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Characterization of the antibody response in vaccinated mice protected against Coxsackievirus B3-induced myocarditis.

Adolescent male CD-1 mice can be rendered resistant to coxsackievirus B3 (CVB3m)-induced myocarditis following inoculation as neonates with a single dose of a temperature-sensitive mutant virus (ts1), derived from the prototype parent virus (CVB3m). Previously, anti-CVB3 neutralizing antibodies were not detected in sera of adolescent ts1 vaccinees by a standard plaque-reduction assay (Gauntt et al 1983. Infect. Immun. 39:851). However, a more sensitive cytopathic effects-reduction assay permitted detection of low titers of anti-CVB3m neutralizing antibodies of the IgG class prior to challenge with CVB3m. Following CVB3m challenge, serum anti-CVB3m neutralizing antibody titers of ts1 vaccines declined on days 1-2 post-inoculation (p.i.) then increased over the next 6 days. The neutralizing antibodies were of both the IgG and IgM classes. Normal mice challenged with CVB3 did not produce detectable serum anti-CVB3m neutralizing antibody until day 4 p.i. and by 8 days p.i. the neutralizing antibody was only of the IgM class. Thus, adolescent murine ts1 vaccines mount a secondary antibody response to CVB3m in both neutralizing IgG and IgM, but resistance to CVB3m-induced myocarditis is due to the presence of low levels of anti-CVB3m IgG neutralizing antibody in serum at the time of challenge with CVB3m.

Animals↗
PubMed1994

A temperature-sensitive mutation of the temperature-regulated SerH3 i-antigen gene of Tetrahymena thermophila: implications for regulation of mutual exclusion.

The Ser genes of Tetrahymena thermophila specify alternative forms of a major cell surface glycoprotein, the immobilization or i-antigen (i-ag). Regulation of i-ag expression assures that at least one i-ag gene is expressed at all times. To learn more about the regulatory system and the possible role of i-ag itself, we studied SerH3-ts1, a temperature-sensitive allele of the temperature-regulated SerH3 gene normally expressed from 20-36 degrees. In homozygotes grown at the nonpermissive temperature (> 32 degrees), H3 is not present on the cell surface, but the gene continues to be transcribed until its 36 degrees cutoff. H3 formed at the permissive temperature is stable at nonpermissive temperatures, indicating that SerH3-ts1 is temperature-sensitive for synthesis rather than function. At nonpermissive temperatures, the S i-ag is expressed in place of H3. This result suggests that normal H protein may play a role in regulating S expression. SerH3-ts1 was isolated following mutagenesis with N-methyl-N'-nitro-N-nitrosoguanidine (MNNG). Sequencing of SerH3-ts1 revealed a single A --> G transition at nucleotide 473, resulting in the substitution of glycine for aspartate. The affected residue is conserved in the internal repeats comprising the H protein, and the charge difference correlates with changes in electrophoretic mobility of the H3 protein.

Animals↗
PubMed2005

Thermococcus coalescens sp. nov., a cell-fusing hyperthermophilic archaeon from Suiyo Seamount.

A cell-fusing hyperthermophilic archaeon was isolated from hydrothermal fluid obtained from Suiyo Seamount of the Izu-Bonin Arc. The isolate, TS1(T), is an irregular coccus, usually 0.5-2 microm in diameter and motile with a polar tuft of flagella. Cells in the exponential phase of growth fused at room temperature in the presence of DNA-intercalating dye to become as large as 5 microm in diameter. Fused cells showed dark spots that moved along in the cytoplasm. Large cells with a similar appearance were also observed upon culture at 87 degrees C, suggesting the occurrence of similar cell fusions during growth. Transmission electron microscopy revealed that cells in the exponential phase possessed a thin and electron-lucent cell envelope that could be lost subsequently during culture. The fragile cell envelope must be related to cell fusion. The cells grew at 57-90 degrees C, pH 5.2-8.7 and at NaCl concentrations of 1.5-4.5 %, with the optima being 87 degrees C, pH 6.5 and 2.5 % NaCl. The isolate was an anaerobic chemo-organotroph that grew on either yeast extract or tryptone as the sole growth substrate. The genomic DNA G+C content was 53.9 mol%. Phylogenetic analysis based on 16S rRNA gene sequencing indicated that the isolate was closely related to Thermococcus species. However, no significant DNA-DNA hybridization was observed between genomic DNA of strain TS1(T) and phylogenetically related Thermococcus species. We propose that isolate TS1(T) represents a novel species, Thermococcus coalescens sp. nov., with the name reflecting the cell fusion activity observed in the strain. The type strain is TS1(T) (=JCM 12540T=DSM 16538T).

