Terminal Knobs Research Articles

A universal chromosome identification system for maize and wild Zea species.

Maize was one of the first eukaryotic species in which individual chromosomes can be identified cytologically, which made maize one of the oldest models for genetics and cytogenetics research. Nevertheless, consistent identification of all 10 chromosomes from different maize lines as well as from wild Zea species remains a challenge. We developed a new technique for maize chromosome identification based on fluorescence in situ hybridization (FISH). We developed two oligonucleotide-based probes that hybridize to 24 chromosomal regions. Individual maize chromosomes show distinct FISH signal patterns, which allow universal identification of all chromosomes from different Zea species. We developed karyotypes from three Zea mays subspecies and two additional wild Zea species based on individually identified chromosomes. A paracentric inversion was discovered on the long arm of chromosome 4 in Z. nicaraguensis and Z. luxurians based on modifications of the FISH signal patterns. Chromosomes from these two species also showed distinct distribution patterns of terminal knobs compared with other Zea species. These results support that Z. nicaraguensis and Z. luxurians are closely related species.

Genomic and epigenomic immunity in common bean: the unusual features of NB-LRR gene family.

In plants, a key class of genes comprising most of disease resistance (R) genes encodes Nucleotide-binding leucine-rich repeat (NL) proteins. Access to common bean (Phaseolus vulgaris) genome sequence provides unparalleled insight into the organization and evolution of this large gene family (∼400 NL) in this important crop. As observed in other plant species, most common bean NL are organized in cluster of genes. However, a particularity of common bean is that these clusters are often located in subtelomeric regions close to terminal knobs containing the satellite DNA khipu. Phylogenetically related NL are spread between different chromosome ends, suggesting frequent exchanges between non-homologous chromosomes. NL peculiar location, in proximity to heterochromatic regions, led us to study their DNA methylation status using a whole-genome cytosine methylation map. In common bean, NL genes displayed an unusual body methylation pattern since half of them are methylated in the three contexts, reminiscent of the DNA methylation pattern of repeated sequences. Moreover, 90 NL were also abundantly targeted by 24 nt siRNA, with 90% corresponding to methylated NL genes. This suggests the existence of a transcriptional gene silencing mechanism of NL through the RdDM (RNA-directed DNA methylation) pathway in common bean that has not been described in other plant species.

Myb-binding Protein 1a (Mybbp1a) Regulates Levels and Processing of Pre-ribosomal RNA

Ribosomal RNA gene transcription, co-transcriptional processing, and ribosome biogenesis are highly coordinated processes that are tightly regulated during cell growth. In this study we discovered that Mybbp1a is associated with both the RNA polymerase I complex and the ribosome biogenesis machinery. Using a reporter assay that uncouples transcription and RNA processing, we show that Mybbp1a represses rRNA gene transcription. In addition, overexpression of the protein reduces RNA polymerase I loading on endogenous rRNA genes as revealed by chromatin immunoprecipitation experiments. Accordingly, depletion of Mybbp1a results in an accumulation of the rRNA precursor in vivo but surprisingly also causes growth arrest of the cells. This effect can be explained by the observation that the modulation of Mybbp1a protein levels results in defects in pre-rRNA processing within the cell. Therefore, the protein may play a dual role in the rRNA metabolism, potentially linking and coordinating ribosomal DNA transcription and pre-rRNA processing to allow for the efficient synthesis of ribosomes.

Trichurispora wellgundis n. g., n. sp. (Apicomplexa: Eugregarinida: Hirmocystidae) Parasitizing Adult Water Scavenger Beetles, Tropisternus collaris (Coleoptera: Hydrophilidae) in the Texas Big Thicket

Trichurispora wellgundis n. g., n. sp. (Apicomplexa: Eugregarinida: Hirmocystidae) is described from the adults of the water scavenger beetle Tropisternus collaris (Coleoptera: Hydrophilidae) collected from B A Steinhagen Lake in the Cherokee Unit of the Big Thicket National Preserve, Tyler County, Texas, U.S.A. Trichurispora is distinguished from known genera of Hirmocystidae by a distinct ''trichurisiform'' oocyst that is hesperidiform in outline, comprising a fusiform oocyst with shallowly ovoid terminal knobs or caps. Oocyst residua are present but confined to a central fusiform residuum vacuole. Adult and larval hydrophilid beetles represent distinctly different opportunities for parasite colonization and diversification. Gregarines have been reported from both adult and larval hydrophilid beetles, but no species and no genus is reported from both adult and larval hosts. In fact, gregarine taxic richness is often more disparate between adult and larval beetles of the same species than between host beetle species. This is the first report of a septate gregarine from an adult hydrophilid beetle in the Nearctic.

