Search PubMed⌕ Search

Biomedical subjects

R H Singer

Publications and source records attributed to R H Singer.

At least 19 recordsLinked to original sources
PubMed1992

The cytoskeleton and mRNA localization.

The localization of mRNA appears to facilitate protein sorting, so that proteins are synthesized in specific cellular regions. The spatial information on the mRNA may be transduced by proteins that recognize specific localizing sequences on the 3' end and then chaperone the mRNA, presumably along filaments, to its destination. Additional sequences such as poly(A), or the nascent chains of cytoskeleton-associated proteins, may then anchor mRNAs on the cytoskeleton.

Animals↗
PubMed1992

Determinants of mRNA localization.

RNA localization provides a mechanism for protein targeting within developing or differentiating cells. Specific cis-acting sequences on mRNA mediate this process. Such 'localizer' or 'zipcode' nucleic acid sequences have been restricted to the 3' untranslated region of several mRNAs. The presence of genetic information denoting a spatial component of translation adds a new dimension to gene expression.

Animals↗
PubMed1992

Poly(A) RNA codistribution with microfilaments: evaluation by in situ hybridization and quantitative digital imaging microscopy.

The distribution of poly(A) RNA has been visualized in single cells using high-resolution fluorescent in situ hybridization. Digital imaging microscopy was used to quantitate the signal in various cellular compartments. Most of the poly(A) signal remained associated with the cellular filament systems after solubilization of membranes with Triton, dissociation of ribosomes with puromycin, and digestion of non-poly(A) RNA with ribonuclease A and T1. The actin filaments were shown to be the predominant cellular structural elements associating with the poly(A) because low doses of cytochalasin released about two-thirds of the poly(A). An approach to assess the extent of colocalization of two images was devised using in situ hybridization to poly(A) in combination with probes for ribosomes, membranes, or F-actin. Digital imaging microscopy showed that most poly(A) spatially distributes most significantly with ribosomes, slightly less with F-actin, and least of all with membranes. The results suggest a mechanism for anchoring (and perhaps moving) much of the cellular mRNA utilizing the interaction between actin filaments and poly(A).

Actin Cytoskeleton↗
PubMed1992

Self-reported functional status predicts change in level of care in independent living residents of a continuing care retirement community.

OBJECTIVE: To test the hypothesis that self-reported functional status predicts change in level of care from independent to dependent in residents of a continuing care retirement community (CCRC). DESIGN: Two-year longitudinal descriptive study of change in level of care and survival. SUBJECTS: One hundred fifty-two residents in the independent-living unit of a non-profit CCRC. Mean age at initial evaluation was 82.3 years, SD 6.2. MEASUREMENT: Predictor variables assessed at baseline were age, sex, physician estimate of functionally significant disease, self-reported functional status, and performance-based hand function. Criterion variables collected at 2-year follow-up were level of care (independent/dependent) and survival (alive/dead). MAIN RESULTS: Self-reported functional status (P less than 0.01) and age (P less than 0.05) were significant predictors of change in level of care in a logistic regression analysis containing all predictor variables. No variable predicted survival. CONCLUSIONS: Self-reported functional status may help to predict dependency in older adults in good health who have few markers of dependency risk.

Activities of Daily Living↗
PubMed1991

Requirement of microfilaments in sorting of actin messenger RNA.

Specific messenger RNAs (mRNAs) can be sequestered within distinct cellular locations, but little is known about how this is accomplished. The participation of the three major cellular filaments in the localization of actin mRNA was studied in chicken embryo fibroblasts. Movement of actin mRNA to the cell periphery and maintenance of that regionalization required intact microfilaments (composed of actin) but not microtubules or intermediate filaments. The results presented here suggest that actin-binding proteins may participate in mRNA sorting.

Actin Cytoskeleton↗
PubMed1991

The GERI-AIMS. Reliability and validity of the arthritis impact measurement scales adapted for elderly respondents.

