BamHI Restriction Sites Research Articles

The Pathway for the Production of Inositol Hexakisphosphate in Human Cells

The yeast and Drosophila pathways leading to the production of inositol hexakisphosphate (InsP(6)) have been elucidated recently. The in vivo pathway in humans has been assumed to be similar. Here we show that overexpression of Ins(1,3,4)P(3) 5/6-kinase in human cell lines results in an increase of inositol tetrakisphosphate (InsP(4)) isomers, inositol pentakisphosphate (InsP(5)) and InsP(6), whereas its depletion by RNA interference decreases the amounts of these inositol phosphates. Expression of Ins(1,3,4,6)P(4) 5-kinase does not increase the amount of InsP(5) and InsP(6), although its depletion does block InsP(5) and InsP(6) production, showing that it is necessary for production of InsP(5) and InsP(6). Expression of Ins(1,3,4,5,6)P(5) 2-kinase increases the amount of InsP(6) by depleting the InsP(5) in the cell, and depletion of 2-kinase decreases the amount of InsP(6) and causes an increase in InsP(5). These results are consistent with a pathway that produces InsP(6) through the sequential action of Ins(1,3,4)P(3) 5/6-kinase, Ins(1,3,4,6)P(4) 5-kinase, and Ins(1,3,4,5,6)P5 2-kinase to convert Ins(1,3,4)P(3) to InsP(6). Furthermore, the evidence implicates 5/6-kinase as the rate-limiting enzyme in this pathway.

Characterization of Arabidopsis Fluoroacetate-resistant Mutants Reveals the Principal Mechanism of Acetate Activation for Entry into the Glyoxylate Cycle

The toxic acetate analogue monofluoroacetic acid was employed to isolate Arabidopsis tDNA-tagged plants deficient in their ability to utilize or sense acetate. Several tDNA-tagged lines were isolated, including two that were determined to be allelic to an EMS-mutagenized line denoted acn1 for ac non-utilizing. Following conventions, the tDNA-tagged mutants were designated acn1-2 and acn1-3. Both mutants displayed identical behavior to acn1-1 on a variety of fluorinated and nonfluorinated organic acids, indicating that resistance was specific to fluoroacetate. Thermal asymmetric interlaced PCR identified the sites of tDNA insertion in both mutants to be within different exons in a gene, which encoded a protein containing an AMP-binding motif. Reverse transcription-PCR confirmed that the gene was not expressed in the mutants, and quantitative reverse transcription-PCR showed that the gene is expressed in imbibed seeds and increases in amount during establishment. The wild type AMP-binding protein cDNA was cloned and expressed in Escherichia coli, and the expressed protein was purified by nickel chelate chromatography. The enzyme was identified as an acyl-CoA synthetase that was more active with acetate than butyrate and was not active with fatty acids longer than C-4. The enzyme was localized to peroxisomes by enzymatic analysis of organellar fractions isolated by sucrose density gradient centrifugation. Labeling studies with [(14)C]acetate showed that acn1 seedlings, like those of the isocitrate lyase mutant icl-1 (isocitrate lyase), are compromised in carbohydrate synthesis, indicating that this enzyme is responsible for activating exogenous acetate to the coenzyme A form for entry into the glyoxylate cycle.

The Repressor Element Silencing Transcription Factor (REST)-mediated Transcriptional Repression Requires the Inhibition of Sp1

The terminal differentiation of neuronal and pancreatic beta-cells requires the specific expression of genes that are targets of an important transcriptional repressor named RE-1 silencing transcription factor (REST). The molecular mechanism by which these REST target genes are expressed only in neuronal and beta-cells and are repressed by REST in other tissues is a central issue in differentiation program of neuronal and beta-cells. Herein, we showed that the transcriptional factor Sp1 was required for expression of most REST target genes both in insulin-secreting cells and neuronal-like cells where REST is absent. Inhibition of REST in a non-beta and a non-neuronal cell model restored the transcriptional activity of Sp1. This activity was also restored by trichostatin A indicating the requirement of histone deacetylases for the REST-mediated silencing of Sp1. Conversely, exogenous introduction of REST blocked Sp1-mediated transcriptional activity. The REST inhibitory effect was mediated through its C-terminal repressor domain, which could interact with Sp1. Taken together, these data show that the inhibition of Sp1 by REST is required for the silencing of its target genes expression in non-neuronal and in non-beta-cells. We conclude that the interplay between REST and Sp1 determines the cell-specific expression of REST target genes.

