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The nrdB gene of bacteriophage T4 codes for the small subunit of ribonucleotide reductase and contains a 598 base pair self splicing intron which is closely related to other group I introns of T4 and eukaryotes. Previously, the nrdB intron mutations, presumably causing splicing defects of the nrdB transcript, were isolated and mapped in or near the nrdB intron. In this communication, we have isolated 181 hydroxylamine-induced revertants for the above primary nrdB intron mutations by strategic usage of the white halo phenotype. Also, we mapped these revertant mutations by marker rescue with subclones of the nrdB gene. Some of the second site mutations were mapped to regions predicted by the secondary structure model of the nrdB intron. To investigate the involvement of protein factors facilitating splicing of the nrdB transcript, we attempted to isolate extragenic revertants of td-nrdB double mutants by utilizing hydroxylamine mutagenesis. Mapping and sequencing of the suppressor mutations in the ...

The nrdB gene of bacteriophage T4 codes for the small subunit of ribonucleotide reductase and contains a 598 base pair self splicing intron which is closely related to other group I introns of 14 and alkaxyotes. The screening, isolation and mapping of the nrbB intron mutations was conducted by the strategic usage of the white halo phenotype exhibited by T4 mutants defective in dyhydrofolate reductase or thymidylate synthase. We have isolated 159 hydroxylamine-induced nrdB mutants, determined which mutations are in nrdB by marker rescue with clones of the nrdB gene and have mapped these mutations by marker rescue using subclones of the nrdB intron. Thirty out of the 159 nrdB mutations are in or near the intron. These mutations cluster towards the ends, mainly the 3′ end. We have performed deletion mapping to further map mutations in the 3′ end of the intron. The mutations map in regions of conserved structural elements, thus supporting secondary structure predictions similar to those of the well studied td intron in the T4 gene coding for thymidylate synthase.

The nrd B gene of bacteriophage T4 codes for the small subunit of ribonucleotide reductase and contains a 598 nuclelotide group 1 self splicing intron. In order to study the functional domains for self-splicing of this intron, 23 nrd B splicing defective intron mutants were analysed for both sequence and functional changes. These mutants cluster towards the ends in regions of conserved structural elements of the intron. These 23 mutants have single base changes at 14 different sites. Interestingly two of these sites that seemed to map within the intron are actually located on the flanking exon sequences on both sides of the intron. A high frequency (4/12) of the mutation sites are in bases not thought to be base-paired in the standard model of group I intron structure. The mutation sites in pairing regions P3, P7, P8, P9 and between P6[3′] and P7[5′] are identical to changes found in the well studied td (encoding dTMP synthase) intron. However, five new mutation sites (S61, SL1, S29, SL11, SL196 and SL126) are unique to the nrd B intron and disrupt self-splicing. A mutation (S61) in the P7.1 pairing region is especially significant because no mutations have been found in this pairing, thus defining a new sub-domain essential for RNA splicing. Like the td intron, the mutation site in P9 of the nrd B intron is a hot spot for mutations, but unlike td , the nrd B intron does not show a mutational hot spot in the P6[5′] region. Our molecular dissection of the nrd B intron also supports the P9.0 and P10 pairings that have been postulated to help form a complex tertiary structure required to give the RNA sequence its catalytic activity: particularly 3′ splice site selection, cleavage and exon ligation.

The detailed distribution and characterization of 51 hydroxylamine (HA)-induced and 59 nitrous acid (NA)-induced mutations in the intron-containing bacteriophage T4 thymidylate synthase (td) gene is reported here. Mutations were mapped in 10 regions of the td gene by recombinational marker rescue using plasmid or M13 subclones of the td gene. Phage crosses using deletion mutants with known breakpoints in the 3' end of the td intron subdivided HA and NA mutations which mapped in this region. At least 31 of the mutations map within the 1-kb group I self-splicing intron. Intron mutations mapped only in the 5' and 3' ends of the intron sequence, in accordance with the hypothesis that the 5' and 3' domains of the T4 td intron are essential for correct RNA splicing. RNA sequence analysis of a number of mapped td mutations has identified two intron nucleotides and one exon nucleotide where both HA- and NA-induced mutations commonly occur. These three loci are characterized by a GC dinucleotide, with the mutations occurring at the cytosine residue. Thus, these data indicate at least three potential sites of both HA- and NA-induced mutagenic hotspot activity within the td gene.