We adapted the Arthritis Impact Measurement Scales (AIMS) for use with frail elderly respondents. The new instrument, called the GERI-AIMS, generates both generic and arthritis-specific impairment scores, to control for comorbid conditions that are commonly found in an older population. An analysis of scores obtained on 438 elderly persons (mean age 76) indicated that more than half of the total disability measured by the generic GERI-AIMS scores is related to arthritis. The GERI-AIMS scales have standardized-item alpha levels greater than 0.70 and correlate well with clinical measures of arthritis severity. Information gained by using the GERI-AIMS has important public policy implications, insofar as information on both generic and disease-specific functional impairment is needed to establish priorities for basic research, epidemiologic, and intervention studies.

Aged↗
PubMed1991

Distribution of myosin heavy chain mRNA in embryonic muscle tissue visualized by ultrastructural in situ hybridization.

We have localized myosin heavy chain (MHC) mRNAs in cells of intact embryonic chick muscle using high resolution in situ hybridization. Blocks of muscle were aldehyde-fixed prior to detergent treatment and hybridized with a biotinated cDNA probe, followed by colloidal gold-labeled antibodies, before embedment. Labeling was determined to represent MHC mRNA by extensive quantitative comparisons of electron micrographs from experimental and four different types of control samples. MHC mRNA was localized primarily to peripheral regions of 14-day chick pectoral muscle cells, where the majority of developing myofibrils were found. MHC mRNAs were consistently associated with the nonmyofibrillar cytoskeletal filaments which had diameters ranging from 4 to 10 nm. They were often oriented parallel to the longitudinal axis of the cell. The resolution of the ultrastructural approach allowed us to demonstrate that the mRNA molecules visualized were not directly associated with myofilaments, suggesting that nascent chains read from those messages do not assemble directly into myofilaments simultaneous with translation.

Actin Cytoskeleton↗
PubMed1991

Localizing DNA and RNA within nuclei and chromosomes by fluorescence in situ hybridization.

The enormous potential of in situ hybridization derives from the unique ability of this approach to directly couple cytological and molecular information. In recent years, there has been a surge of success in powerful new applications, resulting from methodologic advances that bring the practical capabilities of this technology closer to its theoretical potential. A major advance has been improvements that enable, with a high degree of reproducibility and efficiency, precise visualization of single sequences within individual metaphase and interphase cells. This has implications for gene mapping, the analysis of nuclear organization, clinical cytogenetics, virology, and studies of gene expression. This article discusses the current state of the art of fluorescence in situ hybridization, with emphasis on applications to human genetics, but including brief discussions on studies of nuclear DNA and RNA organization, and on applications to clinical genetics and virology. Although a review of all of the literature in this field is not possible here, many of the major contributions are summarized along with recent work from our laboratory.

Animals↗
PubMed1991

Spatial organization of nucleic acid sequences within cells.

High-resolution in situ hybridization has allowed the distribution of specific nucleic acids to be visualized within cells. This has revealed the extent to which nucleic acids exhibit intracellular spatial organization. Hybridization of nonisotopically labeled probes can be detected with high resolution using fluorescence or alkaline phosphatase for light microscopy or colloidal gold for electron microscopy. Results of this approach have shown that messenger RNAs for specific proteins, nuclear transcripts, or the gene for those transcripts all can exhibit non-random intracellular locations. Actin mRNA can be seen localized in the periphery of motile cells, just proximal to the lamellipodia. Highly localized distributions of EBV primary transcripts can be visualized in some cases creating an elongated track of specific RNA. These nuclear transcripts could be seen associated with the nuclear matrix. Further evidence indicates that the genes from which specific RNAs derive may themselves show defined spatial organization. This work sheds light on a level of cellular organization indicating that nucleic acid sequences contain spatial positioning information and therefore can influence the genesis of cellular structure and function.

Animals↗
PubMed1990

Interphase and metaphase resolution of different distances within the human dystrophin gene.

Fluorescence in situ hybridization makes possible direct visualization of single sequences not only on chromosomes, but within decondensed interphase nuclei, providing a potentially powerful approach for high-resolution (1 Mb and below) gene mapping and the analysis of nuclear organization. Interphase mapping was able to extend the ability to resolve and order sequences up to two orders of magnitude beyond localization on banded or unbanded chromosomes. Sequences within the human dystrophin gene separated by less than 100 kb to 1 Mb were visually resolved at interphase by means of standard microscopy. In contrast, distances in the 1-Mb range could not be ordered on the metaphase chromosome length. Analysis of sequences 100 kb to 1 Mb apart indicates a strong correlation between interphase distance and linear DNA distance, which could facilitate a variety of gene-mapping efforts. Results estimate chromatin condensation up to 1 Mb and indicate a comparable condensation for different cell types prepared by different techniques.