EVI1 Abrogates Interferon-α Response by Selectively Blocking PML Induction

EVI1 is an oncogene frequently associated with chronic and acute myeloid leukemia. In hematopoietic cells, EVI1 impairs several pathways including proliferation, differentiation, and apoptosis. Interferon-alpha (IFN-alpha) is a powerful cytokine that controls the immune response and limits the expansion of several tissues including bone marrow. These properties contribute to the effectiveness of IFN-alpha in the treatment of many neoplastic disorders especially chronic myeloid leukemia. We report here that in murine hematopoietic progenitors the expression of EVI1 completely abrogates the antiproliferative and apoptotic effects of IFN-alpha. EVI1 does not repress the JAK/STAT signaling pathway or the activation of many IFN-responsive genes. On the contrary, EVI1 prolongs the phosphorylation of STAT1 and the activation of an IFN-dependent reporter gene. However, EVI1 specifically represses the IFN-dependent induction of the tumor suppressor PML and blocks the apoptotic pathways activated by PML. We show that the position of the ISRE, which is located within the first exon of PML, is critical to block PML induction by IFN-alpha. The relocation of the ISRE to a position upstream of the transcription start site is sufficient to re-establish the response to IFN in the presence of EVI1. Our data suggest that stabilized STAT1 phosphorylation and prolonged binding of the STAT1 complex to the first exon could impair PML transcription and inhibit the activation of PML-dependent apoptotic pathways resulting in loss of IFN response. These results point to a novel mechanism utilized by an oncogene to escape normal cell response to growth-controlling cytokines.

E2A-PBX1 Interacts Directly with the KIX Domain of CBP/p300 in the Induction of Proliferation in Primary Hematopoietic Cells

The E2A gene encodes DNA-binding transcription factors, called E12 and E47, involved in cell specification and maturation. E2A is also involved in a chromosomal translocation that leads to the expression of an oncogenic transcription factor called E2A-PBX1 in cases of acute leukemia. In the work described here, we elucidate the interaction between E2A-PBX1 and transcriptional co-activators. We confirm that the E2A portion can interact with CBP and PCAF and map required elements on E2A and CBP. On CBP, the interaction involves the KIX domain, a well characterized domain that mediates interactions with several other oncogenic transcription factors. On E2A, the interaction with CBP requires conserved alpha-helical domains that reside within activation domains 1 and 2 (AD1 and AD2, respectively). Using purified, recombinant proteins, we show that the E2A-CBP interaction is direct. Notwithstanding the previously demonstrated ability of AD1 and AD2 to function independently, some of our findings suggest functional cooperativity between these two domains. Finally, we show that the CBP/p300-interactive helical domains of E2A are important in the induction of proliferation in cultured primary bone marrow cells retrovirally transduced with E2A-PBX1. Our findings suggest that some aspects of E2A-PBX1 oncogenesis involve a direct interaction with the KIX domain of CBP/p300.

