Yellow Mutant Research Articles

Yellow senescence and incomplete testa mutants in Viciafaba L.*

Yellow senescence and incomplete testa mutants in Viciafaba L.*

The Evolution of Gene Regulation Underlies a Morphological Difference between Two Drosophila Sister Species

Understanding the mechanisms underlying the morphological divergence of species is one of the central goals of evolutionary biology. Here, we analyze the genetic and molecular bases of the divergence of body pigmentation patterns between Drosophila yakuba and its sister species Drosophila santomea. We found that loss of pigmentation in D. santomea involved the selective loss of expression of the tan and yellow pigmentation genes. We demonstrate that tan gene expression was eliminated through the mutational inactivation of one specific tan cis-regulatory element (CRE) whereas the Tan protein sequence remained unchanged. Surprisingly, we identify three independent loss-of-function alleles of the tan CRE in the young D. santomea lineage. We submit that there is sufficient empirical evidence to support the general prediction that functional evolutionary changes at pleiotropic loci will most often involve mutations in their discrete, modular cis-regulatory elements.

Photosystem 2 photochemistry and pigment composition of a yellow mutant of rice (Oryza sativa L.) under different irradiances

A yellow leaf colouration mutant (named ycm) generated from rice T-DNA insertion lines was identified with less grana lamellae and low thylakoid membrane protein contents. At weak irradiance [50 µmol(photon) m-2 s-1], chlorophyll (Chl) contents of ycm were ≈20 % of those of WT and Chl a/b ratios were 3-fold that of wild type (WT). The leaf of ycm showed lower values in the actual photosystem 2 (PS2) efficiency (ΦPS2), photochemical quenching (qP), and the efficiency of excitation capture by open PS2 centres 1 (Fv'/Fm') than those of WT, except no difference in the maximal efficiency of PS2 photochemistry (Fv/Fm). With progress in irradiance [100 and 200 µmol(photon) m-2 s-1], there was a change in the photosynthetic pigment stoichiometry. In ycm, the increase of total Chl contents and the decrease in Chl a/b ratio were observed. ΦPS2, qP, and Fv'/Fm' of ycm increased gradually along with the increase of irradiance but still much less than in WT. The increase of xanthophyll ratio [(Z+A)/(V+A+Z)] associated with non-photochemical quenching (qN) was found in ycm which suggested that ycm dissipated excess energy through the turnover of xanthophylls. No significant differences in pigment composition were observed in WT under various irradiances, except Chl a/b ratio that gradually decreased. Hence the ycm mutant developed much more tardily than WT, which was caused by low photon energy utilization independent of irradiance.

Ultrastructural and molecular characterization of altered plastids in nuclear gene controlled yellow stripe mutant of Pennisetum americanum

The ultrastructural and molecular biological studies were conducted to elucidate the changes in the nuclear gene controlled plastid alterations in yellow stripe mutant of Pennisetum americanum. The plastids in yellow tissue were bound by a double membrane envelope and no internal thylakoid membrane differentiation, whereas plastid development was normal in the green tissue. The nuclear gene apparently influences the individual plastids as evidenced by the presence of heteroplastidic cells. Ribosomal RNAs were extracted from green and yellow seedlings with homozygous recessive and heterozygous genotypes. Plastid specific 23S and 16S rRNAs were absent in the yellow seedlings irrespective of the genotype. The presence of disorganized lamellar membranes in the rRNA/ribosome deficient plastids indicate that they are synthesized outside the plastid compartment. Restriction endonuclease analysis revealed no detectable differences in fragmentation pattern of the plastid DNAs from green and reverted green seedlings. This is consistent with the suggestion that the nuclear gene is not inducing any change in the plastid genome size and in the restriction enzyme recognition sites.

Differential Ras Activation between Caveolae/Raft and Non-Raft Microdomains

Although the consequences of Ras activation have been studied extensively in the context of oncogenesis, its regulation in physiological modes of signal transduction is not well understood. A fluorescent indicator, Raichu-Ras, was fused to the C-terminal hypervariable regions of H-Ras and K-Ras to create indicators for Ras activation within caveolae/rafts (Raichu-tH) and non-raft domains (Raichu-tK) of the plasma membrane, respectively. Raichu-tH was also found abundantly in endomembranes. To monitor Ras activation with high spatial resolution, it is imperative to observe sectioned images of the signals. We have developed a wide-field fluorescence microscope equipped with a digital micromirror device (DMD) to acquire optically sectioned images using fringe projection. This system provides reliable signals from fluorescence resonance energy transfer (FRET) between cyan and yellow mutants of green fluorescent protein. We have used this system to demonstrate that, upon stimulation with growth factors, the two indicators are activated in spatially and temporally unique patterns.

