Genetic fine structure of the glucosyltransferase gene of bacteriophage T2

14 mutants of T2, which carried mutations in the gene coding for glucosyltransferase, were isolated. Although ambers were not selected for, six mutants appeared to be of the amber type. These mutants, as well as another twelve, 5 missense and 7 amber, were located and a genetic map was constructed. The amber and non-amber mutations were not equally distributed over the gt gene. The part transcribed first carried mainly amber mutations; the tail part contained only non-amber mutations. A possible relation between the non-random location of the two kinds of mutation and functional differences within the enzyme is discussed. No intragenic complementation could be demonstrated. The recombination frequencies of amgt mutants are reduced to about two-thirds if crosses are performed under conditions where the DNA of the mutants remains unglucosylated.     

Directional alignment of two Salmonella phage P22 maps constructed from temperature sensitive or Amber mutants

Summary Two linkage maps of Salmonella phage P22, one constructed with temperature sensitive mutants and the other from amber mutants, were aligned with respect to directional sequence of the genes. Alignment was determined by use of a three-factor cross.The results are in agreement with two independent complementation tests conducted between temperature sensitive and amber mutants which had been employed in constructing the linkage maps.     

Mapping of temperature sensitive mutants of bacteriophage phi 29

Summary Temperature-sensitive mutants of eleven complementation groups of phage 29 have been mapped by means of two-factor crosses. The results show the existence of a single non-circular linkage map. Cistrons expressed early after infection are clustered at the left end of the map.     

Genetic and transfection studies with B. subtilis phage SP 50

Summary 27 temperature sensitive mutants of phage SP 50 were isolated after hydroxylamine or nitrous acid mutagenesis, classified by complementation into groups of functional identity, and mapped by two factor crosses. These mutants, together with the plaque type and functional mutants previously isolated by Rottländer (1966), could be arranged in a linear map comprising a total of 2×92,1 recombination units.     

Genetic analysis of bacteriophage phi 29 of Bacillus subtilis: integration and mapping of reference mutants of two collections.

Reference mutants of Bacillus subtilis phage phi 29 of the Madrid and Minneapolis collections were employed to construct a genetic map. Suppressor-sensitive and temperature-sensitive mutants were assigned to 17 cistrons by quantitative complementation. Three-factor crosses were used to assign an unambiguous order for the 17 cistrons. Recombination frequencies determined by two-factor crosses were used to construct a linear genetic map of 24.4 recombination units. The genes were numbered sequentially from left to right (1 to 17) according to their relative map position.     

Studies on the In Vitro Assembly of Bacteriophage φ80 and φ80-Lambda Hybrids

Suppressor-sensitive (sus) mutants of bacteriophage 80 defective in late functions were classified, by means of in vitro assembly tests, into two complementation groups: head donors and tail donors. Each group of mutants was subdivided, by means of two-factor crosses, into six cistrons. Deletion mapping revealed clustering of tail and also of head cistrons. The two clusters were located in the left arm of vegetative 80 (the tail specifying cluster being distal). In vitro cross complementation between 80 and lambda sus mutants revealed that whereas lambda heads could quite efficiently bind 80 tails to form viable phage, the union of 80 heads and lambda tails was very much less efficient. Deletion mapping of the 80 sus mutants, using both 80 and i⁸⁰h^λ deletion lysogens indicated congruent gross gene arrangement in the two related bacteriophages.     

Mapping missense and nonsense mutations in genecI of bacteriophage lambda: Marker effects

Summary Amber and missense mutations in genecI of bacteriophage lambda were mapped by reciprocal four-factor crosses, selecting recombinants between the outside markers (N amber andO amber). Distances betweencI missense mutations were additive. SeveralcIamber mutants recombined with othercI mutations with a higher-frequency than expected from the map location. Multiple exchanges in theN-O region occurred at a frequency greater than expected by chance. This high negative interference was especially marked in crosses with thecIamber mutations that were strong recombiners.A newind mutation,ind2, was found neartsU51, to the left of the previously-knownindl mutation, which is located almost in the center of genecI. The mutationc50 maps to the right oftsU50 andc71. Mutationsc60, andts71, which differ in phenotype, are apparently at the same site.     

