The maxillary palp of Drosophila : ultrastructure and physiology depends on the lozenge gene

The ultrastructure and physiology of the maxillary palp of Drosophila melanogaster have been studied in wild-type and lozenge mutants. Olfactory physiology in the maxillary palp is shown to depend upon the lozenge(lz) gene. Reduced response amplitudes were recorded for all odorants tested, and the physiological defect was shown to map to the lz locus. The structure of the maxillary palp sensilla is described by scanning electron microscopy (SEM) at high magnification, initially in the wild-type. A linear arrangement of pores, connected by furrows, was found in one class of sensilla, the basiconic sensilla. In the lz ³ mutant, morphological alterations in the basiconic sensilla and duplications of sensilla are documented by SEM. The correlation of structural abnormalities in the lz sensilla and physiological abnormalities in odorant response are consistent with an olfactory role for the basiconic sensilla of the maxillary palp. Accepted: 10 September 1996     

Intragenic somatic recombination in the lozenge cistron of Drosophila

Summary In Drosophila melanogaster intragenic mitotic recombination between the two lozenge alleles, lz ³⁶ and lz ^y4, separated from each other by 0.14 meiotic recombination units, was observed. Among 48 725 females of the genotype w ⁺lz³⁶/w lz^y4 which had been irradiated by a dose of 1000 r X-rays as larvae 41–47 hours after oviposition, a total of 11 faceted eye spots (not lz) were induced. All 11 spots were w ⁺, none w. Possible reasons for the lack of the expected w, faceted spots were checked. Inversion of the X chromosome which would suppress recombination between the w and lz loci was not involved. Gene order of lz ³⁶-lz^y4-kinetochore was confirmed by meiotic recombination test. Nonautonomy of lz gene action was not a factor, as tested by gynanders which showed that lz ^y4 and lz ^s were autonomous. Possibility of reverse mutation was not likely as shown by the large scale control experiments. Gene conversion is suggested as a likely mechanism for the lack of the expected w, faceted spots although the possibility of unequal crossing over induced by X-rays can not be excluded, nor can the preferential z-segregation hypothesis.     

Are the T-DNA Mutants Amenable to Standard Recombination Analysis?

Genetic analysis with T-DNA mutants often brings difficulties resulting from instability of the transgenic phenotype. In this work three different Arabidopsis thaliana T-DNA embryonic lethals and one T-DNA morphological mutant were analyzed in F2 progeny after 15 different crosses with marker lines for individual chromosomes. F2 analysis of 44 segregation ratios revealed segregation distortion of similar character consisting in abnormal excess of nontransgenic plants to the detriment of transgenic ones. We quantified this phenotypic drift (d) on the basis of phenotypic ratios given the respective formulas. The d values indicate the rate of F1 gametes which loose the T-DNA mutation or ability of its expression. The obtained d value were relatively high, 0.4 to 0.9 for individual crosses. It makes the standard recombination analysis with insertional mutants very problematic or even impossible.     

Phenol oxidase activity and the Lozenge locus of Drosophila melanogaster

We have found that the phenol oxidase activity in 50-hr Drosophila melanogaster pupae is much greater than that of adult flies. The mutants lz and lz ^g have all of the phenol oxidase components present in wild type, whereas the mutant tyr-1 has all of the wild-type components but the activity of each component is greatly reduced in comparison with wild-type activity. The newly discovered lozenge allele, lz ^rfg, lacks all phenol oxidase activity.Predoctoral fellow supported by Grant GM 1974 from the National Institute of General Medical Sciences, National Institutes of Health.The Oak Ridge National Laboratory is operated for the U.S. Atomic Energy Commission by Union Carbide Corporation.     

A new gene (madI) involved in the phototropic response of Phycomyces

Summary Only eight genes are known to be involved in the phototropic response of Phycomyces (madA-H). Mutants affected in these genes have played a major role in the analysis of photosensory transduction processes in this system. A set of new mutants isolated by Alvarez et al. (1989) that are unable to bend towards dim unilateral blue light were studied by complementation and recombination. Two of these mutants have mutations in madE, one has a mutation in madF and one is a double madE madF mutant. The three remaining mutants tested did not complement each other and showed positive complementation with strains carrying mutations in the genes madA, madB, and madC, indicating that they carried mutations in a new gene designated madI. Recombination analysis showed that madI is unlinked to madA, madB and madC.     

Genetic analysis of leaf form mutants from the Arabidopsis Information Service collection.

