White Sectors Research Articles

Analysis of Mutator-induced mutations in the iojap gene of maize.

The recessive nuclear mutation iojap (ij) in maize produces striped plants with normal chloroplasts in green sectors and poorly developed chloroplasts in the white sectors. The ij mutation is also characterized by an array of additional phenotypic affects which suggests a pivotal role for Ij in chloroplast development. The Ij gene from maize has been isolated; however, the sequence has not provided information on the action of the Ij gene product. As an extension of the analysis of the Ij gene we have generated a number of independent transposon insertion mutations using the Robertson's Mutator (Mu). Insertions were found to be distributed throughout the 5' half of the gene and all showed a similar striping pattern to that of the original ij mutation, ij-ref. While several of the new mutant alleles produced ij-homologous transcripts none produced detectable levels of the Ij polypeptide product. Chloroplast development in some cells appears to be able to proceed in the absence of the Ij polypeptide.

Analysis ofMutator-induced mutations in theIojap gene of maize

The recessive nuclear mutationiojap (ij) in maize produces striped plants with normal chloroplasts in green sectors and poorly developed chloroplasts in the white sectors. Theij mutation is also characterized by an array of additional phenotypic affects which suggests a pivotal role forIj in chloroplast development. TheIj gene from maize has been isolated; however, the sequence has not provided information on the action of theIj gene product. As an extension of the analysis of theIj gene we have generated a number of independent transposon insertion mutations using the Robertson'sMutator (Mu). Insertions were found to be distributed throughout the 5′ half of the gene and all showed a similar striping pattern to that of the originalij mutation,ij-ref. While several of the new mutant alleles producedij-homologous transcripts none produced detectable levels of the Ij polypeptide product. Chloroplast development in some cells appears to be able to proceed in the absence of the Ij polypeptide.

Epigenetic effects on yeast transcription caused by mutations in an actin-related protein present in the nucleus.

Gene inactivation can result when a delta element of the Ty1 transposon inserted into the yeast HIS4 promoter (his4-912delta) alters the transcription initiation site. Previous work has identified mutations that suppress this transcriptional defect by restoring the transcription start site to the native position, and these mutations have been implicated in transcriptional regulation and chromatin structure. We show that in a sin4 mutant such suppression is incompletely penetrant, such that genetically identical yeast cells (sin4 his4-912delta) show either of two distinct phenotypic states, His+ or His-. To study this type of potential epigenetic control of gene expression, we constructed a strain with ADE2 expression under the control of the his4-912delta promoter, as colony color provides a convenient assay for ADE2 expression. We isolated mutations in the ACT3 gene that show variegated expression of this ADE2 reporter. The act3 his4delta-ADE2 colonies display both white and red sectors, showing that the two different phenotypes are possible in a single colony. The two phenotypic states can be inherited during clonal growth, yet are reversible. Analysis of RNA isolated from individual colonies of either red or white color demonstrates that it is the state of the promoter, as either On or Off, that is inherited and is responsible for the colony color. An act3 mutation also affects expression of the HIS4 and LYS2 genes; thus, Act3p is not a delta element-specific transcriptional regulator. Immunofluorescence microscopy experiments demonstrate that the Act3p protein is present in the nucleus. Act3p shows clear homology to actin, and possible roles for an actin-related protein in transcription are discussed.

Ca2+ Binding to Calmodulin and Its Role in Schizosaccharomyces pombe as Revealed by Mutagenesis and NMR Spectroscopy

As a first step toward identifying the important structural elements of calmodulin from Schizosaccharomyces pombe, we examined the ability of heterologous calmodulins and Ca(2+)-binding site mutant S. pombe calmodulins to replace the essential cam1+ gene. A cDNA encoding vertebrate calmodulin allows growth of S. pombe. However, calmodulin from Saccharomyces cerevisiae does not support growth even though the protein is produced at high levels. With one exception, all mutant S. pombe calmodulins with one or more intact Ca(2+)-binding sites allow growth at 21 degrees C. A mutant containing only an intact Ca(2+)-binding site 3 fails to support growth, as does S. pombe calmodulin with all four Ca(2+)-binding sites mutated. Several of the mutant proteins confer a temperature-sensitive phenotype. Analysis of the degree of temperature sensitivity allows the Ca(2+)-binding sites to be ranked by their ability to support fission yeast proliferation. Site 2 is more important than site 1, which is more important than site 4, which is more important than site 3. A visual colony color screen based on the fission yeast ade1+ gene was developed to perform these genetic analyses. To compare the Ca(2+)-binding properties of individual sites to their functional importance for viability, Ca2+ binding to calmodulin from S. pombe was studied by 1H NMR spectroscopy. NMR analysis indicates a Ca(2+)-binding profile that differs from those previously determined for vertebrate and S. cerevisiae calmodulins. Ca(2+)-binding site 3 has the highest relative affinity for Ca2+, while the affinities of sites 1, 2, and 4 are indistinguishable. A combination of an in vivo functional assay and an in vitro physical assay reveals that the relative affinity of a site for Ca2+ does not predict its functional importance.

