GW body disassembly triggered by siRNAs independently of their silencing activity.

GW bodies (or P-bodies) are cytoplasmic granules containing proteins involved in both mRNA degradation and storage, including the RNA interference machinery. Their mechanism of assembly and function are still poorly known although their number depends upon the flux of mRNA to be stored or degraded. We show here that silencing of the translational regulator CPEB1 leads to their disappearance, as reported for other GW body components. Surprisingly, the same results were obtained with several siRNAs targeting genes encoding proteins unrelated to mRNA metabolism. The disappearance of GW bodies did not correlate with the silencing activity of the siRNA and did not inhibit further silencing by siRNA. Importantly, in most cases, GW bodies were rapidly reinduced by arsenite, indicating that their assembly was not prevented by the inhibition of the targeted or off-target genes. We therefore propose that some siRNA sequences affect mRNA metabolism so as to diminish the amount of mRNA directed to the GW bodies. As an exception, GW bodies were not reinduced following Rck/p54 depletion by interference, indicating that this component is truly required for the GW body assembly. Noteworthy, Rck/p54 was dispensable for the assembly of stress granules, in spite of their close relationship with the GW bodies.

Fast modulation of heat-activated ionic current by proinflammatory interleukin 6 in rat sensory neurons.

The pro-inflammatory cytokine interleukin-6 (IL-6) together with its soluble receptor (sIL-6R) induces and maintains thermal hyperalgesia. It facilitates the heat-induced release of calcitonin gene-related peptide from rat cutaneous nociceptors in vivo and in vitro. Here we report that exposure of nociceptive neurons to the IL-6-sIL-6R complex or the gp130-stimulating designer IL-6-sIL-6R fusion protein Hyper-IL-6 (HIL-6) resulted in a potentiation of heat-activated inward currents (I(heat)) and a shift of activation thresholds towards lower temperatures without affecting intracellular calcium levels. The Janus tyrosine kinase inhibitor AG490, the selective protein kinase C (PKC) inhibitor, bisindolylmaleimide 1 (BIM1), as well as rottlerin, a selective blocker of the PKCdelta isoform, but not the cyclooxygenase inhibitor indomethacin, effectively reduced the effect. Reverse transcription-polymerase chain reaction (RT-PCR) and in situ hybridization revealed expression of mRNA for the signal-transducing beta subunit of the receptor gp130 in neuronal somata, rather than satellite cells in rat dorsal root ganglia. Together, the results suggest that IL-6-sIL-6R acts directly on sensory neurons. It increases their susceptibility to noxious heat via the gp130/Jak/PKCdelta signalling pathway.

The translational regulator CPEB1 provides a link between dcp1 bodies and stress granules.

The cytoplasmic polyadenylation element-binding protein (CPEB) has been characterized in Xenopus laevis as a translational regulator. During the early development, it behaves first as an inhibitor and later as an activator of translation. In mammals, its closest homologue is CPEB1 for which two isoforms, short and long, have been described. Here we describe an additional isoform with a different RNA recognition motif, which is differentially expressed in the brain and ovary. We show that all CPEB1 isoforms are found associated with two previously described cytoplasmic structures, stress granules and dcp1 bodies. This association requires the RNA binding ability of the protein, whereas the Aurora A phosphorylation site is dispensable. Interestingly, the rck/p54 DEAD box protein, which is known as a CPEB partner in Xenopus and clam, and as a component of dcp1 bodies in mammals, is also present in stress granules. Both stress granules and dcp1 bodies are involved in mRNA storage and/or degradation, although so far no link has been made between the two, in terms of neither morphology nor protein content. Here we show that transient CPEB1 expression induces the assembly of stress granules, which in turn recruit dcp1 bodies. This dynamic connection between the two structures sheds new light on the compartmentalization of mRNA metabolism in the cytoplasm.

Fast Ca2+-induced potentiation of heat-activated ionic currents requires cAMP/PKA signaling and functional AKAP anchoring.