Although a vast inventory of morphological mutants of Arabidopsis thaliana is available, only some have been used for genetic studies of leaf development. Such is the case with the Arabidopsis Information Service (AIS) Form Mutants collection, assembled by A. R. Kranz and currently stored at the Nottingham Arabidopsis Stock Centre, which includes a large number of mutant lines, most of which have been little studied. With the aim of contributing to the genetic dissection of leaf ontogeny, we have subjected 57 mutant lines isolated by others to genetic analysis; 47 of which were from the AIS collection. These are characterized by vegetative leaves of abnormal shape or size, and were chosen as candidates for mutations in genes required for leaf morphogenesis. The mutant phenotypes studied were shown to be inherited as single recessive Mendelian traits and were classified into 10 phenotypic classes. These mutant strains were found to fall into 37 complementation groups, 7 of which corresponded to known genes. Results of the phenotypic analysis and data on the genetic interactions of these mutants are presented, and their possible developmental defects discussed. Received: 28 October 1998 / Accepted: 21 February 1999     

A general method for the construction of Escherichia coli mutants by homologous recombination and plasmid segregation

Summary A technique is presented by which mutations can be introduced into the Escherichia coli chromosome by gene replacement between the chromosome and a plasmid carrying the mutant gene. The segregational instability of plasmids in E. coli is used with high efficiency to isolate E. coli mutants. The method should be applicable to construction of mutants for any E. coli chromosomal gene provided it is dispensable, and for any E. coli strain provided it is capable of homologous recombination. The use of the method was demonstrated by constructing E. coli mutants for the glycogen branching enzyme gene (glgB) and the -galactosidase gene (lacZ). The results show that recombination occurs via a reciprocal mechanism indicating that the method should, in a slightly modified form, also be useful in transferring chromosomal mutations onto multicopy plasmids in vivo.     

Hormone-Dependent Insertional Mutants of Arabodopsis thalianawith Reduced Viability and Fertility

We present data on the phenotype identification and genetic analysis of offspring in three lines of dominant morphological mutants of Arabidopsis thalianahaving drastically reduced fertility (a sterile calluslike mutant, a flower mutant, and a dwarf mutant) and in five lines of recessive morphological mutants (four mutants with lethal seedlings and one pigmentation mutant). The mutants were selected from a collection of transgenic plants that had genomes carrying a T-DNA insertion of plasmid vectors pLD3 and pPCVRN4; the collection was created earlier via agrobacterial transformation of germinating seeds. The results presented here were obtained using compensation of hormonal imbalance in the insertional morphological mutants of A. thalianaby exogenous hormones.     

Phenotypic interactions between abscisic acid deficient tomato mutants

Summary A series of double mutant homozygotes have been produced from three wilty tomato mutants; flacca, sitiens and notabilis. The phenotypic interaction between the mutant genes has been studied. The severity of phenotype in the double mutants does not correspond to that predicted from the single mutant homozygotes. The results are discussed in relation to the probable involvement of the mutants in abscisic acid metabolism.     

Isolation and Partial Characterization of Mutants of the Trypanosomatid Crithidia fasciculata and Their Use in Detecting Genetic Recombination1

Crithidia fasciculata was used as a model trypanosomatid to study the possible existence of genetic recombination in this group of protozoa. The approach was based on the ability to select a variety of mutants on agar plates. Following mutagenesis of wild type cells by nitrosoguanidine or ethylmethanesulfonate, stable mutant phenotypes were obtained. These included mutants resistant to the drugs actinomycin D, 6-azauracil, 6-azauridine, and 5-fluorouracil, auxotrophs and colony morphology mutants. Following mixed growth of pairs of drug-resistant mutants on selective media, isolates exhibiting stable recombinant phenotypes were obtained. The data presented suggest that 1) Crithidia undergoes some type of genetic recombination and 2) Crithidia must be diploid at some time during this process.     

Influence of the biogenic amine tyramine on ethanol‐induced behaviors in Drosophila