Cytoskeletal Elements in Bacteria Mycoplasma pneumoniae, Thermoanaerobacterium sp., and Escherichia coli as Revealed by Electron Microscopy

Recently, electron microscopic studies on the eubacteria Mycoplasma pneumoniae, Thermoanaerobacterium sp., and Escherichia coli have revealed the existence of cytoskeletal elements so far unknown in prokaryotes. The wall-less bacterium M. pneumoniae contains, in close vicinity to the inner face of the cytoplasmic membrane, a helically organized lining composed of protein elements that form a regular network of meshes that encloses the entire cytoplasm. Numerous regularly spaced pin-like structural elements, the stalks with terminal knobs, connect the lining with the cytoplasmic membrane. In this bacterium, a specific rod-like structural element is located in the tip region. Occasionally, it is bent or twisted. It consists of two matching blade-like sub-elements. A number of parallel linkers, extending from the edges of the rod, make contact with the lining. The proximal end of the rod is attached to a wheel-like complex. Fibrils originating from the wheel cross the cytoplasm and make contact with the lining. E. coli contains a similar helically organized lining close to the inner face of the cytoplasmic membrane. Groups of ribosomes (polysomes) were seen to be attached to the helical elements of the lining. A feature that is common to both bacteria and to Thermoanaerobacterium sp. appears to be that the lining and the fibrils crossing the cytoplasm contain a high number of copies of the bacterial elongation factor Tu (EF-Tu). This indicates that this protein may play an important role as a structural element in bacterial cytoskeletons. This notion was supported by experiments in which the cytoskeleton in E. coli was destabilized by induced expression of truncated EF-Tu, with the consequence of cell lysis, and by the finding that in vitro polymerization of monomeric EF-Tu into protofilaments was hindered in a mixture of full-size EF-Tu and truncated EF-Tu consisting of domain 3 only. Current research and developmental efforts are aimed at the design of a new class of antibacterial drugs, acting by destabilization of the EF-Tu-containing bacterial cytoskeleton, and of an innovative mode of inducible lysis of recombinant bacteria by controlled destabilization of the EF-Tu-containing cytoskeleton.

The small-subunit processome is a ribosome assembly intermediate.

The small-subunit (SSU) processome is a large ribonucleoprotein required for the biogenesis of the 18S rRNA and likely corresponds to the terminal knobs visualized by electron microscopy on the 5' end of nascent rRNAs. The original purification of the SSU processome of Saccharomyces cerevisiae resulted in the identification of 28 proteins. Here, we characterize 12 additional protein components, including five small-ribosomal-subunit proteins (Rps4, Rps6, Rps7, Rps9, and Rps14) that had previously been copurified. Our multiple criteria for including a component as a bona fide SSU processome component included coimmunoprecipitation with Mpp10 (an SSU processome component), the U3 snoRNA, and the anticipated pre-rRNAs. Importantly, the association of specific ribosomal proteins with the SSU processome suggests that the SSU processome has roles in both pre-rRNA processing and ribosome assembly. These ribosomal proteins may be analogous to the primary or secondary RNA binding proteins first described in bacterial in vitro ribosome assembly maps. In addition to the ribosomal proteins and based on the same experimental approach, we found seven other proteins (Utp18, Noc4, Utp20, Utp21, Utp22, Emg1, and Krr1) to be bona fide SSU processome proteins.

Efecto de la infección por el virus de la rabia sobre la expresión de parvoalbúmina, calbindina y calretinina en la corteza cerebral de ratones.

