Presence of the Dominant Extension Allele ED in Red and Mosaic Cattle

The melanocyte-stimulating hormone receptor (MC1-R) is a central regulator of mammalian coat colour, encoded by the extension locus. In cattle, the dominant extension allele E^D is associated with the production of black pigment in coloured areas. Genotyping of the MC1-R gene in a bull with mosaic expression of red vs. black pigment verified the existence of the E^D allele, in spite of the fact that the majority of the animal is red coloured. No further mutations were found within the E^D variant of the MC1-R gene, which was inherited from a completely red mother (genotype E^D/e).     

Activity of a gene in transplanted oocytes in the annelid,Platynereis

Summary Pteridine eye pigment, indicative of the activity of theor ⁺-allele, was observed inor/or larvae ofPlatynereis, derived from transplantedor ⁺/or oocytes. These heterozygous oocytes had grown up inor/or hosts, themselves deficient in pteridine pigment synthesis. It is therefore concluded that theor ⁺ gene product, responsible for pteridine pigment synthesis in theor/or larvae, had been synthesized by the oocyte genomes.Supported by the Deutsche Forschungsgemeinschaft.     

Tissue microenvironment and the genetic control of hair pigment patterns in mice

This study was conducted to assess microenvironmental variability within integumental tissue of genetically identical mice with respect to a specific cellular response: cyclic synthesis of yellow and black pigment by hair bulb melanocytes. Crosses were performed within and between inbred strains of mice that were isogenic with the exception of a single gene substitution at the agouti locus. Agouti locus genes included the A^vy, A^w, A, a^td, a^t, a^x, a^m, and a alleles. The pigment patterns of dorsal, flank, and ventral hairs of the first and third hair generations and of hairs growing in special integumentary areas such as the pinna, tail, and hind foot were studied. It was found that the amount of yellow pigment synthesized by hair bulb melanocytes within genetically identical mice is both agedependent and conditioned by the integumentary environment. Furthermore, the special integumentary regions produce hairs with a variety of pigment patterns in which the distribution and relative amounts of black and yellow pigments do not necessarily conform to dominance relationships expected among agouti locus alleles as judged by their effects on the pigmentation of the dorsal pelage. We conclude that within genetically uniform integumental tissues, microenvironmental differences occur and are reflected as alterations in the metabolic pattern of differentiated cells.     

Cloning and primary structure of Staphylococcus aureus DNA topoisomerase IV: a primary target of fluoroquinolones

Study of the molecular basis for Legionella pneumophila pathogenicity would be facilitated with an efficient mutagen that can not only mark genomic mutations, but can also be used to reflect gene expression during macrophage infection. A derivative of Jn903, Tn903dlllacZ, is shown to transpose with high efficiency in L. pneumophila. Tn903dlllacZ encodes resistance to kanamycin (Km^R) and carries a 5’truncated lacZ gene that can form translational fusions to L. pneumophila genes upon transposition. The cls-acting Tn903 transposase is supplied outside Tn903dlllacZ, and hence chromosomally integrated copies are stable. Km^R LacZ⁺ insertion mutants of L. pneumophila were isolated and shown by DNA hybridization to carry a single Tn903dlllacZ inserted within their chromosomes at various locations. One particular Km^R LacZ⁺ mutant, AB1156, does not produce the brown pigment (Pig) characteristic of Legionella species. Tn903dlllacZ is responsible for this phenotype since reintroduction of the transposonlinked mutation into a wild-type background results in a Pig phenotype. L. pneumophila pigment production is normally observed in stationary-phase growth of cells in culture, and β-galactosidase activity measured from the pig::lacZ fusion increased during the logarithmic-phase growth and peaked at the onset of stationary phase. Interestingly, pig::lacZ expression also increased during macrophage infection. The pigment itself, however, does not appear to be required for L. pneumophila to grow within or kill host macrophages.     