Overlap of Interaction Domains Indicates a Central Role of the P Protein in Assembly and Regulation of the Borna Disease Virus Polymerase Complex

The active polymerase complex of Borna disease virus is composed of the viral proteins N, P, and L. The viral X (negative regulatory factor) protein acts as a regulator of polymerase activity. Interactions of P with N and X were previously studied, but interactions with L were poorly defined. Using a mammalian two-hybrid system, we observed that L specifically interacts with P but not with N, X, or itself. Mapping of the L-binding domain in the P molecule revealed that it overlaps with two adjacent domains required for multimerization and interaction with N. Competition experiments showed that the interaction between L and P was inefficient when N was present, indicating that L may preferentially interact with free P in infected cells. Interestingly, a multimerization-defective P mutant maintained the ability to interact with L, N, and X but failed to support reporter gene expression from an artificial Borna disease virus minigenome. Furthermore, dominant negative effects on minigenome activity were only observed when P mutants with an intact multimerization domain were used, suggesting that P multimers, rather than monomers, exhibit biological activity. P mutants lacking functional interaction domains for L or N still formed complexes with these viral proteins when wild-type P was available as a bridging molecule, indicating that P multimers have the potential to act as scaffolds on which the RNA polymerase complex is assembled.

Identification and Characterization of a Plasmid in Strain Aeronomas hydrophila IBRB-36 4CPA Carrying Genes for Catabolism of Chlorophenoxyacetic Acids

Results of studying plasmid pAH36 in strain Aeronomas hydrophila IBRB-36 4CPA are presented. Plasmid pAH36 possesses BamHI, PstI, and HindIII restriction sites and is 5.4 kb in size. The plasmid was shown to contain genes for catabolism of chlor-substituted phenoxyacetic acids.

NapF Is a Cytoplasmic Iron-Sulfur Protein Required for Fe-S Cluster Assembly in the Periplasmic Nitrate Reductase

The periplasmic nitrate reductase (Nap) is wide-spread in proteobacteria. NapA, the nitrate reductase catalytic subunit, contains a Mo-bisMGD cofactor and one [4Fe-4S] cluster. The nap gene clusters in many bacteria, including Rhodobacter sphaeroides DSM158, contain an napF gene, disruption of which drastically decreases both in vitro and in vivo nitrate reductase activities. In spite its importance in the Nap system, NapF has never been characterized biochemically, and its role remains unknown. The NapF protein has four polycysteine clusters that suggest that it is an iron-sulfur-containing protein. In the present study, a His(6)-tagged NapF protein was overproduced in Escherichia coli and purified anaerobically. The purified NapF protein was used to obtain polyclonal antibodies raised in rabbit, and cellular fractionation of R. sphaeroides followed by immunoprobing with anti-NapF antibodies revealed that the native NapF protein is located in the cytoplasm. This contrasts with the periplasmic location of the mature NapA. However, NapA could not be detected in an isogenic napF(-) strain of R. sphaeroides. The His(6)-tagged NapF protein displayed spectral properties indicative of Fe-S clusters, but these features were rapidly lost, suggesting cluster lability. However, reconstitution of the Fe-S centers into the apo-NapF protein was achieved in the presence of Azotobacter vinelandii cysteine desulfurase (NifS), and this allowed the recovery of nitrate reductase activity in NapA protein that had previously been treated with 2,2'-dipyridyl to remove the [4Fe-4S] cluster. This activity was not recovered in the absence of NapF. Taking into account the cytoplasmic localization of NapF, the presence of labile Fe-S clusters in the protein, the napF(-) strain phenotype, and the NapF-dependent reactivation of the 2,2'-dipyridyl-treated NapA, we propose a role for NapF in assembling the [4Fe-4S] center of the catalytic subunit NapA.

The H,K-ATPase β Subunit as a Model to Study the Role of N-Glycosylation in Membrane Trafficking and Apical Sorting

The role of N-glycosylation in trafficking of an apical membrane protein, the gastric H,K-ATPase beta subunit linked to yellow fluorescent protein, was analyzed in polarized LLC-PK1 cells by confocal microscopy and surface-specific biotinylation. Deletion of the N-glycosylation sites at N1, N3, N5, and N7 but not at N2, N4, and N6 significantly slowed endoplasmic reticulum-to-Golgi trafficking, impaired apical sorting, and enhanced endocytosis from the apical membrane, resulting in decreased apical expression. Golgi mannosidase inhibition to prevent carbohydrate chain branching and elongation resulted in faster internalization and degradation of the beta subunit, indicating that terminal glycosylation is important for stabilization of the protein in the apical membrane and protection of internalized protein from targeting to the degradation pathway. The decrease in the apical content of the beta subunit was less with mannosidase inhibition compared with that found in the N1, N3, N5, and N7 site mutants, suggesting that the core region sugars are more important than the terminal sugars for apical sorting.

