Random Chemical Mutagenesis Research Articles

Accumulation of O 2-tolerant phenotypes in H 2-producing strains of Chlamydomonas reinhardtii by sequential applications of chemical mutagenesis and selection

The photoproduction of hydrogen by anaerobically induced algae is catalyzed by a bidirectional hydrogenase that is rapidly inactivated by oxygen. We isolated two generations of Chlamydomonas reinhardtii strains with H 2-evolving activities of up to 10 times the O 2-tolerance seen in the wild-type (WT). These isolates were generated by two sequential selections, consisting of random chemical mutagenesis, enrichment for H 2-metabolism clones following exposure to increasing amounts of O 2, and screening using a chemochromic sensor. The selected strains were characterized by two types of assays and classified as those that (a) can evolve H 2 following exposure to O 2 concentrations that inactive the WT strain and (b) in addition, are able to quickly reactivate H 2-production activity once O 2 is removed. These results suggest that O 2-tolerance can be increased by successive rounds of mutagenesis, selection, and screening, demonstrating that the WT phenotype can be improved by genetic means. Other results show that the hydrogenase is less sensitive to O 2 when it is actively catalyzing H 2 evolution.

Isolation and characterization of a mutant Chinese hamster ovary cell line that is resistant to Chlamydia trachomatis infection at a novel step in the attachment process.

Host factors involved in Chlamydia trachomatis pathogenesis were investigated by random chemical mutagenesis of Chinese hamster ovary (CHO-K1) cells followed by selection for clones resistant to chlamydial infection. A clonal mutant cell line, D4.1-3, refractory to infection by the C. trachomatis L2 serovar was isolated. The D4.1-3 cell line appears to be lacking in a previously undescribed temperature-dependent and heparin-resistant binding step that occurs subsequent to engagement of cell surface heparan sulfate by L2 elementary bodies. This novel binding step differentiates the lymphogranuloma venereum (LGV) serovar from other serovars and may contribute the different pathologies associated with LGV and non-LGV strains.

The CtsR regulator of stress response is active as a dimer and specifically degraded in vivo at 37°C

CtsR (class three stress gene repressor) negatively regulates the expression of class III heat shock genes (clpP, clpE and the clpC operon) by binding to a directly repeated heptanucleotide operator sequence (A/GGTCAAA NAN A/GGTCAAA). CtsR-dependent genes are expressed at a low level at 37 degrees C and are strongly induced under heat shock conditions. We performed a structure/function analysis of the CtsR protein, which is highly conserved among low G+C Gram-positive bacteria. Random chemical mutagenesis, in vitro cross-linking, in vivo co-expression of wild-type and mutant forms of CtsR and the construction of chimeric proteins with the DNA-binding domain of the lambda CI repressor allowed us to identify three different functional domains within CtsR: a helix-turn-helix DNA-binding domain, a dimerization domain and a putative heat-sensing domain. We provide evidence suggesting that CtsR is active as a dimer. Transcriptional analysis of a clpP'-bgaB fusion and/or Western blotting experiments using antibodies directed against the CtsR protein indicate that ClpP and ClpX are involved in CtsR degradation at 37 degrees C. This in turn leads to a low steady-state level of CtsR within the cell, as CtsR negatively autoregulates its own synthesis. This is the first example of degradation of a repressor of stress response genes by the Clp ATP-dependent protease.

Screening for and characterization of phospholipase A1 hypersecretory mutants of Tetrahymena thermophila.

