Rapid prenatal diagnosis of spinal muscular atrophy by denaturing high-performance liquid chromatography system.

OBJECTIVE: Use of Denaturing High-Performance Liquid Chromatography (DHPLC) in prenatal diagnosis of spinal muscular atrophy (SMA). METHODS: Thirty-three members of 7 families participated in carrier test and disease detection of SMA. Prenatal genetic diagnosis was performed if both parents were carriers or any family members had SMA. DNA extracted from blood, chorionic villi and amniotic fluid was amplified and used for DHPLC. RESULTS: Twenty SMA carriers, seven SMA affected cases, and six normal individuals were identified. SMA status was demonstrated by genotyping and total copy number determinations of SMN1 and SMN2. Families 1-3 were classified as group one (SMA affecting previously born child). Group two, comprising families 4 and 5, had lost a child due to an unknown muscular disease. Group three (SMA-affected parent) comprised families 6 and 7; carrier testing was done. DHPLC prenatal genetic diagnosis was made in seven pregnancies, one in each family (affected, n=2; carrier, n=3; normal, n=2). Pregnancy was terminated for the two affected fetuses. The others were delivered uneventfully and SMA free. CONCLUSION: DHPLC prenatal diagnosis of SMA and determination of SMA status in adults is possible, and SMN1 and SMN2 copy numbers can be determined.

Splicing factor SPF30 bridges an interaction between the prespliceosome protein U2AF35 and tri-small nuclear ribonucleoprotein protein hPrp3.

Spliceosome assembly is a dynamic process involving the sequential recruitment and rearrangement of small nuclear ribonucleoproteins (snRNPs) on a pre-mRNA substrate. Here we identify several spliceosome protein interactions with different domains of human splicing factor SPF30 that have the potential to mediate the addition of the tri-snRNP to the prespliceosome. In particular, we show that the C-terminal tails of SmD1, SmD3, and the protein Lsm4 interact with the central Tudor domain of SPF30. We identify a novel interaction between the N-terminal domain of SPF30 and U2AF35, a prespliceosome protein that has a role in recognizing the 3' splice site and recruiting U2 snRNP. We also show that the C terminus of SPF30 interacts with a middle domain of hPrp3, a component of U4/U6 di-snRNP and the tri-snRNP. Importantly, we show that the U2AF35 and hPrp3 interactions with SPF30 can occur simultaneously, thereby potentially linking 3' splice site recognition with tri-snRNP addition. Finally, we note that SPF30 and its partner-interacting domains are not conserved in yeast, suggesting this interaction network may play an important role in the complex splicing observed in higher eukaryotes.

Identification and purification of CREB like protein in Candida albicans.

cAMP response element binding protein (CREB) belongs to ATF/CREB family of transcription factors, which are bonafide targets of cAMP-PKA signalling pathway in mammalian cells. CREB is known to regulate the genes involved in transcription, cell cycle, cell survival, neurotransmitter, growth factors and immune regulation. But there is no evidence of presence of ATF/CREB family members in Candida albicans. In the present study, CREB like transcription factor has been identified and purified in C. albicans. The putative CREB was observed to have different molecular mass (47 kDa) as compared to its mammalian counterpart (43 kDa). Both forms of CREB (CREB and phosphorylated CREB) were detected in C. albicans and phosphorylation of CREB was found to be a function of cAMP levels and protein kinase A activity within this organism. CREB protein was purified by sequence-specific CRE-DNA affinity chromatography. Purified CREB exhibited characteristic CRE binding activity as revealed by electrophoretic mobility shift assay and gave reactivity with CREB antibodies. CREB protein was phosphorylated by purified catalytic subunit of PKA under in vitro conditions. To the best of our knowledge, this study reports for the first time identification of CREB like protein as an important component of cAMP signalling pathway in C. albicans.

Purification of CREB to apparent homogeneity: removal of truncation products and contaminating nucleic acid.

The cAMP response element binding protein (CREB) is a mammalian transcription factor which regulates the expression of many cellular genes. CREB is commonly expressed in Escherichia coli and purified by heat-extraction followed by affinity chromatography. We have discovered that although this purification yields a reasonably pure product which is active in DNA-binding and functional assays, it contains a large amount of nucleic acid as well as CREB truncation products and other polypeptides. Consequently, this CREB is inadequate for use in biophysical studies including crystallography, and spectroscopic analysis such as analytical ultracentrifugation, FRET, and circular dichroism. We revised the purification protocol to incorporate expression in the Rosetta host strain, nuclease treatment, and denaturing/high salt size-exclusion chromatography. We typically obtain 10mg of CREB per liter of culture media that is 99% homogenous, free of nucleic acid, and amenable to biophysical studies. Comparison of CREB from the original and revised protocols shows similar affinities for the cAMP response element (CRE) but small differences in their secondary structures when assayed by limited proteolysis and circular dichroism.

