Shoot-bud regeneration in subcultured callus of engelmann spruce

Summary Callus cultures ofPicea engelmannii (Parry, Engelmann spruce) were initiated and established from mature embryos cultured on von Arnold and Eriksson’s medium (AE) supplemented with N⁶-benzyladenine (10μM) and naphthalene acetic acid (10 μM). Cultures were maintained by subculture at 3-to-4-wk intervals. After three subcultures, callus was transferred to AE medium with only N⁶-benzyladenine (25 μM). Adventitious buds appeared on the surface of the callus after 2-to 4-wk and grew to adventitious shoots on AE medium without growth hormones or on AE medium with kinetin (0.1 μM). Shoot-forming capacity was maintained through 7 further subcultures. This study was supported by the Natural Sciences and Engineering Research Council of Canada grant G1438 to T. A. Thorpe and D. I. Dunstan.     

Xanthine dehydrogenase (XDH) cross-reacting material in mutants of Drosophila melanogaster deficient in XDH activity

Rocket immunoelectrophoresis was used to estimate xanthine dehydrogenase cross-reacting material (XDH-CRM) in strains containing the cin ¹ and cin ⁹ mutant genes, which are deficient in XDH enzymatic activity. CRM levels were determined as percentages of CRM in the Oregon-R wild-type strain. The mutant strains contain 72 and 76% of Oregon-R CRM, respectively. CRM levels in strains containing the XDH-deficient mutant genes lxd and mal are 93 and 105%, respectively. The high levels of CRM in these four mutant strains indicate that the primary effects of the mutant genes are on the function of XDH protein rather than its accumulation.This research was supported by a grant from the Natural Sciences and Engineering Research Council of Canada to L. W. B.     

Analysis of a recessive mutation on the Y chromosome of the fly Caliphora erythrocephala

Summary In a strain of the mutantw (yellow eye colour, sex-linked and sex-limited to the females) ofCalliphora erythrocephala yellow-eyed males have been found. In connexion with the mechanism of sex-determination in this fly, their origin is explained by an alteration on the Y-chromosome (one of the III^rd large chromosomes) in form of either a pre-meiotic cross-over or a spontaneous mutation on thew ⁺-locus. For being recessive, it can become manifest only in thew strain.     

A setup for incubating plant cultures under continuous flow of gases

Summary This note describes a simple experimental system for incubating plant cell and tissue cultures under conditions in which the gaseous environment within the culture flasks is continuously flushed out by air or gas mixtures of known composition. Two of the major problems in maintaining such cultures, namely, contamination and desccation of the agar-solidified medium, can be overcome by using this setup. Furthermore, with modifications, it can be adapted for commercial micropropagation projects. This research was supported by Natural Sciences and Engineering Research Council of Canada grant A-6467 to T. A. Thorpe. The authors thank R. W. Joy IV for drawing Fig. 1B.     

The genetics of a new mutable allele at the white locus in Drosophila melanogaster

Summary The genetics of a third case of high mutation frequency at the white locus in Drosophila melanogaster has been analyzed. The new mutable allele, w ^+u, mutates from a wild-type to a white-eyed phenotype in both males and females. The mutational event is 1) premeiotic, 2) not associated with crossingover, 3) sensitive to genetic modification, and 4) restricted to germinal tissue. The only mutants produced by w ^+u are deletions of the white locus. These deficiencies include subsites 4 and 5 of the white locus, but are cytologically unobservable. The mutable allele itself maps to subsite 4.The mutational properties of w ^+u are unlike those of the other highly mutable white alleles which have been interpreted in terms of phage-like controlling elements. Rather, the properties of w ^+u favor a model based on the premature termination of chromosome replication near the terminus of a replicon which leads to a chromosome deficient for the material between the point of premature termination and the end of the replicon.Supported by NIH predoctoral traineeship GM-150 and by NIH research grant GM-07428 to Dr. W. K. Baker.From a dissertation submitted to the Division of Biological Sciences of The University of Chicago in partial fulfillment of the requirements for the degree of Doctor of Philosophy.     

