Proceedings ... annual meeting, Electron Microscopy Society of America, Aug 1, 1991
In situ hybridization is a powerful tool for the localization of DNA/RNA sequences in nuclei and ... more In situ hybridization is a powerful tool for the localization of DNA/RNA sequences in nuclei and chromosomes. The introduction of nonisotopic labelling methodologies in conjunction with fluorescent or enzyme-linked detection have resulted in a dramatic increase in the application of this technique at the light microscope (LM) level and has placed it in a pivotal role in cell biology, development and genetics. Development of equivalent mapping protocols at the EM level offers increased spatial resolution. We have combined the use of nonisotopic probes with invmunogold labelling to investigate eukaryotic genome organization at high resolution.Metaphase chromosomes released from mitotically-arrested cells are deposited on gold EM grids by centrifugation through a sucrose cushion. After fixation (0.1% glutaraldehyde, 20 min) and DNA denaturation, chromosomes are hybridized to cloned probes enzymatically labelled with biotin-dUTP, digoxigenin-dUTP, dinitrophenyl-dUTP or covalently coupled to N-acetoxyacetoaminofluorene. Hybrid sites typically are detected by a two-step antibody incubation and 1-30 nm colloidal gold particles.
Publisher Summary This chapter discusses the electron microscopic in situ hybridization (EMISH) t... more Publisher Summary This chapter discusses the electron microscopic in situ hybridization (EMISH) technique for nucleic acid sequence localization. In situ hybridization is a pivotal genome mapping technique that provides the cytological location of a cloned sequence. The development of equivalent mapping techniques at the electron microscope (EM) level present the opportunity to determine the relative map positions of sequences. The EM localization is well suited for mapping sequences on small chromosomal structures and for subnuclear localization in small nuclei, such as yeast. It uses biotin-substituted probes and immunogold tagging of hybrid sites. EMISH is applied successfully to the localization of DNA and RNA sequences in both whole-mount metaphase chromosomes and nuclei in organisms from yeast to man. The methodology is analogous to that used at the light microscope (LM) level with a few modifications. The main source of background in EMISH appears to be the nonspecific binding of antibodies to grid films. In addition to modifying EMISH, the use of primer extension in the presence of biodUTP after hybridization of an oligonucleotide to chromosome preparations is being investigated.
It is possible to combine hybridization to specimens on electron‐microscope grids of nucleic‐acid... more It is possible to combine hybridization to specimens on electron‐microscope grids of nucleic‐acid probes labelled nonisotopically with immunogold detection of hybrid sites to map the position of target sequences rapidly and precisely. The basic technique is described, and examples are provided to illustrate the types of questions which can be approached in the general area of higher‐order chromosome organization and function. A combination of two differentially labelled probes and two different‐sized gold particles permits the simultaneous detection of closely linked or interspersed sequences.
Two different Trypanosoma cruzi polypeptides, with masses of 70 and 68 kDa were purified and char... more Two different Trypanosoma cruzi polypeptides, with masses of 70 and 68 kDa were purified and characterized in this work. These two polypeptides designated PAR 1 and PAR 2, respectively, co-purified during each step of the isolation procedure and were found to be located exclusively in T. cruzi flagella by indirect immunofluorescence. A pre-embedding immunoelectron microscopy procedure, with a gold-tagged secondary antibody, permitted direct identification of PAR 2 as a component of the T. cruzi paraflagellar rod. PAR 1 and PAR 2 were found to be immunologically distinct and showed no cross-reactivity with actin, tubulin, intermediate filament proteins, or other proteins present in mammalian cells. The results presented indicate that PAR 1 and PAR 2 are the major components of T. cruzi paraflagellar filaments, and that these filaments have no counterpart in mammalian cells.