We have described a procedure for the isolation of mutants of Tetrahymena thermophila with hypersecretion of phospholipase A1 (PLA1). Using random chemical mutagenesis, uniparental cytogamy, genetic crossing and a new, fast and effective screening procedure, four PLA1-hypersecretory mutants were isolated. The screening procedure is based on the formation of a halo appearing around cylindrical holes in a lecithin-containing agar plate filled with cell-free supernatants. About 3,940 clones were tested with this procedure in primary screening for hypersecretory features, of which 60 putative hypersecretory mutants were isolated, subcloned and tested in a secondary screening. Of these, four selected mutants showed 1.8-2.2 more PLA1 activity in the cell-free supernatants compared to the wild-type strain CU 438.1. Hypersecretion was only observable for PLA1; no increased activity for two other lysosomal enzymes could be detected. These hypersecretory mutants of T. thermophila can be very useful for increasing the yield of PLA1 in fermentation processes. This is particularly relevant because, in contrast to other phospholipases, PLA1 is not available on the commercial market for fine chemicals and little is known about the role of PLA1 in cell signaling and metabolism.

Evidence for Interactions between Helices 5 and 8 and a Role for the Interdomain Loop in Tetracycline Resistance Mediated by Hybrid Tet Proteins

An interdomain hybrid Tet protein consisting of a class C alpha domain and a class B beta domain (Tet(C/B)) lacks detectable efflux ability and provides only minimal levels of resistance to tetracycline (Tc) (3 microg/ml) compared with intact class B (256 microg/ml) and class C (64 microg/ml). Twenty-one independently isolated mutants of the Tet(C/B) protein with increased Tc resistance were generated by random chemical mutagenesis. Nine mutants with a Glu substitution for Gly-152 in helix 5 of the class C alpha domain produced a resistance of 48 microg/ml, whereas another 9 with an Asp replacement of Gly-247 in helix 8 of the class B beta domain mediated resistance at 32 microg/ml. The third type of mutation, found in 3 mutants expressing 24 microg/ml resistance, was a S202F replacement in the putative interdomain cytoplasmic loop of Tet(C/B). The latter underscores a previously unappreciated function of the interdomain cytoplasmic loop. All three types of Tet(C/B) mutant proteins were expressed in amounts comparable with that of the original protein and demonstrated restored energy-dependent efflux of tetracycline. Site-directed mutational analysis demonstrated that a Gly-247 to Asn mutation could also facilitate Tc resistance by the Tet(C/B) hybrid, and a negatively charged side chain at position 152 was required for Tet(C/B) activity. These mutations appear to promote the necessary functional interactions between the interclass domains that do not occur in the Tet(C/B) hybrid protein and suggest a direct association between helix 5 and helix 8 in the function of Tet efflux proteins.

Glycine Betaine-assisted Protein Folding in a lysAMutant of Escherichia coli

Osmoprotectants exogenously supplied to a hyperosmotic culture medium are efficiently imported and amassed by stressed cells of Escherichia coli. In addition to their evident role in the recovery and maintenance of osmotic balance, these solutes should play an important role on the behavior of cellular macromolecules, for example in the process of protein folding. Using a random chemical mutagenesis approach, a conditional lysine auxotrophic mutant was obtained. The growth of this mutant was restored by addition of either lysine or osmoprotectants including glycine betaine (GB) in the minimal medium. The growth rate increased proportionally with the augmentation of the intracellular GB concentration. The mutation was located in the lysA gene and resulted in the substitution of the Ser at position 384 by Phe of the diaminopimelate decarboxylase (DAPDC), which catalyzes the conversion of meso-diaminopimelate to L-lysine. We purified both the wild type DAPDC and the mutated DAPDC-sf and demonstrated that GB was capable of activating DAPDC-sf in vitro, thus confirming the in vivo results. Most importantly, we showed that the activation was correlated with a conformational change of DAPDC-sf. Taken together, these results show, for the first time, that GB may actively assist in vivo protein folding in a chaperone-like manner.

Improved thermostability of the North American firefly luciferase: saturation mutagenesis at position 354.

