Multiple requirements for nematode spliced leader RNP function in trans-splicing.

The 5' exon donor in nematode trans-splicing, the SL RNA, is a small (approximately 100 nt) RNA that resembles cis-spliceosomal U snRNAs. Extensive analyses of the RNA sequence requirements for SL RNA function have revealed four essential elements, the core Sm binding site, three nucleotides immediately downstream of this site, a region of Stem-loop II, and a 5' splice site. Although these elements are necessary and sufficient for SL RNA function in vitro, their respective roles in promoting SL RNA activity have not been elucidated. Furthermore, although it has been shown that assembly of the SL RNA into an Sm RNP is a prerequisite for function, the protein composition of the SL RNP has not been determined. Here, we have used oligoribonucleotide affinity to purify the SL RNP and find that it contains core Sm proteins as well as four specific proteins (175, 40, 30, and 28 kDa). Using in vitro assembly assays; we show that association of the 175- and 30-kDa SL-specific proteins correlates with SL RNP function in trans-splicing. Binding of these proteins depends upon the sequence of the core Sm binding site; SL RNAs containing the U1 snRNA Sm binding site assemble into Sm RNPs that contain core, but not SL-specific proteins. Furthermore, mutational and thiophosphate interference approaches reveal that both the primary nucleotide sequence and a specific phosphate oxygen within a segment of Stemloop II of the SL RNA are required for function. Finally, mutational activation of an unusual cryptic 5' splice site within the SL sequence itself suggests that U5 snRNA may play a primary role in selecting and specifying the 5' splice site in SL addition trans-splicing.

Production, characterization, and expression of neuropeptide Y by human pheochromocytoma.

Neuropeptide Y (NPY) levels are increased in plasma and tumors of patients with pheochromocytoma. The present study was designed to evaluate plasma and tissue NPY levels simultaneously as well as to study its release and expression in patients with either adrenal or extraadrenal pheochromocytomas. Plasma NPY levels were higher (P < 0.01) in patients with adrenal tumors than in matched normal subjects and patients with extraadrenal tumors. NPY levels were also higher (P < 0.05) in adrenal than in extraadrenal tumors. Bioactive NPY (1-36) was the predominant form in plasma and tumors of patients with adrenal pheochromocytomas. In contrast, patients with extraadrenal pheochromocytomas had an abundance of NPY fragments. NPY mRNA was abundant in 11 of 13 adrenal tumors but in only 1 of 6 extraadrenal tumors. Moreover, NPY was coreleased with NE with manipulation of adrenal but not extraadrenal tumors. These findings indicate that increased NPY gene expression in adrenal pheochromocytomas accounts for the greater biosynthesis and storage of NPY in these tumors and that increased release of NPY results in elevated plasma NPY. Factors regulating NPY gene expression in pheochromocytoma and the role of NPY in the clinical manifestations of the disease remain to be elucidated.

Most mRNAs in the nematode Ascaris lumbricoides are trans-spliced: a role for spliced leader addition in translational efficiency.

Some pre-mRNAs in nematodes are processed by trans-splicing. In this reaction, a 22-nt 5' terminal exon (the spliced leader, SL) and its associated 2,2,7-trimethylguanosine cap are acquired from a specialized Sm snRNP, the SL RNP. Although it has been evident for many years that not all nematode mRNAs contain the SL sequence, the prevalence of trans-spliced mRNAs has, with the exception of Caenorhabditis elegans, not been determined. To address this question in an organism amenable to biochemical analysis, we have prepared a message-dependent protein synthesis system from developing embryos of the parasitic nematode, Ascaris lumbricoides. Using this system, we have used both hybrid-arrest and hybrid-selection approaches to show that the vast majority (80-90%) of A. lumbricoides mRNAs contain the SL sequence and therefore are processed by trans-splicing. Furthermore, to examine the effect of SL addition on translation, we have measured levels of protein synthesis in extracts programmed with a variety of synthetic mRNAs. We find that the SL sequence itself and its associated hypermethylated cap functionally collaborate to enhance translational efficiency, presumably at the level of initiation of protein synthesis. These results indicate that trans-splicing plays a larger role in nematode gene expression than previously suspected.

Functional characterization of ribozymes expressed using U1 and T7 vectors for the intracellular cleavage of ANF mRNA.