[Identification of the novel host factor that interacts with HIV-1 integrase and analysis of the expression profile of the interactor in vivo].

Human immunodeficiency virus type 1 (HIV-1) is one of the lentiviruses, and unlike other retroviruses, HIV-1 is capable of infecting not only dividing cells but also non-dividing cells. It has been strongly suggested that this property is due to the viral mechanism which facilitates the integration of the viral genome cDNA into the host chromosome more efficiently than other retroviruses. HIV-1 integrase (IN) is a viral protein which catalyzes insertion of the viral genome cDNA into the host cell chromosome. However, it has been suggested that HIV-1 IN also plays putative roles at the steps prior to integration such as uncoating, reverse transcription and nuclear transport of the viral cDNA. In this study, we tried to identify the novel host factor which interacted with HIV-1 IN using two different kinds of yeast two hybrid methods: the conventional yeast two hybrid method and the mating method. First, the full-length cDNA fragment of HIV-1 IN was amplified using polymerase chain reaction (PCR), and the amplified products were ligated into pGBT 9 vector as bait. Plasmid vectors expressing human lymphocytes cDNA library and HeLa cDNA library were used as prey plasmid in the conventional yeast two hybrid method and the mating method, respectively. These plasmids were transformed into the corresponding yeast strain cells, and several positive clones were isolated. As a result, a known gene product was identified as a candidate. Further analysis revealed that this protein was expressed in HeLa, 293 T cells, primary macrophages and activated T lymphocytes, and that suppression of this protein expression affected HIV-1 replication.

Phospho-CREB and other phospho-proteins: improved recovery from brain tissue.

During attempts to quantify levels of phosphorylated cAMP response element binding protein (CREB-P) in guinea pig brain stem auditory nuclei by Western blotting, we compared the decay of CREB-P levels when tissues were homogenized in traditional Lysis buffer containing detergents or in 50 mM Tris-HCl buffer containing 0.32 M sucrose. The decay of CREB-P levels was retarded considerably in the Tris-Sucrose medium as compared to the Lysis buffer. Similarly, the levels of two other phospho-proteins, extracellular regulated kinases (ERK1/2-P) and stress activated protein/Jun-N-terminal kinase (SAP/JNK-P), were better preserved by the Tris-Sucrose medium. These findings imply that the detergents typically present in the Lysis buffer may disrupt organelles and increase the exposure of soluble phospho-proteins to hydrolyzing enzymes. In contrast, such exposure was probably minimized in the Tris-Sucrose medium, which is thought to preserve organelle integrity.

Identification of a functional CRE in the promoter of Fukuyama congenital muscular dystrophy gene fukutin.

We isolated a fragment of the fukutin gene promoter from differentiated human NT2 cells using chromatin immunoprecipitation technique with an anti-CREB antibody. This fragment contained a CRE-like sequence and here we describe its functional validation. The results showed that the element was functional in vitro and in vivo and that CREB in neurons was involved in the transcriptional regulation of the fukutin gene. Moreover, its expression in neurons was regulated by cAMP and calcium ions, known triggers of CREB phosphorylation. To our knowledge, this is the first report on the regulation of fukutin gene by transcription factor CREB in response to the signals generated by synaptic activity. The true biological function of fukutin, the gene responsible for Fukuyama-type congenital muscular dystrophy and mental retardation, is at present not known. However, it has been suggested that it might possess glycosyltransferase activity and its intracellular localization within the Golgi structures is consistent with this function. As such, fukutin might play a significant role in post-translational modification of synaptic proteins in neuronal cells.

Candidate cis-elements for human renin gene expression in the promoter region.

