The central region of the synaptonemal complex inBlaps cribrosa studied by electron microscope tomographySchmekel, Karin; Wahrman, Jacob; Skoglund, Ulf; Daneholt, Bertil
doi: 10.1007/BF00650893pmid: 8149808
The synaptonemal complex (SC) in the beetleBlaps cribrosa contains a highly organized central element (CE), two flanking lateral elements (LEs), and a number of regularly spaced transverse filaments (TFs) crossing the central region. The CE is built like a ladder with two longitudinal components running in parallel and a number of regularly spaced transverse CE components, briding the two longitudinal components. The CE is multi-layered with the ladders of the individual layers more or less in register. Essentially every TF originates in one of the LEs, crosses the CE through a transverse CE component and reaches the opposite LE; every transverse CE component in a given layer corresponds to one, and only one, TF. In a CE layer, short irregular pillars form the junctions between the transverse and longitudinal CE components. Adjacent pillars are connected to each other by fine fibrous bridges: the two pillars in the same transverse CE component are linked, and so are the pillars along each longitudinal component, and also more occasionally adjacent pillars in separate CE layers. It is proposed that a TF with the two associated short pillars represents the structural unit in the central region. The ordered structure of the CE is accomplished by linking adjacent pillars to each other into the well-defined three-dimensional organization of the CE.
The three-dimensional structure of the central region in a synaptonemal complex: a comparison between rat and two insect species,Drosophila melanogaster andBlaps cribrosaSchmekel, Karin; Skoglund, Ulf; Daneholt, Bertil
doi: 10.1007/BF00650894pmid: 8149809
The highly ordered central region of the synaptonemal complex (SC) inBlaps cribrosa has recently been studied by electron microscope tomography (EMT), and a simple three-dimensional model presented. Using the same experimental approach we have now compared the central region inBlaps with the central regions inDrosophila melanogaster and rat. In all three species, the SCs exhibit a central element (CE) flanked by two lateral elements (LEs). The central region between the two LEs is crossed by transverse filaments (TFs). TheBlaps CE element is the most ordered one with a well-defined ladder-like structure with two longitudinal components bridged by a number of regularly spaced transverse components, the rungs of the ladder. At the junctions between the longitudinal and transverse components there are prominent dense structures. The CE is multi-layered with the ladders of the separate layers in approximate register. InDrosophila the transverse CE components are as distinct and well organized as inBlaps, while in rat they are present but are less frequent and less well ordered. The longitudinal CE components inDrosophila are often fragmented and even more so in rat. The tomographic analysis revealed that in all three species the central region contains the same structural units: a single TF associated with two short pillars (or globules), which correspond to the junction structures. A fibrous lattice connects the two pillars/globules on the same TF forming the transverse CE component and those on adjacent TFs forming the longitudinal CE component; fibers between pillars/globules also link consecutive CE layers together. In the longitudinal component the number of fibrous bridges between the pillars/globules is related to the conspicuousness of the longitudinal component, i.e.Blaps has most,Drosophila almost as many, and rat considerably fewer bridges. We conclude that the central region in rat,Drosophila andBlaps contains the same basic structural unit but the degree of order and concentration of the units differ: a higher density seems to be accompanied by a higher order within the CE.
A new type of ribonucleoprotein constituent of the polytene nucleus of the salivary glands ofChironomus thummi andCh. tentansVázquez-Nin, G.; Echeverría, O.; Rouelle-Rossier, V.; Fakan, S.
doi: 10.1007/BF00650895pmid: 8149810
Using electron spectroscopic imaging, a new type of small granular structural constituent has been observed in the extrachromosomal zone of the polytene nucleus of the salivary gland cells ofChironomus thummi andChironomus tentans. These granules appear isolated or in small clumps and are often seen to be connected with surrounding thin fibrils. They are stained by the EDTA procedure, which is preferential for nuclear ribonucleoprotein (RNP) constituents, and by the bismuth oxynitrate method for visualizing phosphorylated compounds. The granules are 15–23 nm in diameter and are digested by prolonged post-embedding RNAse hydrolysis. These structural elements contain the highest concentration of phosphorus in the interchromosomal space as revealed by electron energy loss spectroscopy. The small granules exhibit several morphological and cytochemical features in common with interchromatin granules, but they are not labeled with antibodies directed against extranucleolar small nuclear RNPs (snRNPs), as are interchromatin granules.
