Localization of the T-DNA on marker chromosomes in transformed tobacco cells by in situ hybridization

Summary Chromosome and molecular analyses were conducted on tobacco cells which had been transformed by the T-DNA of the Ti-plasmid. These analyses showed that there were specific chromosome rearrangements in the transformed cells (marker chromosomes). There was a positive correlation between the number of marker chromosomes per cell and the oncogenic potential of the transformed cells. However, we show, using the Southern hybridization method, that the T_L fragment of T-DNA, but not the T_R, clearly hybridizes with nuclear DNA. In situ hybridization was used to locate the insertion site of T-DNA: the hybridization signal was found on a small metacentric chromosome. This chromosome may occur single or translocated onto other chromosomes, to make marker chromosomes. Thus, by locating the T-DNA, we have confirmed the correlation between the marker chromosomes and the oncogenic potential.     

Cellular localization of a pollen-specific mRNA by in situ hybridization and confocal laser scanning microscopy

Summary The application of confocal laser scanning microscopy together with in situ hybridization experiments in tobacco pollen enabled a detailed localization of a pollen-specific mRNA. The three-dimensional distribution of this specific mRNA over the whole pollen grain was reconstructed by means of optical sections of one specimen.     

Localization of the calcium release channel gene in cattle and horse by in situ hybridization: evidence of a conserved synteny with glucose phosphate isomerase

In situ hybridization techniques were used to localize regionally the calcium release channel (CRC) gene on cattle and horse chromosomes, using a porcine CRC cDNA probe. In cattle, the hybridization signal peaked on the 18q23-q26 bands and in horse on the 10pter region. Previous studies have shown that the glucose phosphate isomerase (GPI) gene localizes at the same site in both species, indicating that the two loci are syntenic. As CRC and GPI are syntenic in human, pig and mouse, the present results in cattle and horse represent another example of synteny conservation in the evolution of mammalian chromosomes.     

Localization of the ß-subunit of follicle stimulating hormone in cattle and sheep by in situ hybridization

The locus for the beta-subunit of the follicle stimulating hormone gene (FSHB) has been determined in both cattle and sheep by in situ hybridization of a bovine and an ovine cDNA probe, respectively, to metaphase chromosomes. Our results show that the FSHB locus is on cattle chromosome 15 in the region of bands q24-qter and in sheep on the cytogenetically homologous chromosome 15, also in the region q24-qter. The mapping of the FSHB gene in cattle together with the location of other genes (CAT, HBB and PTH) previously found to be syntenic in cattle and on human chromosome 11p, defines an evolutionarily conserved synteny. The localization of the FSHB gene to a cytogenetically homologous region in cattle and sheep is consistent with the hypothesis of extensively conserved chromosome structure in these two species.     

Multidrug resistance gene expression is controlled by steroid hormones in the secretory epithelium of the uterus

The multidrug resistance (mdr) gene family has been shown to encode a membrane glycoprotein, termed the P-glycoprotein, which functions as a drug efflux pump with broad substrate specificity. This multigene family is expressed in a tissue-specific fashion in a wide variety of normal and neoplastic tissues. The regulation of mdr gene expression in normal tissues is not understood. We have recently shown that mdr mRNA and the P-glycoprotein increases dramatically in the secretory luminal and glandular epithelium of the gravid murine uterus. This observation has suggested that mdr gene expression in the uterus is controlled by the physiologic changes associated with pregnancy. This report now demonstrates that mdr mRNA and P-glycoprotein are induced at high levels in the uterine secretory epithelium by the combination of estrogen and progesterone, the major steroid hormones of pregnancy. This regulation of mdr gene expression in the uterus does not require any other contribution from the fetus or placenta. The data indicate that this gene locus is hormonally responsive to estrogen and progesterone in the uterine secretory epithelium, suggesting an important and physiologically regulated role during pregnancy.     

An in situ hybridization protocol for planarian embryos: monitoring myosin heavy chain gene expression

The monitoring of gene expression is fundamental for understanding developmental biology. Here we report a successful experimental protocol for in situ hybridization in both whole-mount and sectioned planarian embryos. Conventional in situ hybridization techniques in developmental biology are used on whole-mount preparations. However, given that the inherent lack of external morphological markers in planarian embryos hinders the proper interpretation of gene expression data in whole-mount preparations, here we used sectioned material. We discuss the advantages of sectioned versus whole-mount preparations, namely, better probe penetration, improved tissue preservation, and the possibility to interpret gene expression in relation to internal morphological markers such as the epidermis, the embryonic and definitive pharynges, and the gastrodermis. Optimal fixatives and embedding methods for sectioning are also discussed. A. Cardona and J. Fernández have contributed equally to this work.     