Characterization of Recombinant, Membrane-attached Full-length Prion Protein

An abnormal isoform, PrP(Sc), of the normal cellular prion protein (PrP(C)) is the major component of the causative agent of prion diseases. Both isoforms were found to possess the same covalent structures, including a C-terminal glycosylphosphatidylinositol anchor, but different secondary and tertiary structures. In this study, a variant of full-length PrP with an unpaired cysteine at the C terminus was recombinantly produced in Escherichia coli, covalently coupled to a thiol-reactive phospholipid, and incorporated into liposomes to serve as a model for studying possible changes in structure and stability of recombinant PrP upon membrane attachment. Covalent coupling of PrP to liposomes did not result in significant structural changes observable by far-UV circular dichroism. Moreover, limited proteolysis experiments failed to detect changes in the stability of liposome-bound PrP relative to soluble PrP. These data suggest that the requirement of raft localization for the PrP(C) to PrP(Sc) conversion, observed previously in cell culture models, is not because of a direct influence of raft lipids on the structure and stability of membranebound PrP(C) but caused by other factors, e.g. increased local PrP concentrations or high effective concentrations of membrane-associated conversion factors. The availability of recombinant PrP covalently attached to liposomes provides the basis for systematic in vitro conversion assays with recombinant PrP on the surface of membranes. In addition, our results indicate that the three-dimensional structure of mammalian PrP(C) in membranes is identical to that of recombinant PrP in solution.

Hox Transcription Factor Ultrabithorax Ib Physically and Genetically Interacts with Disconnected Interacting Protein 1, a Double-stranded RNA-binding Protein

The Hox protein family consists of homeodomain-containing transcription factors that are primary determinants of cell fate during animal development. Specific Hox function appears to rely on protein-protein interactions; however, the partners involved in these interactions and their function are largely unknown. Disconnected Interacting Protein 1 (DIP1) was isolated in a yeast two-hybrid screen of a 0-12-h Drosophila embryo library designed to identify proteins that interact with Ultrabithorax (Ubx), a Drosophila Hox protein. The Ubx.DIP1 physical interaction was confirmed using phage display, immunoprecipitation, pull-down assays, and gel retardation analysis. Ectopic expression of DIP1 in wing and haltere imaginal discs malforms the adult structures and enhances a decreased Ubx expression phenotype, establishing a genetic interaction. Ubx can generate a ternary complex by simultaneously binding its target DNA and DIP1. A large region of Ubx, including the repression domain, is required for interaction with DIP1. These more variable sequences may be key to the differential Hox function observed in vivo. The Ubx.DIP1 interaction prevents transcriptional activation by Ubx in a modified yeast one-hybrid assay, suggesting that DIP1 may modulate transcriptional regulation by Ubx. The DIP1 sequence contains two dsRNA-binding domains, and DIP1 binds double-stranded RNA with a 1000-fold higher affinity than either single-stranded RNA or double-stranded DNA. The strong interaction of Ubx with an RNA-binding protein suggests a wider range of proteins may influence Ubx function than previously appreciated.

Alternative splicing of transcripts of the transposon Restless is maintained in the foreign host Neurospora crassa and can be modified by introducing mutations at the 5' and 3' splice sites.