We have used random chemical mutagenesis and a simple genetic screen to generate and isolate a thermostable mutant of luciferase from the North American firefly (Photinus pyralis). A single G-to-A transition mutation, resulting in the substitution of a glutamate for a lysine residue at position 354 in the protein sequence, was shown to be responsible for this enhanced thermostability. Replacement of Glu-354 with all possible amino acid residues was achieved using directed mutagenesis, and produced mutant enzymes with a range of thermostabilities. The mutations E354K and E354R conferred the largest increases in thermostability, suggesting that side-chain size and hydrophobicity, as well as charge, may also be important contributors to the overall thermostability of the polypeptide chain at this position. Unusually for such mutations, biochemical studies suggest that this position is on the surface of the protein and exposed to solvent.

Conversion from Farnesyl Diphosphate Synthase to Geranylgeranyl Diphosphate Synthase by Random Chemical Mutagenesis

Prenyltransferases catalyze the consecutive condensation of isopentenyl diphosphate (IPP) with allylic diphosphates to produce prenyl diphosphates whose chain lengths are absolutely determined by each enzyme. In order to investigate the mechanisms of the consecutive reaction and of the determination of ultimate chain length, a random mutational approach was planned. The farnesyl diphosphate (FPP) synthase gene of Bacillus stearothermophilus was subjected to random mutagenesis by NaNO2 treatment to construct libraries of mutated FPP synthase genes on a high-copy plasmid. From the libraries, the mutants that showed the activity of geranylgeranyl diphosphate (GGPP) synthase were selected by the red-white screening method (Ohnuma, S.-i., Suzuki, M., and Nishino, T. (1994) J. Biol. Chem. 268, 14792-14797), which utilized carotenoid synthetic genes, phytoene synthase, and phytoene desaturase, to visualize the formation of GGPP in vivo. Eleven red positive clones were identified from about 24,300 mutants, and four (mutant 1, 2, 3, and 4) of them were analyzed for the enzyme activities. Results of in vitro assays demonstrated that all these mutants produced (all-E)-GGPP although the amounts were different. Each mutant was found to contain a few amino acid substitutions: mutant 1, Y81H and L275S; mutant 2, L34V and R59Q; mutant 3, V157A and H182Y; mutant 4, Y81H, P239R, and A265T. Site-directed mutagenesis showed that Y81H, L34V, or V157A was essential for the expression of the activity of GGPP synthase. Especially, the replacement of tyrosine 81 by histidine is the most effective because the production ratios of GGPP to FPP in mutant 1 and 4 are the largest. Based on prediction of the secondary structure, it is revealed that the tyrosine 81 situates on a point 11 approximately 12 A apart from the first DDXXD motif, whose distance is similar to the length of hydrocarbon moiety of FPP. These data might suggest that the aromatic ring of tyrosine 81 blocks the chain elongation longer than FPP. Comparisons of kinetic parameters of the mutated and wild type enzymes revealed several phenomena that may relate with the change of the ultimate chain length. They are a decrease of the total reaction rate, increase of Kmfor dimethylallyl diphosphate, decrease of Vmax for dimethylallyl diphosphate, and allylic substrate dependence of Km for IPP.

Acquisition of the ts phenotype by a chemically mutagenized cold-passaged human respiratory syncytial virus vaccine candidate results from the acquisition of a single mutation in the polymerase (L) gene.

A cold-passaged (cp) temperature-sensitive (ts) mutant of human respiratory syncytial virus designated RSV cpts-248 was previously derived by random chemical mutagenesis of the non-ts mutant cp-RSV that possesses one or more host range mutations. We previously demonstrated in rodents and seronegative chimpanzees that the cpts-248 virus is more attenuated than cp-RSV and is more stable genetically than previously isolated RSV ts mutants. In the present study, we determined that the acquisition of the ts phenotype and the increased attenuation of the cpts-248 virus are associated with a single nucleotide substitution at nucleotide 10,989 that results in a change in the coding region (amino acid position 831) of the polymerase gene. The identification of this attenuating ts mutation is important because cpts-248 was used as the parent virus for the generation of a number of further attenuated mutants that are currently being evaluated as candidate vaccine strains in clinical trials in infants. Furthermore, technology now exists to rationally design new vaccine candidates by incorporating multiple attenuating mutations, such as the one identified here, into infectious viruses that are genetically stable and appropriately attenuated.