Isolation and Preliminary Characterization of Temperature-Sensitive Mutants of Rhizobiophage C

By using mutagenesis induced by N-methyl-N'-nitro-N-nitrosoguanidine, 150 temperature-sensitive mutants of rhizobiophage c were isolated. All were able to form plaques at 14 C but not at 29 C. They were classified into 21 complementation groups. Representative temperature-sensitive mutants from each complementation group were analyzed with regard to gene function. The approximate time of expression of the genes defined by the mutants was measured by temperature-shift experiments. Most genes began to be expressed at 4.0 to 7.5 h after infection at 14 C. Four genes were found which were expressed 2.5 to 3.5 h after infection. Some mutants showed no DNA synthesis at 29 C; some showed a delay in lysis. Some produced apparently normal particles after infection and lysis at 29 C; others produced various types of defective particles. Some mutants showing no DNA synthesis at 29 C nevertheless caused lysis at the normal time together with the production of phage structural components. Representative mutants from each complementation group were mapped by using two-factor crosses. A preliminary genetic map of phage c was constructed from the data.     

Genome structures in a lysogenic Rhizobium meliloti system

Summary The genome structure of the temperateRhizobium meliloti phage and the attachment site of this phage on the host chromosome were examined by genetic means. The heat-sensitive mutants used in 2 and 3 point crosses gave a linear chromosome map. There was no evidence for map circularity. The immunity region has a distal position on the phage chromosome. The functional grouping of the used 23 phage mutants was made byin vivo andin vitro complementation tests and 20 cistrons were obtained. The cistrons, near to the immunity region, were identified as early genes, the remaining ones as morphogenetic cistrons. The latter inin vitro complementation tests gave two complementing groups, presumably as head and tail donors. The attachment site of the prophage on the host chromosome was localized by pulse mutagen treatments in synchronously replicating cultures. The sequence of markers are O-str — hs — att ¹⁶⁻³ — T.     

Genetic mapping of the G101 bacteriophage (Pseudomonas aeuruginosa by temperature-sensitive mutations

Temperature-sensitive (ts) mutations of the G101 phage were isolated after mutagenesis with hydroxylamine. A complementation analysis of 61ts mutants showed that these mutants may be divided into at least 12 complementation groups. Twots mutants probably originated in genes which control lytic functions of the G101 phage. It was shown by three factor crosses that all of the 12ts mutations tested are localized on that side of the “c” region where the probablecI repressor gene is positioned. Sevents mutations is closely linked to thecI ₂₆ clear marker, three exhibit a closer linkage and two do not exhibit any linkage withcI. All mutations isolated until now can be arrange linearly. According to the present knowledge the preliminary genetic map of the G101 phage is linear.     

Temperature-sensitive mutants of frog virus 3: biochemical and genetic characterization

Nineteen frog virus 3 temperature-sensitive mutants were isolated after mutagenesis with nitrosoguanidine and assayed for viral DNA, RNA, and protein synthesis, as well as assembly site formation at permissive (25 degrees C) and nonpermissive (30 degrees C) temperatures. In addition, mutants were characterized for complementation by both quantitative and qualitative assays. Based on the genetic and biochemical data, the 19 mutants, along with 9 mutants isolated earlier, were ordered into four phenotypic classes which define defects in virion morphogenesis (class I), late mRNA synthesis (class II), viral assembly site formation (class III), and viral DNA synthesis (class IV). In addition, we used two-factor crosses to order 11 mutants, comprising 7 complementation groups, onto a linkage map spanning 77 recombination units.     

Genetic analysis of nitrate reductase deficient mutants of Nicotiana plumbaginifolia: Evidence for six complementation groups among 70 classified molybdenum cofactor deficient mutants

Summary A total of 70 cnx mutants have been characterized from a collection of 211 nitrate reductase deficient (NR^-) mutants isolated from mutagenized Nicotiana plumbaginifolia protoplast cultures after chlorate selection and regeneration into plants. They are presumed to be affected in the biosynthesis of the molybdenum cofactor since they are also deficient for xanthine dehydrogenase activity but contain NR apoenzyme. The remaining clones were classified as nia mutants. Sexual crosses performed between cnx mutants allowed them to be classified into six independent complementation groups. Mutants representative of these complementation groups were used for somatic hybridization experiments with the already characterized N. plumbaginifolia mutants NX1, NX24, NX23 and CNX103 belonging to the complementation groups cnxA, B, C and D respectively. On the basis of genetic analysis and somatic hybridization experiments, two new complementation groups, cnxE and F, not previously described in higher plants, were characterized. Unphysiologically high levels of molybdate can restore the NR activity of cnxA mutant seedlings in vivo, but cannot restore NR activity to any mutant from the other cnx complementation groups.     