The regulation of renin gene expression, the rate-limiting enzyme of the system, is thought to be fundamental to the total system. Previously, we mapped six putative cis-elements in the promoter region of the human renin gene with nuclear proteins from human chorionic cells and human renal cortex by DNase I protection assay (footprint A-F). Each footprint contains Ets motif like site (A), HOXñPBX recognition sequence (B), unknown sequence as DNA binding consensus (C), CRE (D), COUP-TFII (ARP-1) motif like site (E), and AGE3 like site (F). Footprint D has been characterized by means of functional studies as the genuine human renin gene CRE interacting with CREB in cooperation with the site of footprint B. To obtain further clues to the specific expression in the promoter region, these putative cis-elements were conducted to a consensus-specific binding assay to compare renin-producing and non-renin-producing cells by EMSA and electromobility super-shift assay. Different sequence-specific DNA/protein binding was obtained among the different cell lines with footprint B site, with COUP-TFII (ARP-1) motif like site and possibly with footprint F site. The results implicate these putative cis-elements and each corresponding trans-factor in the specific expression of the human renin gene in the promoter region. Further functional characterization of these elements would provide important data for a better understanding of human renin gene expression.

The AmCREB gene is an ortholog of the mammalian CREB/CREM family of transcription factors and encodes several splice variants in the honeybee brain.

The transcription factor CREB (cAMP response element binding protein) is required for the switch from short-term to long-term synaptic plasticity and from short-term to long-term memory. Its activity is regulated by the cAMP-dependent signalling cascade, which has been shown to play a crucial role in the honeybee's long-term memory formation. To elucidate the role of the CREB in honeybee memory formation we analysed a CREB-homologous gene, AmCREB, which is expressed as several transcripts in the honeybee brain. Eight transcripts have been identified (AmCREB 1-8) that are generated by alternate splicing. One antibody generated against a subset of these variants reveals a cytosolic localization in the mushroom body alpha-lobes, the glomeruli of the antennal lobes, the protocerebral lobes, the central complex and in the optical lobes.

Phosphatase regulation of gene expression during development of the palate.

In mammalian cells, including those of the embryonic palate, the level of phosphorylation of cellular proteins at any given time reflects the activities of protein kinases and protein phosphatases. Both protein phosphatase-1 (PP-1) and PP-2A inhibit cAMP-mediated increases in transcription by dephosphorylating CREB at ser-133. Western blot analysis indicated that protein phosphatase 1 (PP-1) was expressed constitutively in palatal tissue during its development. Expression of PP-2A was regulated developmentally with maximal expression on gestational day (gd) 14. Densitometric scanning revealed a 30% increase in expression from gd 13 to gd 14. Virtually all phosphatase activity in the tissue extracts could be inhibited by 5 microM okadaic acid, demonstrating that PP-1 and PP-2A account for all detectable ser/thr protein phosphatase activity present in the developing palate. Moreover, no significant differences in PP-1 and PP-2A activities were observed during the period of palate development. Treatment of primary cultures of murine embryonic palate mesenchymal (MEPM) cells with forskolin (20 microM) to elevate intracellular cAMP levels, resulted in a time-dependent increase in CREB ser-133 phosphorylation and a corresponding time dependent decrease in PP-1 and PP-2A levels. Moreover, treatment of MEPM cells with okadaic acid resulted in a dramatic increase in basal CREB ser-133 phosphorylation. This suggests that PP-1 activity may contribute to transcriptional regulation of CREB and that PP-1 and PP-2A are regulated differentially by cAMP. Treatment of MEPM cells with TGF beta 1 (1 ng/ml) under conditions of TGF beta-induced CREB phosphorylation resulted in no effect on the expression of either PP-1 or PP-2A proteins and no significant alterations in total basal protein phosphatase activity. These results demonstrate that transcriptional regulation of CREB in embryonic palatal issue is dependent on the coordinate activity of specific kinases and phosphatases.

Identification of mRNA/protein (mRNP) complexes containing Puralpha, mStaufen, fragile X protein, and myosin Va and their association with rough endoplasmic reticulum equipped with a kinesin motor.