Genetic instability in Drosophila melanogaster: Transpositions of the white gene and their role in the phenotypic expression of the zeste gene

Summary Seven independent transpositions of the w ⁺ gene have been recovered as derivatives of two separate direct tandem duplications of the white locus. The transpositions map to discrete sites on both major autosomes. Five transpositions were employed to study the role of w ⁺ gene dos-age on zeste (z) gene expression. Each transposition generates a unique zeste phenotype; one transposition is not predictive for another. A functional allele of zeste, z ^77h, responds to w ⁺ gene dosage contrary to the z response.Supported by NIH grant GM22221     

Reversal effect of the genetic background on some small-eye mutants,with special reference to gene action of morphogenetic mutants inDrosophila

The facet number of heterozygous females of theBar-eye mutant ofDrosophila melanogaster varies from ±154 to ±665 according to the full eye strains combined with the mutant. TheBar character is commonly recognized as a semidominant, but its manifestation varies from nearly complete dominance to recessiveness according to the mode of the heterozygous combination. Virtually the same is true of the mutantLobe ² which is usually acknowledged as a complete dominant, but may be manifested as an incompletely dominant gene. Thew ^e andb gp strains decrease the eye size of the heterozygotes withBar, but increase the eye size of the heterozygotes withLobe ².Co-isogenic strains ofBar, bar-3 andeyeless ² mutants with Oregonisogenic increase the facet number to degrees 1.4–1.8 times of the original strains. However, the co-isogenicLobe ² strain with the same Oregonisogenic strain reduces the facets completely, and makes virtually all individuals eye-less. These observations indicate that the same genetic background may give an opposite effect to different small-eye mutants. Analysis was carried out for modifying genes ofbar-3. Modifiers were detected on all large chromosomes, besides a recessive major gene located on the 2nd chromosome of Oregon-isogenic strain.A part of this work was performed in the Division of Biology, California Institute of Technology, Pasadena.     

Aldehyde oxidase cross-reacting material in the Aldox n,cin, mal,and lxd mutants of Drosophila melanogaster

Rocket immunoelectrophoresis was used to estimate aldehyde oxidase cross-reacting material (AO-CRM) in larval hemolymph and adult fly extracts in mutants with reduced AO enzymatic activity. Hemolymph of larvae homozygous for Aldox ⁿ, which is a mutation of the presumed structural gene for AO, contains 30% of the wild-type CRM. The demonstration of AO-CRM in Aldox ⁿ larval hemolymph is surprising since this genotype has been reported to lack CRM. By contrast, adult Aldox ⁿ flies lack detectable CRM. The other AO-deficient mutants that were examined are cin, mal, and lxd; each has appreciable levels of CRM in both larval hemolymph and adult extracts. Detection of CRM in these mutants helps to clarify conflicting reports in the literature.This research was supported by a grant from the Natural Sciences and Engineering Research Council of Canada to L.W.B.     

Mechanism of suppression in Drosophila. V. Localization of the purple mutant of Drosophila melanogaster in the pteridine biosynthetic pathway

The suppressible eye color mutant purple (pr) of Drosophila melanogaster is known to be unable to synthesize a wild-type complement of pteridine eye pigments. This study measures the reduced levels of drosopterins, sepiapterin, and an unidentified presumed pteridine in pr and pr ^bw. Pteridine analyses in double mutants combining pr with one of three other eye color mutants sepia, Henna-recessive³, and prune², suggest that the metabolic block in pr occurs prior to sepiapterin biosynthesis. Measurements of GTP and GTP cyclohydrolase in pr showed wild-type levels and indicate the metabolic block in pr to be at one of the steps converting dihydroneopterin triphosphate to sepiapterin. Quantitation of pteridines in suppressed purple [su(s) ²; pr and pr; su(pr) ^e3] shows restoration of pteridines to wild-type or nearly wild-type levels.T. G. W. is a predoctoral trainee supported by Grant GM 1974 from the National Institute of General Medical Sciences, National Institutes of Health.The Oak Ridge National Laboratory is operated by Union Carbide Corporation for the U.S. Energy Research and Development Administration.     