Structural Characterization of a Blue Chromoprotein and Its Yellow Mutant from the Sea Anemone Cnidopus Japonicus

Green fluorescent protein (GFP) and its relatives (GFP protein family) have been isolated from marine organisms such as jellyfish and corals that belong to the phylum Cnidaria (stinging aquatic invertebrates). They are intrinsically fluorescent proteins. In search of new members of the family of green fluorescent protein family, we identified a non-fluorescent chromoprotein from the Cnidopus japonicus species of sea anemone that possesses 45% sequence identity to dsRed (a red fluorescent protein). This newly identified blue color protein has an absorbance maximum of 610 nm and is hereafter referred to as cjBlue. Determination of the cjBlue 1.8 A crystal structure revealed a chromophore comprised of Gln(63)-Tyr(64)-Gly(65). The ring stacking between Tyr(64) and His(197) stabilized the cjBlue trans chromophore conformation along the Calpha2-Cbeta2 bond of 5-[(4-hydroxyphenyl)methylene]-imidazolinone, which closely resembled that of the "Kindling Fluorescent Protein" and Rtms5. Replacement of Tyr(64) with Leu in wild-type cjBlue produced a visible color change from blue to yellow with a new absorbance maximum of 417 nm. Interestingly, the crystal structure of the yellow mutant Y64L revealed two His(197) imidazole ring orientations, suggesting a flip-flop interconversion between the two conformations in solution. We conclude that the dynamics and structure of the chromophore are both essential for the optical appearance of these color proteins.

Importance of colour in the reaction of passerine predators to aposematic prey: experiments with mutants of Pyrrhocoris apterus (Heteroptera)

Persistent questions concerning the warning coloration of unpalatable insects address whether the bright aposematic colour itself or its combination with a species-specific dark pattern is the key factor in their protection against insectivorous birds, and how chromatic polymorphism originates and is maintained in aposematics. In the present study, these questions were tested experimentally, using the birds Parus major, Parus caeruleus, Erithacus rubecula, and Sylvia atricapilla as predators, and chromatically polymorphic firebug Pyrrhocoris apterus: red wild form, white, yellow, and orange mutants (all four of them with the same black melanin pattern, the mutants differing in colour of pteridine pigments only) and the nonaposematic brown-painted wild form as prey. The results show that a specific colour is essential for the birds to recognize the specific aposematic prey; the melanin pattern is not sufficient. White mutants were no better protected than nonaposematic firebugs; red wild-type and orange mutants were equally well protected against all bird species; and the reaction of birds to yellow mutants was species-specific. An evolutionary scenario of ‘recurrent recessive mutations’ is formulated to explain the origin of colour polymorphism in some aposematics.

Signal propagation from membrane messengers to nuclear effectors revealed by reporters of phosphoinositide dynamics and Akt activity.

Among various second messengers, phosphatidylinositol 3,4,5-triphosphate (PIP3) and phosphatidylinositol 3,4-bisphosphate [PI(3,4)P2] regulate a variety of cellular processes, such as cell survival, polarization, and proliferation. Many of these functions are achieved via activation of serine/threonine kinase Akt. To investigate the spatiotemporal regulation of these lipids, we constructed a genetically targetable phosphoinositide (PI) indicator by sandwiching pleckstrin homology (PH) domain of Akt and a "pseudoligand" containing acidic amino acid residues, between cyan and yellow mutants of GFP. In living cells, elevations in PIP3 and PI(3,4)P2 by growth factor-induced activation of phosphatidylinositol 3-kinase (PI3K) resulted in a change in fluorescence resonance energy transfer (FRET) between the fluorescent proteins, increasing yellow to cyan emission ratios by 10-30%. This response can be reversed by inhibiting PI3K and abolished by mutating the critical residues responsible for PI binding. Differential dynamics of PIs were observed at plasma membrane of NIH 3T3 cells, stimulated by various growth factors. On the other hand, the nuclear targeted indicator showed no response within an hour after platelet-derived growth factor stimulation, suggesting that no appreciable amounts of accessible PIP3 and PI(3,4)P2 were produced in the nucleus. Furthermore, simultaneous imaging of a plasma membrane-targeted PI indicator and a nuclear-targeted Akt activity reporter revealed a gradual and sustained accumulation of Akt activity in the nucleus after rapid and transient production of PIP3 and PI(3,4)P2 at plasma membrane in the same cell. Thus, signal propagation from the lipid messengers at plasma membrane to the effectors in the nucleus is precisely controlled by kinases as well as lipid and protein phosphatases.