Some clinical features of rabies and experimental evidence from cell culture and laboratory animals suggest impairment of gabaergic neurotransmission. Several types of gabaergic neurons occur in the cerebral cortex. They can be identified by three neuronal markers: the calcium binding proteins (CaBPs) parvalbumin (PV), calbindin (CB) and calretinin (CR). Rabies virus spreads throughout the cerebral cortex; however, rabies cytopathic effects on gabaergic neurons are unknown. The expression of calcium-binding proteins (CaBPs) parvalbumin (PV), calbindin (CB) and calretinin (CR) was studied in the frontal cortex of mice. The effect of gabaergic neurons was evaluated immunohistochemically. The distribution patterns of CaBPs in normal mice and in mice infected with 'fixed' or 'street' rabies virus were compared. PV was found in multipolar neurons located in all cortical layers except layer I, and in pericellular clusters of terminal knobs surrounding the soma of pyramidal neurons. CB-immunoreactivity was distributed in two cortical bands. One was composed of round neurons enclosed by a heavily labeled neuropil; this band corresponds to supragranular layers II and III. The other was a weakly stained band of neuropil which contained scattered multipolar CB-ir neurons; this corresponds to infragranular layers V and VI. The CR-ir neurons were bipolar fusiform cells located in all layers of cortex, but concentrated in layers II and III. A feature common to samples infected with both types of viruses was a more intense immunoreactivity to PV in contrast to normal samples. The infection with 'street' virus did not cause additional changes in the expression of CaBPs. However, the infection with 'fixed' virus produced a remarkable reduction of CB-immunoreactivity demonstrated by the loss of CB-ir neurons and low neuropil stain in the frontal cortex. In addition, the size of CR-ir neurons in the cingulate cortex was decreased.

Structure of slowly adapting pulmonary stretch receptors in the lung periphery.

Pulmonary sensory receptors are the initiating sites for lung reflexes; however, little is known about their structure, especially the relationship between the structure and function of these receptors. Using a novel approach (combining electrophysiological and morphological techniques), we examined the structures of the typical slowly adapting pulmonary stretch receptors (SARs) located in the lung periphery. We recorded SAR activities in the cervical vagus nerve, identified the receptive field, dissected the SARs in blocks, fixed and processed these blocks for immunohistochemical staining using anti-Na+/K+-ATPase, and examined the blocks under a confocal microscope. These SAR structures have multiple endings that have terminal knobs. Some structures that are located in the airway walls have terminal knobs buried in smooth muscle. Others are in the most peripheral part of the lung, and their terminal knobs have no obvious relation to smooth muscle, suggesting that muscle contraction may not be a direct factor for SAR activation.

A large nucleolar U3 ribonucleoprotein required for 18S ribosomal RNA biogenesis.

Although the U3 small nucleolar RNA (snoRNA), a member of the box C/D class of snoRNAs, was identified with the spliceosomal small nuclear RNAs (snRNAs) over 30 years ago, its function and its associated protein components have remained more elusive. The U3 snoRNA is ubiquitous in eukaryotes and is required for nucleolar processing of pre-18S ribosomal RNA in all organisms where it has been tested. Biochemical and genetic analyses suggest that U3 pre-rRNA base-pairing interactions mediate endonucleolytic pre-rRNA cleavages. Here we have purified a large ribonucleoprotein (RNP) complex from Saccharomyces cerevisiae that contains the U3 snoRNA and 28 proteins. Seventeen new proteins (Utp1 17) and Rrp5 were present, as were ten known components. The Utp proteins are nucleolar and specifically associated with the U3 snoRNA. Depletion of the Utp proteins impedes production of the 18S rRNA, indicating that they are part of the active pre-rRNA processing complex. On the basis of its large size (80S; calculated relative molecular mass of at least 2,200,000) and function, this complex may correspond to the terminal knobs present at the 5' ends of nascent pre-rRNAs. We have termed this large RNP the small subunit (SSU) processome.

Horizontal cell connections with short-wavelength-sensitive cones in macaque monkey retina.

