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1.
Summary Two cDNA probes complementary to fetal rat brain tau cDNA were produced by the polymerase chain reaction (PCR) and labelled
by digoxigenin-11-dUTP incorporation during the PCR elongation step. These probes were tested for thein situ localization of tau mRNAs in sections of rat cerebellum. The hybridization signal was consistent with the known localization
of brain tau mRNAs, showing the validity of cDNA probes labelled by digoxigenin during the PCR.
Using these probes, anin situ hybridization protocol was established and optimized for the localization of tau-related mRNAs in sections of pancreas. The
aim was to determine whether these mRNAs were expressed in the exocrine or the endocrine part of the pancreas. A positive
signal was found only in the exocrine part of the pancreas, and was distributed exclusively in the cytoplasm of acinar cells.
The results described here are the first evidence for a specific expression of tau-related proteins in the exocrine pancreas. 相似文献
2.
《Experimental mycology》1989,13(3):212-222
cDNA clones representing mRNAs abundantly expressed during fruiting ofSchizophyllum commune were used to detect the cellular localization of these mRNAs in freeze-microtome sections of developing fruit bodies. An 18 S rRNA clone was isolated and used as a probe for total RNA. Both RNA and DNA probes with different labels were found suitable but the procedure finally adopted involvedin situ hybridization with nick-translated biotinylated DNA probes. To permit the probes to permeate the cell walls it was necessary to treat the sections with RNasedepletedTrichoderma harzianum wall-lytic enzymes before hybridization. Hybridization at different developmental stages showed that the specific mRNAs were abundantly expressed in specific areas of the fruit bodies. 相似文献
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In situ hybridization of atrial natriuretic peptide mRNA in the endothelial cells of human umbilical vessels 总被引:1,自引:0,他引:1
Wen Qin Cai Giorgio Terenghi Philippe Bodin Geoffrey Burnstock Julia M. Polak 《Histochemistry and cell biology》1993,100(4):277-283
The localization of mRNA encoding preproatrial natriuretic peptide (ANP) was investigated in cultured human umbilical vein endothelial cells (HUVEC) and tissue preparations of umbilical vein and artery. The techniques used were in situ hybridization and in situ hybridization combined with immunocytochemistry, using 32P-radiolabelled and non-radioactive digoxigenin labelled complementary RNA probes. Human ANP mRNAs are mainly localized in the endothelial cells of the umbilical vein and, to a lesser extent, in the endothelial cells of the umbilical artery. The autoradiographic labelling and the intensity of digoxigenin staining were significantly reduced by treatment with RNase before in situ hybridization. This study provides unequivocal evidence for the expression of the ANP gene in the endothelial cells of human umbilical vessels, confirming that these endothelial cells have the ability to synthesize this peptide. The functional significance of the presence of the ANP mRNA in the endothelial cells of human umbilical vessels is discussed. 相似文献
6.
Ken-Ichi Iyama Mitsuhiko Kitaoka Misako Monda Yoshifumi Ninomiya Masando Hayashi 《The Histochemical journal》1994,26(11):844-849
Summary Collagen types II and X mRNAs have been demonstrated simultaneously in newly formed hypertrophic chondrocytes of embryonic chick vertebral cartilage using a double-fluorescence in situ hybridization technique. Digoxigenin- and biotin-labelled type-specific collagen II and X cDNA probes were used. In the embryonic chick vertebra at stage 45, two different fluorescence signals (Fluorescein isothiocyanate and Rhodamine) - one for collagen type II mRNA, the other for type X mRNA - showed differential distribution of the two collagen mRNAs in the proliferating and hypertrophic chondrocyte zones. Several layers of newly formed hypertrophic chondrocytes expressing both collagen types II and X genes were identified in the same section as two different fluorescent colour signals. Low levels of fluorescent signals for collagen type II mRNA were also detected in the hypertrophic chondrocyte zone. Cytological identification of maturing chondrocyte phenotypes, expressing collagen mRNAs, is easier in sections processed by non-radioactive in situ hybridization than in those subjected to radioactive in situ hybridization using 3H-labelled cDNA probes.This study demonstrates that double-fluorescence in situ hybridization is a useful tool for simultaneously detecting the expression of two collagen genes in the same chondrocyte population. 相似文献
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I. Mirgain C. Hagnere G. A. Green C. Harf H. Monteil 《World journal of microbiology & biotechnology》1992,8(1):30-38
Mercury-resistance genes were detected byin situ hybridization using new synthetic oligonucleotide probes specific formerA andmerB genes according to the published sequences of the corresponding enzymes. These DNA probes were used for the detection of specific mercury-resistant microorganisms isolated from the Rhine River which had been polluted 3 years previously in 1986. Mercuric reductase and organomercurial lyase genes persist in the bacterial genome even after the disappearance of the pollutant but are absent in axenic amoebae. A total of 49 bacterial isolates showed DNA homologies with the32P-labelled DNA probes and 15 free-living amoebae were selected due to their harboured symbiotic mercury-resistant bacteria. 相似文献
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Regulation of myosin heavy-chain gene expression during skeletal-muscle hypertrophy. 总被引:3,自引:1,他引:2
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Changes in the myosin phenotype of differentiated muscle are a prominent feature of the adaptation of the tissue to a variety of physiological stimuli. In the present study the molecular basis of changes in the proportion of myosin isoenzymes in rat skeletal muscle which occur during compensatory hypertrophy caused by the combined removal of synergist muscles and spontaneous running exercise was investigated. The relative amounts of sarcomeric myosin heavy (MHC)- and light (MLC)-chain mRNAs in the plantaris (fast) and soleus (slow) muscles from rats was assessed with cDNA probes specific for different MHC and MLC genes. Changes in the proportion of specific MHC mRNA levels were in the same direction as, and of similar magnitude to, changes in the proportion of myosin isoenzymes encoded for by the mRNAs. No significant changes in the proportion of MLC proteins or mRNA were detected. However, high levels of MLC3 mRNA were measured in both normal and hypertrophied soleus muscles which contained only trace amounts of MLC3 protein. Small amounts of embryonic and neonatal MHC mRNAs were induced in both muscles during hypertrophy. We conclude that the change in the pattern of myosin isoenzymes during skeletal-muscle adaptation to work overload is a consequence of changes in specific MHC mRNA levels. 相似文献
10.