Hammerhead ribozymes targeted to various GUC or GUA sites on rat atrial natriuretic factor (ANF) mRNA were developed. The catalytic activity of ribozymes to four of these sites, synthesized by transcription off synthetic oligodeoxynucleotide duplexes, was studied in detail. In vitro, ribozyme-mediated cleavage was highly Mg(2+)-dependent, and at concentrations approaching those found intracellularly, the rate but not the extent of cleavage was markedly reduced. To test for cellular activity, synthetic genes encoding the ribozymes were cloned between the initiation and termination sequences of the U1snRNA gene or between the T7RNA polymerase promoter and terminator sequences in pSP64. Both constructs had defined initiation and termination sequences to minimize transcript size and for message stability. In vitro the addition of T7 or U1 terminator sequences had variable effects on catalytic activity, presumably due to structural interactions between the ribozyme and the added sequence. The ribozyme-encoding plasmids were cotransfected with an expression plasmid containing a rat ANF cDNA into COS-1 cells using a liposome method, which provided high-level transfection efficiency. Quantitation of ANF mRNA by RNase protection showed marked decreases in ANF transcript levels with both the U1- and the T7-expressed ribozymes directed at three of the four sites on ANF mRNA. With all constructs, target accessibility, determined in vitro, was a more important determinant of intracellular ANF mRNA cleavage than catalytic activity per se. ANF mRNA cleavage was not merely due to an antisense effect, since a mutant construct that was catalytically inactive but could still bind produced less cleavage than the corresponding wild-type ribozyme construct. These findings indicate that both U1 and T7 vector systems provide efficient ribozyme expression for the intracellular cleavage of target mRNA.

Serial measurements of FEV1 over 12 years in a patient with alpha-1-protease inhibitor deficiency:influence of augmentation therapy and infections.

In patients with severe alpha-1-protease inhibitor (alpha 1-Pi) deficiency forced expiratory volume in 1 s (FEV1) is an accepted parameter to monitor the progression of emphysema. In a patient with severe alpha 1-Pi deficiency (PiZZ) more than 1,000 FEV1 measurements were performed over a period of 12 years. FEV1 dramatically decreased initially (delta FEV1 > 500 ml/year), but stabilized after augmentation therapy was instituted. Three years later, the FEV1 decreased again abruptly; the deterioration was paralleled by an increasing number of severe bronchopulmonary infections. This nonlinear decline implies a positive influence of augmentation therapy and a deleterious effect of bronchopulmonary infections in the disease (p < 0.0005). Daily variation of FEV1 in infection-free intervals exceeded 30% and were thus higher than the mean decrease in FEV1 per year. In this instance, FEV1 measurements performed once or twice per year may reveal a deterioration, whereas the change of FEV1 is still within the range of spontaneous variation. More frequent measurements of FEV1 can be useful to minimize the influence of high intraindividual variation.

Isolation of cDNA clones and tissue expression of rat ral A and ral B GTP-binding proteins.

cDNA clones encoding the low molecular weight GTP-binding proteins ral A (951 bp) and ral B (2073 bp), including the entire coding region (618 bp), were isolated from a rat PC12 pheochromocytoma library. Northern analyses demonstrated that both ral A and ral B are widely expressed in rat tissues. Two ral A transcripts of 1.1 and 2.9 kb were observed in most tissues in varying proportions. The 1.1 kb ral A band of testes was further shown to be composed of two closely migrating species. In contrast to these findings, a single ral B transcript of 2.3 kb was detected in most tissues. Steady-state levels of ral A transcripts appear greater than ral B. Quantitatively, the testes exhibited the highest ral A and ral B mRNA levels, with lower levels observed in the brain, adrenal and pituitary glands, kidney and ovary. Ral mRNA levels were lowest in muscle tissue, particularly skeletal muscle.

