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PubMed1999

BMP-4 inhibits neural differentiation of murine embryonic stem cells.

Members of the transforming growth factor-beta superfamily, including bone morphogenetic protein 4 (BMP-4), have been implicated as regulators of neuronal and glial differentiation. To test for a possible role of BMP-4 in early mammalian neural specification, we examined its effect on neurogenesis in aggregate cultures of mouse embryonic stem (ES) cells. Compared to control aggregates, in which up to 20% of the cells acquired immunoreactivity for the neuron-specific antibody TuJ1, aggregates maintained for 8 days in serum-free medium containing BMP-4 generated 5- to 10-fold fewer neurons. The action of BMP-4 was dose dependent and restricted to the fifth through eighth day in suspension. In addition to the reduction in neurons, we observed that ES cell cultures exposed to BMP-4 contained fewer cells that were immunoreactive for glial fibrillary acidic protein or the HNK-1 neural antigen. Furthermore, under phase contrast, cultures prepared from BMP-4-treated aggregates contained a significant proportion of nonneuronal cells with a characteristic flat, elongated morphology. These cells were immunoreactive for antibodies to the intermediate filament protein vimentin; they were rare or absent in control cultures. Treatment with BMP-4 enhanced the expression of the early mesodermal genes brachyury and tbx6 but had relatively little effect on total cell number or cell death. Coapplication of the BMP-4 antagonist noggin counteracted the effect of exogenous BMP-4, but noggin alone had no effect on neuralization in either the absence or presence of retinoids. Collectively, our results suggest that BMP-4 can overcome the neuralizing action of retinoic acid to enhance mesodermal differentiation of murine ES cells.

Animals↗
PubMed2006

rAAV-mediated shRNA ameliorated neuropathology in Huntington disease model mouse.

Huntington disease (HD) is a fatal progressive neurodegenerative disorder associated with expansion of a CAG repeat in the first exon of the gene coding the protein huntingtin (htt). Although the feasibility of RNA interference (RNAi)-mediated reduction of htt expression to attenuate HD-associated symptoms is suggested, the effects of post-symptomatic RNAi treatment in the HD model mice have not yet been certified. Here we show the effects of recombinant adeno-associated virus (rAAV)-mediated delivery of RNAi into the HD model mouse striatum after the onset of disease. Neuropathological abnormalities associated with HD, such as insoluble protein accumulation and down-regulation of DARPP-32 expression, were successfully ameliorated by the RNAi transduction. Importantly, neuronal aggregates in the striatum were reduced after RNAi transduction in the animals comparing to those at the time point of RNAi transduction. These results suggest that the direct inhibition of mutant gene expression by rAVV would be promising for post-symptomatic HD therapy.

Adenoviridae↗
PubMed1991

Messenger RNA sequence and expression of rat pancreatitis-associated protein, a lectin-related protein overexpressed during acute experimental pancreatitis.

Rat pancreatitis-associated protein (PAP) is an additional protein appearing in pancreatic juice after induction of prancreatic inflammation. Its messenger RNA was cloned and sequenced from pancreas. The deduced amino acid sequence revealed that PAP was synthetized as a preprotein with, in its mature form, a predicted molecular weight of 16,630. A search in protein data bases revealed a marked homology with the carbohydrate binding region of animal lectins; no hemagglutination activity could be shown for PAP, but the protein induced extensive bacterial aggregation. In healthy rats, the very low level of PAP expression in pancreas could be increased up to 4-fold by physiological stimuli such as chronic hormonal or cholinergic stimulation of pancreatic secretion and adaptation of rats to a carbohydrate-rich diet. By contrast, induction of acute experimental pancreatitis by retrograde injection of sodium taurocholate resulted in dramatic overexpression. Pancreatic concentration of PAP mRNA increased more than 300 x within 12 h whereas concentrations of mRNAs encoding major secretory proteins such as amylase decreased. PAP overexpression persisted during the 2 days of the acute phase and then returned to the control level during pancreatic recovery. PAP mRNA could not be evidenced in liver, stomach, salivary glands, brain, kidney, or testis. Its pattern of expression during severe pancreatic aggression suggests that it might be a stress protein involved in the control of bacterial proliferation.

Acute Disease↗
PubMed1984

Regulation of nuclear binding of the avian oviduct progesterone receptor. Changes during estrogen-induced oviduct development, withdrawal, and secondary stimulation.