Generation of Pmel-dependent conditional and inducible Cre-driver mouse line for melanocytic-targeted gene manipulation

Conditional and inducible gene targeting using Cre/loxP-mediated recombination is a powerful reverse genetics approach used to study spatiotemporal gene functions in specified cell types. To enable temporal gene manipulation in the melanocyte lineage, we established a novel inducible Cre-driver mouse line by targeting an all-in-one tetracycline/doxycycline (Dox)-inducible Cre expression cassette into the Pmel locus (Pmel^{P2A-TetON3G-TRE3G-iCre}), a gene locus preferentially expressed in pigment cells. By crossing these Cre-driver mice with a strong Cre-reporter mouse line, Gt(ROSA)26Sor^{tm9(CAG-tdTomato)Hze}, we show the effectiveness of the Pmel^{P2A-TetON3G-TRE3G-iCre} mouse line in facilitating Dox-inducible Cre/loxP recombination in a wide variety of pigment cell lineages including hair follicle melanocytes and their stem cells. Furthermore, to demonstrate proof of concept, we ablated Notch signaling postnatally in the Pmel^{P2A-TetON3G-TRE3G-iCre} mice. In agreement with the previously reported phenotype, induced ablation of Notch signaling in the melanocyte lineage resulted in premature hair graying, demonstrating the utility of the Pmel^{P2A-TetON3G-TRE3G-iCre} allele. Therefore, the Pmel^{P2A-TetON3G-TRE3G-iCre} mouse line is suitable for assessing gene functions in melanocytes using an in vivo inducible reverse genetics approach. Furthermore, we unexpectedly identified previously unrecognized PMEL-expressing cells in non-pigmentary organs in the mice, suggesting unanticipated functions of PMEL other than melanosome formation.     

Identification, cloning and analysis of theAspergillus niger genepacC, a wide domain regulatory gene responsive to ambient pH

A wide domain regulatory gene implicated in modulating gene expression in response to ambient pH has been cloned and sequenced from the industrially useful filamentous fungusAspergillus niger. This gene,pacC, is able to restore apacC ⁺ phenotype toA. nidulans pacC ^c 11 andpacC ^c 14 mutants with respect to extent of conidiation, conidial pigment intensity and acid phosphatase regulation. ThepacC gene ofA. niger comprises three exons, encodes a three-zinc-finger protein of 677 amino acids, and shows pH-dependent regulation of expression: mRNA levels are elevated under alkaline conditions and considerably reduced under acidic conditions. The occurrence of PacC consensus binding targets within the sequences upstream ofpacC may indicate autoregulation.     

Volatile general anesthetics reveal a neurobiological role for the white and brown genes of Drosophila melanogaster

Molecular and cellular evidence argues that a heterodimer between two ABC transporters, the White protein and the Brown protein, is responsible for pumping guanine into pigment‐synthesizing cells of the fruit fly, Drosophila melanogaster. Previous studies have not detected White or Brown outside pigment‐synthesizing cells nor have behavioral effects of null mutants been reported, other than those that are visually dependent. Nevertheless, we show here that exposure to the volatile general anesthetic (VGA) enflurane reveals a difference in neuromuscular performance between wild‐type flies and those that carry a null allele in either the white or brown gene. Specifically, in a test of climbing ability, w¹¹¹⁸ or bw¹ flies are much less affected by enflurane than are congenic controls. Altered anesthetic sensitivity is still observed when visual cues are reduced or eliminated, arguing that white and brown contribute to neural function outside the eye. This hypothesis is supported by the detection of white message in heads of flies that are genetically altered so as to lack pigment‐producing cells. The w¹¹¹⁸ or bw¹ mutations also alter the response to a second VGA, halothane, albeit somewhat differently. Under some conditions, the combination of w¹¹¹⁸ with another mutation that affects anesthesia leads to a drastically altered phenotype. We consider several ways by which diminished transport of guanine could influence neural function and anesthetic sensitivity. Published 2001 John Wiley & Sons, Inc. J Neurobiol 49: 339–349, 2001     

Unexpected Conservation of the X-Linked Color Vision Gene in Nocturnal Prosimians: Evidence from Two Bush Babies

Bush babies have had a long history of nocturnal life and it would be interesting to know whether their color vision genes have become degenerate. Therefore, we used PCR techniques to sequence the X-linked pigment gene of two of these nocturnal prosimians: Galago senegalensis and Otolemur garnettii. Southern hybridization of genomic DNA of G. senegalensis showed a single X-linked pigment gene. Interestingly, the deduced pigment sequences of the two bush babies are identical. By comparing the X-linked pigments of bush baby, human, squirrel monkey, and marmoset, 38 variable positions were identified. At those positions that may cause a spectral shift, the bush baby pigment has identical or biochemically similar residues to those of the marmoset cone pigment with a spectral peak of 543 nm. This result is consistent with the estimate of 544–545 nm for the spectral peak of the X-linked pigment of Otolemur crassicaudatus, which is closely related to Otolemur garnettii. The neighbor-joining tree of mammalian X-linked pigments showed a significantly shorter branch in the bush baby lineage than in other primate lineages. A relative rate test showed that the nonsynonymous substitution rate of the bush baby X-linked pigment gene is about three times slower than that of the human red pigment gene, though the synonymous substitution rates of the two genes are similar. The slower nonsynonymous rate in the bush baby lineage suggests that the bush baby X-linked pigment gene is under functional constraints, in spite of its nocturnal life. Two radical changes at positions in the intradiskal surface next to the sixth transmembrane domain were observed in the X-linked cone pigment of bush babies but not in other primates. They are changes from Ala to Ser and from Asn to His, which are similar in function to the corresponding residues in rhodopsins. These two changes may be of importance for dim light sensitivity, which is consistent with our proposal that the evolution of the bush baby X-linked pigment gene is under selective pressure. In addition, the 2.5% divergence in introns 2 and 5 of the X-linked pigment gene between the two bush babies supports their classification into two separate genera. Received: 30 November 1996 / Accepted: 17 June 1997     