Proceedings, annual meeting, Electron Microscopy Society of America, 1991
In situ hybridization is a powerful tool for the localization of DNA/RNA sequences in nuclei and ... more In situ hybridization is a powerful tool for the localization of DNA/RNA sequences in nuclei and chromosomes. The introduction of nonisotopic labelling methodologies in conjunction with fluorescent or enzyme-linked detection have resulted in a dramatic increase in the application of this technique at the light microscope (LM) level and has placed it in a pivotal role in cell biology, development and genetics. Development of equivalent mapping protocols at the EM level offers increased spatial resolution. We have combined the use of nonisotopic probes with invmunogold labelling to investigate eukaryotic genome organization at high resolution.Metaphase chromosomes released from mitotically-arrested cells are deposited on gold EM grids by centrifugation through a sucrose cushion. After fixation (0.1% glutaraldehyde, 20 min) and DNA denaturation, chromosomes are hybridized to cloned probes enzymatically labelled with biotin-dUTP, digoxigenin-dUTP, dinitrophenyl-dUTP or covalently couple...
Proceedings, annual meeting, Electron Microscopy Society of America, 1995
The availability of nonradioactive methods to label nucleic acids an the resultant rapid and grea... more The availability of nonradioactive methods to label nucleic acids an the resultant rapid and greater sensitivity of detection has catapulted the technique of in situ hybridization to become the method of choice to locate of specific DNA and RNA sequences on chromosomes and in whole cells in cytological preparations in many areas of biology. It is being applied to problems of fundamental interest to basic cell and molecular biologists such as the organization of the interphase nucleus in the context of putative functional domains; it is making major contributions to genome mapping efforts; and it is being applied to the analysis of clinical specimens. Although fluorescence detection of nucleic acid hybrids is routinely used, certain questions require greater resolution. For example, very closely linked sequences may not be separable using fluorescence; the precise location of sequences with respect to chromosome structures may be below the resolution of light microscopy(LM); and the ...
Chromosome microdissection is a very valuable approach for the construction of detailed specific ... more Chromosome microdissection is a very valuable approach for the construction of detailed specific chromosome libraries. Metaphase chromosomes from both murine and human origin were immobilized by centrifugation onto a glass coverslip previously coated with a thin matrix. Reversible immobilization was obtained using an alginate matrix which exhibits a sol-gel transition in the presence of a counter-ion and a chelating agent. The matrix was selected for its optical qualities permitting imaging, chromosome banding and laser-assisted micromanipulations using focused laser beams. Whole chromosomes and chromosome fragments were cut and released from the matrix using the LaserScissors, a laser microdissecting system mounted onto an inverted microscope. Chromosomes or chromosome fragments were then transported towards the tip of a glass micropipette by means of optical trapping using the LaserTweezers. They were recovered individually with the CellSelector, a microsyringe pump, and made available for DNA amplification using PCR. We are currently in the process of characterizing specific chromosome fragments. Routine recovery of single chromosomes and sub-chromosomal regions should find applications for the mapping of genomes of several species including human and mouse. Reversible immobilization procedures and laser assisted micromanipulations of metaphase chromsomes should be beneficial to the Human Genome Project.
Nucleic acid sequences can be localized on chromosomes in the electron microscope after hybridiza... more Nucleic acid sequences can be localized on chromosomes in the electron microscope after hybridization with a biotinylated DNA probe followed by detection with a primary antibiotin antibody and a secondary antibody coupled to colloidal gold. Hybridization probes can also be labelled with alternative ligands such as N-acetoxy-2-acetylaminofluorene (AAF), Dinitrophenyl-dUTP and Digoxigenin-dUTP. Multiple labelling is possible if these differently modified DNA probes are used in conjunction with colloidal gold preparations of varying particle sizes. A substantial signal amplification can be achieved by incubating preparations with successive cycles of primary antibiotin antibody followed by a biotinylated secondary antibody. Detection is with Streptavidin-gold, and in the case of highly and moderately repeated sequences, the signal is visible in the light microscope. Detailed protocols are given for EM in-situ hybridization to whole mount metaphase chromosomes and include instructions n...