The progesterone receptors from various stages of estrogen induced oviduct development, estrogen withdrawal, and secondary stimulation with estrogen were examined. The progesterone receptors were characterized for their biological function (i.e. capacity for nuclear translocation, nuclear binding, and effects on RNA polymerase II activity) as well as certain physical properties. The progesterone receptors from the undeveloped or partially developed oviducts (0 to 8 days of estrogen treatment) displayed little or no nuclear translocation and binding in vivo or in vitro. Similarly, progesterone showed little or no effect in vivo on RNA polymerase II activity at the early stages of development. As development progressed from 8 to 12 days of estrogen treatment, the above parameters rapidly increased to maximal levels and plateaued through day 23 of estrogen treatment. A marked decrease in these parameters occurred within 1 day of estrogen withdrawal. The reverse series of events occurred during secondary estrogen stimulation of 10-day-old withdrawn chicks. While the receptor concentrations increased rapidly to maximum values by 2 days of restimulation, receptor function did not return until day 4. Similarly, the effects of progesterone on RNA polymerase II activity reached maximal values by day 4. The progesterone receptor isolated from oviducts during development, estrogen withdrawal, and restimulation, displayed similar patterns of cell-free binding to chromatin and nucleoacidic protein as that observed in vivo supporting the nativeness of the in vitro binding assay. In contrast, the cell-free binding of these same progesterone receptor to pure DNA were not similar to the in vivo binding, i.e. no patterns (differences) in progesterone receptor binding were observed. These data support that protein DNA complexes and not pure DNA represent the native acceptor sites for oviduct progesterone receptor. Comparison of the progesterone receptor between the functional and nonfunctional states revealed no differences in the steroid affinity for the receptor, in the apparent pI of the species, or in the sedimentation of the receptor under high salt conditions. However, the nonfunctional receptors consistently displayed a deficiency in one of the two monomer molecular species (the B species) as determined by isoelectric focusing. These results suggest that both monomer species of progesterone receptor are required for biological activity. Interestingly, the 7S "aggregate" species of the progesterone receptor was constantly detected even when only one of the monomer species was present.(ABSTRACT TRUNCATED AT 400 WORDS)

Aging↗
PubMed1988

The cyclic nucleotide phosphodiesterase of Dictyostelium discoideum: the structure of the gene and its regulation and role in development.

The cyclic nucleotide phosphodiesterase (phosphodiesterase) of Dictyostelium discoideum plays an essential role in development by hydrolyzing the cAMP used as a chemoattractant by aggregating cells. We have studied the biochemistry of the phosphodiesterase and a functionally related protein, the phosphodiesterase inhibitor protein, and have cloned the cognate genes. A 1.8-kb and a 2.2-kb mRNA are transcribed from the single-phosphodiesterase gene. The 2.2-kb mRNA comprises the majority of the phosphodiesterase mRNA found in differentiating cells and is transcribed only during development from a promoter at least 2.5 kb upstream of the translational start site. The 1.8-kb phosphodiesterase mRNA is detected at all stages of growth and development, is present at lower levels than the developmentally induced mRNA, and is transcribed from a site proximal to the protein-coding region. The phosphodiesterase gene contains a minimum of three exons, and a 2.3-kb intron, the longest yet reported for this organism. We have shown that the pdsA gene and four fgd genes affect the accumulation of the phosphodiesterase mRNAs, and we believe that these loci represent a significant portion of the genes regulating expression of the phosphodiesterase. The phosphodiesterase gene was introduced into cells by transformation and used as a tool to explore the effects of cAMP on the terminal stages of development. In cells expressing high levels of phosphodiesterase activity, final morphogenesis cannot be completed, and differentiated spore and stalk cells do not form. We interpret these results to support the hypothesis that cAMP plays an essential role in organizing cell movements in late development as well as in controlling the aggregation of cells in the initial phase of the developmental program.

3',5'-Cyclic-AMP Phosphodiesterases↗
PubMed2003

Retention of NMDA receptor NR2 subunits in the lumen of endoplasmic reticulum in targeted NR1 knockout mice.