Nonsense mutants of Serratia phage Kappa

Summary Auxotrophs of Serr. marcescens HY, which behaved like nonsense mutants when tested according to Whitfield et al. (1966), were induced to revert to anauxotrophy. Some of the revertants (called su ⁺), together with the parental auxotrophs (called su ^-), allowed to isolate conditional-lethal (sus) mutants of phage Kappa, which produce infectious progeny only in su ⁺ bacteria. All su ⁺ mutants of Serr. marcescens HY were identified as nonsense suppressors using su ⁺ _amber, su ⁺ _ochre, and su ^- strains of Salm. typhimurium as references and the flagella-specific phage Chi as the main tool to connect the Salmonella system with that of Serratia.After treatment of Kappa with three different mutagens 128 sus mutants were isolated which comprise at least 19 complementation groups. 18 sus mutants, representing different cistrons, and the unselective markers c1, c2, and c49 were mapped mainly by two-factor crosses. Reciprocal three-factor crosses of the general type a x bz and az x b (i.e. with outside markers) revealed a circular linkage map of an estimated maximum length of 90 RU (recombination units). Joined rescue of outside markers, e.g. sus ⁺ A94 and e49, from UV-irradiated phage supported the assumption of circular gene linkage. Some data indicate that certain regions of the phage genome might have a higher chance to recombine than others.Abbreviations moi multiplicity of infection - eop efficiency of plating - RU recombination units - MR marker rescue     

Genetic fine structure and complementation at the albino locus in spider mites (Tetranychus species: Acarina)

Two mutations inTetranychus urticae and nine inTetranychus pacificus, all originating spontaneously, block the production of red and yellow carotenoid pigments in these spider mite species. Inter-mutant crosses were carried out to study complementation and recombination relationships between the mutations. InT. urticae, the two albino mutants complement one another completely, i.e., crosses between them produce wild-type hybrid females; while they recombine with a frequency of 0.05%. Of the nine mutants inT. pacificus, fivep mutants in general are complementary to a high degree with foura mutants.p mutants fail to complement one another, while somea mutants are mutually complementary to a slight degree. Scoring the degree of complementation produced by all possible combinations of mutants permits the construction of a linear complementation map. Certain combinations, however, are exceptional to such a representation. Moreover, marked reciprocal differences in complementation indicate that maternal effects are involved, implying that the albino locus may control more than one enzymatic step. Attempts to derive a genetic map were impeded by the absence of suitable linked markers, by a pronounced maternal effect (high pigmentation) in the haploid F₂ males, and by the appearance of pseudowild type F₂ males. The given genetic sequence, although comparable in a limited fashion to the complementation map, is considered tentative. Pink types appeared in crosses with certainp mutants. These were due to mutation at a separate locus, called rose, and seem to involve the production of pink pigments in an alternative or substitute pathway. A scheme attempting to orientate the present state of understanding of pigmentation in spider mites is presented.This work forms part of a thesis submitted in fulfillment of the requirements for the Ph. D. degree at the University of Amsterdam (1969).     

Genetic complementation and enzyme correlates at the locus encoding the last two steps of de novo pyrimidine biosynthesis in Drosophila melanogaster

Summary Eight mutations of the rudimentary-like (r-l) locus were isolated following mutagenesis with ethylmethanesulfonate and inter se crosses revealed three basic complementation groups, using the wing phenotype as an index of complementation. One group consists of three entirely noncomplementing mutants that each specify severe reductions in levels of both r-l-encoded enzymes, orotate phosphoribosyltransferase (OPR-Tase) and orotidylate decarboxylase (ODCase). The other two groups consist of complementing mutants, such that any member of one group fully complements all members of the other group. One of these groups consists of two mutants that each specify severly reduced OPRTase, but normal ODCase. The other group consists of three mutants that specify severe OPRTase and OD-Case reductions in homoallelic flies, but that appear to contribute OPRTase in certain heteroallelic genotypes. It is concluded that the reciprocal and complementing enzymatic phenotypes of mutants in these two groups account for most instances of genetic trans complementation among r-l mutants. These findings are discussed relative to extant information on OPRTase and OD-Case in animals and an hypothesis is developed that the r-l locus encodes a single polypeptide product that contains both enzyme activities.     