This study describes the connectivity between horizontal cells and short-wavelength-sensitive (SWS) cones in macaque monkey retina. H1 and H2 horizontal cells were either labelled with the carbocyanine dye, DiI, or injected intracellular with Neurobiotin. The retinas were then processed with an antiserum against human SWS cone pigment, which usually stained the entire SWS cone. In these double-labelled retinas, the pattern of connectivity of H1 (n = 91) and H2 (n = 7) cells with SWS cones has been determined. About 85% of the H1 cells examined do not contact SWS cones. The dendritic terminal knobs of five H1 cells that do contact SWS cones were counted. They have, at most, 3% of their dendritic terminal knobs at SWS cones. All H2 cells examined make contact with SWS cones. The dendritic terminal knobs of one H2 cell were counted; about 11% of the dendritic terminal knobs are at the SWS cone. We conclude that horizontal cells in macaque monkey retina show specific patterns of connectivity to SWS cones.

High voltage electron microscopical analysis of synaptic glomeruli in the rat substantia gelatinosa.

Three-dimensional organization of synaptic glomeruli in the rat substantia gelatinosa was investigated using high voltage electron microscopy. In the present study, thiamine monophosphatase activity was used as a marker enzyme for synaptic glomeruli because it has been selectively localized in synaptic glomeruli in the spinal cord. The synaptic region of dense sinusoid axon terminals forming the central part of synaptic glomeruli displayed a rounded appearance. The thiamine monophosphatase activity was irregularly presented on the surface of terminal knobs and preterminals, and weak reaction products were observed in the interior of cross-cut terminal knobs.This study suggested that the primary afferent fiber formed the complicated synapses which are pre- and postsynaptically surrounded by a large number of processes of intradorsal neurons.

Cortico-cortical and subcortico-cortical afferent connection of the rabbit's primary visual cortex. A horseradish peroxidase study.

Neuroanatomical studies were carried out on the visual system of the adult rabbit brain. Either horseradish peroxidase (HRP) or wheat germ agglutinine-horseradish peroxidase (WGA-HRP) was injected into the area occipitalis 1. Several cortico-cortical, ipsi- and contralateral, and subcortico-cortical projections were demonstrated. In the ipsilateral telencephalon several patches of labelled cell groups, some single HRP-positive cell bodies and some labelled fibres were observed in the area retrosplenialis granularis dorsalis, the areas occipitales, the areas temporales, the area perirhinalis, the area entorhinalis, the area praecentralis 1, the regio cingularis 1 and in the regio diagonalis, as well as in the dorsal part of the claustrum. Efferent preterminal fibres and terminal knobs were seen in the nucleus caudatus. Contralaterally, groups of labelled cell bodies and single HRP-positive neurons were found in the area retrosplenialis granularis dorsalis, the areas occipitales and the areas temporales. In the ipsilateral diencephalon, labelled cell bodies were observed in the corpus geniculatum laterale (pars dorsalis and ventralis), the nucleus lateralis thalami, the nucleus reticularis thalami and in nonspecific nuclei of the midline. Contralaterally, very few labelled cell bodies were seen in the nonspecific nuclei of the midline. Some labelled cell bodies were observed in the ipsilateral substantia griseum centrale and in the nucleus reticularis mesencephali. Numerous anterogradely labelled preterminal fibres and terminal knobs but very few labelled cell bodies were seen in the nucleus praetectalis posterior. In the nucleus of the optic tract and in the colliculus superior, numerous labelled fibres could be observed. In the ipsilateral nuclei pontis numerous labelled fibres were detectable.

Peptidergic nociceptive axon visualization in whole-mount preparations of cornea and tympanic membrane in rat

A whole-mount preparation is described for visualizing unmyelinated peptide immunoreactive axons in two specialized tissues, thin enough for tracing axons to their terminal knobs. These tissues, the cornea and the mucosal surface of the tympanic membrane, area known to possess specialized nociceptor innervation and can be used for selecting sensory endings labeled by peroxidase reaction product and embedded in plastic for more detailed analysis. Rat calcitonin gene-related peptide antibody proved a particularly robust primary antibody found in a substantial proportion of sensory unmyelinated axons in a variety of fixation and preparative conditions.