Glutamate is a major excitatory neurotransmitter in the mammalian central nervous system, and the N-methyl-D-aspartate-selective glutamate receptor (NR) consisting of the NR1 subunit and an NR2 or NR3 subunit plays crucial roles in synaptic transmission, plasticity, and learning and memory. By using a knockout mouse strain, in which the NR1 gene deletion is primarily targeted to the CA1 pyramidal cells of the hippocampus, we investigated the in vivo effect of the loss of the NR1 subunit on the cellular expression and intracellular distribution of the NR2 subunits. The NR1 gene deletion had no apparent effect on the levels of NR2A or NR2B mRNA but led to severe reductions of NR2A and NR2B protein in dendrites of CA1 pyramidal cells. This reduced dendritic distribution of the NR2 subunits accompanied their robust accumulation in perikarya, where they were condensed in the lumen of the endoplasmic reticulum as electron-dense granules. These granules were also observed in CA1 pyramidal cells of the control mice but they were much fewer and contained no detectable levels of the NR2 subunit. The effect of the NR1 knockout on intracellular localization of the NR2 subunits was specific in that no such effect was observed for the GluR1 and PSD-95, two other major postsynaptic proteins. These results suggest that the NR1 subunit plays a crucial role in the release of the NR2 subunit from the endoplasmic reticulum in hippocampal pyramidal cells in vivo, and when the NR1 subunit is unavailable, the NR2 subunits are retained and aggregate into intracisternal granules.

Aging↗
PubMed1994

Transfer of Methanolobus siciliae to the genus Methanosarcina, naming it Methanosarcina siciliae, and emendation of the genus Methanosarcina.

A sequence analysis of the 16S rRNA of Methanolobus siciliae T4/M(T) (T = type strain) showed that this strain is closely related to members of the genus Methanosarcina, especially Methanosarcina acetivorans C2A(T). Methanolobus siciliae T4/M(T) and HI350 were morphologically more similar to members of the genus Methanosarcina than to members of the genus Methanolobus in that they both formed massive cell aggregates with pseudosarcinae. Thus, we propose that Methanolobus siciliae should be transferred to the genus Methanosarcina as Methanosarcina siciliae.

Methanosarcina↗
PubMed1990

Role of matrix protein in cytopathogenesis of vesicular stomatitis virus.

The matrix (M) protein of vesicular stomatitis virus (VSV) plays an important structural role in viral assembly, and it also has a regulatory role in viral transcription. We demonstrate here that the M protein has an additional function. It causes visible cytopathic effects (CPE), as evidenced by the typical rounding of polygonal cells after VSV infection. We have analyzed a temperature-sensitive mutant of the M protein of VSV (tsG33) which is defective in viral assembly and which fails to cause morphological changes of the cells after infection at the nonpermissive temperature (40 degrees C). Interestingly, this defect in viral assembly as well as the CPE were reversible. Microinjection of antisense oligonucleotides which specifically inhibit M protein translation also inhibited the occurrence of CPE. Most importantly, when cells were transfected with a cDNA encoding the temperature-sensitive M protein of tsG33, no CPE was observed at the nonpermissive temperature. However, when these cells were shifted to the permissive temperature (32 degrees C), they rounded up and detached from the dish. These results demonstrate that M protein in the absence of the other viral proteins causes rounding of the cells, probably through a disorganization of the cytoskeleton. The absence of CPE at the nonpermissive temperature is correlated with an abnormal dotted staining pattern of M in these cells, suggesting that the mutant M protein may self-aggregate or associate with membranes rather than interact with cytoskeletal elements.

Animals↗
PubMed1987

Two distinct classes of prestalk-enriched mRNA sequences in Dictyostelium discoideum.

We have isolated cDNA clones derived from three mRNA sequences which are inducible by DIF, the putative stalk-specific morphogen of Dictyostelium. The three mRNA sequences are selectively expressed in cells on the stalk cell pathway of differentiation and we have compared them with previously characterized prestalk-enriched mRNA sequences. We find these latter sequences are expressed without a dependence on DIF, are much less highly enriched in prestalk over prespore cells and are expressed earlier during development than the DIF-inducible mRNA sequences. We propose two distinct mechanisms whereby a mRNA may become enriched in prestalk cells. An apparently small number of genes, represented by those we have isolated, is inducible by DIF and accumulates only in prestalk cells. We suggest that a second class of prestalk-enriched mRNA sequences are induced by cAMP to accumulate in all cells during aggregation and then become enriched in prestalk cells by selective loss from prespore cells.

Base Sequence↗
PubMed1992

Characterization of in vitro translated human mineralocorticoid receptor. Structure and activation.