Mox: a novel modifier of the tomato Xa locus.

We have isolated a novel mutation that caused variegated leaf color in a tomato plant which had multiple maize Ac transposable elements and the tomato Xa allele. Xa is a previously characterized semi-dominant mutation that causes tomato leaves to be bright yellow when heterozygous (Xa/xa+). The mutation responsible for the new phenotype was named Mox (Modifier of Xa). The Mox mutation modified the Xa/xa+ yellow leaf phenotype in two ways: it compensated for the Xa allele resulting in a plant with a wildtype green color, and it caused somatic variegation which appeared as white and yellow sectors on the green background. Somatic variegation was visible only if the plant contained both the Mox and Xa loci. Genetic studies indicated that the Mox locus was linked in repulsion to Xa and that the Mox locus was genetically transmitted at a reduced frequency through the male gamete. Molecular characterization of the Ac elements in lines segregating for Mox identified an Ac insertion that appeared to cosegregate with Mox variegation. We propose a model in which the Mox mutation consists of a duplication of the xa+ allele and subsequent Ac-induced breakage of the duplicated region causes variegation.

Nuclear—organelle interactions: the immutans variegation mutant of Arabidopsis is plastid autonomous and impaired in carotenoid biosynthesis

The immutans (im) variegation mutant of Arabidopsis thaliana contains green- and white-sectored leaves due to the action of a nuclear recessive gene. The mutation is somatically unstable, and the degree of sectoring is influenced by light and temperature. Whereas the cells in the green sectors contain normal chloroplasts, the cells in the white sectors are heteroplastidic and contain non-pigmented plastids that lack organized lamellar structures, as well as small pigmented plastids and/or rare normal chloroplasts. This indicates that the plastids in im white cells are not affected equally by the nuclear mutation and that the expression of immutans is 'plastid autonomous'. In contrast to other variegation mutants with heteroplastidic cells, the defect in im is not maternally inherited. immutans thus represents a novel type of nuclear gene-induced variegation mutant. It has also been found that the white tissues of immutans accumulate phytoene, a non-colored C40 carotenoid intermediate. This suggests that immutans controls, either directly or indirectly, the activity of phytoene desaturase (PDS), the enzyme that converts phytoene to zeta-carotene in higher plants. However, im is not the structural gene for PDS. A secondary effect of carotenoid deficiency, both in immutans and in wild-type plants treated with a herbicide that blocks carotenoid synthesis, is an increase in acid ribonuclease activity in white tissue. It is concluded that the novel variegation generated by the immutans mutation should offer great insight into the complex circuitry that regulates nuclear-organelle interactions.

Correlation between polyploidy and auxotrophic segregation in the imperfect yeast Candida albicans

In order to clarify the relationship between polyploidization and the capability of phenotypic switching in the imperfect yeast Candida albicans, two types of variants were isolated as segregants from a fusant, which produced a proportion of the cell population with a higher ploidy than the rest, either in a temperature-dependent or -independent manner, when incubated at low (28 degrees C) and high (37 degrees C) temperatures. In the case of the temperature-dependent type of variants, high-ploidy cells appeared at 37 degrees C but rarely at 28 degrees C. This phenotype was named Pldts (temperature-sensitive polyploidization), and the temperature-independent phenotype was called Pld-. The appearance of high-ploidy cells in the culture of the Pldts strain at 37 degrees C was accompanied by a significant increase in the frequency of auxotrophic variants; these variants probably occur as a result of segregation of auxotrophic markers from the heterozygous to the homozygous state. Both Pldts and Pld- phenotypes were recessive in a fusion with a Pld+ parent. An adenine auxotrophic marker (ade1) was introduced into a Pldts strain in a heterozygous state, and the individual high-ploidy cells of this strain, grown at 37 degrees C, were micromanipulated to form colonies, which consisted of red and white sectors appearing at high frequency on a pink background. When the ade1 auxotrophy was introduced into Pld- strains, frequently sectored colonies were produced. These results suggested an increased level of chromosome missegregation in both types of Pld mutants. Analyses by pulsed-field gel electrophoresis of Ade-segregants, derived from a micromanipulated high-ploidy cell of a Pld(ts) strain, suggested the occurrence of nonreciprocal recombination, some of which includes chromosome loss.

