The mitotic centromeric protein MEI-S332 and its role in sister-chromatid cohesion

Faithful segregation of sister chromatids during cell division requires properly regulated cohesion between the sister centromeres. The sister chromatids are attached along their lengths, but particularly tightly in the centromeric regions. Therefore specific cohesion proteins may be needed at the centromere. Here we show that Drosophila MEI-S332 protein localizes to mitotic metaphase centromeres. Both overexpression and mutation of MEI-S332 increase the number of apoptotic cells. In mei-S332 mutants the ratio of metaphase to anaphase figures is lower than wild type, but it is higher if MEI-S332 is overexpressed. In chromosomal squashes centromeric attachments appear weaker in mei-S332 mutants than wild type and tighter when MEI-S332 is overexpressed. These results are consistent with MEI-S332 contributing to centromeric sister-chromatid cohesion in a dose-dependent manner. MEI-S332 is the first member identified of a predicted class of centromeric proteins that maintain centromeric cohesion. Received: 11 December 1998; in revised form: 4 August 1999 / Accepted: 13 August 1999     

Mapping and genomic characterization of the gene encoding diacylglycerol kinase γ (DAGK3): assessment of its role in dominant optic atrophy (OPA1)

The family of diacylglycerol kinases (DAGKs) is known to play an important role in signal transduction linked to phospholipid turnover. In the fruitfly Drosophila melanogaster, a human DAGK ortholog, DGK2, was shown to underlie the phenotype of the visual mutant retinal degeneration A (rdgA). Previously, the gene encoding a novel member of the human DAGK family, termed DAGK3, was cloned and demonstrated to be abundantly expressed in the human retina. Based on these findings we reasoned that DAGK3 might be an excellent candidate gene for a human eye disease. In the present study, we report the genomic organization of the human DAGK3 gene, which spans over 30 kb of genomic DNA interrupted by 23 introns. In addition, we have mapped the gene locus by fluorescence in situ hybridization to 3q27–28, overlapping the chromosomal region known to contain the gene underlying dominant optic atrophy (OPA1), the most common form of hereditary atrophy of the optic nerve. Mutational analysis of the entire coding region of DAGK3 in 19 unrelated German OPA1 patients has not revealed any disease-causing mutations, therefore excluding DAGK3 as a major cause underlying OPA1. Received: 24 August 1998 / Accepted: 13 October 1998     

Incorporating fluorescent dyes and quantum dots into magnetic microbeads for immunoassays

Microbeads that are both paramagnetic and fluorescently labeled are commercially available in colors spanning the visible spectrum. Although these commercial beads can be bright, polydispersity in both size and fluorescent intensity limit their use in quantitative assays. Very recently, more monodisperse beads have become available, but their large size and surface properties make them less than ideal for some bioassay applications. Here we describe methods to customize commercial nonfluorescent magnetic microparticles with fluorescent dyes and quantum dots (QDs) without affecting their magnetic or surface chemical properties. Fluorescent dyes and 3.3-nm diameter CdSe/ZnS QDs were sequestered within 0.8-micron diameter magnetic beads by swelling the polystyrene matrix of the bead in organic solvent, letting the chromophores partition, and then collapsing the matrix in polar solvents. Chromophore incorporation has been characterized using both UV-visible absorption spectroscopy and fluorescence microscopy, with an average of 3 x 10(8) rhodamine 6G molecules/bead and 6 x 10(4) QDs/bead. The modified beads are uniform in size and intensity, with optical properties comparable to currently available commercial beads. Immunoassay results obtained with our custom fluorescent magnetic microbeads are consistent with those obtained using conventional magnetic microbeads.     

A prevalent POLG CAG microsatellite length allele in humans and African great apes

The human nuclear gene for the catalytic subunit of mitochondrial DNA polymerase (POLG) contains within its coding region a CAG microsatellite encoding a polyglutamine repeat. Previous studies demonstrated an association between length variation at this repeat and male infertility, suggesting a mechanism whereby the prevalent (CAG)₁₀ allele, which occurs at a frequency of >80% in different populations, could be maintained by selection. Sequence analysis of the POLG CAG microsatellite region of more than 1000 human chromosomes reveals that virtually all allelic variation at the locus is accounted for by length variation of the CAG repeat. Analysis of POLG from African great apes shows that a prevalent length allele is present in each species, although its exact length is species-specific. In common chimpanzee (Pan troglodytes) a number of different sequence variants contribute to the prevalent length allele, strongly supporting the idea that the length of the POLG microsatellite region, rather than its exact nucleotide or amino acid sequence, is what is maintained. Analysis of POLG in other primates indicates that the repeat has expanded from a shorter, glutamine-rich sequence, present in the common ancestor of Old and New World monkeys.     

Fibroblasts form a body-wide cellular network

Loose connective tissue forms a network extending throughout the body including subcutaneous and interstitial connective tissues. The existence of a cellular network of fibroblasts within loose connective tissue may have considerable significance as it may support yet unknown body-wide cellular signaling systems. We used a combination of histochemistry, immunohistochemistry, confocal scanning laser microscopy (confocal microscopy), and electron microscopy to investigate the extent and nature of cell-to-cell connections within mouse subcutaneous connective tissue. We found that fibroblasts formed a reticular web throughout the tissue. With confocal microscopy, 30% of fibroblasts processes could be followed continuously from one cell to another. Connexin 43 immunoreactivity was present at apparent points of cell-to-cell contact. Electron microscopy revealed that processes from adjacent cells were in close apposition to one another, but gap junctions were not observed. Our findings indicate that soft tissue fibroblasts form an extensively interconnected cellular network, suggesting they may have important and so far unsuspected integrative functions at the level of the whole body.     

Experimental infection of house finches with Mycoplasma gallisepticum

Mycoplasma gallisepticum (MG) has caused an endemic upper respiratory and ocular infection in the eastern house finch (Carpodacus mexicanus) after the epidemic first described in 1994. The disease has been studied by a number of investigators at a population level and reports describe experimental infection in group-housed MG-free house finches. Because detailed observation and evaluation of individual birds in group-housed passerines is problematic, we studied individually housed house finches that were experimentally inoculated with the finch strain of MG in a controlled environment. To accomplish this, a study was conducted spanning the period of November 2001-April 2002 with 20 MG-free (confirmed by the rapid plate agglutination assay and polymerase chain reaction [PCR] assay) eastern house finches captured in the Cayuga Basin area of central New York (USA) in the summer of 2001. After a period of acclimatization and observation (12 wk), 20 finches were inoculated with a 0.05-ml aliquot of MG (3.24 x 10(5) colony-forming units/ml) via bilateral conjunctival sac instillations. Two additional finches acted as controls and were inoculated in the same manner with preservative-free sterile saline solution. After inoculation, all finches except the controls exhibited clinical signs of conjunctivitis within 2-6 days. The progression of the disease was evaluated by several methods, including PCR, behavioral observations, and physical examination including eye scoring, body weight, and body condition index. Over a period of 21 wk, MG-infected finches developed signs of disease and recovered (80%), developed signs of disease and progressed to become chronically infected (15%), or died (5%). We hypothesize that the high survival rate and recovery of these finches after infection was associated with the use of controlled environmental conditions, acclimatization, a high plane of nutrition, and low stocking (housing) density, all of which are factors documented to be important in the outcome of MG infections in domestic poultry and other species.