The structures of the unactivated and activated mineralocorticoid receptors have been difficult to characterize because of receptor lability and steroid dissociation. Therefore, human mineralocorticoid receptor mRNA was translated in rabbit reticulocyte lysate in the presence and absence of [35S]methionine to compare the structure of [3H]aldosterone-bound and [35S]labeled receptor. In vitro synthesized receptor was able to specifically bind [3H]aldosterone. Unactivated [3H]aldosterone-bound and 60% of unactivated [35S]labeled receptor eluted from DEAE-cellulose with 250 mM salt and had a Rs of 72A. Forty percent of unactivated [35S]labeled receptor eluted from DEAE-cellulose with 100 mM salt and had a Rs of 54A. SDSPAGE showed intact hMR was present in both DEAE-cellulose eluates as three bands between M(r) 110,000-120,000. However, the low salt eluate contained less intact receptor and more lower MW bands. Neither [3H]aldosterone-bound nor [35S]labeled receptor was activated by incubation at 25 degrees C as readily as glucocorticoid receptor studied under identical conditions. Activated [3H]aldosterone-bound receptor eluted from DEAE-cellulose at 100 mM salt and had a Rs of 37A. After activation, 60% of [35S]labeled receptor eluted from DEAE-cellulose with 100 mM salt and had a Rs of 91A. SDS-PAGE of the high and low salt DEAE-cellulose eluates showed that 50% of intact receptor eluted in the low salt peak after activation. These data indicate that: 1. Some in vitro synthesized mineralocorticoid receptor assembles into the heteromeric unactivated form; 2. The remaining intact receptor remains monomeric and unable to bind steroid; 3. Activation causes dissociation of intact receptor from a larger complex; and 4. Activated receptor tends to aggregate.

Aldosterone↗
PubMed2026

Lipid-nanoparticle-mediated base editing of the trabecular meshwork rescues glaucoma in vivo.

Mutations in MYOC, the most common genetic cause of glaucoma, cause misfolded myocilin to accumulate in the endoplasmic reticulum (ER), leading to trabecular meshwork (TM) dysfunction, elevated intraocular pressure, and progressive vision loss. While gene editing offers curative potential, current delivery methods rely on viral vectors, which are limited by inflammation, off-target effects, and poor translatability. Here, we report a nonviral lipid nanoparticle (LNP) platform that enables selective in vivo delivery of mRNA encoding an adenine base editor and single guide RNA (LNP-ABE) to TM cells. A direct comparison of LNP-mCherry with lentiviral GFP revealed that LNPs outperform viral vectors, achieving markedly higher efficiency and greater selectivity for the TM without inducing ocular inflammation. In a Cre-inducible Tg.CreMYOCY437H glaucoma mouse model, LNP-Cre mRNA selectively induced mutant MYOC expression in the TM, faithfully recapitulating key disease features. A single administration of LNP-ABE achieved efficient on-target editing of mutant MYOC, reducing mutant myocilin protein by approximately 46%, decreasing aggregates, alleviating ER stress, and fully rescuing the glaucomatous phenotype in Tg.CreMYOCY437H mice. Importantly, no off-target editing or ocular toxicity was detected. These findings establish LNP-based mRNA delivery as a safe, efficient, and clinically translatable approach for TM-targeted genome editing with broad therapeutic potential in glaucoma.

Animals↗
PubMed2001

Proteome analysis of rat spermatogonia: reinvestigation of stathmin spatio-temporal expression within the testis.

Stathmin is a protein known to be involved in various cell processes including cell proliferation and differentiation. It has already been described in the testis but its recent identification using a proteomic approach in mitotic spermatogenetic stem cells named spermatogonia (Guillaume et al., 2000) has lead us to reinvestigate its expression within the testis. Stathmin and its mRNAs were studied in isolated cells by Western and Northern blots and in situ using immunohistochemistry. We demonstrated that stathmin is indeed expressed in spermatogonia, and that it is also intensively expressed in the meiotic spermatocytes and in the first generations of spermatids. Furthermore, we showed aggregations of the protein in the cytoplasm of the later generations of spermatids preceding its elimination at the time of spermiation. Our Northern blots reveal the presence of two stathmin transcripts of 1.1 and 3.2 kb within the testis from the fetal stage onwards, in spermatogonia, spermatocytes, and spermatids. However, the 3.2 kb RNA transcript was barely detectable in the spermatids. Stathmin expression is known to be associated with microtubule dynamics. Therefore, its expression in the germ line is most probably related to the extremely complex structural cellular rearrangements occurring in germ cells during spermatogenesis. However, the exact role of stathmin and the reason of the existence of two transcripts in the male germ lineage awaits further investigation.

Animals↗
PubMed1992

Differential antisense transcription from the Dictyostelium EB4 gene locus: implications on antisense-mediated regulation of mRNA stability.