A fate map of the Arabidopsis embryonic shoot apical meristem

ABSTRACT We have mapped the fate of cells in the Arabidopsis embryonic shoot apical meristem by irradiating seed and scoring the resulting clonally derived sectors. 176 white, yellow, pale green or variegated sectors were identified and scored for their position and extent in the resulting plants. Most sectors were confined to a fraction of a leaf, and only occasionally extended into the inflorescence. Sectors that extended into the inflorescence were larger, and usually encompassed about a third to a half of the inflorescence circumference. We also find that axillary buds in Arabidopsis are clonally related to the subtending leaf. Sections through the dry seed embryo indicate that the embryonic shoot apical meristem contains approximately 110 cells in the three meristematic layers prior to the formation of the first two leaf primordia. The histological analysis of cell number in the shoot apical meristem, in combination with the sector analysis have been used to construct a map of the probable fate of cells in the embryonic shoot apical meristem.

Sustained and transient properties of chromatic and luminance systems

Reaction times were measured to 450 and 650 nm test increments to examine the temporal behavior of the chromatic and luminance systems. A response-terminated random foreperiod paradigm was employed. Stimuli consisted of chromatic test increments upon backgrounds of varying spatial structure. Conditions were chosen which may preferentially favor the reaction time response being mediated by the chromatic or luminance systems. The temporal properties of the chromatic and luminance systems were demonstrated by the shape of the estimated hazard functions of the reaction time distributions. When the white background was spatially coincident with the test field, the hazard functions showed a relatively small peak. As white sectors were added to the annulus surround (introducing spatial transients between test and background fields), however, the hazard functions became more and more peaked. The hazard functions of the luminance system were estimated by assuming that the chromatic and luminance systems function in parallel. We concluded from the results that the chromatic system may be characterized as a quasi-sustained mechanism and the luminance system as a transient mechanism.

Position effect at S. cerevisiae telomeres: Reversible repression of Pol II transcription

S. cerevisiae chromosomes end with the telomeric repeat (TG 1–3) n. When any of four Pol II genes was placed immediately adjacent to the telomeric repeats, expression of the gene was reversibly repressed as demonstrated by phenotype and mRNA analyses. For example, cells bearing a telomere-linked copy of ADE2 produced predominantly red colonies (a phenotype characteristic of ade2 − cells) containing white sectors (characteristic of ADE2 + cells). Repression was due to proximity to the telomere itself since an 81 bp tract of (TG 1–3) n positioned downstream of URA3 when URA3 was ∼20 kb from the end of chromosome VII did not alter expression of the gene. However, this internal tract of (TG 1–3) n could spontaneously become telomeric, in which case expression of the URA3 gene was repressed. These data demonstrate that yeast telomeres exert a position effect on the transcription of nearby genes, an effect that is under epigenetic control

Intergeneric hybridization betweenMonascus anka andAspergillus oryzae by protoplast fusion

To breed industrially useful strains of a slow-growing, red-pigment-producing strain ofMonascus anka, protoplasts ofM. anka MAK1 (arg) andAspergillus oryzae AOK1 (met, thr) were fused. A mixture of protoplasts prepared from mycelia ofM. anka MAK1 treated with 2% Usukizyme and ofA. oryzae AOK1 treated with 2% Usukizyme and 0.2% NovoZym 234 was incubated with 30% (w/v) polyethylene glycol no. 6000. Heterokaryon fusants complementing the auxotrophies of both mutants were isolated on minimal medium, but segregated into red (MAK1) and white (AOK1) sectors after being cultured on a complete medium. After irradiation with UV light, the fusants gave stable heterozygous diploids that formed long white hyphae. These diploids, which had twice as much DNA in the nucleus as their parents, grew more rapidly than the parent strain YZT1, and produced ethanol earlier than the parents. Production of amylase, protease, and kojic acid by the fusants was intermediate in amount between that of the two parents.

Introduction of a Chimeric Chalcone Synthase Gene into Petunia Results in Reversible Co-Suppression of Homologous Genes in trans.