Twenty-one independent thymidylate synthase deficient (td) mutants were isolated after proflavin mutagenesis of T4D0 phage. A strikingly high proportion of these mutations (17 of 21; 80%) mapped in a small 122 nucleotide (nt) region which spans the 5' splice site of this intron-containing gene. This region comprises only 14% of the total td exon sequence. RNA sequence analysis of these mutants identified a series of frameshift insertion/deletion mutations and indicated a hotspot for proflavin-induced mutations in the 3' end of exon I of the td gene. The mutant sequences at the hotspot site fully support a previously proposed mutagenic mechanism for proflavin-induced mutations in which frameshifts are produced as a consequence of exonuclease or DNA polymerase activity at the 3' ends of nicks in the DNA produced by perturbation of the T4-encoded type II topoisomerase activity by the acridine. Sixteen of the seventeen DNA mutations in the hotspot region can be explained by the model as a consequence of enzymatic processing of nicks at two phosphodiester bonds staggered by 4 base pairs (bp) and located on opposite strands of the DNA. Thus, these mutants exhibit precisely the symmetry expected of topoisomerase-mediated mutagenesis. The DNA sequences of the td hotspot mutants, when considered with the sequences of proflavin-induced mutants in the T4 rIIB and lysozyme genes, confirm the view that proflavin-induced mutations in diverse bacteriophage T4 DNA sequences are all produced by the topoisomerase-dependent mechanisms and do not support the view that classical misalignments in DNA repeats are hotspots for proflavin-induced mutagenesis in T4.

Of 97 nondirected T4 thymidylate synthase-defective ( td ) mutations, 27 were mapped to the intron of the split td gene. Clustering of these intron mutations defined two domains that are functional in splicing, each within approximately 220 residues of the respective splice sites. Two selected mutations, td N57 and td N47, fell within phylogenetically conserved pairings, with td N57 disrupting the exon I-internal guide pairing (P1) in the 5′ domain and td N47 destabilizing the P9 helix in the 3′ domain. A splicing assay with synthetic oligonucleotides complementary to RNA junction sequences revealed processing defects for T4 td N57 and T4 td N47, both of which are impaired in cleavage at the 5′ and 3′ splice sites. Thus prokaryotic genetics facilitates association of specific residue changes with their consequences to splicing.

Two hydroxylamine-induced mutants of bacteriophage T4 defective in modification of host valyl-tRNA synthetase have been isolated by assay of crude extracts for the activity that is characteristic of the wild-type virus. The mutations define a single gene that is situated between the rI and e genes on the T4 genetic map. This new gene is designated vs for valyl-tRNA synthetase. One of the mutations may be of the missense type since it results in the production of a valyl-tRNA synthetase activity that has unusual ureainactivation properties. The other appears to be an amber mutation since the viral enzyme can only be found after infection of cells that are permissive for amber mutations. No differences in growth properties were found between wild type and amber mutant strains on the nonpermissive host. We conclude that the bacteriophage T4 valyl-tRNA synthetase is not essential for viability under prevailing laboratory conditions.

Plating techniques which eliminate T4 plaque formation on Escherichia coli by folate analogue inhibition of dihydrofolate (FH 2 ) reductase (EC 1.5.1.3) allowed the isolation of folate analogue-resistant ( far ) mutants of T4. One class of far mutants overproduces the phage-induced FH 2 reductase. Deoxycytidylate deaminase (EC 3.5.4.12), thymidine kinase (EC 2.7.1.21), and deoxycytidine triphosphatase (EC 3.6.1.12) are also overproduced by 20 min after infection at 37 C. The overproduction of FH 2 reductase by these far mutants is not affected by the absence of DNA synthesis. Other types of mutations that affect the synthesis of early enzymes cause overproduction in the absence of DNA synthesis of some of the above enzymes but not of FH 2 reductase. Therefore, overproducing far mutants apparently have mutations in previously undescribed genes controlling the expression of the T4 genome. Three of four mutants under study map near gene 56, and one maps near gene 52. All of these mutants show delays in DNA synthesis, phage production, and lysis and appear to show decreased levels of RNA synthesis based on the cumulative incorporation of uridine.

Amber mutants of bacteriophage T4 have been isolated that induce thymidine kinase activity only after infection of a strain of Escherichia coli carrying a suppressor mutation. The activity induced when one of these mutants infected this suppressor strain is much more heat sensitive than the activity induced by wild-type T4. This indicates that this amber mutation lies within the structural gene for thymidine kinase. This gene is between fI and v on the standard T4 genetic map. A mutant of tt4 that is unable to induce thymidine kinase activity incorporates only about one-eighth as much thymidine into its DNA as phage that do induce thymidine kinase. This contrasts to the findings that the total thymidine kinase activity in extracts prepared from cells infected with phage able to induce thymidine kinase in only twice as great as the activity in cells infected with the mutant unable to induce the enzyme.