Chromosome Banding↗
PubMed1990

Detection and localization of actin mRNA isoforms in chicken muscle cells by in situ hybridization using biotinated oligonucleotide probes.

We have developed in situ hybridization methodology for nonisotopically labeled oligonucleotide probes to detect cellular mRNA with improved speed, convenience, and resolution over previous techniques. Previous work using isotopically labeled oligonucleotide probes characterized important parameters for in situ hybridization (Anal Biochem 166:389, 1987). Eleven oligonucleotide probes were made to coding and noncoding regions of chick beta-actin mRNA and one oligonucleotide probe to chick alpha-cardiac actin mRNA. All the probes were 3' end-labeled with bio-11-dUTP using terminal transferase, and the labeled probes were hybridized to chicken myoblast and myotube cultures. The hybridized probe was detected using a streptavidin-alkaline phosphatase conjugate. Our assay for the success of probe hybridization and detection was the demonstration of beta-actin mRNA highly localized in the lamellipodia of single cells (Lawrence and Singer, Cell 45:407, 1986) as well as the expression of alpha-cardiac actin mRNA and the repression of beta-actin mRNA in differentiating myoblasts and in myotubes. With the alpha-cardiac probe, we found that this mRNA was distributed all over the cytoplasm of myotubes and differentiated (bipolar) single cells and negative in undifferentiated single cells and at the ends of myotubes. When beta-actin probes were used, two of 11 probes were highly sensitive, and, in pooling them together, the localization of beta-actin mRNA in fibroblastic single cells was evident at the leading edge of the motile cells, the lamellipodium. beta-Actin mRNA was not detected in myotubes except at the ends where contact was made with substrate. This indicates that both beta and cardiac actin mRNA can coexist in the same myotube cytoplasm but at different locations.

Actins↗
PubMed1990

Subcellular localization of low-abundance human immunodeficiency virus nucleic acid sequences visualized by fluorescence in situ hybridization.

Detection and subcellular localization of human immunodeficiency virus (HIV) were investigated using sensitive high-resolution in situ hybridization methodology. Lymphocytes infected with HIV in vitro or in vivo were detected by fluorescence after hybridization with either biotin or digoxigenin-labeled probes. At 12 hr after infection in vitro, a single intense signal appeared in the nuclei of individual cells. Later in infection, when cytoplasmic fluorescence became intense, multiple nuclear foci frequently appeared. The nuclear focus consisted of newly synthesized HIV RNA as shown by hybridization in the absence of denaturation and by susceptibility to RNase and actinomycin D. Virus was detected in patient lymphocytes and it was shown that a singular nuclear focus also characterizes cells infected in vivo. The cell line 8E5/LAV containing one defective integrated provirus revealed a similar focus of nuclear RNA, and the single integrated HIV genome was unequivocally visualized on a D-group chromosome. This demonstrates an extremely sensitive single-cell assay for the presence of a single site of HIV transcription in vitro and in vivo and suggests that it derives from one (or very few) viral genomes per cell. In contrast, productive Epstein-Barr virus infection exhibited many foci of nuclear RNA per cell.

Cell Nucleus↗
PubMed1990

Actin mRNA localizes in the absence of protein synthesis.

Actin mRNA is localized in chicken embryo fibroblasts to the distal regions of leading lamellae, but not within the ruffling edges. In this investigation we have addressed the role of actin translation in this process. The translocation of actin mRNA to the cell periphery was studied by monitoring the distribution of actin mRNA in cells during spreading. Within 90 min, actin mRNA moved from a perinuclear to a peripheral distribution. Formation of lamellipodia preceded actin mRNA localization, indicating that localization is not a prerequisite for this event. Neither puromycin (which dissociates ribosomes from mRNA) nor cycloheximide (which stabilizes ribosomes on mRNA) had any effect on this movement of actin mRNA. Anchoring of actin mRNA was studied using cells with peripherally localized actin mRNA. No change in actin mRNA localization was observed for 30 min in the same inhibitors. These data indicate that the presence of the nascent polypeptide is not necessary for translocation of actin mRNA to the cell periphery, or anchoring at that site. This suggests that the mRNA contains information concerning its spatial distribution within the cytoplasm.