The 2.2 kb mRNA of the Dictyostelium discoideum prespore gene EB4-PSV is constitutively transcribed during growth and development, but the message is only accumulated when cells form aggregates and establish the prespore-prestalk pattern. Disruption of the pattern by mechanical disaggregation results in a rapid loss of the mRNA, while transcription remains nearly unchanged. In early development and after disaggregation, when the mRNA is unstable, a 1.8 kb antisense transcript originating from the same gene locus is detected. This RNA has apparently no coding capacity and is transcriptionally regulated by a promoter located within the translated region of the gene. Excess transcription of antisense RNA in vegetative cells and after disaggregation suggests its involvement in the control of mRNA stability. In agreement with this assumption, the inhibition of RNA synthesis during disaggregation prevents destabilization of the mRNA. This stability regulation of an endogenous mRNA is reminiscent of the loss of specific mRNAs in cells transformed with antisense constructs.

Animals↗
PubMed1965

THE ISOLATION AND PROPERTIES OF CARDIAC RIBOSOMES AND POLYSOMES.

1. A method is described by which good yields of ribosomes and polysomes free of contamination by submitochondrial fragments can be prepared from rat cardiac muscle. These preparations are capable of incorporation of amino acids into protein in vitro. 2. The ribosome preparation consists of 32% of monomeric ribosomes and 68% of ribosomal aggregates or polysomes. The polysome preparation has a decreased monomeric content. Dimers, trimers, tetramers, pentamers and larger components can be differentiated. 3. The polysome aggregate structure is degraded to monomeric ribosomes on incubation with small amounts of ribonuclease or by preparation in the absence of Mg(2+) ions. The degradation in the absence of Mg(2+) ions was not reversible and drastically decreased the incorporation of amino acids in vitro. 4. The cardiac ribosomes contained two major RNA species sedimenting at 19s and 28s in a 1:2.4 ratio. 5. The RNA/protein ratio of cardiac ribosomes and polysomes was consistently lower than that of similar preparations from liver. The concentrations of Na(+) and K(+) ions present during preparation had a great effect on the RNA/protein ratio. 6. Optimum conditions for the incorporation of amino acids into protein in vitro are reported. Cardiac ribosomes have a lower rate of incorporation of amino acids in vitro than liver ribosomes. 7. Heart cell sap is less active than liver cell sap: evidence is presented that a factor, present in liver cell sap and concerned with stimulating the synthesis of the peptide chain, is lacking in heart cell sap. 8. Pulse-labelling of perfused hearts followed by examination of the subcellular structures showed that the ribosomal fraction was the most active in the incorporation of amino acids in vitro.

Amino Acids↗
PubMed2006

Molecular pathways that influence human tau-induced pathology in Caenorhabditis elegans.

Mutations in the gene encoding tau cause frontotemporal dementia with parkinsonism--chromosome 17 type (FTDP-17). In FTDP-17, Alzheimer's disease, and other tauopathies, aggregated hyper-phosphorylated tau forms the neurofibrillary tangles characteristic of these disorders. We previously reported a Caenorhabditis elegans model for tauopathies using human normal and FTDP-17 mutant tau as transgenes. Neuronal transgene expression caused insoluble phosphorylated tau accumulation, neurodegeneration and uncoordinated (Unc) movement. Here we describe a genome-wide RNA-mediated interference (RNAi) screen for genes that modify the tau-induced Unc phenotype. We tested RNAi sequences for 16,757 genes and found 75 that enhanced the transgene-induced Unc phenotype. Forty-six of these genes have sequence similarity to known human genes and fall into a number of broad classes including kinases, chaperones, proteases and phosphatases. The remaining 29 modifiers have sequence similarity only with other nematode genes. To determine if the enhancers are specific for the tau-induced Unc behavior, we exposed several non-tau Unc mutants to tau RNAi enhancer clones. Fifteen enhancers modified phenotypes in multiple Unc mutants, whereas 60 modified only the Unc phenotype in the tau transgenic lines. We also introduced the tau transgene into the background of genetic loss-of-function mutations for a subset of the enhancer genes. Tau transgenic animals homozygous for loss of these enhancer genes exhibited increased impaired motility relative to the tau transgene line alone. This work uncovers novel candidate genes that prevent tau toxicity, as well as genes previously implicated in tau-mediated neurodegeneration.

Animals↗
PubMedDate not supplied

[Interaction of oligoadenylic acids with repolymerized protein of tobacco mosaic virus].

