Cloning of cDNA and amino acid sequence of one of chicken cone visual pigments

The chicken has four kinds of color visual pigments, in addition to rhodopsin. A chicken genomic DNA library was screened with cDNA of human red-sensitive pigment and a chicken genomic DNA fragment including rhodopsin exons 2, 3 and 4, and then a genomic DNA fragment encoding a visual pigment, possibly an iodopsin, was cloned. A cDNA library, constructed from chicken retina mRNA, was screened with the genomic DNA fragment and the cDNA of human red-sensitive pigment, and the cDNA encoding the pigment was cloned. The nucleotide sequence of this cDNA was similar to that of the human red-sensitive pigment, with identities of 78% for the nucleotide sequence and 84% for the amino acid sequence with human red-sensitive pigment.     

Identification of indigo-related pigments produced by Escherichia coli containing a cloned Rhodococcus gene.

Pigments produced by Escherichia coli containing a cloned piece of DNA from Rhodococcus sp. ATCC 21145 were extracted in chloroform and separated into blue and pink components. Evidence from TLC, NMR spectroscopy, absorption spectrum analysis and solubility behaviour suggested that the blue pigment was indigo and the pink pigment was indirubin, a structural isomer of indigo. The proposed pathway for pigment production on LB agar involves the conversion of tryptophan to indole by tryptophanase of E. coli and the oxidation of indole to indigo by the product of the cloned Rhodococcus DNA insert.     

Cloning and expression of Rhodococcus genes encoding pigment production in Escherichia coli

Pigment was produced by Escherichia coli cells carrying recombinant plasmids pNIL100, pNIL200 and pNIL400 containing DNA from Rhodococcus sp. E. coli cells containing pNIL100 or pNIL200 (with DNA inserts from Rhodococcus sp. JL10 and Rhodococcus sp. ATCC 21145 respectively) produced both blue and pink pigments, while cells containing pNIL400 (with a DNA insert from Rhodococcus sp. ATCC 21145) produced only pink pigment. Colonies of E. coli(pNIL100) and E. coli(pNIL200) were dark blue, whereas E. coli(pNIL400) colonies were pink. No pigment was detected in Streptomyces griseus transformants containing pNIL100, pNIL200 or pNIL400. Restriction endonuclease mapping indicated that the cloned DNA fragments were different. The pigment gene(s) in pNIL200 producing both the blue and pink pigments were contained within a 2.8 kb DNA fragment. The pigments produced by E. coli transformants containing pNIL200 were characterized by visible and UV spectroscopy. No similar pigments were detected in Rhodococcus sp. ATCC 21145.     

假单胞杆菌2-萘酸加氧酶基因的克隆与表达

采用Cosmid pLARF1构建了完整的2-萘酸代谢菌2-NAT菌株的基因文库.通过分析基因文库中产生蓝色色素的克隆验证了2-萘酸代谢基因在大肠杆菌体内得到表达,并导致重组细菌产生靛蓝.从产生靛蓝的克隆抽提重组的Cosmid DNA进行酶解分析,发现所有能产生靛蓝的克隆均含有一大小相同的DNA片段.用重组细菌进行靛蓝生物合成的试验展示了微生物法生产靛蓝的美好前景.     

Plasmid-determined 2-hydroxypyridine utilization by Arthrobacter crystallopoietes.

Arthrobacter crystallopoietes has the ability to utilize 2-hydroxypyridine (2-HP) as a source of carbon and nitrogen and forms a blue extracellular pigment when grown in the presence of 2-HP. Ultracentrifugal analyses of pigment producing (Pig+) and pigment nonproducing (Pig-) strains of A. crystallopoietes revealed the presence of plasmid material in both strains. Recovery of plasmid DNA from Pig+ strains is two or three times greater than from Pig- strains. The molecular weight of plasmid DNA recovered from Pig+ strains (62 Mdaltons) is slightly higher than the molecular weight of plasmid DNA from Pig- strains. Consistent with the characterization of plasmid DNA from the two strains is that Pig+ strains contain a 63-Mdalton plasmid encoding 2-HP utilization as well as a cryptic plasmid of very nearly equal molecular weight. Pig- strains contain only the cryptic plasmid.     