Isolation of Transcriptionally Active Mutants of T7 RNA Polymerase that do not Support Phage Growth

Mutants of bacteriophage T7 RNA polymerase defective in functions other than transcription were sought by random chemical mutagenesis of the cloned gene and selection for inability to support the growth of a T7 mutant whose growth is dependent on T7 RNA polymerase supplied by the host cell. About half of the mutant clones appeared unable to make full-length T7 RNA polymerase, many of them producing a truncated protein. Among 116 mutants expressing full-length protein, two-thirds were severely impaired in transcription, but a surprisingly high one-third were able to direct significant transcription in vivo. Both types of mutation were distributed across much of the gene, as determined by a rapid genetic mapping procedure that allows the lethal mutation in each clone to be localized. One mutation (isolated twice) allowed normal gene expression but prevented the formation of mature ends of T7 DNA from concatemers, which normally happens during packaging into phage particles. Thirty-seven of the mutations appeared to increase the sensitivity of the polymerase to inhibition by T7 lysozyme; all were suppressed by mutations in the lysozyme gene, including one suppressor constructed to retain full amidase activity but to be unable to bind T7 RNA polymerase. The two lysozyme-hypersensitive polymerase mutants analyzed in detail showed premature cessation of transcription during infection. Early proteins and those late proteins specified by genes as far right in T7 DNA as genes 8– 9appeared to be produced normally, but expression of genes farther to the right was strongly depressed. DNA replication was depressed about 50% in one of these mutants and 90% in the other, even though the T7 replication proteins were made in normal amounts at the normal time. f2 f2 Abbreviations used: IPTG, isopropyl-β- d-thiogalactopyranoside; aa, amino acid(s).

A novel positive selection for identifying cold-sensitive myosin II mutants in Dictyostelium.

We developed a positive selection for myosin heavy chain mutants in Dictyostelium. This selection is based on the fact that brief exposure to azide causes wild-type cells to release from the substrate, whereas myosin null cells remain adherent. This procedure assays myosin function on a time scale of minutes and has therefore allowed us to select rapid-onset cold-sensitive mutants after random chemical mutagenesis of Dictyostelium cells. We developed a rapid technique for determining which mutations lie in sequences of the myosin gene that encode the head (motor) domain and localized 27 of 34 mutants to this domain. We recovered the appropriate sequences from five of the mutants and demonstrated that they retain their cold-sensitive properties when expressed from extrachromosomal plasmids.

Powerful dominant negative mutants of the human estrogen receptor

We have identified and characterized three human estrogen receptor (ER) mutants, which, at low concentrations, are capable of blocking the intracellular activity of wild type ER. The mutants, a truncated ER (ER1-530), a point mutant (L540Q), and a frameshift (S554fs), were generated by random chemical mutagenesis of the ER hormone binding domain and screened first for low activity in a yeast selection system. In transient co-transfection assays using ER-deficient Chinese hamster ovary cells, all three mutants exhibited less than 10% of the transcription activation activity of wild type ER, and when co-expressed with wild type ER, each of the mutants effectively suppressed the ability of wild type ER to activate transcription of an estrogen-regulated reporter plasmid. When equal amounts of plasmid encoding the ER mutants and wild type ER were used, S554fs, ER1-530, and L540Q suppressed the activity of wild type ER by 80, 55, and 75%, respectively. At a ratio of 1 part S554fs to 10 parts wild type ER, transcription was still inhibited by 40%. Western blot analysis showed that all three mutants were expressed at approximately the same level as wild type ER. Suppression of transcription was specific for ER, since the mutants did not inhibit progesterone receptor-mediated transcription. Not all mutations leading to inactive ER confer the dominant negative phenotype, as five ER mutants rendered transcriptionally inactive by point mutations between residues 516 and 524 of the ER hormone binding domain were poor inhibitors of wild type ER activity. Binding studies showed that the L540Q and S554fs dominant negative mutants bound 17 beta-estradiol with wild type affinity (Kd = 0.3-0.5 nM), whereas ER1-530 exhibited a 15-fold reduction in affinity for estradiol. The three dominant negative ERs showed significant ability to interact with the estrogen response element (ERE) in promoter interference assays, but ER1-530 and S554fs displayed little or no binding to the ERE in gel mobility shift assays where higher affinity for the DNA may be required for the receptor-ERE complex to remain associated during the electrophoresis. These data support the idea that, in all three mutants, it is loss of function of the COOH-terminal transactivation domain which leads to the dominant negative phenotype. S554fs, a powerful dominant negative mutant, is a good candidate for further studies aimed at suppressing the estrogen-dependent growth of human breast cancer cells.