Currently, most organelle isolation procedures rely on physical parameters and centrifugation for... more Currently, most organelle isolation procedures rely on physical parameters and centrifugation for separation. Here, we report the rapid and gentle isolation of a variety of organelles by immunolabeling whole cell lysates with organelle-specific antibodies and streptavidin magnetic particles followed by separation in a magnetic field. Using magnetic immunoabsorption, we have been able to specifically label mouse metaphase chromosomes and a variety of plant organelles, including: amyloplasts, choroplasts and nuclei from whole cell lysates of various plant tissues. We find that the distinct magnetic properties, surface characteristics and mean diameter-size ranges of different particle preparations significantly influence their specific utility for organelle isolations. By using an internal-field magnetic separation device, we have developed a method for quantitative recovery of labeled organelles in microarrays and tested a variety of antibodies to chloroplast outer envelope proteins ...
Publisher Summary This chapter discusses the electron microscopic in situ hybridization (EMISH) t... more Publisher Summary This chapter discusses the electron microscopic in situ hybridization (EMISH) technique for nucleic acid sequence localization. In situ hybridization is a pivotal genome mapping technique that provides the cytological location of a cloned sequence. The development of equivalent mapping techniques at the electron microscope (EM) level present the opportunity to determine the relative map positions of sequences. The EM localization is well suited for mapping sequences on small chromosomal structures and for subnuclear localization in small nuclei, such as yeast. It uses biotin-substituted probes and immunogold tagging of hybrid sites. EMISH is applied successfully to the localization of DNA and RNA sequences in both whole-mount metaphase chromosomes and nuclei in organisms from yeast to man. The methodology is analogous to that used at the light microscope (LM) level with a few modifications. The main source of background in EMISH appears to be the nonspecific binding of antibodies to grid films. In addition to modifying EMISH, the use of primer extension in the presence of biodUTP after hybridization of an oligonucleotide to chromosome preparations is being investigated.
It is possible to combine hybridization to specimens on electron‐microscope grids of nucleic‐acid... more It is possible to combine hybridization to specimens on electron‐microscope grids of nucleic‐acid probes labelled nonisotopically with immunogold detection of hybrid sites to map the position of target sequences rapidly and precisely. The basic technique is described, and examples are provided to illustrate the types of questions which can be approached in the general area of higher‐order chromosome organization and function. A combination of two differentially labelled probes and two different‐sized gold particles permits the simultaneous detection of closely linked or interspersed sequences.
DNA sequences can be mapped on chromosomes at high resolution in the electron microscope after hy... more DNA sequences can be mapped on chromosomes at high resolution in the electron microscope after hybridization with a nonisotopically labeled probe followed by detection with a two-step antibody reaction employing a colloidal gold tag. Hybridization probes can be modified with biotin-dUTP, digoxigenin-dUTP, dinitrophenyl-dUTP, or N-acetoxy-2-acetylaminofluorene (AAF). The availability of different sizes of colloidal gold particles permits the simultaneous detection of several sequences. In addition, low signals can be amplified either with an antibody sandwich scheme or by silver intensification. This technology is applicable both to TEM and SEM preparations of chromosomes, and we have used it to map a number of highly and moderately repeated sequences on whole mount metaphase chromosomes.
We have developed a modification of in situ hybridization at the electron microscope level that p... more We have developed a modification of in situ hybridization at the electron microscope level that permits simultaneous detection of at least two sequences. Probes are labelled with either biotin or AAF and detected with two distinct sizes of colloidal gold. This protocol has been applied to map the positions of Xenopus laevis oocyte-type 5S genes relative to ribosomal precursor genes in several independently derived cell lines. The results for the line TRXO, which expresses some oocyte 5S RNA, indicate that this inappropriate expression is not due to translocation from telomeric sites into the nucleolus organizer, as previously hypothesized. In addition we found that four other Xenopus cell lines, none of which express these genes, also contain distinct 5S oocyte translocations. These results suggest that an alteration in chromosome position is insufficient to result in gene activation and that sequences which are telomeric-proximal are exceptionally prone to translocation.