The dihydrofolate (FH 2 ) reductase specified by the T4 frd gene (previously called the wh gene) is apparently nonessential for phage growth on Escherichia coli because the bacterial FH 2 reductase can partially substitute for the phage enzyme. To study the effect in vivo of folate analogs which specifically inhibit the T4 FH 2 reductase, the enzyme must be made essential for phage production. Partial inhibition of the E. coli FH 2 reductase with the folate analog trimethoprim strongly inhibits phage production by a T4 frd mutant and slightly inhibits wild-type phage production. Adding an additional T4-specific folate analog, a chlorophenyl triazine, strongly inhibits wild-type phage production without preventing the uninfected E. coli cells from multiplying. We have isolated spontaneous mutants of T4 which are capable of producing progeny in the presence of chlorophenyl triazine and another folate analog pyrimethamine. These mutants are designated folate analog resistant ( far ) and have been separated into two general classes. Class I mutants induce T4-specific FH 2 reductases which are less sensitive to the action of the folate analogs than the normal FH 2 reductase. Class II mutants induce an unaltered FH 2 reductase but contain mutations in a variety of T4 genes. Some class II mutants induce more phage FH 2 reductase activity than wild-type T4 and appear to be regulatory mutants.

Mutagenized T4 was screened for hydroxyurea sensitivity on mutant strain Escherichia coli OK305, which was growing slowly on a synthetic medium. Most of the hydroxyurea-sensitive mutants of T4 isolated in this manner are different from those previously described by Warner et al. (1970) and Hercules et al. (1971), and several may be in new cistrons. Most of them produce fewer progeny per infected cell than wild-type T4, even in the absence of hydroxyurea. The first seven mutants studied map at seven distinct sites and affect at least five different cistrons which are scattered throughout the major early region of the T4 genetic map (between gene 38 and rI). At least one of these mutants is defective in the ability to degrade E. coli DNA.

New mutants of T4 have been isolated by using a strain of Escherichia coli lacking thymidine kinase activity. These T4 mutants, designated tk , are able to grow on this E. coli strain under light on plates containing 5-bromodeoxyuridine and were all found to be unable to induce thymidine kinase (ATP: thymidine 5′-phosphotransferase, EC 2.7.1.21). All of these tk mutants fall into one complementation group which maps just to the right of rI on the standard T4 genetic map, far from most other genes coding for enzymes involved in pyrimidine metabolism. The tk mutants grow as well as wild-type T4, indicating that thymidine kinase is a non-essential enzyme.

Hydroxyurea (HU) has been used successfully (Warner el al. , 1970; Hercules et al. , 1971) to select bacteriophage T4 mutants which are deficient in degrading Escherichia coli DNA after infection. Using a different set of plating conditions, HU has also been used to isolate another set of T4 mutants ( hus ) ( Goscin and Hall, 1972 ). All the backcrossed hus mutants degrade bacterial DNA normally. The original hus 13 contained another mutation N. The double mutant hus 13-N degrades E. coli DNA slowly in the absence of phage DNA synthesis, leaving DNA fragments with a molecular weight midway between the sizes of fragments left by mutants defective in genes 46 and 47 and by endonuclease II ( den A) mutants at similar times after infection. Endonuclease IV activity is normal in hus 13-N infected cells. The HU sensitivity of three mutants, hus 3 (which is deficient in exonuclease A), hus 7 and hus 23 is partially due to inefficient utilization of host-derived deoxyribonucleotides as substrates for phage DNA synthesis. This is related to the DNA delay phenotype of these 3 mutants. The mutants hus 7 and hus 23 also appear to package phage DNA into phage particles inefficiently. The backcrossed hus 13 is apparently HU sensitive due to a defect in packaging DNA into phage particles. The HU sensitivity of two other mutants ( hus 19 and hus 20) remains unexplained as these mutants degrade bacterial DNA normally, synthesize phage DNA normally using host-derived deoxyribonucleotides, and package the DNA into phage particles efficiently.

