Artifact‐free objective‐type multicolor total internal reflection fluorescence microscopy with light‐emitting diode light sources—Part I

Total internal reflection fluorescence excitation (TIRF) microscopy allows the selective observation of fluorescent molecules in immediate proximity to an interface between different refractive indices. Objective‐type or prism‐less TIRF excitation is typically achieved with laser light sources. We here propose a simple, yet optically advantageous light‐emitting diode (LED)‐based implementation of objective‐type TIRF (LED‐TIRF). The proposed LED‐TIRF condenser is affordable and easy to set up at any epifluorescence microscope to perform multicolor TIRF and/or combined TIRF‐epifluorescence imaging with even illumination of the entire field of view. Electrical control of LED light sources replaces mechanical shutters or optical modulators. LED‐TIRF microscopy eliminates safety burdens that are associated with laser sources, offers favorable instrument lifetime and stability without active cooling. The non‐coherent light source and the type of projection eliminate interference fringing and local scattering artifacts that are associated with conventional laser‐TIRF. Unlike azimuthal spinning laser‐TIRF, LED‐TIRF does not require synchronization between beam rotation and the camera and can be monitored with either global or rolling shutter cameras. Typical implementations, such as live cell multicolor imaging in TIRF and epifluorescence of imaging of short‐lived, localized translocation events of a Ca²⁺‐sensitive protein kinase C α fusion protein are demonstrated.     

Phenotypic divergence but not genetic distance predicts assortative mating among species of a cichlid fish radiation

The hypothesis of ecological divergence giving rise to premating isolation in the face of gene flow is controversial. However, this may be an important mechanism to explain the rapid multiplication of species during adaptive radiation following the colonization of a new environment when geographical barriers to gene flow are largely absent but underutilized niche space is abundant. Using cichlid fish, we tested the prediction of ecological speciation that the strength of premating isolation among species is predicted by phenotypic rather than genetic distance. We conducted mate choice experiments between three closely related, sympatric species of a recent radiation in Lake Mweru (Zambia/DRC) that differ in habitat use and phenotype, and a distantly related population from Lake Bangweulu that resembles one of the species in Lake Mweru. We found significant assortative mating among all closely related, sympatric species that differed phenotypically, but none between the distantly related allopatric populations of more similar phenotype. Phenotypic distance between species was a good predictor of the strength of premating isolation, suggesting that assortative mating can evolve rapidly in association with ecological divergence during adaptive radiation. Our data also reveals that distantly related allopatric populations that have not diverged phenotypically, may hybridize when coming into secondary contact, e.g. upon river capture because of diversion of drainage systems.     

A unique case of a discontinuous duplication 3q26.1-3q28 resulting from a segregation error of a maternal complex chromosomal rearrangement involving an insertion and an inversion

Until now, few cases of partial trisomy of 3q due to segregation error of parental balanced translocation and segregation of a duplicated deficient product resulting from parental pericentric inversion have been reported so far. Only five cases of chromosomal insertion malsegregation involving 3q region are available yet, thus making it relatively rare. In this case report, we are presenting a unique case of discontinuous partial trisomy of 3q26.1-q28 region which resulted from a segregation error of two insertions involving 3q26.1 to 3q27.3 and 3q28 regions with ~ 21 Mb and ~ 2 Mb sizes, respectively. The maternally inherited insertion was cytogenetically characterized as der(8)(8pter → 8p22::3q26 → 3q27.3::3q28 → 3q28::8p22 → 8qter) and the patient's major clinical features involved Dandy Walker malformation, sub-aortic ventricular septal defect, upslanting palpebral fissures, clinodactyly, hirsutism, and prominent forehead. Besides, a review of the literature involving cases with similar chromosomal imbalances and cases with “3q-duplication syndrome” is also provided.     

Muscle Stem Cells Exhibit Distinct Clonal Dynamics in Response to Tissue Repair and Homeostatic Aging

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Visualization of the terminal structure of rice chromosomes 6 and 12 with multicolor FISH to chromosomes and extended DNA fibers

High-resolution fluorescence in situ hybridization (FISH) on interphase and pachytene nuclei, and extended DNA fibers enabled microscopic distinction of DNA sequences less than a few thousands of base pairs apart. We applied this technique to reveal the molecular organization of telomere ends in japonica rice (Oryza sativa ssp. japonica), which consist of the Arabidopsis type TTTAGGG heptameric repeats and the rice specific subtelomeric tandem repeat sequence A (TrsA). Southern hybridizations of DNA digested with Bal31 and EcoRI, and FISH on chromosomes and extended DNA fibers demonstrated that (1) all chromosome ends possess the telomere tandem repeat measuring 3–4 kb; (2) the subtelomeric TrsA occurs only at the ends of the long arms of chromosomes 6 and 12, and measure 6 and 10 kb, which corresponds to 231 and 682 copies for these sites, respectively; (3) the telomere and TrsA repeats are separated by at most a few thousands of intervening nucleotide sequences. The molecular organization for a general telomere organization in plant chromosomes is discussed.     