Actins↗
PubMed1989

Detection of HIV-1-infected cells from patients using nonisotopic in situ hybridization.

We have demonstrated that a sensitive, nonisotopic in situ hybridization (ISH) assay can be used to detect HIV-infected cells from seropositive, asymptomatic individuals. Our assay is based on the detection of a biotinated HIV DNA probe hybridized to human immunodeficiency virus (HIV)-infected peripheral blood lymphocytes (PBL) using streptavidin and alkaline phosphatase to identify positive cells. This assay is rapid in that it can be performed within a day and is sensitive enough to unambiguously identify a rare, single, positive cell. Patient samples derived from HIV-seropositive hemophiliacs and HIV-seropositive infants were analyzed before and after coculture with normal PBL. The same samples were investigated using a Dupont P24 antigen-capture kit. It was found that ISH always detected the same positive samples as antigen capture, often in shorter times of coculture. In situ hybridization detected over half of our HIV-infected hemophilia patient population as virus positive, whereas the antigen capture assay detected less than one fourth as virus positive. In situ hybridization detected positive cells directly, without coculture, in 12 out of 35 (34%) hemophiliacs and in three out of eight (37%) infants. The speed, sensitivity, and confidence of ISH and nonisotopic detection indicates that it will be useful as a tool for clinical research and diagnosis.

Biotin↗
PubMed1989

Highly localized tracks of specific transcripts within interphase nuclei visualized by in situ hybridization.

Use of in situ hybridization optimized for fluorescent detection of nuclear RNA has revealed a striking localization of specific viral RNAs within nuclei of cells latently infected with EBV. Several hundred kb of specific transcripts is sharply restricted to a small region of the nucleus, frequently in a curvilinear "track". Detection of nuclear RNA was evidenced by hybridization without denaturation, sensitivity to RNAase, inhibition by actinomycin D, and specificity of transcribed sequences. Results indicate that RNA "tracks" extend from an internal genome into the nuclear periphery, and that RNA transport may be coupled to transcription. Localized nRNA is apparent for other viral sequences, different lymphoblastoid cell lines, nuclei prepared by two different methods, and an abundant, nonviral transfected sequence. Implications for understanding nuclear organization and the investigation of gene expression are discussed.

Burkitt Lymphoma↗
PubMed1989

Strategies for ultrastructural visualization of biotinated probes hybridized to messenger RNA in situ.

A progressive development of the application of in situ methodology to ultrastructural procedures has resulted in the ability to detect individual molecules of mRNA with high probability. Beginning with whole-mount cells and then developing myotubes, both in culture and detergent extracted before fixation, we were able to progress to methods which allow detection of mRNA in tissue sections. Initial results confirm that the detection of mRNA in thin-sectioned tissue is very similar to observations on the extracted, cultured cells, and that the same methods of data analysis apply. Current work is devoted to the application of the methodology to other cellular structures, such as the nucleus, and to other tissue-probe systems, such as brain. Acknowledgements. The authors appreciate the skilled help from John McNeil and Shirwin Pockwinse in the laborious and time-consuming preparations of material and photography. FS was on sabbatical leave from the Department of Pathology at Southwestern Medical Center.

Animals↗
PubMed1989

Human type II collagen gene (COL2A1) assigned to chromosome 12q13.1-q13.2 by in situ hybridization with biotinylated DNA probe.

We have made a regional assignment of the type II collagen gene (COL2A1) on human chromosome 12 by means of an in situ hybridization technique with a biotinylated DNA probe. The precise localization of the signal was mapped to the band 12q13.1-q13.2. This result was in agreement with the previous mapping by isotopic in situ hybridization technique (12q13.1-q13.2), but not with the result of Southern hybridization analysis using somatic cell hybrids (12q14.3).

Chromosome Banding↗