Calcium influx and the resulting increase in intracellular calcium concentration ([Ca(2+)](i)) can induce enhanced sensitivity to temperature increases in nociceptive neurons. This sensitization accounts for heat hyperalgesia that is regularly observed following the activation of excitatory inward currents by pain-producing mediators. Here we show that rat sensory neurons express calcium-dependent adenylyl cyclases (AC) using RT-PCR and nonradioactive in situ hybridization. Ionomycin-induced rises in [Ca(2+)](i)-activated calcium-dependent AC and caused translocation of catalytic protein kinase A subunit. Elevation of [Ca(2+)](i) finally resulted in a significant potentiation of heat-activated currents and a drop in heat threshold. This was not prevented in the presence of suramin that nonspecifically uncouples G protein-dependent receptors. The sensitization was, however, inhibited when the specific PKA antagonist PKI(14-22) was added to the pipette solution or when PKA coupling to A kinase anchoring protein (AKAP) was disrupted with InCELLect StHt-31 uncoupling peptide. The results show that heat sensitization in nociceptive neurons can be induced by increases in [Ca(2+)](i) and requires PKA that is functionally coupled to the heat transducer, mostly likely vanilloid receptor VR-1. This calcium-dependent pathway can account for the sensitizing properties of many excitatory mediators that activate cationic membrane currents.

Targeting the kinesin Eg5 to monitor siRNA transfection in mammalian cells.

RNA interference, the inhibition of gene expression by double-stranded RNA, provides a powerful tool for functional studies once the sequence of a gene is known. In most mammalian cells, only short molecules can be used because long ones induce the interferon pathway. With the identification of a proper target sequence, the penetration of the oligonucleotides constitutes the most serious limitation in the application of this technique. Here we show that a small interfering RNA (siRNA) targeting the mRNA of the kinesin Eg5 induces a rapid mitotic arrest and provides a convenient assay for the optimization of siRNA transfection. Thus, dose responses can be established for different transfection techniques, highlighting the great differences in response to transfection techniques of various cell types. We report that the calcium phosphate precipitation technique can be an efficient and cost-effective alternative to Oligofectamine in some adherent cells, while electroporation can be efficient for some cells growing in suspension such as hematopoietic cells and some adherent cells. Significantly, the optimal parameters for the electroporation of siRNA differ from those for plasmids, allowing the use of milder conditions that induce less cell toxicity. In summary, a single siRNA leading to an easily assayed phenotype can be used to monitor the transfection of siRNA into any type of proliferating cells of both human and murine origin.

The determination of free and protein-bound haemoglobin in plasma using a combination of HPLC and absorption spectrometry.

The aim of the study was to develop a method for the determination of haemoglobin in plasma suitable for use to set target values for external quality assessment schemes for this analyte using commercially available test kits and equipment. In the early phase of the method development it became clear that the use of a single method, namely HPLC, would not be possible. However, by combining HPLC and absorption spectrophotometry, both qualitative and quantitative rapid determinations of protein-bound and free haemoglobin were able to be performed on equipment present in most routine clinical chemistry laboratories. The separation of protein-bound and free haemoglobin could be carried out using commercial HPLC equipment for the determination of haemoglobin A1c (HbA1c) without modification of the conditions used. Instead of haemolysed blood, the same volume of plasma (10 microl) was injected. The eluate was not discarded, but collected in 1-minute fractions so that the void volume (protein-bound Hb) and the haemoglobin peaks (free Hb) were available for the colorimetric determination of haemoglobin using the pseudoperoxidase activity of the haem moiety on hydrogen peroxide and a chromogen (3,3',5,5'-tetramethylbenzidine) in concentrated acetic acid and optimal determination at 600 nm. (In this publication at 578 nm due to the use of a spectrophotometer with Hg-discharge lamp and filter). The appearance of a blue colour in the reaction tube or cuvette indicated the presence of haemoglobin. The use of the above chromogen, with its absorption maximum around 600 nm excluded interference from serum components such as bilirubin, which may interfere in the conventional method often used to determine plasma haemoglobin. The method can be used quantitatively by including an aqueous human haemoglobin standard in the run. This elutes from the HPLC column only as free haemoglobin in the concentration range from 0.1 to 10 g/l. Addition of human haemoglobin to haemoglobin-free plasma resulted in the binding of all Hb to plasma proteins up to a concentration between 2 and 3 g/l (void-volume fraction). At higher concentrations free Hb appeared in the 3-5 minute fractions. These observations agree with published data on the scavenging capacity of plasma for Hb released from erythrocytes. The method is rapid, (HPLC-run maximally 6 min, quantitative colorimetric results 5-10 min) precise (inter-assay coefficients of variation < 8%) and suitable for answering the question as to whether the protein-binding (scavenging) system which prevents the nephro- and cerebrotoxic effects of haemoglobin has been saturated or not, an important question in patients with acute haemolysis problems. A qualitative result is obtainable within 10 minutes of injecting the sample into the HPLC-system. The use of this assay in controlling blood transfusion and haemolytic events arising from surgery, intravascular haemolytic bacteria or artificial heart valves can help in rapid corrective action, if needed.

