Preservation of RNA and DNA from mammal samples under field conditions

Ecological and conservation genetics require sampling of organisms in the wild. Appropriate preservation of the collected samples, usually by cryostorage, is key to the quality of the genetic data obtained. Nevertheless, cryopreservation in the field to ensure RNA and DNA stability is not always possible. We compared several nucleic acid preservation solutions appropriate for field sampling and tested them on rat (Rattus rattus) blood, ear and tail tip, liver, brain and muscle. We compared the efficacy of a nucleic acid preservation (NAP) buffer for DNA preservation against 95% ethanol and Longmire buffer, and for RNA preservation against RNAlater (Qiagen) and Longmire buffer, under simulated field conditions. For DNA, the NAP buffer was slightly better than cryopreservation or 95% ethanol, but high molecular weight DNA was preserved in all conditions. The NAP buffer preserved RNA as well as RNAlater. Liver yielded the best RNA and DNA quantity and quality; thus, liver should be the tissue preferentially collected from euthanized animals. We also show that DNA persists in nonpreserved muscle tissue for at least 1 week at ambient temperature, although degradation is noticeable in a matter of hours. When cryopreservation is not possible, the NAP buffer is an economical alternative for RNA preservation at ambient temperature for at least 2 months and DNA preservation for at least 10 months.     

Patterns of wall synthesis inSaccharomyces cerevisiae

Wall formation inSaccharomyces cerevisiae seems to be the result of two main patterns of wall material deposition: (i) around the whole periphery of the cell in nonbudding ones, and (ii) mainly at the tip of the daughter cell or at the cross wall that separates dividing cells. This interpretation has been obtained following experiments in which RNA or protein synthesis has been inhibited. Under these conditions, glucan formation takes place, and wall thickening is probably due to the accumulation of this polysaccharide. Furthermore, once a pattern of wall deposition has been established, it is not modified by inhibition of RNA or protein synthesis.     

Localization of ribosomal cistrons in metaphase chromosomes of Vicia faba (L.)

Tritiated ribosomal RNA (rRNA) was prepared from the roots of Vicia faba after incubation in ³H-uridine. Separation of the nucleic acids by MAK chromatography yielded fractions of specific activity of 4–5 × 10⁵ dpm/μg. 4 + 5S, 18S and 25S RNA fractions were used for cytological hybridization on squash preparations of Vicia faba root tip meristems. Autoradiographs of the 18S and 25S RNA preparations exhibited a clear labelling in the secondary constriction of the satellite (SAT) chromosomes after exposition times of 28 weeks.     

Nuclear genetic activity in B-chromosome rye,in terms of the quantitative interrelationships between nuclear protein,nuclear RNA and histone

The nuclear basis of B-chromosome genetic activity in rye has been investigated using quantitative cytochemical techniques on isolated root tip nuclei. Nuclear DNA amount was found to be directly proportional to B-chromosome number. Relative amounts of total nuclear protein and nuclear RNA however, decreased with increasing numbers of B's but not in a strictly linear fashion. The values were disproportionately low for odd numbered B-classes of plants. Histone protein was found to increase as the number of B's went up, and in this case the values were disproportionately high for odd numbered B-classes. A negative correlation was found between histone and total nuclear protein and histone and nuclear RNA amounts.     

Cytologische Lokalisation von 5S und 18/25S RNA Genorten in Mitose-Chromosomen von Vicia faba

Homologous in situ hybridization with tritiated 4S, 5S and 18/25S RNA from root tip meristems of Vicia faba has been used to study the pattern of distribution of DNA sequences coding for these RNAs in the diploid nuclei. 5S RNA hybridizes to two regions of the satellites of the pair of satellited chromosomes. The sites differ in the level of in situ hybridization implicating different degrees of redundancy. 18/25S RNA hybridization is concentrated to the secondary constriction of these satellite chromosomes. Both, 5S and 18/25S ribosomal RNA gene sites are located on the same pair of chromosomes, but obviously the sequences are not contiguous. An association of 5S RNA cistrons with heterochromatin is assumed. Additional RNA gene sites as well as 4S RNA gene sites are not detectable.     

Microtubules and microfilaments coordinate to direct a fountain streaming pattern in elongating conifer pollen tube tips