The isolation and analysis of one functional type ofpyr-3 mutant inNeurospora

Twenty-seven pyrimidine-requiring mutants were isolated as suppressors of anarg-3 mutant. All 27 are deficient for ATCase activity and show linkage to thecol-4 marker located on linkage group IV. Analyses of prototroph frequencies resulting from crosses of the new mutants to previously mappedpyr-3 mutants indicate that this functional type ofpyr-3 mutant is restricted to one region of the genetic map. Complementation studies with 11 of the new mutants further extend and subdivide the complementation map of thepyr-3 locus.This work was supported in part by National Institutes of Health, Public Health Service Grant GM 15137-01 and by National Science Foundation Grant GB5998.     

Deficiency of double mutant recombinants in crosses of phage T4

Summary Only 1.4% of the double mutant recombinants expected on the basis of wild-type recombination frequencies were observed in the combined data from two-factor crosses between a gene 37 amber mutant, amB280, and eighteen different temperature sensitive mutants which were also defective in gene 37. Similar, though less extreme, deficiencies of double mutant recombinants were observed by Doermann and Parma (1968) for mutants in several other genes. In our amB280xts crosses, frequencies of wild-type recombinants were in reasonably good agreement with those expected from the map positions of the mutants determined in crosses not involving amB280. Wild-type and double mutant recombinants were found at comparable frequencies when each of three other gene 37 amber mutants was crossed to a gene 37 temperature sensitive mutant.Experiments were performed to test whether the deficiency of double mutant recombinants in the amB280xts crosses could be explained by assuming that they occurred primarily in heterozygous particles, where their expression was masked. However, no evidence in support of this explanation was found. Other possible explanations, that the deficiency of double mutants was due to their inviability or the inability of double mutant chromosomes to replicate, were also inconsistent with our observations. The hypothesis considered to most plausibly explain our evidence is that the process by which double mutant recombinant chromosomes are formed is inhibited in the vicinity of a poorly suppressed am mutation.     

Only one gene is required for the glpT-dependent transport of sn-glycerol-3-phosphate in Escherichia coli

Summary Deletion and point mutants defective in the glpT-dependent sn-glycerol-3-phosphate transport system were isolated and located on the Escherichia coli chromosome. They mapped in glpT in the clockwise order gyrA, glpA, glpT at around 48 min on the Escherichia coli linkage map. The mutations within glpT were ordered by deletion mapping, three factor crosses, and by crosses involving transducing bacteriophages carrying glpT-lac operon fusions. Results obtained using these fusion phages indicated that glpT is transcribed in the counterclockwise direction on the E. coli linkage map.Complementation analysis using these mutants revealed only one complementation group. Thus, one gene is necessary and sufficient for the proton motive force-dependent sn-glycerol-3-phosphate transport system.     

Studies of opal mutations in bacteriophage T4B

Hydroxylamine-induced amber and opal mutants are localized on the map of gene 47 of bacteriophage T4B. The matched map of amber and opal mutations showed the presence of four paired sites which seemed to have arisen in the triplet coding for tryptophan.In growth studies o opal mutants in gene 47 in a series of Su⁺ strains the number of strains bearing a gene-suppressor for amber or ochre mutations also had a weak suppressor activity for some opal mutants. This suppressor acitivity is supposedly due to a second mutation in gene Su_uga.A comparative study of the phage yield with amber and opal mutations located in the same (paired) triplet in gene 47 has shown that the suppressor activity depends on the location of the mutant site along the gene.Experiments dealing with the induction of reversions by nitrous acid in amber and opal mutants with mutational sites located in the same triplet of gene 47 (mutant pairs) have shown the essential influence of the nucleotide sequence in the triplet on the frequency of induced reverse mutations at the given site.     

Complementation analysis by somatic hybridisation and genetic crosses of nitrate reductase-deficient mutants of Nicotiana plumbaginifolia

Summary Fusion complementation experiments between nitrate reductase (NR) deficient lines CNX 20, 27, 82 and 103 of Nicotiana plumbaginifolia were performed with the already characterized N. plumbaginifolia mutants nx 1, 24 and 21, belonging respectively to the complementation groups cnx A, B and C. CNX 20 and 82 were identified as belonging to the group of cnx A. CNX 27 complemented with NX 1 and NX 21 but not with NX 24 indicating another B type. The fourth line, CNX 103 showed complementation with CNX 20, NX 21 and NX 24, revealing a fourth cnx complementation group, cnx D, that until now has not been described in higher plants. Genetic crosses inside respectively the NIA and the CNX group, and between NIA and CNX confirmed the fusion complementation results, and showed allelism for the nia mutants