Human PEX19: cDNA cloning by functional complementation, mutation analysis in a patient with Zellweger syndrome, and potential role in peroxisomal membrane assembly

At least 11 complementation groups (CGs) have been identified for the peroxisome biogenesis disorders (PBDs) such as Zellweger syndrome, for which seven pathogenic genes have been elucidated. We have isolated a human PEX19 cDNA (HsPEX19) by functional complementation of peroxisome deficiency of a mutant Chinese hamster ovary cell line, ZP119, defective in import of both matrix and membrane proteins. This cDNA encodes a hydrophilic protein (Pex19p) comprising 299 amino acids, with a prenylation motif, CAAX box, at the C terminus. Farnesylated Pex19p is partly, if not all, anchored in the peroxisomal membrane, exposing its N-terminal part to the cytosol. A stable transformant of ZP119 with HsPEX19 was morphologically and biochemically restored for peroxisome biogenesis. HsPEX19 expression also restored peroxisomal protein import in fibroblasts from a patient (PBDJ-01) with Zellweger syndrome of CG-J. This patient (PBDJ-01) possessed a homozygous, inactivating mutation: a 1-base insertion, A764, in a codon for Met255, resulted in a frameshift, inducing a 24-aa sequence entirely distinct from normal Pex19p. These results demonstrate that PEX19 is the causative gene for CG-J PBD and suggest that the C-terminal part, including the CAAX homology box, is required for the biological function of Pex19p. Moreover, Pex19p is apparently involved at the initial stage in peroxisome membrane assembly, before the import of matrix protein.

In vivo analysis of the 3′ untranslated region of the hepatitis C virus after in vitro mutagenesis of an infectious cDNA clone

Large sections of the 3′ untranslated region (UTR) of hepatitis C virus (HCV) were deleted from an infectious cDNA clone, and the RNA transcripts from seven deletion mutants were tested sequentially for infectivity in a chimpanzee. Mutants lacking all or part of the 3′ terminal conserved region or the poly(U–UC) region were unable to infect the chimpanzee, indicating that both regions are critical for infectivity in vivo. However, the third region, the variable region, was able to tolerate a deletion that destroyed the two putative stem–loop structures within this region. Mutant VR-24 containing a deletion of the proximal 24 nt of the variable region of the 3′ UTR was viable in the chimpanzee and seemed to replicate as well as the undeleted parent virus. The chimpanzee became viremic 1 week after inoculation with mutant VR-24, and the HCV genome titer increased over time during the early acute infection. Therefore, the poly(U–UC) region and the conserved region, but not the variable region, of the 3′ UTR seem to be critical for in vivo infectivity of HCV.

An ethylene-induced cDNA encoding a lipase expressed at the onset of senescence

A cDNA clone encoding a lipase (lipolytic acyl hydrolase) expressed at the onset of petal senescence has been isolated by screening a cDNA expression library prepared from carnation flowers (Dianthus caryophyllus). The cDNA contains the lipase consensus sequence, ITFAGHSLGA, and encodes a 447-amino acid polypeptide with a calculated molecular mass of 50.2 kDa that appears to be a cytosolic protein. Over-expression of the clone in Escherichia coli yielded a protein of the expected molecular weight that proved capable of deesterifying fatty acids from p-nitrophenylpalmitate, tri-linolein, soybean phospholipid, and Tween in both in vitro and in situ assays of enzyme activity. The abundance of the lipase mRNA increases just as carnation flowers begin to senesce, and expression of the gene is also induced by treatment with ethylene. Southern blot analyses of carnation genomic DNA have indicated that the lipase is a single copy gene. The lipase gene is also expressed in carnation leaves and is up-regulated when the leaves are treated with ethylene. Deesterification of membrane lipids and ensuing loss of membrane structural integrity are well established early events of plant senescence, and the expression pattern of this lipase gene together with the lipolytic activity of its cognate protein indicate that it plays a fundamentally central role in mediating the onset of senescence.