Puralpha, which is involved in diverse aspects of cellular functions, is strongly expressed in neuronal cytoplasm. Previously, we have reported that this protein controls BC1 RNA expression and its subsequent distribution within dendrites and that Puralpha is associated with polyribosomes. Here, we report that, following treatment with EDTA, Puralpha was released from polyribosomes in mRNA/protein complexes (mRNPs), which also contained mStaufen, Fragile X Mental Retardation Protein (FMRP), myosin Va, and other proteins with unknown functions. As the coimmunoprecipitation of these proteins by an anti-Puralpha antibody was abolished by RNase treatment, Puralpha may assist mRNP assembly in an RNA-dependent manner and be involved in targeting mRNPs to polyribosomes in cooperation with other RNA-binding proteins. The immunoprecipitation of mStaufen- and FMRP-containing mRNPs provided additional evidence that the anti-Puralpha detected structurally or functionally related mRNA subsets, which are distributed in the somatodendritic compartment. Furthermore, mRNPs appear to reside on rough endoplasmic reticulum equipped with a kinesin motor. Based on our present findings, we propose that this rough endoplasmic reticulum structure may form the molecular machinery that mediates and regulates multistep transport of polyribosomes along microtubules and actin filaments, as well as localized translation in the somatodendritic compartment.

The -180 site of the IL-2 promoter is the target of CREB/CREM binding in T cell anergy.

Anergic T cells display a marked decrease in their ability to produce IL-2 even in the presence of optimal TCR and costimulatory signals. Using IL-2 enhancer/promoter-driven reporter constructs, we have previously identified a region that appears to be a target for cis transcriptional repression in anergy. This region of the promoter, which shares partial homology with a consensus AP-1-binding sequence, is located about -180 bp from the transcriptional start site. In the present study, we demonstrate that cAMP response element-binding protein/cAMP response element modulator (CREB/CREM), activating transcription factor-2/c-Jun, and Jun-Jun/Oct complexes bind to this site. However, the induction of anergy by prolonged stimulation through the TCR led to an increase in binding of only the CREB/CREM complex. Furthermore, the level of binding of this complex appeared to be up-regulated in both resting and restimulated anergic T cells. Finally, an IL-2 promoter-driven reporter construct that contained a mutation that specifically reduced the binding of the CREB/CREM complex displayed a decreased ability to be affected by anergy, while a construct that contained a mutation that decreased the binding of the Jun-Jun/Oct complex was still susceptible to anergy. These findings suggest that the -180 region of the IL-2 promoter is the target of a CREB/CREM transcriptional inhibitor that contributes to the repression of IL-2 production in T cell anergy.

Identification and functional analysis of novel cAMP response element binding protein splice variants lacking the basic/leucine zipper domain.

Two novel cAMP response element binding protein (CREB) splice variants were found by reverse transcription-polymerase chain reaction cloning by using mouse brain RNA as a template. One splice variant, named Delta-14, lacks 14 nucleotides at the beginning of exon 9 of the CREBDelta isoform. The other, named Delta-35, lacks 35 nucleotides at the beginning of exon 8 of CREBDelta. These nucleotide deletions cause frame shifts for codon usage, producing proteins which conserve the major phosphorylation site (Ser(133)) but lack the basic/leucine zipper domain, which is essential for binding to DNA and to other transcription factors. Both variants are widely expressed in peripheral tissues, but are enriched in brain, thymus, and testis. CREBDelta-14 and Delta-35 variant proteins were expressed by using an in vitro translation system and by transfecting into human embryonic kidney 293 cells. Both variants were detected by a CREB antibody that recognizes the CREBDelta amino terminus, but not by an antibody which recognizes the CREBDelta carboxy terminus, as would be predicted based on the frame shift. Activation of the cAMP pathway increased phospho-CREB immunoreactivity, indicating that these variants are substrates of cAMP-dependent protein kinase. In addition, immunocytochemical analysis demonstrated that CREBDelta-14 and Delta-35 are primarily cytosolic, whereas CREBalpha is predominantly in the nucleus. Finally, expression of CREBDelta-14 or Delta-35 decreased cAMP responsive element-chloramphenicol acetyltransferase reporter activity, demonstrating that both can function as repressors of endogenous CREB.

Identification of (CAG)(n) and (CGG)(n) repeat-binding proteins, CAGERs expressed in mature neurons of the mouse brain.