Base Composition↗
PubMed1988

Characterization of efferent T suppressor cells induced by Paracoccidioides brasiliensis-specific afferent T suppressor cells.

Previously, we reported that Paracoccidioides brasiliensis culture filtrate antigen (Pb.Ag) when injected i.v. into mice induces antigen-specific suppressor cells which down-regulate the anti-P. brasiliensis delayed-type hypersensitivity (DTH) response. The suppressor cells are present in both spleens and lymph nodes of Pb.Ag-treated animals and suppress the afferent limb but not the efferent limb of the DTH response to P. brasiliensis. The suppressor cells induced by Pb.Ag are L3T4+ Lyt-1+2- I-J+ T cells and are considered to be equivalent to the Ts1 cells described for other antigen-specific suppressor cell pathways. This report provides data which show that Ts1 cells induced by Pb.Ag or a soluble factor derived from Ts1 cells (TsF1) stimulates the production of second-order or efferent suppressor cells. The second-order suppressor cells are detectable in spleens and lymph nodes of mice 7 days after injection of Ts1 cells or TsF1 and are specific in suppressing the paracoccidioidal DTH response. In addition, the second-order suppressor cells are T cells with an L3T4- Lyt-2+ I-J+ phenotype and are effective in suppressing only the efferent limb of the P. brasiliensis DTH response. On the basis of the characteristics defined in this study, the paracoccidioidal second-order suppressor cells are equivalent to the Ts2 cells described for other antigen-specific suppressor-cell pathways. Thus, the suppressive circuit induced by Pb.Ag is similar to the suppressor-cell pathways that regulate the DTH responses to azobenzenearsonate, 4-hydroxy-3-nitrophenyl acetyl, lysozyme, and Cryptococcus neoformans antigen. We propose that such a suppressor-cell circuit as defined here with the murine model could be responsible for the depressed cell-mediated immune responses observed in paracoccidioidomycosis patients who have antigen circulating in their sera.

Animals↗
PubMed2006

Trapping of a spiral-like intermediate of the bacterial cytokinetic protein FtsZ.

The earliest stage in bacterial cell division is the formation of a ring, composed of the tubulin-like protein FtsZ, at the division site. Tight spatial and temporal regulation of Z-ring formation is required to ensure that division occurs precisely at midcell between two replicated chromosomes. However, the mechanism of Z-ring formation and its regulation in vivo remain unresolved. Here we identify the defect of an interesting temperature-sensitive ftsZ mutant (ts1) of Bacillus subtilis. At the nonpermissive temperature, the mutant protein, FtsZ(Ts1), assembles into spiral-like structures between chromosomes. When shifted back down to the permissive temperature, functional Z rings form and division resumes. Our observations support a model in which Z-ring formation at the division site arises from reorganization of a long cytoskeletal spiral form of FtsZ and suggest that the FtsZ(Ts1) protein is captured as a shorter spiral-forming intermediate that is unable to complete this reorganization step. The ts1 mutant is likely to be very valuable in revealing how FtsZ assembles into a ring and how this occurs precisely at the division site.

Amino Acid Sequence↗
PubMed1983

Influenza virus temperature-sensitive cap (m7GpppNm)-dependent endonuclease.