Proceedings ... annual meeting, Electron Microscopy Society of America, Aug 1, 1991
In situ hybridization is a powerful tool for the localization of DNA/RNA sequences in nuclei and ... more In situ hybridization is a powerful tool for the localization of DNA/RNA sequences in nuclei and chromosomes. The introduction of nonisotopic labelling methodologies in conjunction with fluorescent or enzyme-linked detection have resulted in a dramatic increase in the application of this technique at the light microscope (LM) level and has placed it in a pivotal role in cell biology, development and genetics. Development of equivalent mapping protocols at the EM level offers increased spatial resolution. We have combined the use of nonisotopic probes with invmunogold labelling to investigate eukaryotic genome organization at high resolution.Metaphase chromosomes released from mitotically-arrested cells are deposited on gold EM grids by centrifugation through a sucrose cushion. After fixation (0.1% glutaraldehyde, 20 min) and DNA denaturation, chromosomes are hybridized to cloned probes enzymatically labelled with biotin-dUTP, digoxigenin-dUTP, dinitrophenyl-dUTP or covalently coupled to N-acetoxyacetoaminofluorene. Hybrid sites typically are detected by a two-step antibody incubation and 1-30 nm colloidal gold particles.
Publisher Summary This chapter discusses the electron microscopic in situ hybridization (EMISH) t... more Publisher Summary This chapter discusses the electron microscopic in situ hybridization (EMISH) technique for nucleic acid sequence localization. In situ hybridization is a pivotal genome mapping technique that provides the cytological location of a cloned sequence. The development of equivalent mapping techniques at the electron microscope (EM) level present the opportunity to determine the relative map positions of sequences. The EM localization is well suited for mapping sequences on small chromosomal structures and for subnuclear localization in small nuclei, such as yeast. It uses biotin-substituted probes and immunogold tagging of hybrid sites. EMISH is applied successfully to the localization of DNA and RNA sequences in both whole-mount metaphase chromosomes and nuclei in organisms from yeast to man. The methodology is analogous to that used at the light microscope (LM) level with a few modifications. The main source of background in EMISH appears to be the nonspecific binding of antibodies to grid films. In addition to modifying EMISH, the use of primer extension in the presence of biodUTP after hybridization of an oligonucleotide to chromosome preparations is being investigated.
It is possible to combine hybridization to specimens on electron‐microscope grids of nucleic‐acid... more It is possible to combine hybridization to specimens on electron‐microscope grids of nucleic‐acid probes labelled nonisotopically with immunogold detection of hybrid sites to map the position of target sequences rapidly and precisely. The basic technique is described, and examples are provided to illustrate the types of questions which can be approached in the general area of higher‐order chromosome organization and function. A combination of two differentially labelled probes and two different‐sized gold particles permits the simultaneous detection of closely linked or interspersed sequences.
Two different Trypanosoma cruzi polypeptides, with masses of 70 and 68 kDa were purified and char... more Two different Trypanosoma cruzi polypeptides, with masses of 70 and 68 kDa were purified and characterized in this work. These two polypeptides designated PAR 1 and PAR 2, respectively, co-purified during each step of the isolation procedure and were found to be located exclusively in T. cruzi flagella by indirect immunofluorescence. A pre-embedding immunoelectron microscopy procedure, with a gold-tagged secondary antibody, permitted direct identification of PAR 2 as a component of the T. cruzi paraflagellar rod. PAR 1 and PAR 2 were found to be immunologically distinct and showed no cross-reactivity with actin, tubulin, intermediate filament proteins, or other proteins present in mammalian cells. The results presented indicate that PAR 1 and PAR 2 are the major components of T. cruzi paraflagellar filaments, and that these filaments have no counterpart in mammalian cells.