Comparative studies on the nature and distribution of pigments from two thermophilic bacteria

Summary The pigments of two strains of extremely thermo-resistant bacteria were extracted and examined spectrophotometrically. Both strains contain -carotene as the main pigment according to the spectral characteristics of the pigment in various solvents. According to quantitative data Thermus aquaticus contains more than one hundred times as much of the pigment than is found in strain YT-G, representing almost 80% of the total lipid. The carotene of strain YT-G is destroyed by hot saponification, while that of T. aquaticus resists that procedure without alterations. T. aquaticus contains a small amount of a second pigment with absorption maxima at 370, 400 and 420 m.Solubilization of the membrane of spheroplasts derived from acetate-C¹⁴-labelled cells of T. aquaticus, followed by separation of the crude membrane fraction, showed that about 50% of the C¹⁴ is associated with the membrane fraction. More than 90% of the C¹⁴ is recovered in the yellow supernatant of the acetone-extracted crude membrane fraction, showing that a rapid incorporation of the label into the pigment occurs. The assumption that the pigment is associated with the membrane is supported by submicroscopical observations: T. aquaticus has a well-defined cytoplasmic membrane, and on top of this a sandwiched layer, which is embedded in a very electron-dense material, that we assume to consist of the pigment. Strain YT-G which contains only minute quantities of the same pigment, does not possess this kind of an outer membrane, but shows only a cytoplasmic membrane envelopped by the cell wall.The main experiments were done while on leave to the Ames Research Center, as a recipient of a Senior Post-doctoral Resident Research Associateship of the National Research Council, Washington, D.C., from the Dept. of Exobiology, University of Nijmegen, Driehuizerweg 200, Nijmegen, The Netherlands.     

Unusual development of light-reflecting pigment cells in intact and regenerating tail in the periodic albino mutant of Xenopus laevis

Unusual light-reflecting pigment cells, “white pigment cells”, specifically appear in the periodic albino mutant (a ^p /a ^p ) of Xenopus laevis and localize in the same place where melanophores normally differentiate in the wild-type. The mechanism responsible for the development of unusual pigment cells is unclear. In this study, white pigment cells in the periodic albino were compared with melanophores in the wild-type, using a cell culture system and a tail-regenerating system. Observations of both intact and cultured cells demonstrate that white pigment cells are unique in (1) showing characteristics of melanophore precursors at various stages of development, (2) accumulating reflecting platelets characteristic of iridophores, and (3) exhibiting pigment dispersion in response to α-melanocyte stimulating hormone (α-MSH) in the same way that melanophores do. When a tadpole tail is amputated, a functionally competent new tail is regenerated. White pigment cells appear in the mutant regenerating tail, whereas melanophores differentiate in the wild-type regenerating tail. White pigment cells in the mutant regenerating tail are essentially similar to melanophores in the wild-type regenerating tail with respect to their localization, number, and response to α-MSH. In addition to white pigment cells, iridophores which are never present in the intact tadpole tail appear specifically in the somites near the amputation level in the mutant regenerating tail. Iridophores are distinct from white pigment cells in size, shape, blue light-induced fluorescence, and response to α-MSH. These findings strongly suggest that white pigment cells in the mutant arise from melanophore precursors and accumulate reflecting platelets characteristic of iridophores.     