The first step in influenza viral mRNA synthesis is the endonucleolytic cleavage of heterologous RNAs containing cap 1 (m(7)GpppNm) structures to generate capped primers that are 10 to 13 nucleotides long, which are then elongated to form the viral mRNA chains. We examined the temperature sensitivity of these steps in vitro by using two WSN virus temperature-sensitive mutants, ts1 and ts6, which have a defect in the genome RNA segment coding for the viral PB2 protein. For these experiments, it was necessary to employ purified viral cores rather than detergent-treated virions to catalyze transcription, as preparations of detergent-treated virions contain destabilizing or inhibitory activities which render even the transcription catalyzed by wild-type virus temperature sensitive. Using purified wild-type viral cores, we found that the rates of endonucleolytic cleavage of capped primers and of overall transcription were similar at 39.5 and 33 degrees C, the in vivo nonpermissive and permissive temperatures, respectively. In contrast, the activities of the cap-dependent endonucleases of ts1 and ts6 viral cores at 39.5 degrees C were only about 15% of those at 33 degrees C. The steps in transcription after endonucleolytic cleavage of the capped RNA primer were largely, if not totally, temperature insensitive, indicating that the mutations in the PB2 protein found in ts1 and ts6 virions affect only the endonuclease step. The temperature-sensitive defect is most likely in the recognition of the 5'-terminal cap 1 structure that occurs as a required first step in the endonuclease reaction: the cap-dependent binding of a specific capped primer fragment to ts1 viral cores was temperature sensitive under conditions in which binding to wild-type viral cores was not affected by increasing the temperature from 33 to 39.5 degrees C. Thus, our results establish that the viral PB2 protein functions in cap recognition during the endonuclease reaction.

Endoribonucleases↗
PubMed1987

A Podospora anserina longevity mutant with a temperature-sensitive phenotype for senescence.

A Podospora anserina longevity mutant was identified with a temperature-sensitive phenotype for senescence. This mutant, termed TS1, grew for over 3 m at 27 degrees C, but when shifted to 34 degrees C, it underwent senescence between 10 and 18 cm. A previously described senescence-associated plasmid, alpha senDNA, derived from the mitochondrial genome, was not detected in TS1 at 27 degrees C but was present in senescent cultures at 34 degrees C. A similar result was observed in progeny strains obtained by crossing the TS1 mutant with a wild-type strain. Other mitochondrial excision-amplification DNAs in addition to alpha senDNA were also observed in the senescent cultures. Most were derived from a specific region of the mitochondrial genome. These results provide evidence that alpha senDNA is involved in TS1 senescence and suggest that this plasmid may play a role in the formation of other mitochondrial excision-amplification plasmids.

Aging↗
PubMed1999

Zidovudine [AZT] myotoxicity: quantitative separation of AZT effects on proliferation and differentiation of muscle cells in vitro. Lack of myotoxicity potentiation by retrovirus.

We compared quantitatively the myotoxicity of 3'-azido-2',3'-dideoxythymidine (AZT) against uninfected and ts1 retrovirus infected mouse skeletal muscle (ATCC CRL 1772) cells at different stages of maturation in vitro. The AZT half inhibitory concentration (IC50) for myoblast proliferation was determined for uninfected myoblasts and parallel cultures infected with ts1 virus. The AZT IC50 for muscle cell differentiation was determined in cultures where myoblasts were grown to confluence and then changed to the fusion medium to which AZT was added at increasing concentrations. Creatine kinase activity was used as a marker of muscle cell differentiation and was determined in homogenates after 7 days. Total cellular mitochondrial DNA was analyzed by Southern blotting. The estimated AZT IC50 for muscle cell proliferation (2-5 microM) was significantly less than the AZT IC50 for muscle cell differentiation (100 microM). Infection with ts1 retrovirus did not significantly shift the IC50 for either proliferation or differentiation of muscle cells. Toxic concentrations of AZT did not cause selective depletion of mitochondrial DNA. The myotoxic effects of AZT on myoblast proliferation and muscle cell differentiation in vitro were quantitatively different and were not changed by productive ts1 retrovirus infection of muscle cells. These results suggest that AZT may impair muscle fiber regeneration in the course of retrovirus associated myopathy. The mechanism of AZT myotoxicity was not explained by alterations in total mitochondrial DNA content.

Animals↗
PubMed1999

Thrombospondin-1 acts via IAP/CD47 to synergize with collagen in alpha2beta1-mediated platelet activation.