Genetic interaction between transcription elongation factor TFIIS and RNA polymerase II.

Little is known about the regions of RNA polymerase II (RNAPII) that are involved in the process of transcript elongation and interaction with elongation factors. One elongation factor, TFIIS, stimulates transcript elongation by binding to RNAPII and facilitating its passage through intrinsic pausing sites in vitro. In Saccharomyces cerevisiae, TFIIS is encoded by the PPR2 gene. Deletion of PPR2 from the yeast genome is not lethal but renders cells sensitive to the uracil analog 6-azauracil (6AU). Here, we show that mutations conferring 6AU sensitivity can also be isolated in the gene encoding the largest subunit of S. cerevisiae RNAPII (RPO21). A screen for mutations in RPO21 that confer 6AU sensitivity identified seven mutations that had been generated by either linker-insertion or random chemical mutagenesis. All seven mutational alterations are clustered within one region of the largest subunit that is conserved among eukaryotic RNAPII. The finding that six of the seven rpo21 mutants failed to grow at elevated temperature underscores the importance of this region for the functional and/or structural integrity of RNAPII. We found that the 6AU sensitivity of the rpo21 mutants can be suppressed by increasing the dosage of the wild-type PPR2 gene, presumably as a result of overexpression of TFIIS. These results are consistent with the proposal that in the rpo21 mutants, the formation of the RNAPII-TFIIS complex is rate limiting for the passage of the mutant enzyme through pausing sites. In addition to implicating a region of the largest subunit of RNAPII in the process of transcript elongation, our observations provide in vivo evidence that TFIIS is involved in transcription by RNAPII.

Genetic interaction between transcription elongation factor TFIIS and RNA polymerase II.

Little is known about the regions of RNA polymerase II (RNAPII) that are involved in the process of transcript elongation and interaction with elongation factors. One elongation factor, TFIIS, stimulates transcript elongation by binding to RNAPII and facilitating its passage through intrinsic pausing sites in vitro. In Saccharomyces cerevisiae, TFIIS is encoded by the PPR2 gene. Deletion of PPR2 from the yeast genome is not lethal but renders cells sensitive to the uracil analog 6-azauracil (6AU). Here, we show that mutations conferring 6AU sensitivity can also be isolated in the gene encoding the largest subunit of S. cerevisiae RNAPII (RPO21). A screen for mutations in RPO21 that confer 6AU sensitivity identified seven mutations that had been generated by either linker-insertion or random chemical mutagenesis. All seven mutational alterations are clustered within one region of the largest subunit that is conserved among eukaryotic RNAPII. The finding that six of the seven rpo21 mutants failed to grow at elevated temperature underscores the importance of this region for the functional and/or structural integrity of RNAPII. We found that the 6AU sensitivity of the rpo21 mutants can be suppressed by increasing the dosage of the wild-type PPR2 gene, presumably as a result of overexpression of TFIIS. These results are consistent with the proposal that in the rpo21 mutants, the formation of the RNAPII-TFIIS complex is rate limiting for the passage of the mutant enzyme through pausing sites. In addition to implicating a region of the largest subunit of RNAPII in the process of transcript elongation, our observations provide in vivo evidence that TFIIS is involved in transcription by RNAPII.