Production of active pigment epithelium-derived factor in<Emphasis Type="Italic">E. coli</Emphasis>

Human pigment epithelium-derived factor (PEDF), a neurotrophic factor, is the most potent natural inhibitor of angiogenesis. To produce the active PEDF, the gene coding for the human PEDF protein was expressed in E. coli. The rPEDF protein was expressed at 457 mg l^–1 as a soluble protein. The yield of purified GST fusion protein was 14 mg ll^–1. Purified rPEDF inhibited tube formation in endothelial cells.Revisions requested 30 November 2004; Revisions received 25 January 2005     

Melatonin does not affect the black pigment migration in the crab Neohelice granulata

N-acetyl-5-methoxytryptamine or melatonin is a multifunctional molecule. The main physiological function, at least in vertebrates, is to transduce to the animal the photoperiodic information and regulate rhythmic parameters. But studies have also observed the action of this molecule on pigment migration in ectothermic vertebrates. Thus the aim of this paper was to investigate in vivo and in vitro the influence of melatonin on the pigment migration in melanophores of the crab Neohelice granulate. Injections of melatonin (2 × 10⁻⁹ moles · crab⁻¹) at 07:00 h or 19:00 h did not affect (p > 0.05) the circadian pigment migration of the melanophores in constant darkness. Additionally no significant pigment migration (p > 0.05) was verified in normal and eyestalkless crabs injected with melatonin (10⁻¹⁰–10⁻⁷ moles · crab⁻¹) during the day or night. In the ^{in vitro} assay, the response of melanophores to the pigment-dispersing hormone in eyestalkless crabs injected with melatonin (2 × 10⁻⁹ moles · crab⁻¹) 1 and 12 hours before the observations did not differ (p > 0.05) from the control group (injected with physiological solution). These results suggest that melatonin does not act as a signaling factor for pigment dispersion or aggregation in the melanophores of N. Granulate.     

Production of antibacterial violet pigment by psychrotropic bacterium RT102 strain

The antibacterial action of violet pigment, a mixture of violacein and deoxyviolacein, isolated from phychrotrophic bacterium RT102 strain was examined, and the operational conditions for the effective production of violet pigment were studied. The antibacterial activity of the violet pigment was confirmed for several bacteria such asBacillus licheniformis, Bacillus subtilis, Bacillus megaterium, Staphylococcus aureus, andPseudomonas aeruginosa, and the high concentration of violet pigment, above about 15 mg/L, caused not only growth inhibition but also death of cells. The growth properties of RT102 strain were clarified under various incubation conditions such as pH, temperature, and dissolved oxygen concentration. The maximum violet pigment concentration,i.e. 3.7 g/L, and the maximum productivity of violet pigment,i.e. 0.12 g L⁻¹h⁻¹, were obtained in a batch culture of pH 6, 20°C, and 1 mg/L of dissolved oxygen concentration.     

A ketoreductase gene from Streptomyces mycarofaciens 1748 DNA involved in biosynthesis of a spore pigment

An efficient plasmid transformation system forS. mycarofaciens 1748 has been established. In order to determine the function of MKR gene in S.mycarofaciens 1748, the gene disruption experiment was carried out. For this purpose the plasmid pKC1139 was used. A recombinant strain with white spore appeared, in contrast to the grey-colour spore of S.myarofaciens 1748. This suggested that homologous recombination between plasmidborne MKR gene sequence and the chromosome of S.mycarofaciens 1748 had occurred. A Southern hybridization experiment using a-³²P-labelled MKR gene as probe indicated that the desired integration event had occurred in the recombinant. The result of gene disruption showed that the alteration of this gene in the chromosome of S.mycarofaciens 1748 made sporulating colonies remain white instead of taking on the typical grey colour of sporulating wild type colonies, suggesting that MKR gene is involved in the biosynthesis of a spore pigment. The recombinant strain was incubated with fermentation medium optimised for midecamycin production. A TLC assay showed that the recombinant strain produced midecamycin in quantities comparable to that ofS. mycarofaciens 1748. A pCN8B12 was a clone from genomic library of midecamycin producing strain which contained a 28-kb DNA insert. The 28-kb DNA fragment contained act I-homologous and act III-homologous regions. he PKS (act I-homologous) and MKR (act III-homologous) genes that define spore pigment of midecamycin producing strain were Jocalized by restriction endonuclease digestion with pCN8B12, indicating that they are separated by about 10 kb DNA. The polyketide synthase gene cluster of simila; organization has not been reported yet. Project supported by the National Natural Science Foundation of China.     