Inaccessibility of theEuplotes telomere binding proteinOlins, Ada; Cacheiro, Lucia; Herrmann, Adria; Dhar, Madhu; Olins, Donald
doi: 10.1007/BF00650896pmid: 7512014
The telomere binding protein (TP) from the macronucleus of the ciliateEuplotes eurystomus was purified by removal of tenaciously bound DNA with hydroxylapatite, and the purified TP partially sequences. Rabbit antiserum was generated against a synthetic peptide of 14 amino acids at the amino-terminus of the TP. This antiserum was employed to examine the accessibility of TP antigenic determinants in nuclei and chromatin. Immunofluorescent staining of isolated macronuclei revealed only weak reactivity with specific antiserum. Reactivity within replication bands was demonstrated, and could be augumented by preparation of nuclear scaffolds. Employing a dot immunoblot analysis, the amino-terminal antigenic determinants of TP were revealed after extraction of histone H1 (and some nonhistones). A different aspect of TP inaccessibility was demonstrated by immunoblot analysis of trypsin-treated macronuclei and chromatin; TP was considerably less susceptible to digestion by trypsin than were histones H1 and H3. The relative inaccessibility of TP was not a consequence of chromatin higher-order structure, since soluble macronuclear chromatin in low salt exhibited the same burying of antigenic determinants by dot blot analysis, and the same decreased susceptibility to trypsin, as did isolated nuclei. Electron microscopy of soluble macronuclear chromatin spread in low salt revealed that most telomeres appear unfolded, without stable higher-order structure. The mechanisms for the relative inaccessibility of TP are not yet known, but probably arise as a consequence of the strong interactions of TP with the telomere nucleotide sequence and additional interactions of TP with various chromatin proteins, perhaps including histone H1.
In human chromosomes telomeric regions are enriched in CpGs relative to R-bandsFerraro, Marina; Predazzi, Valentina; Prantera, Giorgio
doi: 10.1007/BF00650897pmid: 8149811
Human chromosomes were in situ nick-translated using as nicking agents the endonucleases MspI (CCGG), its methyl-sensitive isoschizomer HpaII, HaeIII (GGCC), SacII (CCGCGG), EcoRI (GAATTC) and DNaseI. We show that in metaphase chromosomes R-bands are enriched, as compared with G-bands, in the dinucleotide CpG but no more than what is expected on the basis of their relative G+C content. The telomeric regions, on the contrary, besides having a chromatin conformation that is particularly relaxed and accessible to endonucleases, also show an enrichment in CpGs.
Analysis ofDrosophila chromosome4 using pulsed field gel electrophoresisLocke, John; McDermid, Heather
doi: 10.1007/BF00650898pmid: 8149812
Previous estimates of the size ofDrosophila melanogaster chromosome4 have indicated that it is 1% to 4% of the genome or ∼6 Mb. We have used pulsed field gel electrophoresis (PFGE) to separate megabase-sized molecules ofD. melanogaster chromosomal DNA. Southern blots of these gels were probed with DNA fragments from thecubitus interruptus andzfh-2 genes, which are located on chromosome4. They each identify the same-sized distinct band that migrates at approximately 5.2 Mb in DNA preparations from the Kc cell line. We interpret this band to be intact chromosome4. In DNA obtained from embryos of variousD. melanogaster wild-type strains, this chromosome band showed strain-specific size variation that ranged from 4.5 to 5.2 Mb. TheD. melanogaster chromosome4 probes also identified a single, 2.4 Mb band in embryonic DNA fromDrosophila simulans. We conclude thatD. simulans chromosome4 is substantially smaller than that ofD. melanogaster, presumably owing to diffirences in the amount of heterochromatic DNA sequences. Our simple DNA preparation from embryos and PFGE conditions should permit preparative isolation of chromosome4 DNA and will facilitate the molecular mapping of this chromosome.
The spatial arrangement ofAllium triquetrum chain quadrivalents at metaphase I as viewed by confocal microscopyOud, J.; Rickards, G.
doi: 10.1007/BF00650900pmid: 8149814
We examined the three-dimensional arrangement of bivalents and, in particular, a chain of four chromosomes (chain quadrivalent) in the metaphase I spindle of pollen mother cells ofAllium triquetrum by confocal microscopy. Firstly, we show by optical sectioning and three-dimensional image reconstruction that the cooriented pairs of centromeres of all seven bivalents lie virtually parallel to each other in the metaphase I spindle, parallel to the long axis of the spindle. Secondly, we like-wise show that the four centromeres of the chain quadrivalent are aligned in the metaphase I spindle in, essentially, atwo-dimensional array, not in a three-dimensional array, as proposed by some other authors. This two-dimensionality has its basis, we argue, in the principle that poleward directed spindle forces minimise centromere-to-pole distances and therefore align pairs of centromeres connected to opposite poles most axially (vertically) in the spindle. These distances are minimised for the quadrivalent as a whole only when it lies in two dimensions, i.e. in aplane parallel to the spindle axis.