Mating activity of yellow and sepia Drosophila willistoni mutants

By analyzing the mating activity of newly isolated yellow and sepia mutants of Drosophila willistoni no difference in behavior between sepia and wild-type flies were observed, whereas yellow males were less successful than wild-type males when competing for females. These results are in agreement with those reported for other Drosophila species. D. willistoni was different in the 'females-competing' crosses because wild-type males mated more frequently with wild-type females whereas yellow males mated successfully with both phenotypes. These results indicate the complexity of the courtship behavior in D. willistoni and provide data for comparative and evolutionary research into the genus.

Azaphilone Pigments from a Yellow Mutant of the Fungus Monascus kaoliang.

AbstractFor Abstract see ChemInform Abstract in Full Text.

Fluorescent indicators of cAMP and Epac activation reveal differential dynamics of cAMP signaling within discrete subcellular compartments.

Second messenger cAMP regulates many cellular functions through its effectors, such as cAMP-dependent protein kinase (PKA) and Epac (exchange proteins directly activated by cAMP). Spatial and temporal control of cAMP signaling is crucial to differential regulation of cellular targets involved in various signaling cascades. To investigate the compartmentalized cAMP signaling, we constructed fluorescent indicators that report intracellular cAMP dynamics and Epac activation by sandwiching the full-length Epac1 between cyan and yellow mutants of GFP. Elevations of cAMP decreased FRET and increased the ratio of cyan-to-yellow emissions by 10-30% in living mammalian cells. This response can be reversed by removing cAMP-elevating agents and abolished by mutating the critical residue responsible for cAMP binding. Targeting of the reporter to the plasma membrane, where cAMP is produced in response to the activation of beta-adrenergic receptor, revealed a faster cAMP response at the membrane than in the cytoplasm and mitochondria. Simultaneous imaging with targeted cAMP indicator and PKA activity reporter allowed the detection of a much delayed PKA response in the nucleus after the rapid accumulation of cAMP at the plasma membrane of the same cell, despite the immediate presence of a pool of cAMP in the nucleus. Thus, cAMP dynamics and the activation of its effectors are precisely controlled spatiotemporally in vivo.

Azaphilone pigments from a yellow mutant of the fungus Monascus kaoliang

Azaphilone pigments, monascusones A ( 1) and B ( 2), together with two known azaphilones, monascin ( 3) and FK17-P2b2 ( 4), were isolated from the CH 2Cl 2 extract of a yellow mutant of the fungus M. kaoliang grown on rice. Structures of the isolated compounds were elucidated by analyses of spectroscopic data. Monascusone A ( 1), the major metabolite of M. kaoliang, showed no antimalarial (against Plasmodium falciparum), antitubercular (against Mycobacterium tuberculosis H37Ra), and antifungal (toward Candida albicans) activities. Compound 1 exhibited no cytotoxicity against BC (breast cancer) and KB (human epidermoid carcinoma of cavity) cell lines.

Genetic Analysis of Variegation Mutants of Pearl Millet, Pennisetum Glaucum

Intercrosses between different variegated lines of Pennisetum glaucum (L.) R.Br. were made to determine their allelic composition and the number of loci controlling plastid alterations. Self-pollinations of different variegated plants resulted in normal, variegated, and yellow or white progeny. Crosses among yellow stripe mutants (IP 5009, IP 9712, IP 482) resulted in normal and yellow progeny in the F,, and normal and yellow stripe in the F, generations, indicating the complementary interaction of two loci in each cross. Reciprocal crosses between the yellow stripe mutants IP 5009 and IP 13160-1 revealed similarity in their genotypes. Progeny composition from the crosses between the white stripe mutants VCM-36 and GWS-14 indicated their genotypic similarities. Crosses between yellow stripe and white stripe mutants (IP 5009 x VCM-36, IP 482 x VCM-36) indicated differences in their genotypes. Comparison of segregation patterns in the progenies of intercrosses revealed at least 4 independent loci, any one of which in recessive condition leads to mutant phenotype(s) while the development of chlorophyll is accomplished by the complementary interaction of dominant genes at these loci. Among the recessive genotypes in the F,s from intergenotypic crosses, the mutant phenotypes fell short of expectation, indicating differential penetrance in expression.