DNA sequence analysis of spontaneous and γ-radiation (anoxic)-induced lacI mutations in Escherichia coliumuC122::Tn5: differential requirement for umuC at G · C vs. A · T sites and for the production of transversions vs. transitions

Escherihica coliumC122::Tn5 cells were γ-radiated (¹³⁷Cs, 750 Gy, under N₂), and lac-constitutive mutants were produced at 36% of the wild-type level (the umC strain was not deficient in spontaneous mutagenesis, and the mutational spectrum determined by sequencing 263 spontaneous lacI^d mutations was very similar to that for the wild-type strain). The specific nature of the umC strain's partial radiation was determined by sequencing 325 radiation-induced lacI^d mutations. The yields of radiation-induced mutation classes in the umC strain (as a percentage of the wild-type yield) were: 80% for A · T → G · C transitions, 70% for multi-base additions, 60% for single-base deletions, 53% for A · T → C · G transversions, 36% for G · C → A · T transitions, 25% for multi-base deletions, 21% for A · T → T · A transversions, 11% for G · C → C · G transversions, 9% for G · C → T · A transversions and 0% for multiple mutations. Based on these deficiencies and other factors, it is concluded that the umC strain is near-normal for A · T → G · C transitions, single-base deletions and possibly A · T → C · G transversions; is generally deficient for mutagenesis at G · C sites fro transversions, and is grossly deficient in multiple mutations. Damage at G · C sites seems more difficult for translesion DNA synthesis to bypass than damage at A · T sites, and especially when trying to produced a transversion. The yield of G · C → A · T transitions in the umC strain *36% of the wild-type level) argues that a basic sites are involved in no more than 64% of γ-radiation-induced base substitutions in the wild-type strain. Altogether, these data suggest that the UmuC and UmuD′ proteins facilitate, rather than being absolutely required for, translesion DNA synthesis; with the degree of facilitation being dependent both on the nature of the noncoding DNA damage, i.e., at G · C vs A · T sites, and on the nature of the misincorporated base, i.e., whether it induces transversions or transitions.     

Non-morphogenic metabolism during shoot induction in radiata pine cotyledon explants

Summary The effects of excision, light and cytokinin (N⁶-benzyladenine) on¹⁴C-acetate metabolism in cotyledons ofPinus radiata (D. Don) were determined.¹⁴CO₂ was released and the distribution of radioactivity into lipids, sugars, organic acids and amino acids was determined. While light and cytokinin generally caused some increase in metabolism, the effect of excision, i.e., wounding, was most pronounced. Specific metabolites examined (citrate, malate, succinate, alanine, aspartate, glutamate and glutamine) were at least 50% greater in¹⁴C-labeling in excised cotyledons as compared to intact seedlings. This enhancement of wound metabolism would mask possible morphogenically-related changes occurring at that time. This research was supported by the Natural Sciences and Engineering Research Council of Canada Grant A-6467 to T.A. Thorpe.     

The genetic analysis of a large transposing element of Drosophila melanogaster

A member of Ising's family of large transposing elements (TEs) has inserted into, or very near, the crinkled (ck, 2–50) locus. This TE (TE36) carries functional alleles of both the white and roughest loci, and causes a hypomorphic mutation of ck. The TE is visible in polytene chromosomes as a two-banded insertion between 35B9 and 35C1. These bands show homology to foldback (FB) elements by in situ hybridization. All spontaneous losses of TE36 remain mutant for ck and retain sequences homologous to FB at the site of TE's insertion. TE36 carries only one functional copy of w ⁺, by the criterion that z w, TE36/ + flies are wild-type for eye color but z w; TE36/TE36 flies are zeste. This white⁺ gene is dosage compensated since w/Y; TE36/+ males have twice as much eye pigment as w/w; TE36/ + females. A form of the TE that has four polytene chromosome bands and expresses twice as much pigment as TE36 has been recovered. However, its white genes are not suppressed by zeste.     

