, to form oval yeast cells or blastospores (Y-phenotype) and their growth as long filamentous forms, mostly with the appearance of pseudomycelium, giving rise to rough colonies (R phenotype). In order to carry out a genetic complementation analysis, we isolated morphological mutants that carried other genetic markers (nutritional, conditional lethal) adequate for crosses by means of protoplast fusion. Wild-type hybrids of regular mononuclear oval yeast cells and smooth colonies were obtained by crossing pairs of complementing mutants, whereas hybrids from crosses of non-complementing mutants still retained their morphological alterations. Our results define two complementation groups, which represent two genes relevant for dimorphism, whose alteration interferes with the correct transition from blastospores to mycelium.

Characterization of the yeast HEM2 gene and transcriptional regulation of COX5 and COR1 by heme.

The respiratory deficiency of two noncomplementing mutants of Saccharomyces cerevisiae (C41 and N28) has been shown to be due to mutations in HEM2, the structural gene for delta-aminolevulinate dehydratase. The mutants are unable to convert delta-aminolevulinic acid to porphobilinogen and are not complemented by the hem2 mutant GL4 (Gollub, E. G., Liu, K.-P., Dagan, J., Adlersberg, M., and Sprinson, D. B. (1977) J. Biol. Chem. 252, 2846-2854). A gene capable of complementing the respiratory deficiency of C41 and N28 has been cloned by transformation of a hem2 mutant with a recombinant plasmid library of wild type yeast nuclear DNA. The sequence of the protein encoded by the cloned gene exhibits extensive homology to the recently reported sequence of human delta-aminolevulinate dehydratase (Wetmur, J. G., Bishop, D. F., Cantelmo, C., and Desnick, R. J. (1986) Proc. Natl. Acad. Sci. U. S. A. 83, 7703-7707). Several approaches were taken to study the effect of heme on transcription of PET genes known to code for subunit components of respiratory enzymes and of mitochondrial ATPase. The first involved measurements of the steady state levels of mRNAs for subunit 5 of cytochrome oxidase and the beta subunit of F1 ATPase in wild type and in a hem2 mutant. Secondly, transcription of the genes coding for the cytochrome oxidase and ATPase subunits as well as of the COR1 gene coding for the 44-kDa core 1 subunit of coenzyme QH2-cytochrome c reductase was quantitated by fusing the 5'-flanking and part of the coding region of each gene to the lacZ gene of Escherichia coli in vectors capable of integrating into yeast chromosomal DNA. The different lacZ fusions were integrated into nuclear DNA of a wild type strain and of hem2 mutants allowing expression of beta-galactosidase to be studied as a function of intracellular heme. These experiments indicate that the promoters of the genes for subunits of the respiratory complexes are regulated by heme. In contrast, the expression of the ATPase subunit appears to be heme-independent. Because neither subunit 5 of cytochrome oxidase nor the core 1 subunit of coenzyme QH2-cytochrome c reductase are hemoproteins, transcriptional regulation by heme may be a general mechanism for controlling the synthesis of mitochondrial proteins involved in respiration.

Trans-dominant defective mutants of simian virus 40 T antigen.

We constructed a collection of linker insertion mutants in the simian virus 40 (SV40) genome and studied several of these with changes limited to a part of the large T antigen gene corresponding to an amino acid sequence shared with other ATPases. Two of these mutants were found to have a novel phenotype in that they could not be complemented for plaque formation by a late-region deletion mutant. These two mutants, in contrast to other mutants in this region, were able to transform rat cells in culture at a frequency close to that of the wild-type gene. The noncomplementing mutants were found to be potent inhibitors of SV40 DNA replication despite the presence of wild-type T antigen in the transfected cells. This inhibition was shown to be the result of the introduced mutations in the large T antigen gene. We conclude that the large T antigens of the noncomplementing mutants can act as inhibitors of SV40 DNA replication.

A Drosophila melanogaster mutant resistant to a chemical analog of juvenile hormone

Methoprene, a chemical analog of juvenile hormone, is toxic when applied to late third-instar larvae of Drosophila melanogaster. Using an ethyl methane sulfonate mutagenesis screen, we have selected two noncomplementing mutants, one of which is nearly 100 times more resistant than wild-type to either methoprene or juvenile hormone III topically applied or incorporated into the diet. The mutation, named methoprene-tolerant ( Met), also confers resistance to methoprene-induced pseudotumor formation in larvae as well as to juvenile hormone III- or methoprene-induced vitellogenic oocyte development in adult females. Met adults show little or no cross-resistance to four other insecticides. The mutation was mapped by recombination to a location 35.4 on the X-chromosome and uncovered by chromosomes deficient for the region 10C2–10D4. Complementation was observed between Met and a lethal allele of the RNA polymerase II locus, which is also found in this region. Since the Met mutation also confers resistance to methoprene-induced abnormalities in adult cuticle formation, the autonomy of Met expression could be evaluated in flies mosiac for this mutation. Autonomous expression of Met was found both in abdominal cuticle as well as in external male genitalia. The characteristics of Met are consistent with those expected of a mutant having altered juvenile hormone reception in target tissue.

Protein synthesis in yeast. Identification of an altered elongation factor in thermolabile mutants of the yeast Saccharomyces cerevisiae.

Cell-free extracts from the wild type yeast strain (A364A) and from a group of noncomplementing mutants that are conditionally defective in translation were preincubated at a restrictive temperature prior to incubation at a permissive temperature for protein synthesis. Results of these experiments showed that upon exposure to the restrictive temperature (39 degrees C), all five of the noncomplementing mutants lost ability to incorporate amino acid into protein. The wild type parent strain retained better than 80% of the activity under identical conditions of heat treatment. Mutant extracts could be revived to incorporate amino acid by the addition of the purified yeast elongation factor 3. Factors 1 and 2 had no effect. The heat-treated extract from one mutant did not supplement the activity of the other mutant. Although all five of the mutants were inactivated by preincubation at 39 degrees C, each showed a variable rate and extent of thermolability. Heat-treated mutant extracts were fully active in polyphenylalanine synthesis with liver ribosomes but not with the yeast ribosomes. Since liver ribosomes do not require factor 3, this assay then confirms that factor 3 is the thermolabile component in this group of noncomplementing mutants.