New And Interesting Orchid Records for Tanzania

The orchid taxa Brachycorythis ovata subsp. schweinfurthii, Habenaria arianae and Habenaria disparilis are newly recorded for Tanzania, while Brachycorythis congoensis is newly recorded for the floral region T7. A stable, pure yellow mutant of Disa erubescens is recorded for the first time.

Reducing the Environmental Sensitivity of Yellow Fluorescent Protein: MECHANISM AND APPLICATIONS

Yellow mutants of the green fluorescent protein (YFP) are crucial constituents of genetically encoded indicators of signal transduction and fusions to monitor protein-protein interactions. However, previous YFPs show excessive pH sensitivity, chloride interference, poor photostability, or poor expression at 37 degrees C. Protein evolution in Escherichia coli has produced a new YFP named Citrine, in which the mutation Q69M confers a much lower pK(a) (5.7) than for previous YFPs, indifference to chloride, twice the photostability of previous YFPs, and much better expression at 37 degrees C and in organelles. The halide resistance is explained by a 2.2-A x-ray crystal structure of Citrine, showing that the methionine side chain fills what was once a large halide-binding cavity adjacent to the chromophore. Insertion of calmodulin within Citrine or fusion of cyan fluorescent protein, calmodulin, a calmodulin-binding peptide and Citrine has generated improved calcium indicators. These chimeras can be targeted to multiple cellular locations and have permitted the first single-cell imaging of free [Ca(2+)] in the Golgi. Citrine is superior to all previous YFPs except when pH or halide sensitivity is desired and is particularly advantageous within genetically encoded fluorescent indicators of physiological signals.

Plastids undifferentiated, a nuclear mutation that disrupts plastid differentiation in Zea mays L.

Photosynthetic development in any plant requires the intracellular co-ordination of chloroplast and nuclear gene expression programs. In this report, we investigate the role of a nuclear gene in photosynthetic development by examining C4 photosynthetic differentiation in a yellow mutant of maize (Zea mays L.). The plastids undifferentiated (pun) mutation disrupts plastid biogenesis in both bundle sheath and mesophyll cells, at an early developmental stage and in a light-independent manner. Chloroplast thylakoids are disrupted in the mutant and both membrane-associated and soluble chloroplast-encoded proteins accumulate at much reduced levels. The observed plastid morphology is consistent with a general defect in chloroplast biogenesis that is most likely exerted at the post-translational level. Despite aberrant chloroplast development, nuclear photosynthetic genes are expressed normally in pun mutants. Thus, neither functional chloroplasts nor the Pun gene product are required to establish nuclear photosynthetic gene expression patterns in maize.

Spatiotemporal dynamics of guanosine 3',5'-cyclic monophosphate revealed by a genetically encoded, fluorescent indicator.

To investigate the dynamics of guanosine 3',5'-cyclic monophosphate (cGMP) in single living cells, we constructed genetically encoded, fluorescent cGMP indicators by bracketing cGMP-dependent protein kinase (cGPK), minus residues 1-77, between cyan and yellow mutants of green fluorescent protein. cGMP decreased fluorescence resonance energy transfer (FRET) and increased the ratio of cyan to yellow emissions by up to 1.5-fold with apparent dissociation constants of approximately 2 microM and >100:1 selectivity for cGMP over cAMP. To eliminate constitutive kinase activity, Thr(516) of cGPK was mutated to Ala. Emission ratio imaging of the indicators transfected into rat fetal lung fibroblast (RFL)-6 showed cGMP transients resulting from activation of soluble and particulate guanylyl cyclase, respectively, by nitric oxide (NO) and C-type natriuretic peptide (CNP). Whereas all naive cells tested responded to CNP, only 68% responded to NO. Both sets of signals showed large and variable (0.5-4 min) latencies. The phosphodiesterase (PDE) inhibitor 3-isobutyl-1-methylxanthine (IBMX) did not elevate cGMP on its own but consistently amplified responses to NO or CNP, suggesting that basal activity of guanylate cyclase is very low and emphasizing the importance of PDEs in cGMP recycling. A fraction of RFL cells showed slowly propagating tides of cGMP spreading across the cell in response to delocalized application of NO. Biolistically transfected Purkinje neurons showed cGMP responses to parallel fiber activity and NO donors, confirming that single-cell increases in cGMP occur under conditions appropriate to cause synaptic plasticity.