The zinc finger transcription factor, MOK2, negatively modulates expression of the interphotoreceptor retinoid-binding protein gene, IRBP.

The human and murine MOK2 orthologue genes encode Krüppel/TFIIIA-related zinc finger proteins, which are factors able to recognize both DNA and RNA through their zinc finger motifs. MOK2 proteins have been shown to bind to the same 18-base pair (bp)-specific sequence in duplex DNA. This MOK2-binding site was found within introns 7 and 2 of human PAX3 and interphotoreceptor retinoid-binding protein (IRBP) genes, respectively. As these two genes are expressed in the brain as MOK2, we have suggested that PAX3 and IRBP genes are two potentially important target genes for the MOK2 protein. In this study, we focused our attention on IRBP as a potential MOK2 target gene. Sequence comparison and binding studies of the 18-bp MOK2-binding sites present in intron 2 of human, bovine, and mouse IRBP genes show that the 3'-half sequence is the essential core element for MOK2 binding. Very interestingly, 8-bp of this core sequence are found in a reverse orientation, in the IRBP promoter. We demonstrate that MOK2 can bind to the 8-bp sequence present in the IRBP promoter and repress its transcription when transiently overexpressed in retinoblastoma Weri-RB1 cells. In the IRBP promoter, it appears that the TAAAGGCT MOK2-binding site overlaps with the photoreceptor-specific CRX-binding element. We suggest that MOK2 represses transcription by competing with the cone-rod homeobox protein (CRX) for DNA binding, thereby decreasing transcriptional activation by CRX. Furthermore, we show that Mok2 expression in the developing mouse and in the adult retina seems to be concordant with IRBP expression.

Involvement of the proinflammatory cytokines tumor necrosis factor-alpha, IL-1 beta, and IL-6 but not IL-8 in the development of heat hyperalgesia: effects on heat-evoked calcitonin gene-related peptide release from rat skin.