原位杂交检测microRNA-205在乳腺癌中的表达

目的探讨microRNA-205表达与乳腺恶性病变的关系。方法乳腺疾病及癌组织芯片原位杂交分析microRNA-205的表达;实时定量RT-PCR方法检测正常乳腺细胞株、恶性程度不同的乳腺癌细胞株中microRNA-205的表达。结果原位杂交分析显示,36例正常与良性乳腺病变中,33例(91.67%)表达阳性;36例乳腺癌中,23例(63.89%)表达阳性。microRNA-205的表达在乳腺正常与良性病变中的表达较恶性病变中高且有统计学差异(P=0.011),但与乳腺癌TNM分期、临床分期无关(P0.05)。实时定量RT-PCR结果显示,四个高度恶性乳腺癌细胞株(MDA-MB-231、HS578T、BT549和SUM159PT)中microRNA-205的表达较永生化正常乳腺上皮细胞株MCF10A和四个低度恶性细胞株(MDA-MB-468、T-47D、ZR-75-1和SKBR3)中为低(P0.05)。结论原位杂交适用于microRNA-205的表达分析;组织芯片标本原位杂交与乳腺细胞株实时定量RT-PCR分析结果提示,microRNA-205可能参与了乳腺癌的发生、发展,并随着乳腺癌的演进呈下调趋势。     

RNA原位杂交实用技术

利用互补RNA为探针进行原位杂交是分析组织或细胞内RNA分布的行之有效的方法 ,通过对mRNA分布的研究可以了解特定基因的表达情况。原位杂交技术过程较长 ,操作繁琐 ,从而在一些实验中不能得到很好的使用 ,为此本文根据我们过去的实际操作经验对该技术中的一些使用技巧作简要的介绍。     

In situ hybridization to somatic metaphase chromosomes of potato

Summary An in situ hybridization procedure was developed for mitotic potato chromosomes by using a potato 24S rDNA probe. This repetitive sequence hybridized to the nucleolar organizer region (NOR) of chromosome 2 in 95%–100% of the metaphase plates. Another repetitive sequence (P5), isolated from the interdihaploid potato HH578, gave a ladderpattern in genomic Southern's of Solanum tuberosum and Solanum phureja, but not in those of Solanum brevidens and two Nicotiana species. This sequence hybridized predominantly on telomeric and centromeric regions of all chromosomes, although chromosomes 7, 8, 10 and 11 were not always labeled clearly.     

Tentative chromosomal localization of the bovine major histocompatibility complex by in situ hybridization

Summary. A genetic region, most likely the major histocompatibility complex, was assigned to bands q13–23 of cattle chromosome 23 by in situ hybridization using a cloned DNA sequence of a class I gene of the pig major histocompatibility complex.     

A methacrylate embedding procedure developed for immunolocalization on plant tissues is also compatible with in situ hybridization.

A recently described method that uses methacrylate embedding of aldehyde fixed plant tissues allows the immunolabelling of a range of antigens (Baskin et al. 1992). We have tested whether the same embedding procedure is also compatible with in situ hybridization. For this purpose we have used 2- 5 μm sections of methacrylate embedded plantlets of Arabidopsis thaliana. After removal of the resin the sections were prepared for in situ hybridization following standard procedures. Three different digoxygenin (dig)-labelled probes were used, recognizing RNAs coding for the chlorophyll a/b binding protein cab-140, the β-tubulin tub5 and meri a member of the meri-5 family. Each of the probes shows the labelling pattern expected from the literature. Moreover, the method allows a good structural preservation of very fragile tissues, in contrast to paraffin embedding. We conclude that methacrylate embedding, allowing both immunolabelling and in situ hybridization with high resolution and structural preservation, offers a high potential for the functional analysis of genes and proteins in plant development. This is especially true for Arabidopsis thaliana, a widely used model species where it seems to be the method of choice.     

Genomic reorganization and disrupted chromosomal synteny in the siamang (Hylobates syndactylus) revealed by fluorescence in situ hybridization

We employed in situ hybridization (“chromosome painting”) of chromosome-specific DNA libraries of all human chromosomes to establish homologies between the human and siamang karyotypes (Hylobates syndactylus, 2n = 50). Numerous intra- and interchromosomal rearrangements have led to a massive reorganization of the siamang karyotype. There have been a minimum of 33 translocations. The 24 siamang autosomes are composed of 60 recognizable segments that show DNA homology to regions of the 22 human autosomes. Only two autosomes have not been involved in translocations. The siamang presents a case, in a primate closely related to humans, in which chromosome morphology and synteny are highly disturbed in a manner similar to that encountered among rodents. © 1995 Wiley-Liss, Inc.     

