Molecular Characterization of the Mouse Tyrosinase Gene:Pigment Cell-Specific Expression in Transgenic Mice

Tyrosinase is the key enzyme in melanin synthesis, and is expressed in the pigment epithelium of the retina, a cell layer derived from the optic cup; and in neural crest-derived melanocytes of skin, hair follicle, choroid, and iris. The tyrosinase gene has been cloned and shown to map to the well-characterized c-locus (albino locus) of the mouse. Subsequent studies demonstrated that a functional tyrosinase minigene was able to rescue the albino phenotype in transgenic mice. The transgene was expressed in a cell type-specific manner in skin and eye. During development of the mouse, the tyrosinase gene is expressed in the pigment epithelium of the retina as early as day 10.5 of gestation. In the hair follicle, tyrosinase gene expression is detected from day 16.5 onwards. This cell-type–specific expression is largely reproduced in transgenic mice. Our results suggest that sequences in the immediate vicinity of the mouse tyrosinase gene are sufficient to provide cell type-specificity and developmental regulation in melanocytes and the pigment epithelium.     

Isolation and characterization of tetranucleotide microsatellite markers in a mouth‐brooding haplochromine cichlid fish (Pseudocrenilabrus multicolor victoriae) from Uganda

Eight tetranucleotide microsatellite loci were isolated from the haplochromine cichlid fish, Pseudocrenilabrus multicolor victoriae, an important model species for studies in respiratory ecology, conservation, and evolution. We surveyed variation at these loci in 23 individuals from western Uganda, finding four to 19 alleles per locus and an average expected heterozygosity of 0.8575. These microsatellite loci will be used to examine gene flow and population structure in Ugandan P. m. victoriae.     

The Torradovirus‐specific RNA2‐ORF1 protein is necessary for plant systemic infection

Tomato apex necrosis virus (ToANV, species Tomato marchitez virus, genus Torradovirus, family Secoviridae) causes a severe tomato disease in Mexico. One distinctive feature of torradoviruses compared with other members of the family Secoviridae is the presence of an additional open reading frame (ORF) in genomic RNA2 (denominated RNA2‐ORF1), located upstream of ORF2. RNA2‐ORF2 encodes a polyprotein that is processed into a putative movement protein and three capsid proteins (CPs). The RNA2‐ORF1 protein has homologues only amongst other torradoviruses and, so far, no function has been associated with it. We used recombinant and mutant ToANV clones to investigate the role of the RNA2‐ORF1 protein in various aspects of the virus infection cycle. The lack of a functional RNA2‐ORF1 resulted in an inability to systemically infect Nicotiana benthamiana and tomato plants, but both positive‐ and negative‐strand RNA1 and RNA2 accumulated locally in agroinfiltrated areas in N. benthamiana plants, indicating that the RNA2‐ORF1 mutants were replication competent. Furthermore, a mutant with a deletion in RNA2‐ORF1 was competent for virion formation and cell‐to‐cell movement in the cells immediately surrounding the initial infection site. However, immunological detection of the ToANV CPs in the agroinfiltrated areas showed that this mutant was not detected in the sieve elements even if the surrounding parenchymatic cells were ToANV positive, suggesting a role for the RNA2‐ORF1 protein in processes occurring prior to phloem uploading, including efficient spread in inoculated leaves.     

Gene expression profiling: opening the black box of plant ecosystem responses to global change

The use of genomic techniques to address ecological questions is emerging as the field of genomic ecology. Experimentation under environmentally realistic conditions to investigate the molecular response of plants to meaningful changes in growth conditions and ecological interactions is the defining feature of genomic ecology. Because the impact of global change factors on plant performance are mediated by direct effects at the molecular, biochemical, and physiological scales, gene expression analysis promises important advances in understanding factors that have previously been consigned to the 'black box' of unknown mechanism. Various tools and approaches are available for assessing gene expression in model and nonmodel species as part of global change biology studies. Each approach has its own unique advantages and constraints. A first generation of genomic ecology studies in managed ecosystems and mesocosms have provided a testbed for the approach and have begun to reveal how the experimental design and data analysis of gene expression studies can be tailored for use in an ecological context.     

Soil organic matter dynamics during 80 years of reforestation of tropical pastures

Our research takes advantage of a historical trend in natural reforestation of abandoned tropical pastures to examine changes in soil carbon (C) during 80 years of secondary forest regrowth. We combined a chronosequence approach with differences in the natural abundance of ¹³C between C3 (forest) and C4 (pasture) plants to estimate turnover times of C in the bulk soil and in density fractions. Overall, gains in secondary forest C were compensated for by the loss of residual pasture-derived soil C, resulting in no net change in bulk soil C stocks down to 1 m depth over the chronosequence. The free light fraction (LF), representing physically unprotected particulate organic matter, was most sensitive to land-use change. Reforestation replenished C in the free LF that had been depleted during conversion to pastures. Turnover times varied with model choice, but in general, soil C cycling rates were rapid for the 0–10 cm depth, with even the heavy fraction (HF) containing C cycling in decadal time scales. Turnover times of C in the free LF from the 0–10 cm depth were shorter than for the occluded and HFs, highlighting the importance of physical location in the soil matrix for residence time in the soil. The majority of the soil C pool (82±21%) was recovered in the mineral-associated density fraction. Carbon-to-nitrogen ratios and differences in natural abundance ¹⁵N of soil organic matter (SOM) showed an increasing degree of decomposition across density fractions with increasing mineral association. Our data show that the physical distribution of C in the soil has a large impact on soil C turnover and the ability of soils to maintain SOM stocks during land-use and land-cover change.     