Inheritance patterns of new genetic markers and occurrence of spontaneous mosaicism in the monogenic blowfly Chrysomya rufifacies (Diptera: Calliphoridae)

 Four new genetic markers for Chrysomya rufifacies, a fly with maternal sex determination, were characterized. The markers include one body colour mutant, black body (bl), and three eye colour mutants, brown eye (br), apricot eye (ap), and red eye (w ^r ). Two of the latter, br and w ^r , turn out to be sex linked, the others behave as autosomal genes belonging to different linkage groups. w ^r is a hypomorphic and w an apomorphic mutation of the white gene, w/w is epistatic to br/br and to ap/ap. A preliminary genetic linkage map with the sex realizer F′/f  and the loci br and w residing in homomorphic sex chromosomes is established. Evidence is presented that crossing over is absent in the male sex. The possible causes of the spontaneous appearance of mosaics for eye colour observed among individuals heterozygous for recessive genes are discussed. Received: 18 March 1996/Accepted: 9 August 1996     

Regeneration of plantlets from mature embryos of western larch

Summary Larix occidentalis Nutt. can be micropropagated using whole excised mature embryos. The basal medium for induction (over 70% response) was half-strength Quoirin and LePoivre salts with Schenk and Hildebrandt organics and N⁶-benzyladenine singly or with kinetin at a final concentration of 10μM. Bud elongation was best on half-strength Schenk and Hildebrandt or Bornman’s mineral salts, and elongated shoots could be maintained on either half-strength Quoirin and LePoivre or Schenk and Hildebrandt formulations containing 0.05% activated charcoal, 2% sucrose, and 0.7% agar. Axillary buds developed naturally on elongating juvenile shoots, but a high sucrose treatment (6%) for 2 wk enhanced the number produced. Very good rooting (80 to 100%) was obtained by pulsing shoots for 2 to 3 h in a solution of indole butyric acid (1 mM, pH 4.5–5.0), or by planting shoots in peat-vermiculite moistened with basal medium containing 5μM α-naphthalene acetic acid, pH 5.0. Rooted shoots were hardened before transfer to the greenhouse, and initially were kept at low light and high humidity after transfer to ex vitro conditions. This research was supported initially by a National Research Council of Canada, Division of Energy Contract, and subsequently by a Natural Sciences and Engineering Research Council of Canada strategic grant to T. A. Thorpe.     

A new RNA synthesis mutant of E. coli

A temperature-sensitive mutant of E. coli is described. At the nonpermissive temperature, the capacity for RNA and protein synthesis decreases logarithmically in the mutant. The mutant is unable to support the growth of f2 or T7 virus, even at the permissive temperature. The temperature-sensitive mutation maps approximately 1 away from rif ^r in E. coli and therefore affects a gene previously undescribed. The temperature sensitivity is suppressed by sublethal concentrations of rifampicin. Moreover, in rif ^r T^s double mutants, the T ^s mutation suppresses rif ^r and vice versa. The partially purified RNA polymerases from mutant and wild-type cells have different temperature and salt optima.This research was supported by Public Health Service grant GM-14368 from the National Institute of General Medical Sciences and by grant IN-29 from the American Cancer Society. One of us (D.P.) is a predoctoral trainee, supported by a National Science Foundation Graduate Traineeship Program and by a National Institutes of Health Predoctoral Research Fellowship. S. Marshall is supported by LASBAU.     

Properties of the nonlethal recombinational repair deficient mutants of bacteriophage T4

Summary The rate at which ³H thymidine is incorporated into DNA is increased in T4w-infected cells compared to wild-type when measured late in infection under conditions of low thymidine concentration. This increased DNA synthesis is sensitive to hydroxyurea but not to mitomycin C, and can be prevented by the addition of chloramphenicol early in infection. Also, DNA replicative intermediates isolated from T4w-infected cells late in infection sediment significantly faster than those isolated from wild-type-infected cells. In contrast, DNA replicative intermediates isolated from T4x-or T4y-infected cells sediment more slowly than those produced by wild-type T4. Cells coinfected with wild-type T4⁺ and T4x, y or w; or T4w and T4x or y, produce wild-type DNA replicative intermediates. Cells coinfected with T4x and T4y produce more slowly sedimenting DNA replicative intermediates. Cells coinfected with T4w and wild-type T4 show wild-type rates of DNA synthesis while cells coinfected with T4w and T4x or T4y show increased rates of DNA synthesis over that observed with wild-type alone.     