The primary transcript of the transposon Restless from Tolypocladium inflatum undergoes an unusual mechanism of alternative splicing by employing either of two 3' "CAG" splice sites. These are separated by only four nucleotides, thus generating two different splice products, which differ in their coding capacity. To analyse whether this alternative splicing occurs in its natural host exclusively, we introduced the transposon into the heterologous host Neurospora crassa. In addition to the wild -type transposon sequence, transposon sequences mutagenised in vitro with modified 5' and 3' intron splice sites were generated. RNA was isolated from transformants and RT-PCR was performed with specific oligonucleotides flanking the intron sequence. Alternative splicing was analysed, employing a simple test procedure based on the convenient presence of a BamHI restriction site between both splice sites. The ratio of alternative splicing seems to be influenced by both the 5' and the 3' splice site, as mutations at either position influence the ratio of alternative splice products. At the 5' splice site, mutating the first "C" has a strong effect on the ratio of alternative splicing, while mutating the second "C" has little or no effect. Similarly, at the 3' splice site, only mutations at the first 3' "CAG" change the ratio of alternative splicing. It appears that alternative splicing of the Restless intron is not host-specific, but is influenced by the intron splice site sequences themselves.

Human S100B Protein Interacts with the Escherichia coli Division Protein FtsZ in a Calcium-sensitive Manner

S100B is a small, dimeric EF-hand calcium-binding protein abundant in vertebrates. Upon calcium binding, S100B undergoes a conformational change allowing it to interact with a variety of target proteins, including the cytoskeletal proteins tubulin and glial fibrillary acidic protein. In both cases, S100B promotes the in vitro disassembly of these proteins in a calcium-sensitive manner. Despite this, there is little in vivo evidence for the interaction of proteins such as tubulin with S100B. To probe these interactions, we studied the expression of human S100B in Escherichia coli and its interaction with the prokaryotic ancestor of tubulin, FtsZ, the major protein involved in bacterial division. Expression of S100B protein in E. coli results in little change in FtsZ protein levels, causes a filamenting bacterial phenotype characteristic of FtsZ inhibition, and leads to missed rounds of cell division. Further, S100B localizes to positions similar to those of FtsZ in bacterial filaments: the small foci at the poles, the mid-cell positions, and between the nucleoids at regular intervals. Calcium-dependent physical interaction between S100B and FtsZ was demonstrated in vitro by affinity chromatography, and this interaction was severely inhibited by the competitor peptide TRTK-12. Together these results indicate that S100B interacts with the tubulin homologue FtsZ in vivo, modulating its activity in bacterial cell division. This approach will present an important step for the study of S100 protein interactions in vivo.

Glycodelin A, not glycodelin S, is apoptotically active. Relevance of sialic acid modification.

Glycodelin, previously known as PP14 (placental protein-14), is a kernel lipocalin secreted by the glandular epithelium of the endometrium upon progesterone stimulation and by the seminal vesicles. The isoform of the protein present in female reproductive tissue, glycodelin A (GdA), and the male counterpart, glycodelin S (GdS), have identical amino acid sequences, but strikingly different N-linked glycans. It is well documented in literature that GdA is an immunosuppressive protein, and we have shown that this activity is due to its ability to induce apoptosis in activated T cells. The precise role of GdS in seminal plasma is not known. In this study, we report that GdS is not apoptotically active. We observe that the apoptotic activity requires the presence of sialic acid residues on the complex glycans, as in the case of GdA; however, complex glycans of GdS are non-sialylated. We have expressed the wild-type protein in Pichia pastoris, which does not add sialic acid to the secreted proteins, and confirmed our observations that the protein is apoptotically inactive in the non-sialylated form. Our results indicate that differential glycosylation modulates the function of the different glycodelin isoforms.