Integrin-associated protein (IAP; or CD47) is a receptor for the cell binding domain (CBD) of thrombospondin-1 (TS1). In platelets, IAP associates with and regulates the function of alphaIIbbeta3 integrin (Chung et al, J Biol Chem 272:14740, 1997). We test here the possibility that CD47 may also modulate the function of platelet integrin alpha2beta1, a collagen receptor. The CD47 agonist peptide, 4N1K (KRFYVVMWKK), derived from the CBD, synergizes with soluble collagen in aggregating platelet-rich plasma. 4N1K and intact TS1 also induce the aggregation of washed, unstirred platelets on immobilized collagen with a rapid increase in tyrosine phosphorylation. The effects of TS1 and 4N1K on platelet aggregation are absolutely dependent on IAP, as shown by the use of platelets from IAP-/- mice. Prostaglandin E1 (PGE1) prevents 4N1K-dependent aggregation on immobilized collagen but does not inhibit the 4N1K peptide stimulation of alpha2beta1-dependent platelet spreading. Finally, a detergent-stable, physical association of IAP and alpha2beta1 integrin is detected by coimmunoprecipitation. These results imply a role for IAP and TS1 in the early activation of platelets upon adhesion to collagen.

Alprostadil↗
PubMed1992

Ocular infection with a murine neurovirulent retrovirus does not cause retinal degeneration.

The developing eyes of CFW/D mice inoculated at birth with a neurovirulent mutant (ts1) of Moloney murine leukemia virus (MoMuLV), nonneurovirulent wild type (wt) MoMuLV, and conditioned virus-free medium were studied comparatively by immunohistochemistry, lectin histochemistry and light microscopy. Cellular targets for viral antigen expression in the eye were identical in both ts1 and wt MoMuLV-infected mice. Viral antigen first was observed in endothelial cells of the retina and subsequently spread in a spatial and temporal pattern consistent with normal vascularization of the developing retina. The virus also was observed in (1) epithelial cells of the bulbar and palpebral conjunctiva, ora ciliaris retinae, and lacrimal gland; (2) endothelial cells of the ciliary body, iris, choroid, and sclera; (3) amacrine cells of the retina; and (4) smooth muscle cells and endothelia of the periocular muscle. Although ts1 MoMuLV induced a spongiform encephalopathy in the brain and spinal cord, structural lesions were not observed in the retina or other ts1 MoMuLV-infected ocular structures; differentiation of the retina was normal. The lectin Ricinus communis agglutinin-I (RCA-I) labeled (1) endothelial cells of the hyaloid vessels, tunica vasculosa lentis, retina, ciliary body, iris, choroid, and sclera; (2) epithelial cells of the cornea, bulbar and palpebral conjunctiva, ora ciliaris retinae, and lacrimal gland; (3) smooth muscle cells and endothelia of the periocular muscle; (4) inner segments of the photoreceptor layer; and (5) amacrine cells of the retina.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1985

Regulation of cell-mediated immunity in cryptococcosis. III. Characterization of second-order T suppressor cells (Ts2).

Frequently, patients with systemic cryptococcosis have depressed or absent in vivo or in vitro cell-mediated immune responses to cryptococcal antigen. In addition, these patients have relatively high levels of circulating cryptococcal antigen, which is generally considered indicative of a poor prognosis. Because acquired cell-mediated immunity is an important host defense mechanism in cryptococcosis, we have been studying the effects of cryptococcal antigen on such responses. Using a murine model, we have demonstrated that cryptococcal antigen given i.v. to CBA/J mice, to simulate antigen levels found in patients, can trigger the production of a series of suppressor cells that specifically inhibit anticryptococcal cell-mediated immune responses. Briefly, cryptococcal antigen induces afferent suppressor and/or suppressor inducer (Ts1) cells in the lymph nodes of mice, and the Ts1 cells, or a soluble factor derived therefrom (TsF1), stimulate the production of second-order or efferent suppressor (Ts2) cells. This report provides data that demonstrate that the Ts2 cells were induced in spleens from cyclophosphamide (Cy)-resistant precursors in response to cryptococcal Ts1 cells or TsF1 and that a proliferative process sensitive to Cy was not required for Ts2 induction. The Ts2 cells were detectable 6 days after Ts1 injection, were specific in suppressing the cryptococcal delayed-type hypersensitivity (DTH) response, were T cells with an Lyt-1-,2+,Ia+ (I-J+) phenotype, were effective only on the efferent limb of the cryptococcal DTH response, and mediated their activity via a soluble factor (TsF2). Thus far, the suppressive pathway induced by cryptococcal antigen has characteristics more like those of the suppressor circuits described for the DTH responses to the haptens azobenzenearsonate and 4-hydroxy-3-nitrophenyl acetyl than those of the modulating circuits induced by other antigens. We postulate that a suppressive circuit similar to the one we have defined in the mouse model is operating to suppress the in vivo and in vitro cell-mediated immune responses in cryptococcosis patients who have significant amounts of circulating cryptococcal antigen.