The discrimination of high-risk HPV types by in situ hybridization and the polymerase chain reaction
C. S. Herrington S. M. Anderson A. K. Graham J. O'D. McGee 《The Histochemical journal》1993,25(3):191-198
Summary The parameter Tmt has been defined by non-isotopic in situ hybridization and describes the tissue melting temperature (Tmt) of human papillomavirus (HPV) DNA sequences. In this study, multiple in situ hybridization signals for HPV types 16, 31 and 33 in individual archival biopsies hybridized with genomic probes are shown by polymerase chain reactions to be due to cross-hybridization of probe sequences to a single tissue target. Tmt is independent of viral type but depends on the homology between probe and target when using nick-translated whole genomic probes. The difference between Tm and Tmt is not due to the presence of viral capsid protein. Multiple HPV signals in archival material should not therefore be interpreted as indicative of multiple HPV infection unless adequate stringency conditions have been employed or they are present in morphologically distinct areas of the biopsy.Furthermore, extrapolation of calculated DNA homologies to non-isotopic in situ hybridization analysis may not be appropriate. A hybridization signal does not imply probe and target identity: this has implications for HPV typing in clinical material. 相似文献
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A nucleic acid probe for the Herpesvirus anguillae (HVA) Taiwan isolate was constructed using recombinant DNA techniques. This probe consisted of a specific viral DNA fragment (1550 bp) generated by digestion of HVA DNA with the restriction enzyme HindIII, and labeled non‐radioactively with digoxigenin (DIG). The probe was used to detect the HVA genome from HVA‐infected cell cultures and tissue specimens prepared from infected eels, using either dot blot or in situ hybridizations. 相似文献
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The human embryonic myosin alkali light chain gene: use of alternative promoters and 3'' non-coding regions. 总被引:4,自引:2,他引:2
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M Rotter K Zimmerman A Poustka N Soussi-Yanicostas A Starzinski-Powitz 《Nucleic acids research》1991,19(7):1497-1504
Recently we have found evidence that the human embryonic myosin alkali light chain (MLC1 emb) gene has two functional promoters and that its mRNAs exhibit heterogeneity in their 3'untranslated regions (UTR). To study this more in detail we have isolated and characterized the human MLC1emb gene. We focussed in particular on 2 kilobases of 5'flanking region and the alternative 3'UTRs. RNA primer extension and S1 mapping analyses revealed that the MLC1emb gene can indeed be driven either by a proximal or a distal promoter, both in fetal and adult cardiac tissue. These MLC1emb RNAs can contain either the proximal or distal 3'UTR. In contrast to this, in fetal as well as adult masseter muscle MLC1emb mRNA is predominantly transcribed from the proximal promoter and contains mainly the distal 3'UTR. These results explain the known heterogeneity of MLC1emb mRNAs. Finally, we present evidence that the murine MLC1emb gene also contains a functional distal promoter element which has hitherto been undetected. 相似文献
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Odile Cohen-Haguenauer Paul J. R. Barton Nguyen Van Cong Arlette Cohen Michel Masset Margaret Buckingham Jean Frézal 《Human genetics》1989,81(3):278-282
Summary In all eukaryotes, myosin plays a major role in the maintenance of cell shape and in cellular movement; in association with actin and other contractile proteins it is also a major structural component of the muscle sarcomere. Several isoforms of myosin alkali light chain have been identified, associated with different muscle types. We have recently localized the gene encoding the fast skeletal muscle alkali light-chain isoforms MLC1F and MLC3F (HGM symbol, MYL1) to human chromosome 2q32.1-qter (Cohen-Haguenauer 1988). We present here the chromosomal assignment of two loci encoding the ventricular muscle isoform MLC1V (equivalent to the slow skeletal muscle isoform MLC1Sb) and the atrial muscle isoform MLC1A (equivalent to the fetal isoform MLC1emb) using a panel of 25 independent man-rodent somatic cell hybrids. The MLC1V gene (HGM symbol, MYL3) was mapped to human chromosome 3 using a human full-length cDNA probe that hybridizes to a single major human TaqI 2.8-kb fragment. The MLC1A probe (HGM symbol, MYL4) was a 360-bp mouse cDNA fragment that gave a distinct signal with human DNA using low stringency conditions of hybridization and washings and after presaturation of the Southern blots with rodent DNA. A single PstI 7.8-kb fragment gives an intense signal, and its presence correlates with the presence of chromosome 17 among the hybrids. These data are in keeping with the localizations of the MLC1V gene to mouse chromosome 9, and of the MLC1A gene to mouse chromosome 11, which share some markers in common with human chromosomes 3 and 17 respectively. 相似文献
14.