Electron microscopy of map 2 (microtubule-associated protein 2)

In this study we demonstrate by the platinum-replication technique that MAP 2 (microtubuleassociated protein 2) is a long, thin, flexible structure. The contour length of the molecule is variable ranging up to a maximum of about 185 nm. The mass per unit length is about 1630 daltons/ nm, approximately the same as a double helix. Circular dichroism of purified MAP 2 showed, however, that the alpha helix content is actually very low. The MAP 2 strands sometimes have either one or two terminal knobs. There was very little difference in the appearance of the MAP 2 prepared either with or without the inclusion of a 100°C treatment. We also present images of taxol-stabilized microtubules decorated with MAP 2 molecules showing that the latter often extend outward from the microtubule wall as thin filaments up to 80 or 90 nm in length.

Zea diploperennis

The cytogenetic analysis of Zea diploperennis, diploid perennial teosinte (n = 10), Is presented and compared cytologically to that of the tetraploid perennial teosinte. Pachytene chromosome morphology and meiosis of the F1 hybrid of diploperennis and the WMT maize stock show a paracentric inversion in chromosome 5 and a terminal inversion in chromosome 9. Both the diploid and tetraploid perennials have the same small terminal knobs (or prominent telomeres) on otherwise knobless chromosomes and both have the chromosome 9 inversion.

Adsorption complex of filamentous fd virus.

Abstract We have directly visualized what appears to be the adsorption apparatus of a filamentous virus. Electron microscopy reveals at least three knobs, located specifically at one end of the virion, which is more tapered than is the other end. The knobs are attached to the virion tip by connecting stems that are too thin to be seen in most preparations. The protease subtilisin digests away the adsorption protein from the virus, as shown by electrophoretic analysis, and it removes the knobs seen by electron microscopy. Resistance of virion DNAs to endonucleases, even after removal of the terminal knobs with subtilisin, indicates that the thin stems do not consist of DNA. The size of the knobs indicates that each is principally a monomer of the viral adsorption protein; each stem is presumed to be a connecting protein bridge.

Congenital fibre type disproportion: A morphometric study (4 cases)

Four children with congenital fibre type disproportion were described. It was shown that their type I fibres were at least 12% smaller than the type II fibres. There was no increase in the terminal innervation ratio (TIR), but a decreased number of terminal knobs was observed in the biopsy of one child. The distribution of fibre types in the biopsy of another child bears out the notion that the abnormalities as seen in the biopsy can be traced back to the spine.

Fluorescence microscopy of the avian (<i>Gallus domesticus</i>) carotid body

Sterba's technique to detect neurosecretory substances was applied to carotid body tissue, surrounding branchial derivatives, ganglion nodosum and the carotid arterial wall. Mainly a yellowish-green, a yellowish-red and a bright yellow fluorescence was observed as well as greenish-yellow fibres with varicosities and terminal knobs. The metachromatic reaction of the technique was briefly discussed.

Non-nucleolar transcription complexes of rat liver as revealed by spreading isolated nuclei.

Miller's technique was applied to isolated nuclei of rat liver. Both the usual nucleolar and non-nucleolar transcription complexes were visualized. In addition, an unusual type of putative non-ribosomal transcription unit was revealed. It was charcaterized by a high density of the lateral ribonucleoprotein (RNP) fibrils. Although these particular units exhibited a regular increase of fibril lengths, the length of the transcript-covered deoxyribonucleoprotein (DNP) fibres and the morphological aspect of the RNP fibrils distinguished them from the nucleolar 'Christmas-tree'-like figures. The linear and granular configuration of the transcripts and the absence of terminal knobs made them similar to non-nucleolar nascent RNP fibrils.

Nematophagous fungi: Hohenbuehelia, the perfect state of Nematoctonus

A predatory Nematoctonus species (N90) isolated from farmyard soil in Ontario produced gill-forming basidiocarps of Hohenbuehelia on potato dextrose agar. In the presence of nematodes, discharged basidiospores germinated to produce short aerial stalks with terminal adhesive knobs. Germinated basidiospores adhered to the cuticle of nematodes to initiate monokaryotic infection cycles. Multiple basidiospore infections on a single host resulted in dikaryotization in the host body. Crossing single basidiospore cultures demonstrated bipolar sexuality for this species of Hohenbuehelia. The implications of these findings are discussed.