Proinflammatory cytokines contribute to the development of inflammatory and neuropathic pain and hyperalgesia in many in vivo models. The rat skin model was used to investigate the effects of proinflammatory cytokines on the basal and heat-evoked release of calcitonin gene-related peptide from nociceptors in vitro. In contrast to the excitatory effects of cytokines observed in vivo, none of the cytokines tested evoked any calcitonin gene-related peptide (CGRP) release at normal skin temperature of 32 degrees C. However, the cytokines IL-1beta, tumor necrosis factor (TNF)-alpha, and IL-6 but not IL-8 induced a pronounced and transient sensitization of the heat-evoked CGRP release from nociceptors in vitro. This heat sensitization was dose dependent, with EC(50) for IL-1 beta of 2.7 ng/ml and for TNF-alpha of 3.1 ng/ml. The maximum IL-1 beta effect reached almost 600% of the heat-evoked release, and the maximum TNF-alpha effect induced a rise in CGRP release of 350%. In contrast to IL-1 beta and TNF-alpha, IL-6 did not induce heat sensitization when applied alone but was only effective in the presence of soluble IL-6 receptor. This suggests a constitutive expression of signaling receptors for TNF and IL-1 beta and the signal transduction molecule gp130 but not IL-6 receptor or IL-8 receptor. Furthermore, the acute cytokine signaling observed in the present study was independent of transcriptional pathways because sensitization occurred on short latency in vitro and under conditions that excluded chemotactic accumulation of immune cells from blood vessels. Our results demonstrate that interleukins may play an important role in the initiation of heat hyperalgesia in inflammation and neuropathy.

Role of [Ca2+]i in the ATP-induced heat sensitization process of rat nociceptive neurons.

In inflamed tissue, nociceptors show increased sensitivity to noxious heat, which may account for heat hyperalgesia. In unmyelinated nociceptive afferents in rat skin in vitro, a drop of heat threshold and an increase in heat responses were induced by experimental elevation of intracellular calcium ([Ca2+]i) levels with the calcium ionophore ionomycin (10 microM). Similar results were obtained in experiments employing [Ca2+]i release from preloaded "caged calcium" (NITR-5/AM) via UV photolysis. In both cases, sensitization was prevented by preventing rises in [Ca2+]i with the membrane-permeant calcium chelator BAPTA-AM (1 mM). No pronounced change of mechanical sensitivity was observed. Heat-induced membrane currents (Iheat) were investigated with patch-clamp recordings, and simultaneous calcium measurements were performed in small sensory neurons isolated from adult rat dorsal root ganglia (DRG). Ionomycin-induced rises in [Ca2+]i resulted in reversible sensitization of Iheat. In the same subset of DRG neurons, the endogenous algogen ATP (100 microM) was used to elevate [Ca2+]i, which again resulted in significant sensitization of Iheat. In correlative recordings from the skin-nerve preparation, ATP induced heat sensitization of nociceptors, which again could be blocked by preincubation with BAPTA-AM. Rises in [Ca2+]i in response to inflammatory mediators, e.g., ATP, thus appear to play a central role in plastic changes of nociceptors, which may account for hypersensitivity of inflamed tissue.

Expression of the release factor eRF1 (Sup45p) gene of higher eukaryotes in yeast and mammalian tissues.

Polypeptide chain termination in eukaryotic cells is mediated in part by the release factor eRF1 (Sup45p). We have isolated and characterised cDNAs encoding this translation factor from Syrian hamster (Mesocricetus auratus) and human (Homo sapiens) Daudi cells. Comparison of the deduced amino acid sequence of these new eRF1 (Sup45p) sequences with those published for Saccharomyces cerevisiae, Arabidopsis thaliana, Xenopus laevis and human indicates a high degree of amino acid identity across a broad evolutionary range of species. Both the 5' and 3' UTRs of the mammalian eRF1 (Sup45p)-encoding cDNAs show an unusually high degree of conservation for non-coding regions. In addition, the presence of two different lengths of 3' UTR sequences in the mammalian eRF1 (Sup45p) cDNAs indicated that alternative polyadenylation sites might be used in vivo. Northern blot analysis demonstrated that eRF1 (Sup45p) transcripts of differing length, consistent with the use of alternative polyadenylation sites, were detectable in a wide range of mammalian tissues. The Xenopus, human and Syrian hamster eRF1 (Sup45p) cDNAs were shown to support the viability of a strain of S cerevisiae carrying an otherwise lethal sup45::HIS3 gene disruption indicating evolutionary conservation of function. However, the yeast strains expressing the heterogenous eRF1 (Sup45p) showed a defect in translation termination as defined by an enhancement of nonsense suppressor tRNA activity in vivo. Western blot analysis confirmed that Xenopus eRF1 (Sup45p) was primarily ribosome-associated when expressed in yeast indicating that the ribosome-binding domain of eRF1 (Sup45p) is also conserved.