大鼠胃卵泡刺激素及其受体的免疫荧光定位及原位杂交

目的探讨大鼠胃组织中是否表达卵泡刺激素（FSH）及其受体（FSHR）,为进一步研究FSH对消化系统的功能调节提供理论依据。方法选取SD大鼠20只,雌雄不拘,经腹腔注射戊巴比妥钠麻醉成功后,用4％多聚甲醛先快后慢灌流固定2h,开腹取胃组织置于300g／L蔗糖液中直至组织沉底,恒冷箱切片机切成厚度为6／zm的组织切片用于单标记免疫荧光定位研究。另一部分胃组织放入4％多聚甲醛室温固定6—8h,按石蜡包埋程序包埋后制成4pm石蜡切片用于原位杂交研究。结果在大鼠胃底腺的壁细胞和主细胞呈FSH和FSHR免疫反应阳性,阳性物质分布于细胞质,细胞核呈阴性反应;上述细胞同样含有FSH和FSHRmRNA杂交信号,信号物质亦分布于细胞质内,细胞核呈阴性反应。结论FSH及其受体定位于大鼠壁细胞和主细胞,同时大鼠壁细胞和主细胞又能产生FSH及其受体,说明FSH对壁细胞和主细胞功能作用可能是通过旁分泌或自分泌的作用来实现的。     

Expression of a chimaeric granule-bound starch synthase-GUS gene in transgenic potato plants

Granule-bound starch synthase is the key enzyme in amylose synthesis. The regulation of this gene was investigated using a chimaeric gene consisting of a 0.8 kb 5 upstream sequence of the granule-bound starch synthase gene from potato and the -glucuronidase gene which was introduced into potato using an Agrobacterium tumefaciens binary vector system. The chimaeric gene was highly expressed in stolons and tubers, whereas the expression in leaves, stems or roots from greenhouse-grown plants was relatively low. However, leaves from in vitro grown plantlets exhibited an elevated GUS expression. The expression of the chimaeric gene was inducible in leaves by growth on relatively high concentrations of sucrose, fructose and glucose and was about 30- to 50-fold higher than in leaves from greenhouse-grown plants. The granule-bound starch synthase gene is expressed organ-specifically since stolons and tubers showed GUS activities 125- to 3350-fold higher than in leaves. The activities in these two organs are 3- to 25-fold higher than the expression of the CaMV-GUS gene. Histochemical analysis of different tissues showed that only certain regions of leaves and roots express high GUS activities. Stolons and tubers show high expression.     

In situ RNA hybridization using Technovit resin in Arabidopsis thaliana

A protocol is described for RNA in situ hybridization using thin sections prepared by Technovit resin. Technovit is a widely used resin for histological examinations. Since it does not require time-consuming processes such as removal of the resin and can be performed without high temperature treatment, a high resolution of sections could be possible compared to other resins and paraffin. Thin sections (approximately 4 m) were made from inflorescences of Arabidopsis thaliana embedded in Technovit 8100 resin, and in situ hybridization was performed using the protocol described in this article. Hybridization signals were observed using LEAFY and other genes as probes, showing that this resin can be used for in situ analysis. In our experiments, the most important factor for a successful in situ hybridization pattern was to optimize the RNase A concentration after hybridization. We routinely used RNase A at a concentration of 2–5 ng/ml, a concentration much lower than that used for paraffin embedding method. Thus, the use of the Technovit resin for plant tissue embedding results in a faster protocol and greater quality than allowed by paraffin sections.     

Localization of ovulation hormone-like neuropeptide in the central nervous system of the snail Lymnaea stagnalis by means of immunocytochemistry and in situ hybridization

Summary The caudo-dorsal cells (CDC) in the cerebral ganglia of the pond snail Lymnaea stagnalis synthesize the 36-amino acid ovulation hormone (CDCH). We have used immuno-cytochemistry and in situ hybridization to reveal the localization of neurons and axons containing CDCH-like material.A monoclonal antibody to a fragment of CDCH and a cDNA probe encoding CDCH reacted with the CDC-system, with specific cell groups in the cerebral and pleural ganglia, and with individually occurring neurons throughout the central nervous system. The cells in the pleural ganglia, which were found in about 50% of the preparations studied, are considered as ectopic CDC. They are morphologically similar to CDC in their somal dimensions and axonal organization. By means of immuno-electron microscopy it was shown that these neurons contain secretory vesicles that are similar to those of the CDC. The neurons of the bilateral groups occurring in the cerebral ganglia in addition to the CDC are smaller and more intensely stained than the CDC. Axons of these small neurons probably have varicosities located on the CDC axons in the neuropil of the cerebral ganglion, indicating synaptic contacts. Two major axon tracts could be followed from (or toward) the neuropil of the cerebral ganglion. One tract runs from the cerebral gangion via the pleural and parietal ganglia to the visceral ganglion, giving off branches to most nerves emanating from these ganglia. The other tract could be traced through the cerebro-pedal connective to the pedal ganglia. Only in the right pedal ganglion was extensive axonal branching observed. The nerves emanating from this ganglion contained many more immunoreactive axons than those from the left pedal ganglion. A polyclonal antibody raised against the synthetic fragment of CDCH stained, in addition to the neurons and axons revealed with the monoclonal antibody and the cDNA probe, three other major groups of neurons. Two are located in the cerebral ganglion, the other in the left pedal ganglion.The present findings suggest the presence of a system of neurons that contain CDCH or CDCH-like peptides. The role this system may play in the control of egg-laying and egg-laying behaviour is discussed.