Three unusual but cytogenetically similar cases with up to five different cell lines involving structural and numerical abnormalities of chromosome 18.

We report two prenatal and two postnatal diagnosed cases (the latter monozygotic twins) with ring chromosomes after GTG banding. All four, de novo r(18), cases turned out to be more complex after application of high-resolution molecular cytogenetics techniques such as use of fluorescence in situ hybridization, centromeric probes, multicolor banding, and locus-specific probes for chromosome 18. All four cases are mosaics involving chromosome 18 in up to five different cell lines, including 46,r(18); 46,dr(18); 47,r(18)x2; 46,mar(18); and 45,-18. Mosaicism sharing both numerical and structural anomalies is rare, but rings often appear as mosaics due to their mitotic instability. Overall, patients with ring chromosome 18 usually share clinical features of 18q- syndrome and, less frequently, those of 18p- syndrome. High-resolution molecular cytogenetics techniques were useful in the characterization of cases with dynamic mosaicism and in establishing the relationship between loss or gain of chromosomal material and the phenotype.     

Mosaic small supernumerary marker chromosome 1 at amniocentesis: Prenatal diagnosis,molecular genetic analysis and literature review

We present prenatal diagnosis and molecular cytogenetic analysis of mosaic small supernumerary marker chromosome 1 [sSMC(1)]. We review the literature of sSMC(1) at amniocentesis and chromosome 1p21.1-p12 duplication syndrome. We discuss the genotype–phenotype correlation of the involved genes of ALX3, RBM15, NTNG1, SLC25A24, GPSM2, TBX15 and NOTCH2 in this case.     

Toxic Metabolites of an Unidentified Filamentous Fungus Isolated from Zinnia Leaves

Two ribonucleases (RNases) designated RNase I and RNase II were found in Euphausia superba and isolated by (NH₄)₂SO₄ fractionation, 2 cycles of CM-cellulose chromatography and gel filtration on Sephadex G-100. This procedure resulted in a 2,116-fold purification of RNase I and a 130-fold purification of RNase II. The molecular weight of both purified enzymes was estimated by gel filtration to be 31,500. The isoelectric points were 6.0 (RNase I) and 7.0 (RNase II). Each enzyme hydrolyzed poly A-U, poly U but did not degrade poly G, poly C and DNA. Both enzymes were classified as endonuclease from the hydrolysis product of yeast RNA and poly A. The enzymes were located mainly in the cardiac and pyloric portion of the stomach.     

Multicolor bimolecular fluorescence complementation reveals simultaneous formation of alternative CBL/CIPK complexes in planta

The specificity of intracellular signaling and developmental patterning in biological systems relies on selective interactions between different proteins in specific cellular compartments. The identification of such protein-protein interactions is essential for unraveling complex signaling and regulatory networks. Recently, bimolecular fluorescence complementation (BiFC) has emerged as a powerful technique for the efficient detection of protein interactions in their native subcellular localization. Here we report significant technical advances in the methodology of plant BiFC. We describe a series of versatile BiFC vector sets that are fully compatible with previously generated vectors. The new vectors enable the generation of both C-terminal and N-terminal fusion proteins and carry optimized fluorescent protein genes that considerably improve the sensitivity of BiFC. Using these vectors, we describe a multicolor BiFC (mcBiFC) approach for the simultaneous visualization of multiple protein interactions in the same cell. Application to a protein interaction network acting in calcium-mediated signal transduction revealed the concurrent interaction of the protein kinase CIPK24 with the calcium sensors CBL1 and CBL10 at the plasma membrane and tonoplast, respectively. We have also visualized by mcBiFC the simultaneous formation of CBL1/CIPK1 and CBL9/CIPK1 protein complexes at the plasma membrane. Thus, mcBiFC provides a useful new tool for exploring complex regulatory networks in plants.