Cloning and functional expression of a cDNA encoding a pheromone gland-specific acyl-CoA Δ11-desaturase of the cabbage looper moth, Trichoplusia ni

Desaturation of coenzyme-A esters of saturated fatty acids is a common feature of sex pheromone biosynthetic pathways in the Lepidoptera. The enzymes that catalyze this step share several biochemical properties with the ubiquitous acyl-CoA Δ9-desaturases of animals and fungi, suggesting a common ancestral origin. Unlike metabolic acyl-CoA Δ9-desaturases, pheromone desaturases have evolved unusual regio- and stereoselective activities that contribute to the remarkable diversity of chemical structures used as pheromones in this large taxonomic group. In this report, we describe the isolation of a cDNA encoding a pheromone gland desaturase from the cabbage looper moth, Trichoplusia ni, a species in which all unsaturated pheromone products are produced via a Δ11Z-desaturation mechanism. The largest ORF of the ≈1,250-bp cDNA encodes a 349-aa apoprotein (PDesat-Tn Δ11Z) with a predicted molecular mass of 40,240 Da. Its hydrophobicity profile is similar overall to those of rat and yeast Δ9-desaturases, suggesting conserved transmembrane topology. A 182-aa core domain delimited by conserved histidine-rich motifs implicated in iron-binding and catalysis has 72 and 58% similarity (including conservative substitutions) to acyl-CoA Δ9Z-desaturases of rat and yeast, respectively. Northern blot analysis revealed an ≈1,250-nt PDesat-Tn Δ11Z mRNA that is consistent with the spatial and temporal distribution of Δ11-desaturase enzyme activity. Genetic transformation of a desaturase-deficient strain of the yeast Saccharomyces cerevisiae with an expression plasmid encoding PDesat-Tn Δ11Z resulted in complementation of the strain’s fatty acid auxotrophy and the production of Δ11Z-unsaturated fatty acids.

Cloning of the cDNA encoding the urotensin II precursor in frog and human reveals intense expression of the urotensin II gene in motoneurons of the spinal cord

Urotensin II (UII) is a cyclic peptide initially isolated from the caudal neurosecretory system of teleost fish. Subsequently, UII has been characterized from a frog brain extract, indicating that a gene encoding a UII precursor is also present in the genome of a tetrapod. Here, we report the characterization of the cDNAs encoding frog and human UII precursors and the localization of the corresponding mRNAs. In both frog and human, the UII sequence is located at the C-terminal position of the precursor. Human UII is composed of only 11 amino acid residues, while fish and frog UII possess 12 and 13 amino acid residues, respectively. The cyclic region of UII, which is responsible for the biological activity of the peptide, has been fully conserved from fish to human. Northern blot and dot blot analysis revealed that UII precursor mRNAs are found predominantly in the frog and human spinal cord. In situ hybridization studies showed that the UII precursor gene is actively expressed in motoneurons. The present study demonstrates that UII, which has long been regarded as a peptide exclusively produced by the urophysis of teleost fish, is actually present in the brain of amphibians and mammals. The fact that evolutionary pressure has acted to conserve fully the biologically active sequence of UII suggests that the peptide may exert important physiological functions in humans.

Molecular cloning of a cytochrome P450 taxane 10β-hydroxylase cDNA from Taxus and functional expression in yeast

The early steps in the biosynthesis of Taxol involve the cyclization of geranylgeranyl diphosphate to taxa-4(5),11(12)-diene followed by cytochrome P450-mediated hydroxylation at C5, acetylation of this intermediate, and a second cytochrome P450-dependent hydroxylation at C10 to yield taxadien-5α-acetoxy-10β-ol. Subsequent steps of the pathway involve additional cytochrome P450 catalyzed oxygenations and CoA-dependent acylations. The limited feasibility of reverse genetic cloning of cytochrome P450 oxygenases led to the use of Taxus cell cultures induced for Taxol production and the development of an approach based on differential display of mRNA-reverse transcription-PCR, which ultimately provided full-length forms of 13 unique but closely related cytochrome P450 sequences. Functional expression of these enzymes in yeast was monitored by in situ spectrophotometry coupled to in vivo screening of oxygenase activity by feeding taxoid substrates. This strategy yielded a family of taxoid-metabolizing enzymes and revealed the taxane 10β-hydroxylase as a 1494-bp cDNA that encodes a 498-residue cytochrome P450 capable of transforming taxadienyl acetate to the 10β-hydroxy derivative; the identity of this latter pathway intermediate was confirmed by chromatographic and spectrometric means. The 10β-hydroxylase represents the initial cytochrome P450 gene of Taxol biosynthesis to be isolated by an approach that should provide access to the remaining oxygenases of the pathway.