Stable analogues of cyclic AMP but not cyclic GMP sensitize unmyelinated primary afferents in rat skin to heat stimulation but not to inflammatory mediators, in vitro.

The aim of this investigation was to evaluate the role played by cyclic nucleotides in the transduction of inflammatory pain and hyperalgesia. Unmyelinated afferents (n = 79) were exposed to stable analogues of cyclic AMP and cyclic GMP, to inflammatory mediators and to Methylene Blue, an inhibitor of guanylyl cyclase. Analogues of cyclic AMP at a concentration of 1 mM (n = 9) but not 10 microM (n = 16) sensitized nociceptor responses to noxious heat and enhanced interstimulus activity. In addition. mechanical thresholds were moderately, but significantly lowered after superfusion of the cyclic AMP analogue (1 mM). Addition of 10 microM cyclic AMP analogue to a mixture of excitatory inflammatory mediators (serotonin, histamine, bradykinin and prostaglandin E2, 10 microM each) did not further increase nociceptor activity (n = 15), in contrast to a previous report that cAMP sensitized bradykinin responses. Cyclic GMP analogues (10 microM, 1 mM) did not alter heat sensitivity or mechanical thresholds of polymodal C-fibres, nor did they enhance the ongoing activity that resulted from repeated heat stimulation. After inhibition of guanylyl cyclase with Methylene Blue, cyclic GMP analogues (1-10 microM) did not alter nociceptor responses evoked by application of the mixture of inflammatory mediators. The findings indicate that polymodal nociceptor sensitization and excitation is independent of cyclic GMP. Cyclic AMP can obviously contribute to the increased heat sensitivity of inflamed tissue, whereas cyclic GMP might be of importance in the recruitment of "silent" nociceptors.

Topical acetylsalicylate attenuates capsaicin induced pain, flare and allodynia but not thermal hyperalgesia.

The effect of acetylsalicylic acid (ASA) on capsaicin-evoked activation of cutaneous nociceptors was tested in a double blind study in 10 volunteers. Capsaicin (2% in ethanol) was applied topically for 30 min. Topical ASA (0.25 g/ml) reduced pain intensity and axon reflex flare size. Also, areas of secondary hyperalgesia to light touch and pin-prick were diminished. In contrast, capsaicin-induced heat hyperalgesia was unaffected by ASA. It is concluded that ASA counteracts the excitatory effects of capsaicin on nociceptors and mechanical hyperalgesia but not its sensitizing action to heat.

An apparent autocrine mechanism amplifies the dexamethasone- and retinoic acid-induced expression of mouse lipocalin-encoding gene 24p3.

We have isolated, sequenced and characterized the mouse 24p3 gene. The 24p3 protein is a member of the lipocalin family comprising secreted transporters of hydrophobic ligands. The 24p3 cDNA had been initially isolated during a search for genes overexpressed during a SV40-induced mitotic reaction [Hraba-Renevey et al., Oncogene 4 (1989) 601-608]. 24p3 comprises six exons, five introns and 793 bp of 5' regulatory region. The transcription start point (tsp) was identified by primer extension. Putative regulatory elements, including a TATA-like box and two glucocorticoid responsive core elements (GRE), have been mapped in the 5'-flanking region. Based on this observation, we examined the effect of a glucocorticoid (dexamethasone, Dex) on 24p3 expression. Dex induced the expression of 24p3 dramatically in the absence of de novo protein synthesis. This activation was further amplified by an apparent autocrine mechanism. Similar results were obtained with retinoic acid. Using the cat reporter gene system, we have shown that the 5'-flanking region of 24p3 confers Dex inducibility. Furthermore, we have identified a 43-bp region of the 24p3 promoter required for the Dex responsiveness. The biological implications are discussed in light of these results.