The Influence of Nitrogen, Light and Water Stress on CO2 Exchange and Organic Acid Accumulation in the Tropical C3-CAM Tree, Clusia minor

The carbon balance and changes in leaf structure in Clusia minorL., were investigated in controlled conditions with regardto nitrogen supply and responses to low and high photosyntheticallyactive radiation (PAR). Nitrogen deficiency and high PAR ledto the production of smaller leaves with higher specific leafdry weight (SLDW) and higher leaf water content, but with lowerchlorophyll content. Nitrogen and PAR levels at growth alsoaffected CO₂ exchange and leaf area. In – N conditions,total daily net CO₂ uptake and leaf area accumulation were slightlyless for high-PAR-grown plants. In contrast, high-PAR-grownplants supplied with nitrogen showed about a 4-fold higher totaldaily CO₂ uptake and about twice the total leaf area of low-PAR-grownplants. Although total daily net CO₂ uptake of +N plants wasonly slightly higher than –N plants under the low PARlevel, –N plants produced almost three times more leafarea but with lower SLDW. Under well-watered conditions, low-PAR-grownplants showed only CO₂ evolution during the night and malicacid levels decreased. However, there was considerable night-timeaccumulation of titratable protons due to day/night changesin citric acid levels. High-PAR-grown plants showed net CO₂uptake, malate and citrate accumulation during the dark period.However, most of the CO₂ fixed at night probably came from respiratoryCO₂. Positive night-time CO₂ exchange was readily observed forlow-PAR-grown plants when they were transferred to high PARconditions or when they were submitted to water stress. In plantsgrown in high and low PAR, CAM leads to a substantial increasein daily water use efficiency for water-stressed plants, althoughtotal net CO₂ uptake decreased.     

Investigating South American biogeographic history using patterns of skull shape variation on Cerdocyon thous (Mammalia: Canidae)

Patterns of geographic variation of the canid Cerdocyon thous have historically been obscured by its remarkable intraspecific morphological variability. The observed distribution is highly associated with phytophysiognomy, a feature considered highly dynamic along geological time. In the present study, we tested whether vegetation distribution during the Holocene Glacial Maximum of South America (HGM) explains the patterns of morphological variation within Cerdocyon thous . The species was divided in groups according to paleohabitats that could support their presence during the HGM, and then tested for differences in skull morphometrics. The results obtained demonstrate that the climatic changes during the HGM influenced the population structure of this species, resulting in the establishment of geographical groups with different degrees of morphological cohesion. Higher morphological cohesion found in the Northern group might be explained by the marked discontinuity between its geographical range and the rest of the species'distribution. The Eastern and Southern morphological divergence is less striking and, although this could be related to past vegetation distribution, the disappearance of those barriers leads to a population structure that could be slowly breaking down.  © 2009 The Linnean Society of London, Biological Journal of the Linnean Society , 2009, 98 , 77–84.     

Identification and Characterization of Epithelial Cells in Mammalian Tissues by Immunofluorescence Microscopy Using Antibodies to Prekeratin

The occurrence of intermediate-sized filaments containing prekeratin-like proteins ('cytokeratins') has been examined in various organs of rat and cow by electron microscopy and by immunofluorescence microscopy on frozen sections using antibodies to defined constitutive proteins of various types of intermediate-sized filaments (prekeratin, vimentin, desmin). Positive cytokeratin reaction and tonofilament-like structures have been observed in the following epithelia: epidermis; ductal, secretory, and myoepithelial cells of sweat glands; mammary gland duct; myoepithelial cells of lactating mammary gland; milk secreting cells of cow; ductal, secretory, and myoepithelial cells of various salivary glands; tongue mucosa; bile duct; excretory duct of pancreas; intestinal mucosa; urothelium; trachea; bronchi; thymus reticulum, including Hassall corpuscles; mesothelium; uterus; and ciliated cells of oviduct. None of the epithelial cells mentioned has shown significant reaction with antibodies to vimentin, the major component of the type of intermediate-sized filaments predominant in mesenchymal cells. The widespread, if not general occurrence of cytokeratin filaments in epithelial cells is emphasized, and it is proposed to use this specific structure as a criterion for true epithelial character or origin.     

A Two-Dimensional Separation of Proteins from Chloroplast Thylakoids and Other Membranes

• 1 A two-dimensional, highly reproducible, analytical separation procedure for Triton/urea extractable proteins, using isoelectric focusing in the first dimension and gelelectrophoresis with sodium dodecylsulfate (SDS) in the second, is described. The separation of erythrocyte membrane proteins compares the reliability with that of published procedures.
• 2 From the thylakoid membranes of the green alga Chlamydomonas reinhardii and of spinach, two-dimensional maps of the Triton/urea extractable proteins were established, containing 58 and 33 peptide spots, respectively, which were characterized by their molecular weights and apparent isoelectric points. In Chlamydomonas some of the spots could be attributed to the photosystem I and II particles. A few major spots in the maps of the thylakoid proteins from the two non-related plants were found at the same relative positions.
• 3 A one-dimensional electrophoretic comparison of the SDS and the Triton/urea-extractable proteins showed that in the Triton/urea extract of thylakoid membranes some proteins of the photosystem II region, and in that of erythrocyte membranes, some components of the band 3 and 5 region are reduced or missing.