Sensitive fluorescence in situ hybridization signal detection in maize using directly labeled probes produced by high concentration DNA polymerase nick translation

The signal produced by fluorescence in situ hybridization (FISH) often is inconsistent among cells and sensitivity is low. Small DNA targets on the chromatin are difficult to detect. We report here an improved nick translation procedure for Texas red and Alexa Fluor 488 direct labeling of FISH probes. Brighter probes can be obtained by adding excess DNA polymerase I. Using such probes, a 30 kb yeast transgene, and the rp1, rp3 and zein multigene clusters were clearly detected.     

A tale of two centromeres—diversity of structure but conservation of function in plants and animals

The structural and functional aspects of two specific centromeres, one drawn from the animal kingdom (Drosophila) and the other from the plant kingdom (maize), are compared. Both cases illustrate an epigenetic component to centromere specification. The observations of neocentromeres in Drosophila and inactive centromeres in maize constitute one line of evidence for this hypothesis. Another common feature is the divisibility of centromere function with reduced stability as the size decreases. The systems differ in that Drosophila has no common sequence repeat at all centromeres, whereas maize has a 150-bp unit present in tandem arrays together with a centromere-specific transposon, centromere retrotransposon maize, present at all primary constrictions. Aspects of centromere structure known only from one or the other system might be common to both, namely, the presence of centromere RNAs in the kinetochore as found in maize and the organization of the centromeric histone 3 in tetrameric nucleosomes.     

Analysis of Autosomal Dosage Compensation Involving the Alcohol Dehydrogenase Locus in Drosophila Melanogaster

An example of autosomal dosage compensation involving the expression of the alcohol dehydrogenase (Adh) locus is described. Flies trisomic for a quarter of the length of the left arm of chromosome two, including Adh, have diploid levels of enzyme activity and alcohol dehydrogenase messenger RNA. Subdivision of the compensating trisomic into smaller ones revealed a region that exerts an inverse regulatory effect on alcohol dehydrogenase activity and messenger RNA levels and a smaller region surrounding the structural gene that exhibits a direct gene dosage response. The two opposing effects are of sufficient magnitude that they cancel when simultaneously present resulting in the observed compensation in the larger aneuploid. An Adh promoter-white structural gene fusion construct is affected by the inverse regulatory region indicating that the effect is mediated through the Adh promoter sequences. The role of autosomal dosage compensation in understanding aneuploid syndromes and karyotype evolution in Drosophila species is discussed.     

Interactions of Vestigial and Scabrous with the Notch Locus of Drosophila Melanogaster

Interactions are described between the Notch locus of Drosophila melanogaster, and two other loci, scabrous and vestigial, which respectively affect the eyes and wings. The Notch locus is responsible for mediating decisions of cell fate throughout development in many different tissues. Mutations and duplications of vestigial and scabrous alter the severity of phenotypes associated with Notch mutations and duplications in a manner that is essentially tissue- and allele-specific. These interactions indicate that the products of vestigial and scabrous act in conjunction with Notch to stimulate the differentiation of specific cell types.     

Human alveolar macrophage fibronectin: synthesis, secretion, and ultrastructural localization during gelatin-coated latex particle binding

Human pulmonary alveolar macrophages synthesized and secreted several characteristic high molecular weight proteins for at least 7 d in vitro. Immunoprecipitates of medium and cell lysates from metabolically labeled cultures with specific anti-human plasma fibronectin IgG contained one major labeled polypeptide of molecular weight 440,000 (unreduced) or 220,000 (reduced). An identical polypeptide in conditioned medium from radiolabeled macrophages bound specifically to gelatin-Sepharose, demonstrating that alveolar macrophages synthesized and secreted a molecule immunologically and functionally similar to fibronectin. Fibronectin was the major newly synthesized and secreted polypeptide of freshly harvested alveolar macrophages. Pulse-chase experiments revealed that newly synthesized fibronectin was rapidly secreted into medium, approximately 50 percent appearing by 1 h and 80 percent by 8 h. Immunoperoxidase staining using antifibronectin F(ab’)(2)-peroxidase conjugates revealed the majority of immunoreactive fibronectin to be intracellular, localized to endoplasmic reticulum and Golgi apparatus. No extracellular matrix fibronectin was visualized, and cell surface staining was rarely seen, usually appearing only at sites where cells were closely apposed and not at sites of macrophage-substrate attachment. Similar immunostaining of fibroblast cultures revealed cell surface-associated fibrillar fibronectin. Ultrastructural localization of fibronectin during binding and phagocytosis of gelatin-coated and plain latex particles revealed fibronectin only on gelatin-latex beads and at their cell binding sites. Neigher plain latex beads nor their cell membrane binding sites stained for fibronectin. These results demonstrate that fibronectin is a major product of human alveolar macrophages, is rapidly secreted, and is localized at cell membrane binding sites for gelatin-coated particles. In view of the known binding properties of fibronectin, it may serve as an endogenous opsonic factor promoting the binding of staphylococcus, denatured collagen, fibrin, or other macromolecules to macrophages in the lower respiratory tract.