Genomic organization and expression of the human alpha 1B-adrenergic receptor.

alpha 1-Adrenergic receptors (ARs) are members of the guanine nucleotide-binding protein-coupled receptor superfamily. The genes for all ARs described thus far are intronless. We report here the cloning and the nucleotide sequence of the gene for the human alpha 1B-AR. It consists of two exons and a single large intron of at least 20 kilobases which interrupts the coding region at the end of the putative sixth transmembrane domain. The deduced amino acid sequence of the encoded receptor has a high degree of homology to the cloned hamster, rat, and dog alpha 1B-ARs. To characterize the encoded protein, a fusion gene constructed by splicing together exon 1 and exon 2 was expressed transiently in COS-1 cells. The transfected gene fusion product resulted in the production of an alpha 1B-AR with ligand binding characteristics indistinguishable from those of the expressed hamster alpha 1B cDNA. Evidence that the human alpha 1B-AR gene we have isolated is indeed transcribed is the finding of similar sized (2.8-kilobase) transcripts in human heart and other tissues by Northern blot analysis when either exon 1 or exon 2 is used as a probe. Moreover, using primers designed to span the exon 1/exon 2 boundary, a polymerase chain reaction product generated from single-stranded DNA prepared from human heart mRNA had the exact size and nucleotide sequence predicted for a transcript in which exon 1 is spliced to exon 2. The 5'-flanking region (924 base pairs (bp)) of exon 1 contains neither a TATA box nor a CAAT box but is high in GC content (70%) and contains several Sp1 binding sites (GC boxes), consistent with promoters described for housekeeping genes. The 5'-untranslated region also contains a putative cyclic AMP response element. Primer extension studies and RNase protection assays suggested that there are several potential transcription start sites in most tissues with a predominant site located 173 bp upstream from the translation start site. The 3'-flanking region contains a putative polyadenylation signal (ATTAAA) 492 bp downstream from the stop codon. The genomic organization of the human alpha 1B-AR with a single large intron interrupting its coding region differs from those of other ARs as well as muscarinic and 5-hydroxy-tryptamine receptors, which are intronless. The location of the intron in the human alpha 1B-AR gene is also unique among those members of the G-protein-coupled receptor family that do possess introns.(ABSTRACT TRUNCATED AT 400 WORDS)

The nematode spliced leader RNA participates in trans-splicing as an Sm snRNP.

The trans-spliced leader RNA (SL RNA) of nematodes resembles U snRNAs both in cap structure and in the presence of a consensus Sm binding site. We show here that synthetic SL RNA, synthesized by in vitro transcription, is efficiently used as a spliced leader donor in trans-splicing reactions catalyzed by a cell free extract prepared from developing embryos of the parasitic nematode, Ascaris lumbricoides. Efficient utilization of synthetic SL RNA requires a functional Sm binding site. Mutations within the Sm binding sequence that prevent immunoprecipitation by Sm antisera and prevent cap trimethylation abolish trans-splicing. The effect on trans-splicing is not due to undermethylation of the cap structure.

Trans splicing of nematode pre-messenger RNA in vitro.

In nematodes, a fraction of mRNAs contains a common 22 nucleotide 5' terminal spliced leader (SL) sequence derived by trans splicing. Here, we show that a cell-free extract prepared from developing embryos of the parasitic nematode Ascaris lumbricoides catalyzes accurate and efficient SL addition to a synthetic pre-mRNA at an authentic trans splice acceptor site. SL addition occurs via a trans splicing reaction that proceeds through Y-branched intermediates. The branchpoint is located at either of two adenosine residues located 18 and 19 nucleotides upstream of the splice acceptor site.

Characterization and expression of a spliced leader RNA in the parasitic nematode Ascaris lumbricoides var. suum.

The parasitic nematode Ascaris spp. contains a 22-nucleotide spliced-leader (SL) sequence identical to the trans-SL previously described in Caenorhabditis elegans and other nematodes. The SL comprises the first 22 nucleotides of a approximately 110-base RNA and is transcribed by RNA polymerase II. The SL RNA contains a trimethylguanosine cap and a consensus Sm binding site. Furthermore, the Ascaris SL RNA has the potential to adopt a secondary structure which is nearly identical to potential secondary structures of similar SL RNAs in C. elegans and Brugia malayi.

Organization and expression of 5S rRNA genes in the parasitic nematode, Brugia malayi.

DNA sequence analysis of genes encoding 5S rRNA in the human parasitic nematode Brugia malayi (B. malayi) indicates a surprising degree of heterogeneity. This variation in coding sequence is not accompanied by corresponding heterogeneity in flanking regions which are highly conserved. Six out of eight potential 5S coding regions differed; of these sequence variants, two were abundant in the B. malayi genome. Direct RNA sequence analysis indicated that one of these abundant variants accounts for most if not all of expressed 5S RNA at two stages of development.