We attempted to overexpress chalcone synthase (CHS) in pigmented petunia petals by introducing a chimeric petunia CHS gene. Unexpectedly, the introduced gene created a block in anthocyanin biosynthesis. Forty-two percent of plants with the introduced CHS gene produced totally white flowers and/or patterned flowers with white or pale nonclonal sectors on a wild-type pigmented background; none of hundreds of transgenic control plants exhibited such phenotypes. Progeny testing of one plant demonstrated that the novel color phenotype co-segregated with the introduced CHS gene; progeny without this gene were phenotypically wild type. The somatic and germinal stability of the novel color patterns was variable. RNase protection analysis of petal RNAs isolated from white flowers showed that, although the developmental timing of mRNA expression of the endogenous CHS gene was not altered, the level of the mRNA produced by this gene was reduced 50-fold from wild-type levels. Somatic reversion of plants with white flowers to phenotypically parental violet flowers was associated with a coordinate rise in the steady-state levels of the mRNAs produced by both the endogenous and the introduced CHS genes. Thus, in the altered white flowers, the expression of both genes was coordinately suppressed, indicating that expression of the introduced CHS gene was not alone sufficient for suppression of endogenous CHS transcript levels. The mechanism responsible for the reversible co-suppression of homologous genes in trans is unclear, but the erratic and reversible nature of this phenomenon suggests the possible involvement of methylation.

Response pooling between chromatic and luminance systems

Two experiments were designed to examine interactions of the responses of the chromatic and luminance systems to suprathreshold stimuli. We measured simple reaction times (RT) to eight photometrically matched (1 cd/m 2) wavelengths between 448 and 658 nm. These chromatic test stimuli were incrementally presented on either a spatially coextensive 1.2 or a larger 2° steady white background. Sectors of the outer annulus region (between 1.2 and 2°) could be removed to allow systematic variation of the extent of the spatial contour between test and background fields. When the white background was spatially coincident with the test field, RTs showed trichromatic saturation-like wavelength dependence, with a maximum RT at 572 nm. As white sectors were added to the annulus (introducing spatial transients between test and background fields), RTs became less and less wavelength-dependent, and were nearly wavelength-independent when a full annulus was used. The data were analysed in terms of a two-system RT model for processing chromaticity and luminance.

Intraspecific hybridization of Trichoderma reesei QM 9414 by protoplast fusion using colour mutants

Protoplasts were obtained from mycelia of two colour strains of Trichoderma reesei QM 9414 using a mycolytic enzyme system from Trichoderma viride BIA. Approximately 9–15% of the protoplasts obtained were regenerated on a solid culture medium with a soft agar overlay. When protoplasts from the two colour mutants were treated with an osmotically balanced solution containing 35% poly( ethylene glycol), they fused effectively. The production of well defined white and brown sectors on agar plates demonstrated that the fusants were heterokaryons. The heterokaryons produced cellulase, xylanase and β-glucosidase activities in amounts comparable to those of the parent colour strains or of strain QM 9414. Green conidia were induced from the heterokaryons after treatment with 0.1% d- camphor. This suggested the existence of a parasexual cycle in this species of Trichoderma and presented the possibility that diploidization had occurred.

The plastome mutator of Oenothera continues to function as originally described

Recently, Lindenhahn et al. (1985) hypothesized that the plastome mutator (pm) system in Oenothera originated through contaiminating cross-pollination and that the variegation was an example of hybrid plastome-genome incompatibility. Their evidence was based on restriction pattern analyses of white sectors which showed wild-type plastome III patterns rather than the wild-type plastome I patterns of the green portions of their plants. Their hypothesis does not adequately account for the results which our laboratories have obtained independently; the pm-system of Oenothera continues to generate many new and different plastome mutations following the genetic parameters as published originally (Epp 1973). Our studies support mutator gene function. The restriction pattern of the chloroplast DNA of five newly isolated pm-induced variegation sectors are reported here to show a restriction pattern identical to the green wild-type plastids. The restriction pattern reported by Lindenhahn et al. (1985) for their white sector plastids is different than we would expect from a pm-induced plastome mutation. Their overall analysis did not utilize many of the salient features of the genetics of Oenothera and of the pm-system. The white sectors they observed are probably due to an accidental contamination by plastome III plastids. Suggestions are made for delineating experimentally plastome mutations and hybrid incompatibility. For future analyses, a comparative study of numerous pm-induced sectors is recommended, since the pm-system readily generates many different plastome mutations with independent origins. This comparison would greatly assist in the interpretation of restriction patterns.

Yeast centromere sequences do not confer mitotic stability on circular plasmids containing ARS elements of Tetrahymena thermophila rDNA.

