scholarly journals 087 Localization of ENOD2 Transcript Accumulation in Indeterminant Nodules of Maackia amurensis Rupr. & Maxim. (Amur Maackia)

HortScience ◽  
1999 ◽  
Vol 34 (3) ◽  
pp. 456C-456
Author(s):  
Harry T. Horner ◽  
David J. Hannapel ◽  
William R. Graves ◽  
Carol M. Foster ◽  
David J. Hannapel ◽  
...  
Keyword(s):  
In Situ Hybridization ◽  
Periodic Acid ◽  
Nodule Development ◽  
Coding Region ◽  
Transcript Accumulation ◽  
Mrna Accumulation ◽  
Peripheral Tissues ◽  
Maackia Amurensis ◽  
Early Nodulin

Early nodulin genes, such as ENOD2, play a role in the first stages of nodulation. Although ENOD2 is conserved among nodulating legumes studied to date, its occurrence and activity have not been studied among woody legumes such as Maackia amurensis Rupr. & Maxim. Our objective was to localize MaENOD2 transcripts during nodule development and describe the anatomy of nodules formed on the roots of M. amurensis in relation to ENOD2 mRNA accumulation. Nodules (<1 mm, 1-2 mm, >2 mm in diameter, and mature) were prepared for light microscopy, sectioned, and stained with safranin and fast green for structural contrast or with the periodic acid Schiff's reaction for starch. The location of ENOD2 transcripts was determined by using in situ hybridization with DIG-labeled sense and antisense RNAs transcribed from a 602-bp fragment of the coding region of MaENOD2. Mature nodules from M. amurensis possessed peripheral tissues, a distal meristem, and a central infected region characteristic of indeterminant development. In situ hybridization showed that MaENOD2 transcripts accumulated in the distribution layer and uninfected cells of the central symbiotic region. Amyloplasts that contained starch grains were identified in these tissues and in the inner parenchyma of the nodule. Throughout nodule development, transcripts were restricted to areas with high levels of stored starch that surrounded cells actively fixing N2. Our results suggest that ENOD2 in M. amurensis may be a cell wall component of tissues that regulate nutrient flow to and from sinks, such as symbiotic regions of a nodule. These data may lead to a better understanding of the role of the ENOD2 gene family during nodulation.

scholarly journals Carbon Metabolism in Developing Soybean Root Nodules: The Role of Carbonic Anhydrase

2000 ◽  
Vol 13 (1) ◽  
pp. 14-22 ◽  
Author(s):  
Nektarios Kavroulakis ◽  
Emanouil Flemetakis ◽  
Georgios Aivalakis ◽  
Panagiotis Katinakis
Keyword(s):  
In Situ Hybridization ◽  
Carbonic Anhydrase ◽  
Root Nodules ◽  
Cell Types ◽  
Nodule Development ◽  
Vascular Bundles ◽  
Transcript Accumulation ◽  
Cortical Cells ◽  
Gene Transcripts

A full-length cDNA clone encoding carbonic anhydrase (CA) was isolated from a soybean nodule cDNA library. In situ hybridization and immunolocalization were performed in order to assess the location of CA transcripts and protein in developing soybean nodules. CA transcripts and protein were present at high levels in all cell types of young nodules, whereas in mature nodules they were absent from the central tissue and were concentrated in cortical cells. The results suggested that, in the earlier stages of nodule development, CA might facilitate the recycling of CO2 while at later stages it may facilitate the diffusion of CO2 out of the nodule system. In parallel, sucrose metabolism was investigated by examination of the temporal and spatial transcript accumulation of sucrose synthase (SS) and phosphoenolpyruvate carboxylase (PEPC) genes, with in situ hybridization. In young nodules, high levels of SS gene transcripts were found in the central tissue as well as in the parenchymateous cells and the vascular bundles, while in mature nodules the levels of SS gene transcripts were much lower, with the majority of the transcripts located in the parenchyma and the pericycle cells of the vascular bundles. High levels of expression of PEPC gene transcripts were found in mature nodules, in almost all cell types, while in young nodules lower levels of transcripts were detected, with the majority of them located in parenchymateous cells as well as in the vascular bundles. These data suggest that breakdown of sucrose may take place in different sites during nodule development.

scholarly journals Rapid and efficient generation of PCR-derived riboprobe templates for in situ hybridization histochemistry.