This study investigates how microtubules and microfilaments control organelle motility within the tips of conifer pollen tubes. Organelles in the 30-m-long clear zone at the tip of Picea abies (L.) Karst. (Pinaceae) pollen tubes move in a fountain pattern. Within the center of the tube, organelles move into the tip along clearly defined paths, move randomly at the apex, and then move away from the tip beneath the plasma membrane. This pattern coincides with microtubule and microfilament organization and is the opposite of the reverse fountain seen in angiosperm pollen tubes. Application of latrunculin B, which disrupts microfilaments, completely stops growth and reduces organelle motility to Brownian motion. The clear zone at the tip remains intact but fills with thin tubules of endoplasmic reticulum. Applications of amiprophosmethyl, propyzamide or oryzalin, which all disrupt microtubules, stop growth, alter organelle motility within the tip, and alter the organization of actin microfilaments. Amiprophosmethyl inhibits organelle streaming and collapses the clear zone of vesicles at the extreme tip together with the disruption of microfilaments leading into the tip, leaving the plasma membrane intact. Propyzamide and oryzalin cause the accumulation of membrane tubules or vacuoles in the tip that reverse direction and stream in a reverse fountain. The microtubule disruption caused by propyzamide and oryzalin also reorganizes microfilaments from a fibrillar network into pronounced bundles in the tip cytoplasm. We conclude that microtubules control the positioning of organelles into and within the tip and influence the direction of streaming by mediating microfilament organization.Electronic Supplementary Material Supplementary material is available in the online version of this article at Abbreviations APM Amiprophosmethyl - FITC Fluorescein isothiocyanate - LATB Latrunculin B     

Cllmodulin in tip-growing plant cells,visualized by fluorescing calmodulin-binding phenothiazines

Calmodulin (CaM) was visualized light-microscopically by the fluorescent CaM inhibitors fluphenazine and chlorpromazine, both phenothiazines, during polar tip growth of pollen tubes of Lilium longiflorum, root hairs of Lepidium sativum, moss caulonema of Funaria hygrometrica, fungal hyphae of Achlya spec. and in the alga Acetabularia mediterranea, as well as during multipolar tip growth in Micrasterias denticulata. Young pollen tubes and root hairs showed tip fluorescence; at later stages and in the growing parts of the other subjects the fluorescence was almost uniform. After treatment with cytochalasin B, punctuate fluorescence occurred in the clear zone adjacent to the tip of pollen tubes. The observations indicate that there is CaM in all our tested systems detectable with this method. It may play a key role in starting polar growth. As in pollen tubes, CaM might be in part associated with the microfilament network at the tip, and thus regulate vesicle transport and cytoplasmic streaming.Abbreviations CaM calmodulin - CB cytochalasin B - CTC chlorotetracycline     

NMR identification of the binding surfaces involved in the Salmonella and Shigella Type III secretion tip‐translocon protein–protein interactions

The type III secretion system (T3SS) is essential for the pathogenesis of many bacteria including Salmonella and Shigella, which together are responsible for millions of deaths worldwide each year. The structural component of the T3SS consists of the needle apparatus, which is assembled in part by the protein–protein interaction between the tip and the translocon. The atomic detail of the interaction between the tip and the translocon proteins is currently unknown. Here, we used NMR methods to identify that the N‐terminal domain of the Salmonella SipB translocon protein interacts with the SipD tip protein at a surface at the distal region of the tip formed by the mixed α/β domain and a portion of its coiled‐coil domain. Likewise, the Shigella IpaB translocon protein and the IpaD tip protein interact with each other using similar surfaces identified for the Salmonella homologs. Furthermore, removal of the extreme N‐terminal residues of the translocon protein, previously thought to be important for the interaction, had little change on the binding surface. Finally, mutations at the binding surface of SipD reduced invasion of Salmonella into human intestinal epithelial cells. Together, these results reveal the binding surfaces involved in the tip‐translocon protein–protein interaction and advance our understanding of the assembly of the T3SS needle apparatus. Proteins 2016; 84:1097–1107. © 2016 Wiley Periodicals, Inc.     

Distinguishing features of immature stages of Panchaetothripinae (Thysanoptera: Thripidae) known in New Zealand

Diagnostic morphological characters of the juvenile Panchaetothripinae in New Zealand are illustrated. Keys developed enable colonies with only immature stages to be identified without needing to rear adults. Live larvae or larvae in ethanol are distinguished by the presence of expanded tips of body setae (Parthenothrips dracaenae), the absence of setae at the abdomen tip (Hercinothrips bicinctus), setae at abdomen tip not longer than abdominal tip width (Heliothrips haemorrhoidalis) and abdominal tip setae longer than abdominal tip width (Sigmothrips aotearoana, endemic species). The presence or absence of spine-like setae on abdominal segments 9 and 10, and the number and length of setae on the wing buds, enable identification of pupae. Abdominal spine-like setae were on the prepupa and pupa of H. bicinctus and S. aotearoana, species that pupate off the plant, and are probably defensive structures. This is the first record of spine-like setae on segment 10 of terebrantian pupae.     