Animals↗
PubMed1995

A role for the intermediate affinity IL-2R in the protection against glucocorticoid-induced apoptosis.

Recent work has shown that T lymphocytes undergo apoptosis upon treatment with the glucocorticoid analogue dexamethasone. These cells can be protected from the effect of dexamethasone by interleukin-2 (IL-2) or IL-4. We were interested in analysing whether a transfected cell dependent on three different lymphokines could be protected by them from the effect of dexamethasone. In addition, we took advantage of our cellular system, in which we expressed intermediate- or high-affinity IL-2R independently, to analyse the role of these receptors in the protection from glucocorticoid-induced apoptosis. In this report we show that IL-2 rescues murine T cells expressing exogenous intermediate- (TS1 beta) or high-affinity (TS1 alpha beta) IL-2 receptor (IL-2R) from dexamethasone-induced apoptosis. This result suggests that intermediate-affinity IL-2R alone can replace high-affinity IL-2R for the protection from the effect of dexamethasone. In addition, IL-4 and IL-9 are rescue-factors, as well as IL-2, of glucocorticoid-treated TS1 beta and TS1 alpha beta cells. Our data suggest that the presence of the alpha-chain of the IL-2R is not required for rescue by IL-2 from the effect of dexamethasone. In addition, we show that proliferation is not required for preventing glucocorticoid-induced apoptosis. This result implies a new role for the intermediate-affinity IL-2R.

Animals↗
PubMed1993

Temporal central and peripheral nervous system changes induced by a paralytogenic mutant of Moloney murine leukemia virus TB.

BACKGROUND: The temporal localization of cellular targets for viral replication and the morphopathogenesis of neurodegeneration in the central nervous system (CNS) and peripheral nervous system induced by ts1, a neuropathogenic and lymphocytopathic mutant of Moloney murine leukemia virus-TB, were studied in the highly susceptible FVB/N mouse strain in order to better understand the mechanisms of this neurodegenerative disease. EXPERIMENTAL DESIGN: Newborn FVB/N mice were inoculated intraperitoneally with 0.1 ml of viral suspension containing 10(6) to 10(7) infectious units/ml. The mice were observed daily for clinical signs of disease and killed at specific time points. Their nervous system tissues were collected and processed for light and electron microscopy and for immunohistochemical viral-antigen detection. RESULTS: ts1-Infected FVB/N mice developed a rapidly progressive wasting disease that culminated in hindleg paralysis or paraplegia 30 to 35 days postinoculation (pi). CONCLUSIONS: Clear evidence of CNS lesions involving the cerebellar ventricular system, the grey and white matter of the brain stem and the spinal cord were seen as early as 5 to 10 days pi. These lesions, which began as mild perivascular and paraventricular neuropil spongiform changes and cytoplasmic vacuolation of neuronal and glial cell processes, progressed in severity with time and culminated in almost complete destruction of the white and gray matter in the brain stem and the cervical and lumbar spinal cord. Viruses were detected as early as 5 to 10 days pi in the fourth ventricle choroid plexus and ventricular lumen and budding from endothelial cells within the brain stem and cerebellum. Endothelial, ependymal, microglial, astroglial, and oligodendroglial cells were positive for gp70env. Astroglial and microglial cell proliferation with microglial syncytia formation was detected only within the areas showing spongiform degeneration. Viral replication was consistently high in the capillary endothelial cells of those areas showing spongiform degeneration, whereas in the glial cells, relatively few budding viruses were present. Neurodegeneration was accompanied by demyelinization within the CNS and peripheral nervous system and by hindleg muscle degeneration and necrosis. Multiple cellular targets for ts1 viral infection and replication were detected within the nervous system. The presence of budding virus and the immunodetection of viral antigen in the choroid plexus and ependymal cells of the fourth ventricle and the central canal of the spinal cord demonstrated that cerebrospinal fluid as well as blood can disseminate virus within the CNS. Pathologic and functional changes within the blood-brain barrier and glial system probably account for the neuronal necrosis and spongiform changes that result in paralysis induced by ts1 infection.