The biogenic amine tyramine has been implicated in drug‐induced behavior. The Drosophila inactive mutant is characterized by reduced tyramine and octopamine levels and is defective in cocaine sensitization. To test whether there is an overlap in the use of the amine neurotransmitter system in ethanol‐ and cocaine‐induced behaviors, mutant analyses were extended to the phenotypic characterization of inactive and other mutants effecting the tyramine and octopamine neurotransmitter system. The inactive mutant displays increased ethanol sensitivity and is impaired in the initial startle response upon ethanol application. Furthermore, this mutant fails to regulate its alcohol‐induced hyperactivity properly. In contrast to the defects seen after cocaine application, inactive mutants develop normal ethanol tolerance and sensitize to the locomotor activating effect of ethanol. The tyramine‐β‐hydroxylase mutant (TβH) with increased tyramine and depleted octopamine levels displays normal ethanol sensitivity, a startle repression, and hyperactivates more in response to ethanol. In addition, TβH mutants fail to develop a tolerance to the hyperactivating effect of ethanol. Ethanol‐induced sensitization does not seem to be impaired in either mutant, suggesting that tyramine is not required for this process. The comparative analysis of the phenotypes associated with inactive and TβH mutants suggests that the fine tuning of ethanol‐induced hyperactivity can be correlated with different tyramine levels. Defects in other aspects of ethanol‐induced behaviors might be due to different molecules or mechanisms. © 2005 Wiley Periodicals, Inc. J Neurobiol, 2005     

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.     

Isolation and characterization of Paracoccus denitrificans mutants with increased conjugation frequencies and pleiotropic loss of a (nGATCn) DNA-modifying property

A selection scheme was devised to isolate Paracoccus denitrificans mutants with increased recipient qualities in transfer experiments, using broad host range plasmids. In some of the mutants obtained, a DNA modifying activity that prevents the activity of the restriction endonucleases BamHI and BglII on isolated P. denitrificans DNA had simultaneously been lost. From a detailed analysis of the restriction properties of the enzymes SAU3 AI, MboI and DpnI, it was concluded that a subset of GATC sequences in P. denitrificans DNA may be methylated at an unusual position. It was concluded that P. denitrificans possesses at least one potent host-dependent restriction/modification system which affects conjugation. In addition to the class of restriction-defective mutants, at least one other class of enhanced transfer mutants with unknown defect(s) was isolated. Strains, in which the two mutant classes were combined, exhibited transfer frequencies which were significantly higher than strains containing either mutation alone. Such double mutant strains appeared to be well suited for future experiments like complementation analysis, transposon mutagenesis and gene replacement by homologous recombination.     

Development of an efficient gene targeting system in Colletotrichum higginsianum using a non-homologous end-joining mutant and Agrobacterium tumefaciens-mediated gene transfer

The hemibiotrophic ascomycete Colletotrichum higginsianum is the casual agent of anthracnose disease of cruciferous plants. High efficiency transformation by Agrobacterium tumefaciens-mediated gene transfer has been established for this fungus. However, targeted gene mutagenesis through homologous recombination rarely occurs in C. higginsianum. We have identified and disrupted the C. higginsianum homologue of the human Ku70 gene, ChKU70, which encodes a protein that plays a role in non-homologous end-joining for repair of DNA breaks. chku70 mutants showed a dramatic increase in the frequency of integration of introduced exogenous DNA fragments by homologous recombination without any detectable phenotypic defects. This result demonstrates that the chku70 mutant is an efficient recipient for targeted gene mutagenesis in C. higginsianum. We have also developed a novel approach [named direct repeat recombination-mediated gene targeting (DRGT)] for targeted gene disruption through Agrobacterium tumefaciens-mediated gene transfer. DRGT utilizes homologous recombination between repeated sequences on the T-DNA flanking a partial fragment of the target gene. Our results suggest that DRGT in the chku70 mutant background could be a useful tool for rapid isolation of targeted gene disruptants in C. higginsianum.     

Effect of recF, recJ, recN, recO and ruv mutations on ultraviolet survival and genetic recombination in a recD strain of Escherichia coli K12

Summary DNA repair and recombination were investigated in a recD mutant of Escherichia coli which lacked the nuclease activity of the RecBCD enzyme. The resistance of this mutant to ultraviolet (UV) light was shown to be a function of recJ. A recD recJ double mutant was found to be more sensitive to UV radiation than a recB mutant, whereas recD and recJ single mutants were resistant. Recombination in conjugational crosses with Hfr donors was also reduced in recD recJ strains, but the effect was modest in comparison with the sensitivity to UV. Within certain limits, mutations in recF, recN, recO, lexA and ruv did not affect sensitivity to UV and recombination in a recD mutant any more than in a recD ⁺ strain. The possibility that recD and recJ provide overlapping activities, either of which can promote DNA repair and recombination in the absence of the other, is discussed.     