The trinucleotide repeats (CAG)(n) and (CGG)(n) have been shown to be expanded in responsible genes of several human hereditary neurological disorders. In studies of mice, we previously identified two homologous single-stranded (ss)(CAG) and ss(CGG) repeat-binding proteins, CAGER-1 (44 kDa) and CAGER-2 (40 kDa) (CAG-element-recognizing proteins). The specific binding activities of these proteins were predominantly detected in the mouse brain. We have isolated the cDNAs encoding CAGER-1 and CAGER-2 and found that they were identical to previously reported cDNAs for Puralpha and Purbeta, respectively. Puralpha of 28 kDa was previously identified as a replication-origin-binding protein that is ubiquitously expressed in proliferating cells. We show that the transcripts of CAGERs increase after birth and are detected at high levels in the adult mouse brain but at very low or virtually undetectable levels in other mouse tissues. Biochemical properties and molecular weights are different between CAGERs and Puralpha/beta. Immunostaining with specific antibodies against CAGERs indicates that CAGERs in the mouse brain reside in nonproliferating neurons but not in proliferating glia. We conclude that CAGERs and Puralpha/beta are unrelated proteins, and CAGERs are neuronal single-stranded sequence-binding proteins in the mouse brain. Misassignment of cDNAs is described.

ZPK inhibits PKA induced transcriptional activation by CREB and blocks retinoic acid induced neuronal differentiation.

Zipper Protein Kinase (ZPK) is a leucine zipper protein localized to the nucleus which exhibits serine-threonine kinase activity and is associated with the stress dependent signal transduction pathway. ZPK forms heterodimers with leucine zipper containing transcription factors such as the cyclic AMP responsive element binding protein (CREB) and Myc. Furthermore ZPK phosphorylates both Myc and CREB. Overexpression of ZPK in NTera-2 human teratocarcinoma cells results in inhibition of PKA induced transcriptional activation by CREB and prevents retinoic acid induced differentiation of the cells to neurons. Our results suggest that ZPK stifles neural differentiation of NT-2 cells partly due to its inhibitory effect on CREB function.

Lack of c-Jun activity increases survival to cisplatin.

Antineoplasic agents such as cisplatin and adriamycin execute their pharmacological role by triggering apoptosis. We have studied the mechanism of apoptosis induction by cisplatin and adriamycin. Both drugs activated JNK with slow and persistent kinetics. Adriamycin activated caspase-3 before the rise in JNK activity, while the response to cisplatin occurs hours after JNK activation. The increase in JNK activity was necessary for cisplatin-mediated apoptosis but it was dispensable for adriamycin-induced cell death. Cells derived from c-jun knock out mice were more resistant to cisplatin cell death than normal cells, but no difference was observed in response to adriamycin. Activation of JNK and cell death by cisplatin is mediated by the MEKK1/SEK1 cascade, since expression of dominant negative expression vectors of these kinases blocked both processes. p38 was also activated by cisplatin with similar kinetics as JNK. AP-1 complexes were activated by cisplatin including mainly c-jun/ATF-2 heterodimers suggesting that AP-1-dependent transcription partially mediated cisplatin-induced apoptosis.

The absence of a major Ca2+ signaling pathway in GABAergic neurons of the hippocampus.

The Ca2+/calmodulin-dependent protein phosphatase 2B or calcineurin (CN) participates in several Ca2+-dependent signal transduction cascades and, thus, contributes to the short and long term regulation of neuronal excitability. By using a specific antibody to CN, we demonstrate its absence from hippocampal interneurons and illustrate a physiological consequence of such CN deficiency. Consistent with the lack of CN in interneurons as detected by immunocytochemistry, the CN inhibitors FK-506 or okadaic acid significantly prolonged N-methyl-D-aspartate channel openings recorded in the cell-attached mode in hippocampal principal cells but not those recorded in interneurons. Interneurons were also devoid of Ca2+/calmodulin-dependent protein kinase IIalpha, yet many of their nuclei contained the cyclic AMP-responsive element binding protein. On the basis of the CN and Ca2+/calmodulin-dependent protein kinase IIalpha deficiency of interneurons, entirely different biochemical mechanisms are expected to govern Ca2+-dependent neuronal plasticity in interneurons versus principal cells.

Involvement of a single-stranded DNA binding protein, ssCRE-BP/Pur alpha, in morphine dependence.

We have purified a nuclear protein from mouse cerebella that binds to single-stranded oligo-DNA of cAMP response element and is modulated by morphine treatment. Isolation of the cDNA clone showed that the nuclear protein (ssCRE-BP) was identical to Pur alpha, a DNA binding protein for single-stranded purine-rich sequences that was originally isolated as a replication factor. ssCRE-BP/Pur alpha and mRNA were abundant in the brain. The levels of ssCRE-BP/Pur alpha and the transcript were not changed by chronic morphine treatment, however, the levels of an activator of ssCRE-BP/Pur alpha, which is necessary for the DNA binding, may be modulated by the treatment.