We have found that mutations in phage T4 genes 41 (five of five) and 61 (three of three) cause resistance to the folate analogue pyrimethamine that inhibits T4 dihydrofolate (FH2) reductase. These genes code for subunits of a T4 primase and are part of a putative T4 replication complex. In contrast to many previously isolated folate analogue-resistant (Far) T4 mutants, these T4 primase mutants do not overproduce FH2 reductase nor do they alter its primary structure. A new mutant with a single lesion in gene 41 was isolated which proved resistant to the folate analogue at 30° and was lethal at 42°. This mutant induced normal levels of FH2 reductase (encoded by the frd gene) and appeared to have normal expression of other T4 genes at 30°. Like other mutations in gene 41, tsP129 reduced phage-induced DNA synthesis to about 15% that of wild-type T4 as measured by thymidine incorporation under restrictive conditions. Double mutants carrying mutations in genes 41 and 61, 41 and frd or 61 and frd showed allele-specific suppression suggesting that the products of these genes interact. We suggest that abnormal interactions between components of the replication complex and a DNA precursor synthesizing complex cause folate analog resistance by allosterically altering the T4 FH2 reductase.

Some spontaneously occurring bacteriophage T4 mutants ( far mutants) were able to form plaques in the presence of concentrations of folate analogues that completely inhibit plaque formation by wild-type phage T4. Some of these far mutants were shown to be ribonucleoside diphosphate (RDP) reductase (EC 1.17.4.1) deficient, and some independently isolated RDP reductase-deficient mutants ( nrd mutants) were shown to be folate analogue resistant. The map positions of the RDP reductase-deficient far mutants were shown to be within the genes controlling the phage-induced RDP reductase activity.

The organization of genes into exons separated by introns may permit rapid evolution of protein-coding sequences by exon shuffling. Introns could provide non-coding targets for recombination, which would then give rise to novel combinations of exons. Evidence to support this theory is indirect and consists of examples of homologous domains of protein structure encoded in different genes, with introns in conserved positions at the boundaries of these domains. Here, we report the first direct evidence for exon shuffling. Two spontaneous deletion mutations of phage T4 have been characterized by sequencing, and they are clearly the result of recombination between homologous regions of two self-splicing group I introns. As a result of the recombination, exons of different genes are transcribed together, with a hybrid intron between them. One of these introns is proficient in self-splicing.

T4 induces dCMP deaminase activity in Escherichia coli B. Mutants ( cd ) of T4 unable to induce this activity have been isolated at a high frequency by hydroxylamine treatment. No selection procedure was needed other than an enzymatic assay of extracts of phage-infected cells. The isolation of these mutants indicates that at least one T4 cistron controls the production of dCMP deaminase activity. Studies of one cd mutant suggest that either it makes no dCMP deaminase or else one that is structurally altered. The cd + function does not appear to be essential for T4 growth in E. coli B nor does it significantly affect the production of the other early enzymes dTMP synthetase and dCMP hydrooxymethylase.

Genetic mapping of a T4 mutant ( cd ) deficient in the ability to induce dCMP deaminase in Escherichia coli B has shown that it is very closely linked to a mutant ( td ) deficient in the ability to induce dTMP synthetase. The finding that these genes controlling sequential biosynthetic reactions are very closely linked and probably adjacent to each other suggests that each of these genes may be part of a larger unit of genetic organization, possibly a unit of coordinate expression. A new type of T4 mutant ( wh ) has been found. The three genes appear to be contiguous and in the order cd, td, wh . Like the cd and td functions, the wh function does not appear to be essential for growth of T4 in E. coli B. Preliminary analysis of one wh mutant suggests a defect in the ability to induce synthesis of dihydrofolate reductase.

Seventeen non-directed td- (thymidylate synthase-deficient) splicing-defective mutations isolated in phage T4 were localized within the catalytic core of the ribozyme. All of the mutations occur in conserved structural elements that form part of the td intron core secondary structure. Remarkably, seven of the seventeen independently isolated mutations clustered in the dinucleotide 5' element (P6[5']) of the putative two-base-pair P6 stem. An analysis of this region was undertaken by site-directed mutagenesis of the plasmid-borne td gene, leading to the following findings: First, the short P6 pairing in the td secondary structure model was verified with appropriate P6[5'] and P6[3'] compensatory mutations. Second, all P6[5'] and P6[3'] mutants are defective in the first step of splicing, guanosine-dependent 5' splice site cleavage, whereas their activity at the 3' splice site is variable. Third, residual in vitro splicing activity of the mutants altered on only one side of the P6 pairing is correlated with the ability to form an alternative two-base-pair P6 stem. Fourth, the degree to which the compensatory mutants have their splicing activity restored is highly condition-dependent. Restoration of phenotype of the compensatory P6[5']:[3'] constructs is weak under stringent in vitro conditions as well as in vivo. This sequence specificity is consistent with phylogenetic conservation of the P6 pairing elements in group I introns, and suggests either structural constraints on the P6 stem or a dual function of one or both pairing elements.