Molecular evolution of human visual pigment genes

By comparing the published DNA sequences for (a) the genes encoding the human visual color pigments (red, green, and blue) with (b) the genes encoding human, bovine, and Drosophila rhodopsins, a phylogenetic tree for the mammalian pigment genes has been constructed. This evolutionary tree shows that the common ancestor of the visual color pigment genes diverged first from that of the rhodopsin genes; then the common ancestor of the red and green pigment genes and the ancestor of the blue pigment gene diverged; and finally the red and green pigment genes diverged from each other much more recently. Nucleotide substitutions in the rhodopsin genes are best explained by the neutral theory of molecular evolution. However, important functional adaptations seem to have occurred twice during the evolution of the color pigment genes in humans: first, to the common ancestor of the three color pigment genes after its divergence from the ancestor of the rhodopsin gene and, second, to the ancestor of the red pigment gene after its divergence from that of the green pigment gene.     

A Streptomyces avermitilis gene encoding a 4-hydroxyphenylpyruvic acid dioxygenase-like protein that directs the production of homogentisic acid and an ochronotic pigment in Escherichia coli.

A 1.5-kb genomic fragment isolated from Streptomyces avermitilis that directs the synthesis of a brown pigment in Escherichia coli was characterized. Since pigment production in recombinant E. coli was enhanced by the addition of tyrosine to the medium, it had been inferred that the cloned DNA might be associated with melanin biosynthesis. Hybridization studies, however, showed that the pigment gene isolated from S. avermitilis was unrelated to the Streptomyces antibioticus melC2 determinant, which is the prototype of melanin genes in Streptomyces spp. Sequence analysis of the 1.5-kb DNA that caused pigment production revealed a single open reading frame encoding a protein of 41.6 kDa (380 amino acids) that resembled several prokaryotic and eukaryotic 4-hydroxyphenylpyruvate dioxygenases (HPDs). When this open reading frame was overexpressed in E. coli, a protein of about 41 kDa was detected. This E. coli clone produced homogentisic acid (HGA), which is the expected product of the oxidation of 4-hydroxyphenylpyruvate catalyzed by an HPD, and also a brown pigment with characteristics similar to the pigment observed in the urine of alkaptonuric patients. Alkaptonuria is a genetic disease in which inability to metabolize HGA leads to increasing concentrations of this acid in urine, followed by oxidation and polymerization of HGA to an ochronotic pigment. Similarly, the production of ochronotic-like pigment in the recombinant E. coli clone overexpressing the S. avermitilis gene encoding HPD is likely to be due to the spontaneous oxidation and polymerization of the HGA accumulated in the medium by this clone.     

Establishment of pigment cell lineage in embryos of the sea urchin, Hemicentrotus pulcherrimus

In an attempt to estimate the number of pigment precursor cells in sea urchin embryos, DNA synthesis and cell divisions were blocked with aphidicolin from various stages of development. Interestingly, pigment cells differentiated on a normal time schedule, even if the embryos were treated from late cleavage stages on. In most of the embryos treated from 10 h on, 10-15 pigment cells differentiated. Thereafter, the number of pigment cells in the aphidicolin-treated embryos further increased, as the initiation of the treatment was delayed. On the other hand, total cell volumes in the pigment lineage, calculated from the averaged number and diameter of differentiated pigment cells, were almost the same irrespective of the time of the initiation of aphidicolin treatment. This indicated that the increase in the number was caused by divisions of the pre-existing cells in the pigment lineage. Thus, the founder cells that exclusively produce pigment cells could be identified. They are nine times-cleaved blastomeres and specified by 10 h post-fertilization. The obtained results also clarified the division schedule in the pigment lineage; the founder cells divide once (10th) until hatching, and divide once more (11th) by the end of gastrulation.     

Assessment of RAPD marker frequencies over cycles of recurrent selection for pigment concentration and percent solids in red beet (Beta vulgaris L.)

In the past twenty years, betalain pigments found in red beet (Beta vulgaris L.) have been adopted for use as natural red food colorings. Utility and extractability of these compounds is dependent upon native concentrations of both pigment and total dissolved solids. In an effort to develop red beet populations with elevated levels of betalain pigment, recurrent half-sib family selection for high pigment and both high and low solids was practiced for seven cycles. This scheme resulted in the development of a high pigment/high solids and a high pigment/low solids population. A total of 200 randomly chosen oligonucleotide decamer primers were used to amplify genomic DNA of individual plants in the selection scheme for repeatability and clarity of amplification products. Thirty-one primers were chosen on this basis and used to assess random-amplified polymorphic DNA (RAPD) marker frequencies on genomic DNA samples isolated from 47 randomly-chosen individual plants in each of cycles 1, 3, and 6 in both high pigment/high solids and high pigment/low solids. Number of scorable Polymerase Chain Reaction products ranged from 1–10, resulting in a total of 161 RAPD markers. Chi-square and regression analyses were performed to determine the presence/absence of a linear trend in marker frequencies during the selection scheme. Comparisons were made among cycles within high pigment/high solids and high pigment/low solids populations. Significant linear trends were detected in both cases for certain RAPD markers. Formulae were used to test whether observed linear marker frequency trends were due to the effects of selection or random genetic drift. Chi-square tests revealed a subset of markers which exhibited significant frequency changes across cycles changed due to selection. These data demonstrate changes in RAPD marker frequencies with recurrent selection and suggest linkage of RAPD markers to genes controlling pigment in red beet.     