Proceedings, annual meeting, Electron Microscopy Society of America, 1991
In situ hybridization is a powerful tool for the localization of DNA/RNA sequences in nuclei and ... more In situ hybridization is a powerful tool for the localization of DNA/RNA sequences in nuclei and chromosomes. The introduction of nonisotopic labelling methodologies in conjunction with fluorescent or enzyme-linked detection have resulted in a dramatic increase in the application of this technique at the light microscope (LM) level and has placed it in a pivotal role in cell biology, development and genetics. Development of equivalent mapping protocols at the EM level offers increased spatial resolution. We have combined the use of nonisotopic probes with invmunogold labelling to investigate eukaryotic genome organization at high resolution.Metaphase chromosomes released from mitotically-arrested cells are deposited on gold EM grids by centrifugation through a sucrose cushion. After fixation (0.1% glutaraldehyde, 20 min) and DNA denaturation, chromosomes are hybridized to cloned probes enzymatically labelled with biotin-dUTP, digoxigenin-dUTP, dinitrophenyl-dUTP or covalently couple...
Proceedings, annual meeting, Electron Microscopy Society of America, 1995
The availability of nonradioactive methods to label nucleic acids an the resultant rapid and grea... more The availability of nonradioactive methods to label nucleic acids an the resultant rapid and greater sensitivity of detection has catapulted the technique of in situ hybridization to become the method of choice to locate of specific DNA and RNA sequences on chromosomes and in whole cells in cytological preparations in many areas of biology. It is being applied to problems of fundamental interest to basic cell and molecular biologists such as the organization of the interphase nucleus in the context of putative functional domains; it is making major contributions to genome mapping efforts; and it is being applied to the analysis of clinical specimens. Although fluorescence detection of nucleic acid hybrids is routinely used, certain questions require greater resolution. For example, very closely linked sequences may not be separable using fluorescence; the precise location of sequences with respect to chromosome structures may be below the resolution of light microscopy(LM); and the ...
Chromosome microdissection is a very valuable approach for the construction of detailed specific ... more Chromosome microdissection is a very valuable approach for the construction of detailed specific chromosome libraries. Metaphase chromosomes from both murine and human origin were immobilized by centrifugation onto a glass coverslip previously coated with a thin matrix. Reversible immobilization was obtained using an alginate matrix which exhibits a sol-gel transition in the presence of a counter-ion and a chelating agent. The matrix was selected for its optical qualities permitting imaging, chromosome banding and laser-assisted micromanipulations using focused laser beams. Whole chromosomes and chromosome fragments were cut and released from the matrix using the LaserScissors, a laser microdissecting system mounted onto an inverted microscope. Chromosomes or chromosome fragments were then transported towards the tip of a glass micropipette by means of optical trapping using the LaserTweezers. They were recovered individually with the CellSelector, a microsyringe pump, and made available for DNA amplification using PCR. We are currently in the process of characterizing specific chromosome fragments. Routine recovery of single chromosomes and sub-chromosomal regions should find applications for the mapping of genomes of several species including human and mouse. Reversible immobilization procedures and laser assisted micromanipulations of metaphase chromsomes should be beneficial to the Human Genome Project.
Nucleic acid sequences can be localized on chromosomes in the electron microscope after hybridiza... more Nucleic acid sequences can be localized on chromosomes in the electron microscope after hybridization with a biotinylated DNA probe followed by detection with a primary antibiotin antibody and a secondary antibody coupled to colloidal gold. Hybridization probes can also be labelled with alternative ligands such as N-acetoxy-2-acetylaminofluorene (AAF), Dinitrophenyl-dUTP and Digoxigenin-dUTP. Multiple labelling is possible if these differently modified DNA probes are used in conjunction with colloidal gold preparations of varying particle sizes. A substantial signal amplification can be achieved by incubating preparations with successive cycles of primary antibiotin antibody followed by a biotinylated secondary antibody. Detection is with Streptavidin-gold, and in the case of highly and moderately repeated sequences, the signal is visible in the light microscope. Detailed protocols are given for EM in-situ hybridization to whole mount metaphase chromosomes and include instructions n...