The w/w SMART assay of Drosophila melanogaster detects the genotoxic effects of reactive oxygen species inducing compounds

The somatic mutation and recombination w/w⁺ eye assay has been used for genotoxic evaluation of a broad number of chemicals with different action mechanisms yielding high values of sensitivity, specificity and accuracy. The aim of this work was to determine the utility of this assay in the evaluation of reactive oxygen species inducers. For this, we have tested eight compounds: diquat, paraquat, menadione, juglone, plumbagin, streptonigrin, tert-butyl hydroperoxide and 4-nitroquinoline 1-oxide, using the Drosophila Oregon K strain which had previously shown advantageous conditions to test this type of compounds. Diquat was the only chemical for which the results were clearly negative, probably because its high toxicity, whereas indications of a marginal genotoxicity rised for menadione. The remaining compounds were evaluated as positives. The conclusion of these experiments is that the w/w⁺ assay is capable to detect genotoxic effects induced by compounds that generate reactive oxygen species through different action mechanisms.     

Specificity of mutations induced by riboflavin mediated photosensitization in the supF gene of Escherichia coli

Riboflavin-mediated photosensitization has been shown to produce 8-hydroxyguanine (oh⁸Gua) in DNA. We investigated the specificity of mutation of photosensitized supF gene induced in Escherichia coli. The oh⁸Gua repair deficient E. coli mutant mutM and mutY were transformed with plasmid pUB3 carrying the supF gene irradiated with white light in the presence of riboflavin. Under these conditions, riboflavin photosensitization increased the amounts of oh⁸Gua in pUB3 DNA. Three types of a single base substitution occurring at G:C pairs were detected in both wild-type and mutM mutant strains. Almost all base substitutions were transversions to T:A or C:G pairs occurring at a similar extent in both wild-type and mutM strains. Mutations derived from mutY strain transformed with photosensitized DNA were only G:C to T:A transversions. These G:C to T:A transversions observed in the mutY strain were suggested to be the result of mispairing of oh⁸Gua with adenine. Riboflavin-mediated photosensitization may also produce lesions on DNA causing G:C to C:G changes by unknown mechanisms.     

DNA polymerase III-dependent repair synthesis in response to bleomycin in toluene-treated Escherichia coli

Summary Bleomycin (BLM) is an antitumor drug which interacts with and damages DNA. We have reported a repair response dependent on DNA polymerase I in toluene-treated Escherichia coli. We report here that DNA polymerase III can also catalyze a repair response in toluene-treated E. coli following exposure to BLM. Polymerase III-mediated synthesis differs because it is ATP-dependent, whereas polymerase I-mediated repair synthesis is not. Polymerase III repair synthesis is independent of replicative synthesis, as demonstrated in a polA ^-, dnaB _ts strain, or use of Novobiocin to inhibit replication, and replication persists in the presence of repair synthesis. It appears that ATP-dependent repair synthesis in response to BLM is also present in polA ⁺ strains. Repair synthesis does not require the uvrA gene product.This research was supported by Public Health Service grants GM-19122 and GM-24711 from the National Institute of General Medical Sciences, Robert A. Welch Foundation grant Q-543 and American Cancer Society BC-290     

Some observations on variegation inDrosophila

Attempts to modify experimentally the variegated phenotypes of the mutantsz inDrosophila melanogaster andw ^m2 inD. hydei, were largely unsuccessful. Transplanted eyes ofz were found to be more intensely pigmented, but this effect was not influenced by treatment of the imaginal disks or by the host's genotype. Inw ^m2, increased pigmentation was observed after treatment with 5-bromo-uracil.Thew ^m2 phenotype is strongly modified by supernumerary Y chromosomes. One Y shifts the eye colour from yellow to brown, two Y's, to dark red. In the Malpighian tubules, two Y's have a similarly strong effect, but one Y a slight effect only. Malpighian tubules ofw ^m2 larvae were used for a quantitative analysis of mottling. An average clonal cluster size of 1.1 was found. This indicates that the state of pigmentation is determined around the last cell division in the embryo.