We have previously demonstrated that a 657 bp TaqI-XbaI and a 427 bp XbaI-XbaI fragment from the 5' non-transcribed spacer of the extrachromosomal ribosomal DNA of Tetrahymena thermophila function as autonomously replicating sequences (ARS) in Saccharomyces cerevisiae. These fragments are adjacent to each other in a region that encompasses the in vivo origin of bidirectional replication of rDNA. The presence of a yeast centromere (CEN) fragment does not confer mitotic stability on these plasmids. A sensitive yeast colony colour assay (Hieter et al. 1985a) has been used to evaluate the cis-acting effect of each ARS segment on the pattern of inheritance of a plasmid containing CEN5:URA3:SUP4. Colonies of transformed cells obtained both in the presence and absence of selection were red with no detectable white or pink sectors. The lack of sectoring indicates that both plasmids are lost at an extremely high rate, likely due to 1:0 segregation events. We conclude that while these ARS elements confer a high frequency transformation phenotype, they lack a function which is required in cis for the maintenance of mitotic stability in the presence of a centromere. This missing cis-acting function may result in the inability of the plasmids to be brought under the control of cell-cycle regulated replication.

Nicotiana tabacum mutants with chloroplast encoded streptomycin resistance and pigment deficiency

Callus ofNicotiana tabacum SRI, a mutant with maternally inherited streptomycin resistance, was induced from leaf sections. Callus pieces were mutagenised with N-ethyl-N-nitrosourea and inoculated onto a shoot-induction medium on which calli are normally green. White callus sectors were observed in the mutagenised cultures, and white and variegated shoots were regenerated from these sectored calli. The SR1-A10 line regenerated a chimeric shoot with white leaf margins. The chimeric shoot was grafted onto a normal green rootstock, grown into a flowering plant in the greenhouse, and crosses were made. The SRI-A15 line was crossed using flowers formed on albino plants grown in sterile culture. Pigment deficiency was maternally inherited in both lines. Physical mapping of the chloroplast genome of the SR1-A15 mutant by SalI, PstI and BamHI restriction endonucleases did not reveal any difference between the SR1-A15 and the parental SRI chloroplast genomes.

Structural Differences between Green and White Sectors of Variegated Scindapsus aureus Leaves

Abstract Several anatomical and ultrastructural features, as well as some related physiological parameters, have been studied in green and white sectors of Scindapsus aureus variegated leaves. Incidence of structural variations in dark respiration rates of leaves also has been considered. Total chlorophyll content of white sectors is about 2% that of green ones. Dark respiration rate in white sectors is about half that measured in green sectors. There are no differences anatomically between both types of sectors. Plastid number and plastid long axis length are greater in green than in white sectors. Such differences explain why the plastid area to leaf area ratio in white sectors is about half that of green sectors. This ratio has been related to differences in dark respiration rate between green and white sectors. Mutant plastids are typical photobleaching mutants. There seems to be a good correspondence with Knoth's model for plastid degeneration. Mutant plastids have an extensive system of peripheral reticula.

Regulation of flavonoid gene expression in Petunia hybrida: Description and partial characterization of a conditional mutant in chalcone synthase gene expression

White flowers of the Petunia hybrida line W43 accumulate glucosides of 4-coumaric acid and caffeic acid and are able to synthesize anthocyanins from exogeneously supplied naringenin, suggesting that W43 is blocked in a biosynthetic step preceding the formation of naringenin. The cultivar Red Star contains a similar mutation as W43; the genetic background of this cultivar, however, allows the production of considerable amounts of anthocyanins in certain areas of the flower. When grown at reduced light, flowers of Red Star are uniformly coloured, whereas under an enhanced light regime the flowers exhibit alternating white and coloured areas. In white sectors of flowers with a colour pattern virtually no chalcone synthase (CHS) enzyme activity could be demonstrated. The enzymes chalcone isomerase (CHI), UDPG: flavonoid-3-0-glucosyltransferase (3GT) and SAM: anthocyanin methyltransferase (OMT) are present, although at a more or less reduced level. From Western blotting and in vitro translation experiments we infer that the absence of CHS enzyme activity in the white sectors of the cultivar Red Star is due to the absence of translationally active CHS mRNA. The potential use of the mutants for genetic engineering in plants is emphasized.

Placental lead levels

Bleomycin, an antibiotic and antineoplastic drug that inhibits DNA synthesis and causes several types of chromosomal aberration, was found to increase mitotic recombination in Aspergillus nidulans. Heterozygous prototrophic diploid strains grown on media containing bleomycin produced significant increases of yellow and white sectors compared with controls. Further, the increased colour segregants were due to mitotic crossing-over, whereas the non-disjunctional segregants remained at the control level. Bleomycin also induced point mutations in the methionine-suppressor system of the methG1 biA1 strain of Aspergillus nidulans. Conidia treated in suspension with various concentrations of Neomycin increased the methionine-independent mutants 30-fold and more.