1993 ◽  
Vol 41 (5) ◽  
pp. 773-776 ◽  
Author(s):  
J H Sitzmann ◽  
P K LeMotte
Keyword(s):  
In Situ Hybridization ◽  
Coding Region ◽  
Biopsy Material ◽  
Hybridization Histochemistry ◽  
In Situ Hybridization Histochemistry ◽  
Gene Coding ◽  
Suprabasal Cell ◽  
Cell Layers ◽  
Negative Controls

In situ hybridization histochemistry (ISH) using cRNA probes (riboprobes) has become a powerful technique for the examination of gene expression in tissue sections. The construction of plasmid templates for the synthesis of riboprobes with phage RNA polymerases is often a difficult and time-consuming step. We have therefore developed a rapid, efficient, and flexible method to generate totally artificial riboprobe templates by the polymerase chain reaction (PCR). We have made riboprobe templates using self-priming oligonucleotide primers spanning 146 BP of the 3' end of the human cytokeratin 1 (K1) gene coding region flanked by T7 and T3 promoters. These PCR-derived riboprobe templates were used to synthesize 35S-labeled anti-sense riboprobes as well as sense riboprobes as negative controls. The riboprobes were then applied in ISH to human skin sections made from routinely fixed and paraffin-embedded clinical biopsy material. Consistent with published results, we observed strong expression of K1 mRNA in the suprabasal cell layers of the epidermis but only weak to undetectable signals in the basal and cornified cell layers and in the dermis. With this experimental procedure we see no decrease in probe efficiency or quality compared to conventional methods. The use of PCR-derived riboprobe templates for ISH makes it possible to detect expression of any desired gene of known sequence rapidly and efficiently.

scholarly journals Lotus japonicus Contains Two Distinct ENOD40 Genes That Are Expressed in Symbiotic, Nonsymbiotic, and Embryonic Tissues

2000 ◽  
Vol 13 (9) ◽  
pp. 987-994 ◽  
Author(s):  
Emmanouil Flemetakis ◽  
Nektarios Kavroulakis ◽  
Nicolette E. M. Quaedvlieg ◽  
Herman P. Spaink ◽  
Maria Dimou ◽  
...  
Keyword(s):  
Root Nodule ◽  
Vascular System ◽  
Lotus Japonicus ◽  
Mrna Accumulation ◽  
Mesorhizobium Loti ◽  
Embryonic Tissues ◽  
Early Nodulin ◽  
Gene Transcripts ◽  
Young Seed

ENOD40, an early nodulin gene, has been postulated to play a significant role in legume root nodule ontogenesis. We have isolated two distinct ENOD40 genes from Lotus japonicus. The transcribed regions of the two ENOD40 genes share 65% homology, while the two promoters showed no significant homology. Both transcripts encode a putative dodecapeptide similar to that identified in other legumes forming determinate nodules. Both ENOD40 genes are coordinately expressed following inoculation of roots with Mesorhizobium loti or treatment with purified Nod factors. In the former case, mRNA accumulation could be detected up to 10 days following inoculation while in the latter case the accumulation was transient. High levels of both ENOD40 gene transcripts were found in nonsymbiotic tissues such as stems, fully developed flowers, green seed pods, and hypocotyls. A relatively lower level of both transcripts was observed in leaves, roots, and cotyledons. In situ hybridization studies revealed that, in mature nodules, transcripts of both ENOD40 genes accumulate in the nodule vascular system; additionally, in young seed pods strong signal is observed in the ovule, particularly in the phloem and epithelium, as well as in globular stage embryos.

In situ hybridization reveals differential spatial distribution of mRNAs for type I and type II collagen in the chick limb bud

Development ◽  
1988 ◽  
Vol 103 (1) ◽  
pp. 111-118 ◽  
Author(s):  
C.J. Devlin ◽  
P.M. Brickell ◽  
E.R. Taylor ◽  
A. Hornbruch ◽  
R.K. Craig ◽  
...  
Keyword(s):  
In Situ Hybridization ◽  
Limb Development ◽  
Type I Collagen ◽  
Type Ii Collagen ◽  
Limb Bud ◽  
Type I ◽  
Type Ii ◽  
Mrna Accumulation ◽  
Rna Probes

During limb development, type I collagen disappears from the region where cartilage develops and synthesis of type II collagen, which is characteristic of cartilage, begins. In situ hybridization using antisense RNA probes was used to investigate the spatial localization of type I and type II collagen mRNAs. The distribution of the mRNA for type II collagen corresponded well with the pattern of type II collagen synthesis, suggesting control at the level of transcription and mRNA accumulation. In contrast, the pattern of mRNA for type I collagen remained more or less uniform and did not correspond with the synthesis of the protein, suggesting control primarily at the level of translation or of RNA processing.