Currently, most organelle isolation procedures rely on physical parameters and centrifugation for... more Currently, most organelle isolation procedures rely on physical parameters and centrifugation for separation. Here, we report the rapid and gentle isolation of a variety of organelles by immunolabeling whole cell lysates with organelle-specific antibodies and streptavidin magnetic particles followed by separation in a magnetic field. Using magnetic immunoabsorption, we have been able to specifically label mouse metaphase chromosomes and a variety of plant organelles, including: amyloplasts, choroplasts and nuclei from whole cell lysates of various plant tissues. We find that the distinct magnetic properties, surface characteristics and mean diameter-size ranges of different particle preparations significantly influence their specific utility for organelle isolations. By using an internal-field magnetic separation device, we have developed a method for quantitative recovery of labeled organelles in microarrays and tested a variety of antibodies to chloroplast outer envelope proteins ...
Publisher Summary This chapter discusses the electron microscopic in situ hybridization (EMISH) t... more Publisher Summary This chapter discusses the electron microscopic in situ hybridization (EMISH) technique for nucleic acid sequence localization. In situ hybridization is a pivotal genome mapping technique that provides the cytological location of a cloned sequence. The development of equivalent mapping techniques at the electron microscope (EM) level present the opportunity to determine the relative map positions of sequences. The EM localization is well suited for mapping sequences on small chromosomal structures and for subnuclear localization in small nuclei, such as yeast. It uses biotin-substituted probes and immunogold tagging of hybrid sites. EMISH is applied successfully to the localization of DNA and RNA sequences in both whole-mount metaphase chromosomes and nuclei in organisms from yeast to man. The methodology is analogous to that used at the light microscope (LM) level with a few modifications. The main source of background in EMISH appears to be the nonspecific binding of antibodies to grid films. In addition to modifying EMISH, the use of primer extension in the presence of biodUTP after hybridization of an oligonucleotide to chromosome preparations is being investigated.
It is possible to combine hybridization to specimens on electron‐microscope grids of nucleic‐acid... more It is possible to combine hybridization to specimens on electron‐microscope grids of nucleic‐acid probes labelled nonisotopically with immunogold detection of hybrid sites to map the position of target sequences rapidly and precisely. The basic technique is described, and examples are provided to illustrate the types of questions which can be approached in the general area of higher‐order chromosome organization and function. A combination of two differentially labelled probes and two different‐sized gold particles permits the simultaneous detection of closely linked or interspersed sequences.
DNA sequences can be mapped on chromosomes at high resolution in the electron microscope after hy... more DNA sequences can be mapped on chromosomes at high resolution in the electron microscope after hybridization with a nonisotopically labeled probe followed by detection with a two-step antibody reaction employing a colloidal gold tag. Hybridization probes can be modified with biotin-dUTP, digoxigenin-dUTP, dinitrophenyl-dUTP, or N-acetoxy-2-acetylaminofluorene (AAF). The availability of different sizes of colloidal gold particles permits the simultaneous detection of several sequences. In addition, low signals can be amplified either with an antibody sandwich scheme or by silver intensification. This technology is applicable both to TEM and SEM preparations of chromosomes, and we have used it to map a number of highly and moderately repeated sequences on whole mount metaphase chromosomes.
We have developed a modification of in situ hybridization at the electron microscope level that p... more We have developed a modification of in situ hybridization at the electron microscope level that permits simultaneous detection of at least two sequences. Probes are labelled with either biotin or AAF and detected with two distinct sizes of colloidal gold. This protocol has been applied to map the positions of Xenopus laevis oocyte-type 5S genes relative to ribosomal precursor genes in several independently derived cell lines. The results for the line TRXO, which expresses some oocyte 5S RNA, indicate that this inappropriate expression is not due to translocation from telomeric sites into the nucleolus organizer, as previously hypothesized. In addition we found that four other Xenopus cell lines, none of which express these genes, also contain distinct 5S oocyte translocations. These results suggest that an alteration in chromosome position is insufficient to result in gene activation and that sequences which are telomeric-proximal are exceptionally prone to translocation.
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Papers by Sandya Narayanswami