Animals↗
PubMed1997

Radioimmunoscintigraphy with a novel monoclonal antiprostate antibody (E4): an experimental study in nude mice.

BACKGROUND: Prostate cancer is one of the leading causes of death among men, despite achievements in diagnosis and therapy. Radioimmunolocalization and radioimmunotherapy of malignant tumors have demonstrated increasing potential and may become useful tools in the management of prostate cancer. METHODS: Nude mice were inoculated subcutaneously with cells from the poorly differentiated human prostate cancer cell line DU-145. The intact monoclonal antibody (MoAb) E4 and an intact anticytokeratin-8 MoAb, TS1, used for comparison were labeled with 125I and injected intraperitoneally (i.p.) in the mice. Repetitive quantitative scintigraphic recordings were performed during 1 month. The mice were killed at Day 29 after injection of the radiolabeled MoAb. The tumors and the organs were dissected and weighed. The remaining activity was measured in a gamma well counter. One part of the tumor was immediately fixed in Bouin's solution for autoradiography and the other in formaldehyde for microscopy. RESULTS: The study demonstrated significant radioimmunolocalization of the MoAb E4 into the DU-145 prostate tumor tissue in the animal model, with an average radiation dose of 0.08 Gy/MBq in the tumor. TS1 localized preferentially in necrotic parts of the tumor, yielding a tumor dose of 0.02 Gy/MBq. CONCLUSIONS: The MoAb E4 is a promising radiotracer for prostate cancer and may be used in radioimmunotherapy. As in earlier studies, TS1 shows significant radioimmunolocalization into necrotic tumor tissue, which also exists in prostate cancer.

Animals↗
PubMed2002

The combination of external beam radiotherapy and experimental radioimmunotargeting with a monoclonal anticytokeratin antibody.

BACKGROUND: Doses to tumors of up to 80 grays (Gy) have been postulated to eradicate solid experimental tumors with radiommunotargeting, but this value has proved difficult to reach. Combining two treatment modalities, external beam radiotherapy and radioimmunotargeting, could potentially give rise to a number of advantages. METHODS: The purpose of this study was to detect potential benefits with different treatment timing strategies when combining external beam radiotherapy and radioimmunotargeting, with the anticytokeratin monoclonal antibody (MAb) TS1 injected into a nude mouse model carrying subcutaneous human HeLa Hep 2-cell tumors. Cytokeratins are present in necrotic regions within tumors, thereby providing a potential increase in binding sites for TS1 if combined with external beam radiotherapy. External beam radiotherapy was given before, after, and simultaneously with injection of radiolabeled MAb. RESULTS: The highest yields in terms of total accumulated dose (Gy), percentage of injected activity per gram of tumor tissue, and accumulated dose per injected activity (Gy/MBq) were seen in the group receiving external beam radiotherapy prior to MAb-injection. CONCLUSIONS: Enhanced effects may be achievable by combining external beam radiotherapy with experimental radioimmunotargeting using the monoclonal anticytokeratin antibody TS1, if the radiotherapy is given prior to MAb injection.

Animals↗
PubMed1986

Mutations blocking the transport of the influenza virus hemagglutinin between the rough endoplasmic reticulum and the Golgi apparatus.