In situ localization of mRNA for epidermal growth factor in the submandibular gland of the mouse.

1985 ◽  
Vol 33 (12) ◽  
pp. 1235-1240 ◽  
Author(s):  
E W Gresik ◽  
R M Gubits ◽  
T Barka
Keyword(s):  
In Situ Hybridization ◽  
Growth Factor ◽  
Epidermal Growth Factor ◽  
Submandibular Gland ◽  
Cdna Probe ◽  
Coding Region ◽  
Specific Hybridization ◽  
Epidermal Growth ◽  
Convoluted Tubules

Epidermal growth factor (EGF) is a polypeptide originally isolated from the mouse submandibular gland, where it is localized immunocytochemically in cells of the granular convoluted tubules (GCT). cDNAs encoding the precursor of mouse submandibular EGF have been cloned (Scott et al. Science 221:236, 1983; Gray et al. Nature 303:722, 1983). A fragment of one of these clones, pmegf10, containing the EGF coding region, was tritium-labeled by nick-translation and used as a probe for in situ hybridization to EGF mRNA. A specific hybridization signal for EGF mRNA was seen only in mature or developing GCT cells. The intensity of the signal was stronger in glands of intact males than in females or in castrated males. In glands of castrates treated with testosterone, or of intact females treated with triiodothyronine (T3), the signal was comparable to that in intact males. In glands of males treated with T3 the intensity of the signal was stronger than in untreated males. A weak to moderate signal was seen in developing GCT cells of 20-day-old males but not females. Hybridization for 3 days gave a stronger signal than that for 1 day. No signal was seen in either sex at 10 days of age, or in control preparations exposed to labeled DNA of pBR322. The presence of EGF mRNA exclusively in GCT cells provides strong evidence that these cells are the only site of synthesis of EGF in the submandibular gland. In situ hybridization with this cDNA probe will provide a sensitive method to determine possible cellular sites of EGF production outside of the submandibular gland.

scholarly journals Non-isotopic in situ hybridization method for mitochondria in oncocytes.

1994 ◽  
Vol 42 (2) ◽  
pp. 273-276 ◽  
Author(s):  
V A Varma ◽  
C M Cerjan ◽  
K L Abbott ◽  
S B Hunter
Keyword(s):  
Mitochondrial Dna ◽  
In Situ Hybridization ◽  
Coding Region ◽  
Chain Reaction ◽  
Formalin Fixed Paraffin ◽  
Formalin Fixed Paraffin Embedded ◽  
Polymerase Chain ◽  
Endogenous Biotin ◽  
Formalin Fixed

We used in situ hybridization to specifically identify mitochondria in a series of formalin-fixed, paraffin-embedded oncocytic lesions. Digoxigenin-labeled DNA probes were generated by the polymerase chain reaction (PCR), with primers designed to amplify a mitochondrion-specific 154 BP sequence within the ND4 coding region. Probes were hybridized with mitochondrial DNA under stringent conditions. Oncocytes were strongly and consistently stained, reflecting the high copy number of mitochondrial DNA within these cells. Because of the presence of endogenous biotin within mitochondria, digoxigenin is preferable to biotin as a label for detection of mitochondria.

scholarly journals Expression of MsLEC1 Transgenes in Alfalfa Plants Causes Symbiotic Abnormalities

2004 ◽  
Vol 17 (1) ◽  
pp. 16-26 ◽  
Author(s):  
Laurence M. Brill ◽  
Nancy A. Fujishige ◽  
Cheryl A. Hackworth ◽  
Ann M. Hirsch
Keyword(s):  
In Situ Hybridization ◽  
Sinorhizobium Meliloti ◽  
Northern Blot Analysis ◽  
Root Tips ◽  
Mrna Accumulation ◽  
Lectin Gene ◽  
Hybridization Procedure ◽  
Sense Orientation ◽  
Legume Lectins