A 22-nucleotide spliced leader sequence in the human parasitic nematode Brugia malayi is identical to the trans-spliced leader exon in Caenorhabditis elegans.

The mRNAs encoding a 63-kDa antigen in the human parasitic nematode Brugia Malayi contain a spliced leader sequence of 22 nucleotides (nt) that is identical to the trans-spliced leader found on certain actin mRNAs in the distantly related nematode Caenorhabditis elegans. The 22-nt sequence does not appear to be encoded near the 63-kDa genes but is present in multiple copies in several locations within the parasite genome, including the 5S rRNA gene repeat. The 5S-linked copies of the 22-nt sequence are transcribed to yield a 109-nt nonpolyadenylated RNA with the 22-nt leader sequence at its 5' end. We suggest that the 22-nt leader is acquired by 63-kDa antigen mRNAs through trans-splicing. These results indicate that trans-splicing is widespread in nematodes and argue for the functional significance of the 22-nt spliced leader exon in nematode mRNA metabolism.

A multi-copy gene encodes a potentially protective antigen in Brugia malayi.

A genomic library of Brugia malayi was constructed and screened by hybridization with a cDNA clone corresponding to a potentially protective antigen of 63 kDa. The antigen is encoded by a multicopy gene family. Five distinct gene copies were isolated and one was characterized in detail by nucleotide sequence analysis. An apparent pseudogene was also characterized. The organization of genes encoding the antigen is typical of higher eukaryotes in exon/intron organization although the introns have an unusually high A+T content (75%). Organization of the genomic sequence along with S1 nuclease and primer extension analyses indicate that a short untranslated exon is spliced to the 5' end of the mRNAs encoding the antigen.

Cloning and characterization of a potentially protective antigen in lymphatic filariasis.

To facilitate biochemical studies of protective filarial antigens, a lambda gt11 cDNA library was constructed from Brugia malayi adult mRNA and screened with rabbit sera that recognizes a limited set of filarial antigens of approximately 25, 42, 60, and 112 kDa. Antigens of approximately equal to 25 and approximately equal to 60 kDa have been shown previously to induce enhanced clearance of microfilaremia in mice. A 154-base pair clone detected by immunological reactivity was used to isolate by hybridization a nearly full-length cDNA clone of 1.8 kilobases. Nucleotide-sequence analysis indicated that this clone was derived from a mRNA encoding a 63-kDa antigen. A fusion polypeptide containing 37 kDa of the Escherichia coli TrpE protein (anthranilate synthase) and 55 kDa of the cloned protein was recognized in immunoblot experiments with antisera raised against a partially purified preparation of the approximately equal to 60-kDa protective filarial antigen. These data relate the cloned antigen to a potentially protective antigen in lymphatic filariasis.

Building a hierarchy with neural networks: an example-image vector quantization.

Electronic neural networks can perform the function of associative memory. Given an input pattern, the network searches through its stored memories to find which of them best matches the input. Thus the network does a combination of content-addressable search and error correction. The number of random memories that a network can store is limited to a fraction of the number of electronic neurons in the circuit. We propose a method for building a hierarchy of networks that allows the fast parallel search through a list of memories that is too large to store in a single network. We have demonstrated the principle of this approach by an example in image vector quantization.

The determination of pentachlorophenol and tetrachlorophenols in wadden sediment and clams (Mya arenaria) using triethylsulfonium hydroxide for extraction and pyrolytic ethylation.

A method to determine the concentration of pentachlorophenol and tetrachlorophenols in wadden sediments and clams is described. This method involves the extraction of lyophilized specimens with toluene under acidic conditions and the back extraction of the chlorophenols into a methanol/water solution of triethylsulfonium hydroxide. Upon injection of the methanol/water phase into the gaschromatograph a pyrolytic ethylation is performed and the ethylethers of pentachlorophenol and tetrachlorophenols formed thereby are separated in quartz capillary columns and detected by an electron capture detector. Using tribromophenol as internal standard the recovery rates for the chlorophenols were within the range of 76.7 and 98.8%. The method described does not require any evaporation or chromatographic clean-up steps. The detection limit was found to be 2 nmol/kg (approximately 500 ng/kg) for sediment and 0.1 mumol/kg (approximately 25 micrograms/kg) for clams. Its accuracy was verified by gaschromatography-mass spectrometry experiments.