Comparison of different approaches for the incorporation of non-radioactive labels into polymerase chain reaction products 总被引:1,自引:0,他引:1
Different methods for labelling polymerase chain reaction (PCR) products with non-radioactive labels for their detection by hybridization with immobilized DNA probes were compared. The use of digoxigenin (DIG) as a label provided greater sensitivity than biotin in a PCR system targeting the invA gene from Salmonella typhimurium. Incorporation of digoxigenin into amplicons in the form of 5-DIG-labelled oligonucleotide primers resulted in better assay signals and was more economical than DIG-labelled dUTP. 相似文献
15.
The disease resistance response in pea is associated with increased levels of specific mRNAs 总被引:1,自引:1,他引:0
A cDNA library was constructed using poly(A)+RNA fromPisum sativum which had been treated for 8 h with the fungusFusarium solani f. sp.phaseoli. Two thousand four hundred recombinant colonies were screened by differential colony hybridization using32P-labelled cDNAs prepared from RNA extracted from either noninoculated or inoculated pea tissue. cDNA clones were then selected,
which showed greater hybridization with cDNA prepared from pea RNA 8 h post-inoculation than with a cDNA probe from 0 h. Seven
distinct hybridization classes were chosen for further study. Northern blot analyses of total cellular RNAs inoculated for
16 h with eitherF. solani phaseoli or water demonstrated that each cDNA clone selected represents an mRNA species which increases substantially in abundance
during infection.
Results of3H-uridine pulse-labelling experiments suggested that enhanced synthesis is at least partially responsible for the accumulation
of the fungus-inducible mRNAs which hybridized with the clones. 相似文献
16.
Given the demand for improved methods for detecting and characterizing RNA variants in situ, we developed a quantitative method for detecting RNA alternative splicing variants that combines in situ hybridization of fluorescently labeled peptide nucleic acid (PNA) probes with confocal microscopy Förster resonance energy transfer (FRET). The use of PNA probes complementary to sequences flanking a given splice junction allows to specifically quantify, within the cell, the RNA isoform generating such splice junction as FRET efficiency measure. The FRET-based PNA fluorescence in situ hybridization (FP-FISH) method offers a conceptually new approach for characterizing at the subcellular level not only splice variant isoform structure, location, and dynamics but also potentially a wide variety of close range RNA–RNA interactions. In this paper, we explain the FP-FISH technique workflow for reliable and reproducible results. 相似文献
17.
E. Varricchio M.G. Russolillo L. Maruccio S. Velotto G. Campanile M. Paolucci F. Russo 《European journal of histochemistry : EJH》2013,57(1)
Calpains are Ca2+-dependent proteases able to cleave a large number of proteins involved in many biological functions. Particularly, in skeletal muscle they are involved in meat tenderizing during post mortem storage. In this report we analyzed the presence and expression of µ- and m-calpains in two skeletal muscles of the Marchigiana cattle soon after slaughter, using immunocytochemical and immunohistochemical techniques, Western blotting analysis and Casein Zymography. Therefore, the presence and the activity of these proteases was investigated until 15th day post mortem during normal process of meat tenderizing. The results showed m- and µ-calpain immunosignals in the cytoplasm both along the Z disk/I band regions and in the form of intracellular stores. Moreover, the expression level of µ-calpain but not m-calpain decreased after 10 days of storage. Such a decrease in µ-calpain was accompanied by a gradual reduction of activity. On the contrary, m-calpain activity persisted up to 15 days of post mortem storage. Such data indicate that expression and activity of both µ-calpain and m-calpain analyzed in the Marchigiana cattle persist longer than reported in literature for other bovines and may be related to both the type of muscle and breed examined.Key words: m-calpain, µ-calpain, skeletal muscle, Marchigiana cattle, immunohistochemistry, Electron Microscopy. 相似文献
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Developmental regulation of myosin gene expression in mouse cardiac muscle 总被引:19,自引:5,他引:14
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《The Journal of cell biology》1990,111(6):2427-2436
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Magnus Stougaard Jakob S Lohmann Magdalena Zajac Stephen Hamilton-Dutoit Jørn Koch 《BMC biotechnology》2007,7(1):69