A highly conserved eukaryotic protein family possessing properties of polypeptide chain release factor.

The termination of protein synthesis in ribosomes is governed by termination (stop) codons in messenger RNAs and by polypeptide chain release factors (RFs). Although the primary structure of prokaryotic RFs and yeast mitochrondrial RF is established, that of the only known eukaryotic RF (eRF) remains obscure. Here we report the assignment of a family of tightly related proteins (designated eRF1) from lower and higher eukaryotes which are structurally and functionally similar to rabbit eRF. Two of these proteins, one from human and the other from Xenopus laevis, have been expressed in yeast and Escherichia coli, respectively, purified and shown to be active in the in vitro RF assay. The other protein of this family, sup45 (sup1) of Saccharomyces cerevisiae, is involved in omnipotent suppression during translation. The amino-acid sequence of the eRF1 family is highly conserved. We conclude that the eRF1 proteins are directly implicated in the termination of translation in eukaryotes.

Activated human platelets in plasma excite nociceptors in rat skin, in vitro.

Extravascular activation of thrombocytes may contribute to nociceptor excitation and pain, since platelets store and, upon stimulation, release potential algogenic substances such as serotonin, histamine and precursor molecules of bradykinin. To test this hypothesis, a skin-nerve preparation of rat hairy skin, in vitro, was used that allows to record and characterize single afferent nerve fibers. In a first protocol, receptive fields of nociceptive C-fibers, at the corium side of the skin patch, were exposed to adenosine diphosphate (ADP), to heparinized human platelet-rich plasma (PRP) and to PRP activated by ADP. Such activated platelets excited 9/11 units characterized as mechano-heat responsive C-nociceptors (CMH); peak discharges of more than 10 spikes/s were observed. After application of activated PRP, 4/5 high threshold mechanosensitive C-units and 4/5 mechano-cold sensitive C-units became responsive to heat stimulation but only few of these fibers were excited (1/5 in each group). In a second series of experiments the exposure to native PRP was prolonged to test for the effect of spontaneous platelet activation resulting from cutaneous collagen. Prolonged exposure did, but not significantly, enhance fiber discharge. During subsequent exposure to activated PRP, the discharge commenced, on average, after a significant delay of about three minutes. With this protocol 5/7 CMH units were driven by activated platelets. Following both protocols, mechanical (v.Frey) and thermal thresholds of the CMH units were not significantly altered. The findings demonstrate that nociceptors can indeed be driven and sensitized by activated platelets. This pain inducing mechanism may be relevant to certain clinical conditions, and it appears promising to scrutinize the chemical factors involved.

[Hypersalivation as a leading symptom of neoplastic meningiosis in highly malignant non-Hodgkin's lymphoma]. / Hypersalivation als Leitsymptom einer Meningiosis neoplastica bei hochmalignem Non-Hodgkin-Lymphom.

Partial remission of a centroblastic non-Hodgkin's lymphoma, clinical stage IV A, in a 79-year-old man was achieved by six courses of chemotherapy with epirubicin, cyclophosphamide and vincristine. The only residual finding was a palpable small cervical lymphoma. After a treatment pause of about 6 weeks increasing hypersalivation set in which ultimately made food intake impossible and led to a breakdown in the patient's general state. Findings in the region of the head, neck, throat and the base of the skull were unremarkable, but cerebrospinal fluid contained 1300/3 cells, almost all of them lymphoblasts. After five intrathecal injections of at first 15 mg methotrexate and 4 mg dexamethasone each, followed by five more with 40 mg cytarabine added to them, the CSF cell count became normal. At the same time salivation clearly decreased and food intake became once again possible. The patient died 5 months later from hypercalcaemia due to osseous infiltrations. Until his death there was no recurrence of the hypersalivation as the cardinal sign of meningeal carcinomatosis.

Graft-versus-host mortality induced by noncytolytic CD4+ T cell clones specific for non-H-2 antigens.