Molecular basis of abnormal red-green color vision: a family with three types of color vision defects

The molecular nature of three different types of X-linked color-vision defects, protanomaly, deuteranomaly, and protanopia, in a large 3-generation family was determined. In the protanomalous and protanopic males the normal red pigment gene was replaced by a 5' red-3' green fusion gene. The protanomalous male had more red pigment DNA in his fusion gene than did the more severely affected protanopic individual. The deuteranomalous individual had four green pigment genes and one 5' green-3' red fusion gene. These results extend those of Nathans et al., who proposed that most red-green color-vision defects arise as a result of unequal crossing-over between the red and green pigment genes. The various data suggest that differences in severity of color-vision defects associated with fusion genes are caused by differences in crossover sites between the red and green pigment genes. Currently used molecular methodology is not sufficiently sensitive to define these fusion points accurately, and the specific color-vision defect within the deutan or protan class cannot be predicted. The DNA patterns for color-vision genes of female heterozygotes have not previously been described. Patterns of heterozygotes may not be distinguishable from those of normals. However, a definite assignment of the various color pigment gene arrays could be carried out by family study. Two compound heterozygotes for color-vision defects who tested as normal by anomaloscopy were found to carry abnormal fusion genes. In addition, a normal red pigment gene was present on one chromosome and at least one normal green pigment gene was present on the other.(ABSTRACT TRUNCATED AT 250 WORDS)     

Fast-in Situ hybridization and immunoenzymatic color pigment detection of mouse bone marrow micronucleus

The development of a whole mouse genomic DNA probe coupled to color pigment painting detection methodology can accurately verify mouse micronuclei induced by chemicals or drugs leading to a lower probability of potential artifacts. Using color pigment painting detection of probes in conjunction with Wright's Giemsa counterstain instead of the current fluorescence detection technology ensures low cost, high resolution permanent documentation of slides for a particular test compound. The permanent color pigment-detected micronuclei and adjoining counterstain allows slides to be stored for future analysis without enhancing the signal or adding antifading agents that are associated with fluorescence detection. Combining innovative technology such as fast-in situ hybridization of DNA probes with immunoenzymatic color pigment detection provides rapid verification of true micronuclei (DNA containing) within 2-3 hr.     

Carotenoid pigments and the enhanced resistance of Pseudomonas methanolica to the action of ultraviolet radiation

The correlation was studied between the elevated resistance of Pseudomonas methanolica, a facultative methylotroph, to the bactericidal action of UV (254 nm) and the content of a pink pigment in this organism. Absorption spectra of cell extracts containing the pigment were recorded. The ethanol and acetone extracts had two absorption maxima at 420-430 and 500-510 nm, respectively, which was typical of carotenoids. The culture with the pigment and its variant without the pigment had nearly identical survival rates. Therefore, the elevated resistance of P. methanolica cells seems to stem from the activity of DNA reparation cellular systems rather than from the presence of carotenoid pigments in the cells.     

A 195-kb cosmid walk encompassing the human Xq28 color vision pigment genes

By using cosmid walking, we have cloned a 195-kb region from chromosome band Xq28 that encompasses the red and green color pigment genes and 85 kb of flanking sequences. This has allowed us to confirm that the color pigment genes are within very homologous units arranged in tandem array. Each unit contains two BssHII sites and one NruI site that are frequently methylated in male leukocyte DNA. A NotI and an EagI site are present 6 kb upstream from the red pigment gene promoter; the NotI site was shown to be unmethylated in the active X chromosome in leukocytes and may represent a CpG island for the whole cluster. We have identified another CpG island, 61 kb 3' from the last green pigment gene, that is unmethylated in leukocytes on the active X chromosome, but methylated on the inactive X. This island is flanked by sequences conserved in evolution and may thus correspond to an expressed gene. We also describe an informative three-allele restriction fragment length polymorphism within the pigment gene cluster.     