Selection and characterization of randomly produced mutants of gene V protein of bacteriophage M13.

Gene V protein of bacteriophage Ff (M13, f1, fd) is a master regulator of phage DNA replication and phage mRNA translation. It exerts these two functions by binding to single-stranded viral DNA or to specific sequences in the 5' ends of its target mRNAs, respectively. To study the structure/function relationship of gene V protein, M13 gene V was inserted in a phagemid expression vector and a library of missense and nonsense mutants was constructed by random chemical mutagenesis. Phagemids encoding gene V proteins with decreased biological activities were selected and the nucleotide sequences of their gene V fragments were determined. Furthermore, the mutant proteins were characterized both with respect to their ability to inhibit the production of phagemid DNA transducing particles and their ability to repress the translation of a chimeric lacZ reporter gene whose expression is controlled by the promoter and translational initiation signals of M13 gene II. From the data obtained, it can be deduced that the mechanism by which gene V protein binds to single-stranded DNA differs from the mechanism by which it binds to its target sequence in the gene II mRNA.

Random mutagenesis of CSF-1 receptor (FMS) reveals multiple sites for activating mutations within the extracellular domain.

Retroviral vectors containing human FMS protooncogene cDNA were reconfigured to allow single-step excision and reinsertion of restriction fragments encoding short segments of the extracellular domain of the colony-stimulating factor 1 receptor (CSF-1R). Fragments ligated into M13 bacteriophages were subjected to random chemical mutagenesis on both strands and recloned into the parental vector to create libraries of FMS genes containing mutations restricted to predefined target cassettes. Transfection of retroviral vector libraries into NIH/3T3 cells gave rise to transformed foci from which cellular DNA was amplified by the polymerase chain reaction (PCR), using primers flanking the mutagenized target sequences. Amplified fragments from individual primary transformants were recloned into intact FMS vector plasmids, and those with transforming activity were subjected to nucleotide sequence analysis. Alternatively, retroviruses rescued from transformed cells by superinfection with helper virus were used to generate secondary transformants containing unique copies of proviral DNA, whose sequences were determined after PCR amplification. Novel activating mutations were identified within sequences separating the third and fourth immunoglobulin-like loops, as well as within non-covalently stabilized loop 4 of the CSF-1R extracellular domain. Thus, FMS mutations able to convert human CSF-1R to an active oncoprotein are not restricted to those previously identified at codon 301. This approach should be generally applicable for defining activating mutations in related growth factor receptors, including those for platelet-derived growth factor and Steel factor (KIT ligand), in which ligand-independent oncoprotein variants have not been identified.

Thermal stabilities of mutant Escherichia coli tryptophan synthase α subunits

Random chemical mutagenesis, in vitro, of the 5′ portion of the Escherichia coli trpA gene has yielded 66 mutant α subunits containing single amino acid substitutions at 49 different residue sites within the first 121 residues of the protein; this portion of the α subunit contains four of the eight α helices and three of the eight β strands in the protein. Sixty-two of the subunits were examined for their heat stabilities by sensitivity to enzymatic inactivation (52 °C for 20 min) in crude extracts and by differential scanning calorimetry (DSC) with 29 purified proteins. The enzymatic activities of mutant α subunits that contained amino acid substitutions within the α and β secondary structures were more heat labile than the wild-type α subunit. Alterations only in three regions, at or immediately C-terminal to the first three β strands, were stability neutral or stability enhancing with respect to enzymatic inactivation. Enzymatic thermal inactivation appears to be correlated with the relative accessibility of the substituted residues; stability-neutral mutations are found at accessible residual sites, stability-enhancing mutations at buried sites. DSC analyses showed a similar pattern of stabilization/destabilization as indicated by inactivation studies. T m differences from the wild-type α subunit varied ± 7.6 °C. Eighteen mutant proteins containing alterations in helical and sheet structures had T m's significantly lower (−1.6 to −7.5 °C) than the wild-type T m (59.5 °C). In contrast, 6 mutant α subunits with alterations in the regions following β strands 1 and 3 had increased T m's (+1.4 to +7.6 °C). Because of incomplete thermal reversibilities for many of the mutant α subunits, most likely due to identifiable aggregated forms in the unfolded state, reliable differences in thermodynamic stability parameters are not possible. The availability of this group of mutant α subunits which clearly contain structural alterations should prove useful in defining the roles of certain residues or sequences in the unfolding/folding pathway for this protein when examined by urea/guaninidine denaturation kinetic analysis.