UNLABELLED: The relative contribution of individual non-H-2 Ag and of T cell subsets that initiate graft-vs-host reaction (GVHR) as well as the mechanism responsible for histopathologic lesions are still a matter of debate. To address these questions and to favor the selection of T cells primed in vivo against non-H-2 Ag important in GVHR we derived T cell clones from spleens of (DBA/2 x B10.D2)F1 (H-2d) mice developing this reaction after the graft of B10.D2 (H-2d) cells incompatible for numerous non-H-2 Ag plus Mlsa. The pattern of reactivity of eight selected clones against cells from different strains of mice including (BXD)RI strains indicated that one CD4+ clone is specific for Mlsa and seven additional clones (six CD4+ and one CD8+) are specific for four different non-H-2 Ag (Ag.I-IV) and proliferate in an H-2-restricted manner. The same series of experiments suggested that Ag.I and II are poorly polymorphic and allowed to propose the localisation of the genes controlling Ag.I (chromosome 1) and Ag.III (chromosome 4). All the clones show a triple (alpha, beta, gamma) mRNA transcript for TCR but at their surface they express the alpha/beta-heterodimer. The clone specific for Mlsa expresses V beta 6 and that specific for Ag.IV expresses V beta 8.1. Rapid mortality accompanied by clinical and histologic signs of severe GVHR was observed after administration of CD4+ clones (together with host-syngeneic bone marrow) derived early after grafting and specific for Ag.I and II but not after administration of: 1) CD8+ cytolytic clone derived early after grafting and specific for Ag.IV; 2) CD4+ clones derived late after grafting and specific for Ag.III; and 3) CD4+ clone specific for Mlsa. A clear correlation was established between the capacity of CD4+ clones to induce GVHR mortality, to mediate host-specific DTH and to release a high level of TNF. IN CONCLUSION: 1) the reaction against a single non-H-2 Ag is sufficient to provoke lethal GVHR; 2) the capacity to provoke GVHR mortality depends on antigenic specificity and functional properties of the responding clones; 3) the inflammatory process mediated by CD4+ clones may play a major role whereas the specific CD8+ T cell-mediated cytolytic activity is not necessarily lethal.

A gene that encodes a protein consisting solely of zinc finger domains is preferentially expressed in transformed mouse cells.

We describe the cloning and characterization of the mouse MOK-2 gene, a new member of the Krüppel family of zinc finger proteins. Sequencing of both cDNA and genomic clones showed that the predicted MOK-2 protein consists of seven zinc finger domains with only five additional amino acids. The finger domains of MOK-2 are highly homologous to one another but not to those of other zinc finger proteins. MOK-2 is preferentially expressed in transformed cell lines, brain tissue, and testis tissue. Its possible role in cellular transformation is discussed.

SV40-induced expression of mouse gene 24p3 involves a post-transcriptional mechanism.

SV40 and polyoma virus induce a mitotic host reaction in confluent, Go-arrested primary mouse kidney cell cultures. To define the primary effects of infection we constructed a cDNA library corresponding to polyA+ mRNA isolated shortly after onset of polyoma T-antigen synthesis. By differential screening of the library we have isolated and then sequenced cDNA recombinant 24p3; determined by Northern blotting, 24p3 mRNA steady state levels increased in parallel with polyoma and SV40 T-antigen synthesis. Since this rapid and early increase was particularly striking (14-20 fold) in SV40-infected cells, we studied the molecular mechanism of induction in this virus-cell system. We show that wt SV40 large T-antigen is required for the increase in 24p3 mRNA levels. The results tend to exclude that this increase is due to an SV40-induced stabilization of the 24p3 mRNA, or to an SV40-induced stimulation of transcription of the 24p3 gene; they are compatible with the working hypothesis that SV40 large T-antigen increases the efficiency of processing, possibly splicing, of the 24p3 pre-mRNA. The biological implications of these results are discussed.