In Situ Detection of a PCR-Synthesized Human Pancentromeric DNA Hybridization Probe by Color Pigment Immunostaining: Application for Dicentric Assay Automation

We report a low cost and efficient method for synthesizing a human pancentromeric DNA probe by the polymerase chain reaction (PRC) and an optimized protocol for in situ detection using color pigment immunostaining. The DNA template used in the PCR was a 2.4 kb insert containing human alphoid repeated sequences of pancentromeric DNA subcloned into pUC9 (Miller et al. 1988) and the primers hybridized to internal sequences of the 172 bp consensus tandem repeat associated with human centromeres. PCR was performed in the presence of biotin-11-dUTP, and the product was used for in situ hybridization to detect the pancentromeric region of human chromosomes in metaphase spreads. Detection of pancentromeric probe was achieved by immunoenzymatic color pigment painting to yield a permanent image detected at high resolution by bright field microscopy. The ability to synthesize the centromeric probe rapidly and to detect it with color pigment immunostaining will lead to enhanced identification and eventually to automation of various chromosome aberration assays.     

Characterization of a Deep-Sea Sediment Metagenomic Clone that Produces Water-Soluble Melanin in Escherichia coli

To access to the microbial genetic resources of deep-sea sediment by a culture-independent approach, the sediment DNA was extracted and cloned into fosmid vector (pCC1FOS) generating a library of 39,600 clones with inserts of 24–45 kb. The clone fss6 producing red-brown pigment was isolated and characterized. The pigment was identified as melanin according to its physico-chemical characteristics. Subcloning and sequences analyses of fss6 demonstrated that one open reading frame (ORF2) was responsible for the pigment production. The deduced protein from ORF2 revealed significant amino acid similarity to the 4-hydroxyphenylpyruvate dioxygenase (HPPD) from deep-sea bacteria Idiomarina loihiensis. Further study demonstrated that the production of melanin was correlated with homogentistic acid (HGA). The p-hydroxyphenylpyruvate produced by the Escherichia coli host was converted to HGA through the oxidation reaction of introduced HPPD. The results demonstrate that expression of DNA extracted directly from the environment might generate applicable microbial gene products. The construction and analysis of the metagenomic library from deep-sea sediment contributed to our understanding for the reservoir of unexploited deep-sea microorganisms.     

Programmed cell death during pigment gland formation in Gossypium hirsutum leaves

Ultrastructural studies have shown that the formation of pigment glands in Gossypium hirsutum L. leaves is a lysigenous process, originating from a cluster of cells in the ground meristem. Various techniques were used here to investigate whether programmed cell death (PCD) plays a critical role in this developmental process. Nuclei of internal cells in the pigment gland‐forming tissue were TUNEL‐positive and DAPI‐negative, suggesting that DNA cleavage is an early event and complete DNA degradation is a late event. Smeared bands and a lack of laddering after gel electrophoresis indicate that DNA cleavage is random. Ultrastructurally, secretory cells in the pigment glands become distorted, nuclei are densely stained, and chromosomes become condensed until completely degraded at late stages. Vacuoles with electron‐dense bodies and membrane‐bound autophagosomes are seen in both secretory and sheath cells, suggesting that autophagy plays a key role in PCD during cytoplasm degradation. Buckling of cell walls, seen at early stages, later leads to a complete breakdown of the walls. Together, these results suggest that PCD plays a critical role in the lysigenous development of pigment glands in G. hirsutum leaves.     

CHARACTERIZATION OF OCEANIC PHOTOSYNTHETIC PICOEUKARYOTES BY FLOW CYTOMETRY1

To interpret flow cytometric data that are routinely obtained on natural oceanic communities, 23 strains of photosynthetic picoeukaryotes belonging to four classes (Prasinophyceae, Chlorophyceae, Pelagophyceae, and Prymnesiophyceae) and six pigment types were investigated for their light scattering in the forward and right-angle directions, chlorophyll fluorescence, and DNA content as measured by flow cytometry. Cell she was assessed by Coulter counter, and pigment composition was measured by reverse-phase high-performance liquid chromatography. The size and GC% of the nuclear genome of cultured picoeukaryotes was measured from the fluorescence of DNA-specific dyes. Using these two parameters, we could discriminate species within pigment groups. DNA staining of preserved natural samples may also prove useful in discriminating cooccurring populations in situ as long as the communities are not too complex. Using the relationships that we established between size and light-scattering properties of the cells, we estimated equivalent diameters of picoeukaryotes in natural populations to be between 1.3 and 2 μm. Chlorophyll a content was between 6 and 16 fg·cel^?1 as calculated from relationships that we established between chlorophyll a content and red fluorescence of the cultured strains. With respect to size, chlorophyll a content, and pigment composition, Pelagomonas sp. strains (Pelagophyceae) appeared to be the most representative of the natural communities in subtropical ocean waters. In contrast, green coccoid strains, which often outcompete other strains in culture, might only be minor contributors to these communities.