Interaction of oligoadenylic acids (pA) n, n = 3 to 6, with the protein of tobacco mosaic virus has been studied by methods of equilibrium dialysis, gel filtration, and precipitation of the complex by centrifugation. It has been found that oligo(A) forms specific complexes with virus-like helical protein aggregates (HPA), but does not interact neither with double disks nor other small protein aggregates. Interaction becomes stronger when pH and/or ionic strength are decreased. The binding of (pA)3 to HPA decreases with temperature, whereas the binding of longer oligonucleotides increases. The binding constants for the interaction of (pA)3 with HPA in 0,1 M acetate pH 5.50 have been found to be equal 510 +/- 70 at 30 degrees C and 1960 +/- 250 at 0 degrees C. In the case of (pA)3 the binding equilibrium is reached within minutes at T greater than or equal to 20 degrees C; 15 h is needed for the same at 0 degrees C. The binding of oligonucleotides containing four or more residues proceeds at a much lower rate. For instance, in the case of (pA)5 the equilibrium is not reached even within 200 hours at 0 degrees C or 48 hours at 30 degrees C. Two possible mechanisms of interaction are discussed: (1) direct interaction of oligonucleotides with the inner part of protein aggregate through the central hole of the particle due to the fluctuational opening of the V-helices (2) binding of oligonucleotides on the end surfaces of HPA followed by their redistribution along the particle due to the end-to-end association and disruption of particles. A hypothesis concerning the phasing of RNA in the initiation complex is advanced making use of the data of J.J. Steckert and T. M. Schuster.

Adenine Nucleotides↗
PubMed1981

Chemical transformation of cultured skin fibroblasts from humans genetically predisposed to cancer.

Adenomatosis of the colon and rectum (ACR) is an inherited form of cancer. Assuming that phenotypic expressions that appear in cell strains reflect its biological abnormalities, the study of cultured skin fibroblasts derived from individuals with an inherited form of cancer such as ACR provides a unique study for analysis of the oncogenic process. Growth disorders and increased susceptibility to tumor promoters and to transformation by an oncogenic RNA tumor virus have been demonstrated in these skin fibroblasts. We found that human skin fibroblasts (PF) derived from ACR individuals were sensitive to a chemical carcinogen. Cells treated only with various levels of N-methyl-N'-nitro-N-nitrosoguanidine (MNNG) underwent morphological alteration. The morphologically altered cells formed large cell aggregates when suspended in liquid growth medium above an agar base and grew to high saturation densities but did not form colonies in soft agar. Transformed cells were resistant to rechallenge of MNNG (1 microgram/ml) and showed prolonged life span compared to those untreated cells. However, no tumors were produced when cells were inoculated subcutaneously into nude mice. Data suggest that neoplastic transformation of these skin cells by chemical carcinogens is a multi-phase process.

Cell Transformation, Neoplastic↗
PubMed1991

Ultrastructural localization of defined sequences of viral RNA and DNA by in situ hybridization of biotinylated DNA probes on sections of herpes simplex virus type 1 infected cells.

The influence of fixation and enzymatic digestions on the ability of a denatured double-stranded DNA probe to bind specifically to related sequences of RNA and DNA in sections of Lowicryl embedded cells was investigated. Specificity of the hybridization was assessed using a biotinylated cloned subgenomic herpes simplex virus type 1 DNA fragment to localize viral nucleic acids in sections of infected cells. The probe was detected by anti-biotin antibodies and indirect immunogold labeling. Controls indicated that protease digestion of proteins from the section eliminated non-specific binding of the probe and labeling of endogenous biotin. Both formaldehyde and glutaraldehyde fixation retained viral RNA in protease digested sections. Its labeling was randomly and sparsely distributed over the fibrillo-granular network of the infected nucleus and over the ribosome-rich regions of cytoplasm. Labeling of single-stranded portions of viral DNA in protease-RNase digested sections was infrequent. It was located precisely over nucleoids of a few viral nucleocapsids whatever their location in the cell and their stage of maturation. Labeling of double-stranded viral DNA by denaturation of the DNA in the sections of Lowicryl embedded cells was possible after fixation with formaldehyde but not glutaraldehyde. Among several denaturation protocols, 0.5 N NaOH treatment was best for hybridization of both non-encapsidated and encapsidated viral DNA in protease-RNase digested sections. Free viral genomes were detected exclusively within the virus-replicating region of infected nuclei. Labeling of viral nucleoids was independent of their location in the cell. The high percentage of labeled viral nucleoids suggests that the related viral DNA sequence is not aggregated in the nucleoid but is extended and therefore numerous portions of this defined DNA sequence are accessible at the surface of the section for the binding of the probe.

Animals↗