Effects of random mutagenesis upon potato spindle tuber viroid replication and symptom expression

A combination of random chemical mutagenesis plus temperature gradient gel electrophoresis was used to isolate a collection of 57 potato spindle tuber viroid (PSTV) cDNAs containing mutations distributed throughout the entire 359 nucleotide genome. Although the presence of multiple mutations was often associated with a loss of cDNA infectivity, infectious PSTV cDNAs containing as many as four unlinked alterations could be isolated. Several mutations in the pathogenicity domain and left terminal loop were stably maintained in the resulting progeny, but those which affect base pairing in secondary hairpins I and II were either lethal or rapidly reverted to wild-type. One stable C → U substitution which may promote significant structural rearrangement within the right side of the pathogenicity domain had no detectable effect upon symptom expression. The variable domains of several noninfectious mutants contained an A → G substitution which is likely to inhibit the in vitro formation of secondary hairpin II via stabilization of the native structure, and the lethal nature of this mutation was confirmed by oligonucleotide-directed mutagenesis. Several lines of evidence now point toward an essential role for secondary hairpin II in the replication of PSTV and related viroids.

Structure-function studies of interferon-α based on random mutagenesis and expression in vitro

An efficient procedure for random chemical mutagenesis was used to create analogs of human interferon (IFN)-α4. Unique restriction enzyme sites were introduced into the human IFN-α4 gene to enable cassetting of the gene for localized random mutagenesis. Single-stranded IFN-α4 DNA was treated with nitrous acid, followed by second-strand synthesis using reverse transcriptase. A 72 base pair cassette spanning the coding region for amino acid residues 120 to 136 (120–136 region) was isolated and cloned into a phagemid vector adjacent to a GC-rich sequence. A DNA segment comprising the IFN-α4 cassette sequence and the GC clamp was excised and electrophoresed on a denaturing gradient gel, which allowed the separation from unmutated DNA of DNA fragments with single base pair changes. DNA fragments with mobility different from that of the unmutated fragment were pooled and cloned into an expression vector. Using this procedure, mutations were found in the DNA of 48% of the clones analyzed. However, mutations at two “hot spots” accounted for 89% of these clones. Four of the IFN-α4 analogs with mutations in the 120–136 region were expressed in vitro. The antiproliferative activities on human Daudi cells of most of the analogs were less than 0.2% of the activity of unmodified IFN-α4, suggesting that the integrity of the carboxy terminus is important for the antiproliferative activity of human IFN-α4.

Selection and characterization of randomly produced mutants in the gene coding for M1 RNA

The gene for M1 RNA, the catalytic subunit of RNase P of Escherichia coli, was subjected to random chemical mutagenesis in vitro. Mutations were selected by electrophoresis in denaturing gradient gels. Twenty-seven different mutants of the gene for M1 RNA were selected, and in 24 cases the mutations were identified as single base substitutions. The mutant forms of M1 RNA were analyzed in vitro for catalytic activity in the absence and in the presence of the protein subunit of RNase P (C5 protein). The structure of mutant RNAs was probed by limited digestion with ribonuclease T 1; a correlation between reduced catalytic activity of mutant M1 RNAs and perturbations in secondary and tertiary structure was noted in many cases. The results indicate the involvement of specific regions of the M1 RNA molecule in the catalytic function of RNase P, in the binding of the C5 protein, and in substrate binding.