The typology and topography of the tarsal chemoreceptors of the blow flies Calliphora vicina Robineau-Desvoidy and Phormia terranovae Robineau-Desvoidy and the Housefly Musca domestica L

A comparative morphological study concerning typology and topography of chemoreceptors on the prothoracic legs of Calliphora vicina, Phormia terranovae and Musca domestica has been carried out. The typological criteria of Grabowski and Dethier ('54) and Hansen and Heumann ('71) were used. A single criterion, the shape of the tip, was used to define the different types of chemoreceptors. A-hairs have a rhombic pore at the side of the tip; B-hairs have an oval pore at the tip apex and D-hairs have a rectangular pore under an undulated, cap-like structure at the hair tip. A-, B-and D-hairs were found in the tarsomeres of Phormia; in Musca and Calliphora only B- and D-hairs were found. An opening and closing mechanism may operate on the pores of the tips of the chemoreceptors. Chemoreceptors were counted and a topographical map was completed, using SEM-techniques. Topographical maps are of value in electrophysiological and behavioural research, where only a limited optical magnification is possible.     

Morphology,cell growth,and polysaccharide production of <Emphasis Type="Italic">Nostoc flagelliforme</Emphasis> in liquid suspension culture at different agitation rates

Noscoc flagelliforme is a terrestrial macroscopic cyanobacterium with high economic value. Free-living cells that were separated from a natural colony of N. flagelliforme were cultivated in a 20-L photobioreactor for 16 days at five agitation rates with impeller tip speeds at 0.3, 0., 0.8, 1.0, and 1.5 m·s⁻¹. With different impeller tip speeds there were significant differences in the cell growth and polysaccharide production, and different types of cell colonies appeared because of different shear forces caused by agitation. At harvest time, cell concentrations with tip speeds of 0.8 and 1.0 m·s⁻¹ were clearly higher than those with the other three tip speeds, but dry cell weights with the tip speeds of 0.3, 0.5, 0.8, and 1.0 m·s⁻¹ were almost the same. The highest RPS (polysaccharide that released into liquid medium) production was obtained with the tip speeds of 0.8 and 1.0 m·s⁻¹, while the highest EPS (polysaccharide that formed capsule or slime layer) production was obtained with the tip speed of 0.5 m·s⁻¹. The tip speed of 1.5 m·s⁻¹ was harmful for both cell growth and polysaccharide production, indicating that an appropriate shear force was needed in the liquid suspension culture of N. flagelliforme.     

Does Hyphal‐Tip Ensure the Same Allelic Composition at SSR Loci as Monosporic Isolates of Sclerotinia sclerotiorum?

The proper characterization of individual is a basic stage in population genetic studies. In Sclerotinia sclerotiorum, genetic uniformity of an individual can be obtained by isolation of single ascospore; however, hyphal‐tip isolates are commonly used in genetic studies. The aim of this study was to assess whether hyphal‐tip isolates of S. sclerotiorum can be used as surrogate of monoascosporic (monosporic) isolates. Twenty‐eight isolates of S. sclerotiorum were collected from common bean plants with white mold symptoms and were purified by hyphal‐tip or single ascospore. The correspondence between hyphal‐tip and monosporic isolates was assessed through the allelic composition at 10 microsatellite (SSR) loci of the isolates obtained by both methods. For the SSR loci comprised of dinucleotide repeats in 92% of the cases, the difference (di) between the amplicon size values for hyphal‐tip and monosporic isolates was no more than one base pair. For the loci comprised of tetra or pentanucleotide repeats in 89% of the cases, di was no more than one base pair. The same allelic profile was found for hyphal‐tip or single ascospore isolates of S. sclerotiorum. When monosporic isolates cannot be easily obtained, hyphal‐tip can safeguard the genotypic identity of S. sclerotiorum isolates.     

The role of +TIPs in directional tip expansion

Aspergillus nidulans is an ideal model to study nuclear migration and intracellular transport by dynein and kinesin owing to its long neuron‐like hyphae, conserved transport mechanisms, and powerful genetics. In this organism, as in other filamentous fungi, microtubules have been implicated in patterning cell shape through polarized tip growth – the hallmark mode of growth that generates the elongated hyphae. Exactly how microtubules regulate tip growth is incompletely understood and remains a fascinating question for various cell types, such as pollen tubes and root hairs. Zeng et al. (2014) describe important new findings in A. nidulans regarding the role of EBA, the master regulator of microtubule plus end‐tracking proteins, in specifying microtubule dynamics required for directional tip growth at the hyphal tip.     

A Real-Time Quantitative PCR Assay for the Detection of Sphaeropsis sapinea from Inoculated Pinus nigra Shoots

Pinus nigra (Austrian pine) is a tree widely planted in central and southern Europe in the reforestation of areas with poor soils. One of the major constrains of P. nigra is a fungal disease caused by Sphaeropsis sapinea which causes shoot tip dieback and tree mortality. This paper describes a real‐time quantitative PCR assay based on the partial sequence of the small subunit ribosomal RNA of S. sapinea to detect and quantify DNA of the fungal pathogen in S. sapinea inoculated shoots. The study found that real‐time quantitative PCR was a reliable technique to diagnose this disease and suggests that it can also be used to study fungal behaviour in host tissue and particularly to quantify fungal growth in the latent or endophytic phase.