Legume lectins have been proposed to have important symbiotic roles during Rhizobium-legume symbioses. To test this hypothesis, the symbiotic responses of transgenic alfalfa plants that express a portion of the putative alfalfa lectin gene MsLEC1 or MsLEC2 in either the antisense or sense orientation were analyzed following inoculation with wild-type Sinorhizobium meliloti 1021. MsLEC1-antisense (LEC1AS) plants were stunted, exhibited hypernodulation, and developed not only abnormally large nodules but also numerous small nodules, both of which senesced prematurely. MsLEC2-antisense plants were intermediate in growth and nodule number compared with LEC1AS and vector control plants. The symbiotic abnormalities of MsLEC1-sense transgene plants were similar to but milder than the responses shown by the LEC1AS plants, whereas MsLEC2-sense transgene plants exhibited symbiotic responses that were identical to those of vector and nontransgenic control plants. MsLEC1 mRNA accumulation was not detected in nodule RNA by Northern blot analysis but was localized to alfalfa nodule meristems and the adjacent cells of the invasion zone by in situ hybridization; transcripts were also detected in root meristems. A similar spatial pattern of MsLEC2 expression was found by using a whole-mount in situ hybridization procedure. Moreover, mRNAs for an orthologous lectin gene (MaLEC) were detected in white sweetclover (Melilotus alba) nodules and root tips.

scholarly journals Aspartate Aminotransferase in Alfalfa Nodules: Localization of mRNA During Effective and Ineffective Nodule Development and Promoter Analysis

1999 ◽  
Vol 12 (4) ◽  
pp. 263-274 ◽  
Author(s):  
Hirofumi Yoshioka ◽  
Robert G. Gregerson ◽  
Deborah A. Samac ◽  
Kim C. M. Hoevens ◽  
Gian Trepp ◽  
...  
Keyword(s):  
Aspartate Aminotransferase ◽  
Root Nodules ◽  
Critical Role ◽  
Nodule Development ◽  
Vascular Bundles ◽  
Fixed Nitrogen ◽  
Transcript Accumulation ◽  
Specific Expression ◽  
Promoter Deletion Analysis

Aspartate aminotransferase (AAT) plays a critical role in the assimilation of symbiotically fixed nitrogen into aspartate and asparagine in legume root nodules. The enzyme occurs as a cytosolic form (AAT1) and a plastid form (AAT2) in alfalfa nodules. To elucidate the functional role of each isozyme in root nodule metabolism further, in situ hybridization was used to determine the pattern of transcript accumulation from the two genes. AAT2 transcripts were localized to infected cells throughout the symbiotic zone of effective alfalfa nodules; however, expression was reduced in ineffective nodules. The AAT1 gene was expressed in the uninfected cells of the invasion zone and symbiotic zone, the nodule parenchyma, and nodule vascular bundles of both effective and ineffective nodules. The AAT1 and AAT2 promoters were evaluated in transgenic alfalfa plants containing promoter β-glucuronidase (GUS) gene fusions. Histochemical staining patterns agreed with results from in situ localization. The distribution pattern of gene transcripts suggests that AAT1 has a role in maintenance of the O2 diffusion barrier in nodules and that AAT2 plays a major role in assimilation of recently fixed nitrogen. Promoter deletion analysis of the AAT2 promoter revealed that nodule-specific expression was retained in a promoter fragment of 300 bp.

scholarly journals Increased F1/GAP-43 mRNA Accumulation in Gerbil Hippocampus after Brain Ischemia

1995 ◽  
Vol 15 (6) ◽  
pp. 1132-1136 ◽  
Author(s):  
Masafumi Tagaya ◽  
Tomohiro Matsuyama ◽  
Hitoshi Nakamura ◽  
Ryuji Hata ◽  
Souichiro Shimizu ◽  
...  
Keyword(s):  
In Situ Hybridization ◽  
Mrna Expression ◽  
Brain Ischemia ◽  
Pyramidal Neurons ◽  
Granule Cells ◽  
Hippocampal Formation ◽  
Mrna Accumulation ◽  
Synaptic Reorganization ◽  
Intense Signal

To assess whether ischemia could induce GAP-43 mRNA expression, we performed in situ hybridization in gerbil brains that had been subjected to 5 min of global ischemia. In control dentate granule cells, little hybridization was detected in contrast to the intense signal generated by pyramidal neurons of the adult hippocampal formation. After ischemia, we detected a robust GAP-43 signal over hippocampal granule cells at 3 h of reperfusion, persisting through 7 days, and disappearing by 14 days. This demonstrated GAP-43 gene induction after ischemia, and suggests that GAP-43 may be involved in reactive events, including fiber sprouting and synaptic reorganization, that follow ischemia.

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