A Superfamily of Voltage-gated Sodium Channels in Bacteria

NaChBac, a six-alpha-helical transmembrane-spanning protein cloned from Bacillus halodurans, is the first functionally characterized bacterial voltage-gated Na(+)-selective channel. As a highly expressing ion channel protein, NaChBac is an ideal candidate for high resolution structural determination and structure-function studies. The biological role of NaChBac, however, is still unknown. In this report, another 11 structurally related bacterial proteins are described. Two of these functionally expressed as voltage-dependent Na(+) channels (Na(V)PZ from Paracoccus zeaxanthinifaciens and Na(V)SP from Silicibacter pomeroyi). Na(V)PZ and Na(V)SP share approximately 40% amino acid sequence identity with NaChBac. When expressed in mammalian cell lines, both Na(V)PZ and Na(V)SP were Na(+)-selective and voltage-dependent. However, their kinetics and voltage dependence differ significantly. These single six-alpha-helical transmembrane-spanning subunits constitute a widely distributed superfamily (Na(V)Bac) of channels in bacteria, implying a fundamental prokaryotic function. The degree of sequence homology (22-54%) is optimal for future comparisons of Na(V)Bac structure and function of similarity and dissimilarity among Na(V)Bac proteins. Thus, the Na(V)Bac superfamily is fertile ground for crystallographic, electrophysiological, and microbiological studies.

Physical and Functional Interactions between USF and Sp1 Proteins Regulate Human Deoxycytidine Kinase Promoter Activity

Deoxycytidine kinase (EC 2.7.1.74, dCK) is central to drug activity of anticancer and antiviral agents such as cytosine arabinoside (araC) and gemcitabine. HepG2 hepatocellular carcinoma cells were used to study the transcriptional regulation of dCK. 5'-Deletion and site-directed mutagenesis of the dCK upstream region (positions -464 to -27) confirmed the importance of two GC-boxes (positions -317 to -309 and -213 to -206) and two E-boxes (positions -302 to -297 and -278 to -273). In vitro electromobility shift assays with HepG2 nuclear extracts and in vivo chromatin immunoprecipitation assays with HepG2 chromatin extracts confirmed the presence of bound Sp1/Sp3 and USF1/2. Co-transfections in HepG2 cells showed that USF1 and USF2a stimulated and Sp1 repressed promoter activity from a dCK-luciferase reporter gene construct. In Sp- and USF-null Drosophila Mel-2 cells, both Sp1 and USF1 stimulated dCK promoter activity in a dose-dependent manner, however, both Sp3 and USF2a were effectively inert. Combined Sp1 and USF1 showed additive transactivation at lower concentrations of Sp1. Sp1 was inhibitory at higher levels. Stimulation by combined USF1/USF2a with Sp1 was similar to that for USF1 alone with Sp1, whereas transactivation by Sp1 and USF2a without USF1 was synergistic. Physical interactions between USF and Sp proteins were confirmed by immunoprecipitations with Sp- and USF-specific antibodies. Domain mapping of USF1 and USF2a localized the functional interactions between USF and Sp proteins to the DNA binding domain of USF. Identifying the physical and functional interactions between Sp and USF proteins may lead to a better understanding of the basis for differential expression of the dCK gene in tumor cells and may foster strategies for up-regulating dCK gene expression and improving chemotherapy with araC and gemcitabine.

An allele of dihydroflavonol 4-reductase associated with the ability to produce red anthocyanin pigments in potato (Solanum tuberosum L.).

The potato R locus is necessary for the production of red pelargonidin-based anthocyanin pigments in any tissue of the plant, including tuber skin and flower petals. The production of pelargonidins in plants requires the activity of dihydroflavonol 4-reductase (DFR) to catalyze the reduction of dihydrokaempferol into leucopelargonidin. To test the hypothesis that potato R encodes DFR, portions of both dfr alleles were sequenced from a diploid potato clone known to be heterozygous Rr. Sequence comparison revealed a polymorphic BamHI restriction site. The presence or absence of this site was monitored in three diploid populations that segregated for R, as well as in a wide range of tetraploid breeding clones and cultivars, by amplifying a fragment of dfr and digesting the products with BamHI. An identically sized dfr restriction fragment lacking the BamHI site was present in all potato clones that produced red anthocyanin pigments, while the same fragment was absent in many potato clones with white tuber skin and flowers. An independent RFLP test using DraI to detect sequence polymorphism was performed on a subset of the potato clones. This test also revealed dfr-derived bands that were present in all red-colored potatoes and absent in several white clones. The presence of shared restriction fragments in all red-colored potatoes provides strong evidence that R does indeed code for DFR. The data are also consistent with a 48 year-old hypothesis by Dodds and Long, that R was selected just once during the domestication of potato. A cDNA clone corresponding to the red allele of dfr was sequenced and compared to two other alleles. The red allele is predicted to encode a 382 amino acid protein that differs at ten amino acid positions from the gene products of the two alternative alleles. Several of these differences map in a region known to influence DFR substrate specificity in Gerbera.