Recombinant adeno-associated virus type 2-mediated gene delivery into the <Emphasis Type="Italic">Rpe65</Emphasis><Superscript>-/-</Superscript> knockout mouse eye results in limited rescue

Background  

Leber's congenital amaurosis (LCA) is a severe form of retinal dystrophy. Mutations in the RPE65 gene, which is abundantly expressed in retinal pigment epithelial (RPE) cells, account for approximately 10–15% of LCA cases. In this study we used the high turnover, and rapid breeding and maturation time of the Rpe65 ^-/- knockout mice to assess the efficacy of using rAAV-mediated gene therapy to replace the disrupted RPE65 gene. The potential for rAAV-mediated gene treatment of LCA was then analyzed by determining the pattern of RPE65 expression, the physiological and histological effects that it produced, and any improvement in visual function.     

Visualizing bz1 missense suppression in Zea mays: an assay for monocot tRNA expression and utilization

Bombardment of a highly expressed dicot tRNA^ala(GAC) gene into Zea mays bz-E2 or bz-E5 coleoptiles causes suppression of an Ala₄₅₈ →Val missense mutation, visualized by the development of anthocyanin pigment. Missense suppression is blocked by mutation of tRNA^ala(GAC) at a site that prevents aminoacylation by the dicot alanyl-tRNA synthetase, indicating that features identified for expression and utilization of dicot tRNAs also function in monocots. This assay of the expression and utilization of tRNA^ala(GAC) also can be used to study a variety of tRNAs and their genes, most of which can be relatively easily altered to be charged by alanyl tRNA synthetase.     

The role of melanocyte-stimulating hormone (MSH) receptor in bovine coat color determination

The melanocyte-stimulating hormone (MSH) receptor has a major function in the regulation of black (eumelanin) versus red (phaeomelanin) pigment synthesis within melanocytes. We report three alleles of the MSH-receptor gene found in cattle. A point mutation in the dominant allele E ^D gives black coat color, whereas a frameshift mutation, producing a prematurely terminated receptor, in homozygous e/e animals, produces red coat color. The wild-type allele E ⁺ produces a variety of colors, reflecting the possibilities for regulating the normal receptor. Microsatellite analysis, RFLP studies, and coat color information were used to localize the MSH-receptor to bovine Chromosome (Chr) 18.     

Extracellular phycoerythrin-like protein released by freshwater cyanobacteria <Emphasis Type="Italic">Oscillatoria</Emphasis> and <Emphasis Type="Italic">Scytonema</Emphasis> sp.

During growth of the freshwater cyanobacteria, Oscillatoria sp. BTCC/A0004, and Scytonema sp. TISTR 8208, a pink pigment is released into the growth medium. The pigment from each source had a molecular weight of approximately 250 kDa and had adsorption maxima at 560 and 620 nm. These results suggest that pink pigment is a phycoerythrin-like protein. It inhibited the growth of green algae, Chlorella fusca and Chlamydomonas reinhardtii, but not other cyanobacteria or true bacteria. The concentration at which growth inhibition 50% occurred was 0.5, 6 and more than 10 mg ml⁻¹, respectively.     

Molecular characterization of durum and common wheat recombinant lines carrying leaf rust resistance (Lr19) and yellow pigment (Y) genes from Lophopyrum ponticum

Chromosome 7E from Lophopyrum ponticum carries a valuable leaf rust resistant gene designated Lr19. This gene has not been widely used in common wheat breeding because of linkage with the yellow pigment gene Y. This gene tints flour yellow, reducing its appeal in bread making. However, a high level of yellow pigment is desirable in durum wheat breeding. We produced 97 recombinant chromosomes between L. ponticum transfer 7D.7E#1 and its wheat homoeologues, using the ph1b mutation that promotes homoeologous pairing. We characterized a subset of 37 of these lines with 11 molecular markers and evaluated their resistance to leaf rust and the abundance of yellow pigment. The Lr19 gene was mapped between loci Xwg420 and Xmwg2062, whereas Y was mapped distal to Xpsr687, the most distal marker on the long arm of chromosome 7. A short terminal 7EL segment translocated to 7A, including Lr19 and Y (line 1-23), has been transferred to durum wheat by backcrossing. The presence of this alien segment significantly increased the abundance of yellow pigment. The Lr19 also conferred resistance to a new durum leaf rust race from California and Mexico that is virulent on most durum wheat cultivars. The new durum lines with the recombinant 7E segment will be useful parents to increase yellow pigment and leaf rust resistance in durum wheat breeding programs. For the common wheat breeding programs, we selected the recombinant line 1-96, which has an interstitial 7E segment carrying Lr19 but not Y. This recombinant line can be used to improve leaf rust resistance without affecting flour color. The 7EL/7DL 1-96 recombinant chromosome did not show the meiotic self-elimination previously reported for a 7EL/7BL translocation.