Wheat mutants showing altered adult plant disease resistance

Wheat mutants, selected on an altered resistance phenotype to Puccinia striiformis f.sp. tritici, the causal agent of yellow rust, were assessed in greenhouse tests to determine the growth stage at which the mutant phenotype was expressed and which components of yellow rust resistance were altered. Four of the mutants showed reduced levels of yellow rust infection, while three exhibited increased levels of infection. The infection characters altered were those seen after the establishment of hyphal growth, i.e. days after inoculation to sporulation, the percentage of inoculated leaf tissue producing sporulating colonies and the number of spores produced per cm2 of inoculated leaf tissue. The altered resistance phenotype was developmentally regulated in all of the mutants. The changes seen in the infection characters differed for each mutant, suggesting that different genes may have been altered. An altered resistance phenotype to brown rust and/or powdery mildew was also seen for some of the yellow rust mutants.

Color mutants of Monascus sp. KB9 and their comparative glucoamylases on rice solid culture

Monascus sp. KB9 strain was used to convert carbohydrate base agricultural commodities to value added pigments. Mutagenesis of this strain using UV light and successive isolation was found to improve the yield of pigment product. As a result, two hyper pigment productive strains, red and yellow mutants were obtained. In addition, one white mutant was also obtained. These color mutants together with their parental strain were used to compare glucoamylase production in rice solid culture. Initial moisture content of rice at 35–43% is found to affect glucoamylase of these four strains. It is noteworthy that the white mutant which lost its ability to produce pigment and revealed morphology indifference to its parental strain could produce the highest glucoamylase enzyme at 4.07×10 5 U/gdw (units per gram-dry weight). This is about 3, 7, and 16 times higher than the wild type strain, red, and yellow mutants, respectively. The crude enzyme showed its thermophilic property at optimal temperature, 65°C, and its optimal pH, 4.7.

In vitro spectrofluorometric determination of chlorophyll de novo synthesis in chlorophyll-deficient phenotypes of Pennisetum glaucum (L.) R.Br

Blocks in chlorophyll (Chl) biosynthesis in crude extracts of well-defined chlorophyll-deficient seedlings and green siblings from five Pennisetum glaucum (L.) R.Br. genotypes and the ability of these genotypes to relieve such blocks in crude extract mixtures were studied with low temperature (77 K) fluorescence spectroscopy. Dark-adapted green seedling extracts exhibited F657, F685, and F735 peaks corresponding with protochlorophyllide (Pchlide), Chl from photosystem II and from photosystem I, respectively. Extracts of white seedlings from white stripe mutants and an albino mutant exhibited F657 peaks indicative of Pchlide, whereas the extracts of yellow seedlings from yellow stripe mutants exhibited F657 and F678 peaks representing Pchlide and chlorophyllide (Chlide) accumulation, respectively. Addition of their green sibling's extract resulted in the appearance of F685 and F735 peaks, suggesting de novo relief of the blocks and photoreduction of Pchlide to Chl(ide). Incubating a mixture of crude extracts of yellow seedlings from two different yellow stripe mutants did not alter their peaks, indicating a lack of reconstitution because of a common limiting factor in the two yellow seedling sources. Likewise, the mixture of crude extract of yellow seedlings with that of white seedlings from white stripe mutants displayed no Chl peaks. Spontaneous greening in the mixture of crude extracts of yellow seedlings with that of albino mutant and its failure in other reconstitution experiments suggests that Chl synthase in albino seedlings enabled the conversion of Chlide to Chl in vitro. The factors governing the in vitro reconstitution and its implications in genetic complementation are discussed.Key words: chlorophyll-deficient mutants, blocks in chlorophyll biosynthesis, crude extracts, in vitro synthesis of chlorophyll.