Mutants ts1 and ts227 of fowl plague virus have a temperature-sensitive defect in the transport of the hemagglutinin from the rough endoplasmic reticulum to the Golgi apparatus. The primary structure of the hemagglutinin of the mutants and of a number of revertants derived from them has been analysed by nucleotide sequencing. The transport block of the hemagglutinin of ts227 can be attributed to a single amino acid exchange. It involves the replacement of aspartic acid at position 457 by asparagine thereby introducing a new glycosylation site which appears to be located in a cryptic position in the lower part of the hemagglutinin stalk. Attachment of carbohydrate to this site is temperature-dependent. At permissive temperature only a small fraction of the monomers (approximately 30%) is glycosylated in this position, whereas at nonpermissive temperature this is the case with all subunits. The data suggest that under the latter conditions the new oligosaccharide interferes by steric hindrance with the trimerization of the hemagglutinin. The hemagglutinin of ts1 has an essential amino acid exchange at position 275 where serine is replaced by glycine. This substitution may increase the flexibility of the molecule in the hinge region between the globular domain and the stalk. The exchange of a conserved glutamic acid residue at position 398 that is involved in the interaction between different monomers contributes also to the structural instability of the ts1 hemagglutinin. These observations support the notion that the transport of the hemagglutinin from the rough endoplasmic reticulum to the Golgi apparatus depends on trimer assembly.

Animals↗
PubMed1986

Suppression of defective RAS1 and RAS2 functions in yeast by an adenylate cyclase activated by a single amino acid change.

We have constructed the yeast strain TS1, with the RAS2 gene replaced by mutant allele encoding a partially defective gene product, and with an inactive RAS1 gene. TS1 cells accumulate as unbudded cells upon temperature shift from 30 to 37 degrees C, thus showing that the RAS1 and RAS2 gene functions are important for progression through the G1 phase of the cell cycle. After the isolation of revertants able to grow at the nonpermissive temperature, we have found that a chromosomal point mutation can bypass the G1 arrest of TS1 and cdc25 cells, and the lethality of ras1 ras2 mutants. The mutation predicts the replacement of threonine by isoleucine at position 1651 of yeast adenylate cyclase. The RAS-independent, as well as the RAS-dependent adenylate cyclase activity, is increased by the mutation. Like the wild-type enzyme, the RAS-dependent activity of the mutant adenylate cyclase is turned on by the GTP-bound form of the RAS2 protein. The amino acid sequence surrounding the threonine 1651 shows similarity with protein kinase substrates. Possible implications for the function of adenylate cyclase are discussed.

Adenylyl Cyclases↗
PubMed1994

Poly(A) site selection in the yeast Ty retroelement requires an upstream region and sequence-specific titratable factor(s) in vitro.

In the Ty retrotransposon of Saccharomyces cerevisiae, as in most retroelements, the polyadenylation site of the 5' long terminal repeat (LTR) is ignored and the one in the 3' LTR is efficiently used. We examine here the contribution to this poly(A) site selection of the region termed 'U3', corresponding to the upstream non-transcribed portion of the 5' LTR. Using an established assay in vitro, we find that 3' processing is accurate and efficient with an RNA substrate corresponding to most of the LTR, whereas none is detectable with a shorter transcript lacking the U3 region, thus explaining why the 5' poly(A) site is ignored in genomic Ty mRNA. When HIS4 coding RNA, representing 'non-specific' sequence, replaces the U3 region, the Ty polyadenylation site is activated to 50% of the wild-type level. Within one specific region (TS1) in U3, 90-95 nt upstream of the poly(A) site, the change of UAGUAU to UCGCAU reduces processing efficiency by half, to the non-specific level provided by other sequences or by a deletion of the TS1 region. Another region (TS2) near the poly(A) site appears to be independently responsible for the remaining half of the processing activity. Alteration of both TS1 and TS2 eliminates processing entirely. In competition assays, excess unlabeled U3, but not its mutated counterparts, reduces the processing of radiolabeled Ty mRNA, suggesting the involvement of some sequence-specific titratable factor(s) in the whole cell extract for U3-specific activation.(ABSTRACT TRUNCATED AT 250 WORDS)

Binding, Competitive↗