Ac/Ds transposon mutagenesis in Arabidopsis thaliana: mutant spectrum and frequency of Ds insertion mutants

Using a two-component Ac/Ds system consisting of a stabilized Ac element (Ac_c1) and a non-autonomous element (Ds_A), 650 families of plants carrying independent germinal Ds_A excisions/transpositions were isolated. Progenies of 559 of these Ac_c1/Ds_A families, together with 43 families of plants selected for excision/transposition of wild-type (wt)Ac, were subjected to a broad screening program for mutants exhibiting visible alterations. This resulted in the identification of 48 mutants showing a wide variety of mutant phenotypes, including embryo lethality (24 mutants), chlorophyll defects (5 mutants), defective seedlings (2 mutants), reduced fertility (5 mutants), reduced size (3 mutants), altered leaf morphology (2 mutants), dark green, unexpanded rosette leaves (3 mutants), and aberrant flower or shoot morphology (4 mutants). To test whether these mutants were due to transposon insertions, a series of Southern blot experiments was performed on 28 families, comparing in each case several mutant plants with others showing the wild-type phenotype. A preliminary analysis revealed in 4 of the 28 families analyzed a common, novel Ds_A fragment in all mutant plants, which was present only in heterozygous plants with wt phenotype, as expected for Ds_A insertion mutations. These four mutants included two showing embryo lethality, one with dark green, unexpanded rosette leaves and stunted inflorescences, and one with curly growth of stems, leaves and siliques. Further evidence for Ds_A insertion mutations was obtained for one embryo lethal mutant and for the stunted mutant, while in case of the second embryo lethal mutant, the Ds_A insertion could be separated from the mutant locus by genetic recombination.     

Phytotron cultivation of early barley mutants

Conclusions The authors have tried to gather data which permit some information on the between and within locus reactions of induced early barley mutants to different photo- and thermoperiods. Eight mutant cases, showing rather drastic earliness in field cultivation and representing three different gene loci, were examined in phytotron experiments according to routine methods of cultivation. One of the mutants, mat-a ⁸, has been released as an original Swedish barley variety under the name of Svalöf's Mari. In a previous publication (Dormling et al., 1966) this mutant was compared to its parent variety, Svalöf's Bonus, under 30 different climatic conditions. In the present investigation three photoperiods (24, 16 and 8 hours of artificial light) were combined with three suitable thermoperiods (20-15°, 20-10° and 15-10°C).The results indicate that photoperiodic insensitivity, with regard to ear formation and heading capacity, as well as kernel production, is of rather frequent occurrence in connection with drastically early mutants in barley. Four out of eight induced mutants give a more or less pronounced insensitivity. Three of the four insensitive mutants represent locus a, one belongs to locus b. Of the two c-mutants none was insensitive; both were on the contrary pronounced long-day types.Photo- and thermoperiods interact in various ways. This is especially clear in the c-mutants just mentioned, which have a high generative productivity and efficiency at continuous light and high thermoperiods. They produce no grain but considerable vegetative matter at 8 hours of light, independently of thermoperiod, as well as at 16 hours of light with high temperatures. In fact, mutants of loci a and c differ strikingly with regard to their relations to the climatic conditions applied. The insensitive mutant b ¹³ is remarkably similar to the mutant a ¹², but its resemblance to the sensitive mutants b ⁷ and b ¹⁰ of the same locus is evidenced by its high average internode number.It ought to be pointed out here that the mutants of the three gene loci analysed in this study can be distinguished phenotypically with regard to morphological as well as physiological properties, in the field as well as in phytotron cultivation. The c-mutants are especially characteristic. However, there also seem to be clear differences in reaction between the allelic mutants of a locus. In fact, all eight mutants studied seem to react more or less differently.The insensitive mutant a ⁸, which has been released into practice, is also widely used in recombination work, and successful segregates have been isolated. The characteristics of a ⁸, which make the mutant valuable in practice, are also found in phytotron experimentation, specially with regard to earliness, generative efficiency and yield. Also the semidwarf habit and the insensitivity to changes in photo- and thermoperiods readily show up.This investigation has been generously supported by the Swedish Research Council of Forestry and Agriculture.     

Early blocked asporogenous mutants of Bacillus subtilis 168. I. Isolation and characterization of mutants resistant to antibiotic(s) produced by sporulating Bacillus subtilis 168

Summary A number of mutants (abs)-resistant to antibiotic(s) produced by sporulating Bacillus subtilis 168 have been isolated from an early blocked asporogenous mutant (spoA12). At least four classes were recognized according to their phenotypic properties. Genetic analysis has shown that these mutants were neither partial revertants nor suppressor mutants of the spoA gene. Both nonsense and missense mutants of the spoA gene are reverted partially by a secondary mutation which is resistant to antibiotic of B. subtilis 168. Another asporogenous mutant, spoB, whose locus is closely linked to pheA, is also affected by the same abs mutation. The nature of abs mutants is discussed.