WRN Interacts Physically and Functionally with the Recombination Mediator Protein RAD52

Werner syndrome (WS) is a premature aging disorder that predisposes affected individuals to cancer development. The affected gene, WRN, encodes an RecQ homologue whose precise biological function remains elusive. Altered DNA recombination is a hallmark of WS cells suggesting that WRN plays an important role in these pathways. Here we report a novel physical and functional interaction between WRN and the homologous recombination mediator protein RAD52. Fluorescence resonance energy transfer (FRET) analyses show that WRN and RAD52 form a complex in vivo that co-localizes in foci associated with arrested replication forks. Biochemical studies demonstrate that RAD52 both inhibits and enhances WRN helicase activity in a DNA structure-dependent manner, whereas WRN increases the efficiency of RAD52-mediated strand annealing between non-duplex DNA and homologous sequences contained within a double-stranded plasmid. These results suggest that coordinated WRN and RAD52 activities are involved in replication fork rescue after DNA damage.

Bcl-2 Protein Is Required for the Adenine/Uridine-rich Element (ARE)-dependent Degradation of Its Own Messenger

We have shown previously that the decay of human bcl-2 mRNA is mediated by an adenine/uridine-rich element (ARE) located in the 3'-untranslated region. Here, we have utilized a non-radioactive cell-free mRNA decay system to investigate the biochemical and functional mechanisms regulating the ARE-dependent degradation of bcl-2 mRNA. Using RNA substrates, mutants, and competitors, we found that decay is specific and ARE-dependent, although maximized by the ARE-flanking regions. In unfractionated extracts from different cell types and in whole cells, the relative enzymatic activity was related to the amount of Bcl-2 protein expressed by the cells at steady state. The degradation activity was lost upon Bcl-2 depletion and was reconstituted by adding recombinant Bcl-2. Ineffective extracts from cells that constitutively do not express Bcl-2 acquire full degradation activity by adding recombinant Bcl-2 protein. We conclude that Bcl-2 is necessary to activate the degradation complex on the relevant RNA target.

Restriction fragment analysis of the ribosomal DNA of Paratelmatobius and Scythrophrys species (Anura, Leptodactylidae)

Physical maps of the ribosomal RNA gene 28S of species belonging to the genera Paratelmatobius and Scythrophrys were constructed, using five restriction endonucleases. The restriction sites for Bam HI, Bgl II, Bst EII, and Eco RI had similar positions in all species, although there were interspecific differences in the size of the restriction fragments obtained. An additional Pvu II site was found in Scythrophrys specimens from Piraquara (State of Paraná, Brazil) and from São Bento do Sul (State of Santa Catarina, Brazil), but not in the Scythrophrys specimens from Rancho Queimado (State of Santa Catarina, Brazil). This finding is in agreement with the hypothesis regarding the existence of two species in the genus Scythrophrys. On the other hand, the extra Bst EII site considered in the literature to be a synapomorphy for the subfamilies Leptodactylinae and Telmatobiinae was not observed in the genera Paratelmatobius and Scythrophrys, which brings new questions about some taxonomic classifications that include Paratelmatobius in Leptodactylinae and Scythrophrys in Telmatobiinae. Interspecific variation was observed in the size of the restriction fragments analyzed and, in the case of group I Scythrophrys, there was also a variation between the individuals of the two populations. These data suggest that sequencing of the rDNA segments studied here may be useful in phylogenetic studies of the genera Paratelmatobius and Scythrophrys.