Directional cDNA library construction assisted by the in vitro recombination reaction

We report here a new directional cDNA library construction method using an in vitro site-specific recombination reaction, based on the integrase–excisionase system of bacteriophage λ. Preliminary experiments revealed that in vitro recombinational cloning (RC) provided important advantages over conventional ligation-assisted cloning: it eliminated restriction digestion for directional cloning, generated low levels of chimeric clones, reduced size bias and, in our hands, gave a higher cloning efficiency than conventional ligation reactions. In a cDNA cloning experiment using an in vitro synthesized long poly(A)+ RNA (7.8 kb), the RC gave a higher full-length cDNA clone content and about 10 times more transformants than conventional ligation-assisted cloning. Furthermore, characterization of rat brain cDNA clones yielded by the RC method showed that the frequency of cDNA clones >2 kb having internal NotI sites was ∼6%, whereas these cDNAs could not be cloned at all or could be isolated only in a truncated form by conventional methods. Taken together, these results indicate that the RC method makes it possible to prepare cDNA libraries better representing the entire population of cDNAs, without sacrificing the simplicity of current conventional ligation-assisted methods.

FULL-malaria: a database for a full-length enriched cDNA library from human malaria parasite, Plasmodium falciparum

FULL-malaria is a database for a full-length-enriched cDNA library from the human malaria parasite Plasmodium falciparum (http://133.11.149.55/). Because of its medical importance, this organism is the first target for genome sequencing of a eukaryotic pathogen; the sequences of two of its 14 chromosomes have already been determined. However, for the full exploitation of this rapidly accumulating information, correct identification of the genes and study of their expression are essential. Using the oligo-capping method, we have produced a full-length-enriched cDNA library from erythrocytic stage parasites and performed one-pass reading. The database consists of nucleotide sequences of 2490 random clones that include 390 (16%) known malaria genes according to BLASTN analysis of the nr-nt database in GenBank; these represent 98 genes, and the clones for 48 of these genes contain the complete protein-coding sequence (49%). On the other hand, comparisons with the complete chromosome 2 sequence revealed that 35 of 210 predicted genes are expressed, and in addition led to detection of three new gene candidates that were not previously known. In total, 19 of these 38 clones (50%) were full-length. From these obser­vations, it is expected that the database contains ∼1000 genes, including 500 full-length clones. It should be an invaluable resource for the development of vaccines and novel drugs.

HUNT: launch of a full-length cDNA database from the Helix Research Institute

The Helix Research Institute (HRI) in Japan is releasing 4356 HUman Novel Transcripts and related information in the newly established HUNT database. The institute is a joint research project principally funded by the Japanese Ministry of International Trade and Industry, and the clones were sequenced in the governmental New Energy and Industrial Technology Development Organization (NEDO) Human cDNA Sequencing Project. The HUNT database contains an extensive amount of annotation from advanced analysis and represents an essential bioinformatics contribution towards understanding of the gene function. The HRI human cDNA clones were obtained from full-length enriched cDNA libraries constructed with the oligo-capping method and have resulted in novel full-length cDNA sequences. A large fraction has little similarity to any proteins of known function and to obtain clues about possible function we have developed original analysis procedures. Any putative function deduced here can be validated or refuted by complementary analysis results. The user can also extract information from specific categories like PROSITE patterns, PFAM domains, PSORT localization, transmembrane helices and clones with GENIUS structure assignments. The HUNT database can be accessed at http://www.hri.co.jp/HUNT.

The Stanford Microarray Database

The Stanford Microarray Database (SMD) stores raw and normalized data from microarray experiments, and provides web interfaces for researchers to retrieve, analyze and visualize their data. The two immediate goals for SMD are to serve as a storage site for microarray data from ongoing research at Stanford University, and to facilitate the public dissemination of that data once published, or released by the researcher. Of paramount importance is the connection of microarray data with the biological data that pertains to the DNA deposited on the microarray (genes, clones etc.). SMD makes use of many public resources to connect expression information to the relevant biology, including SGD [Ball,C.A., Dolinski,K., Dwight,S.S., Harris,M.A., Issel-Tarver,L., Kasarskis,A., Scafe,C.R., Sherlock,G., Binkley,G., Jin,H. et al. (2000) Nucleic Acids Res., 28, 77–80], YPD and WormPD [Costanzo,M.C., Hogan,J.D., Cusick,M.E., Davis,B.P., Fancher,A.M., Hodges,P.E., Kondu,P., Lengieza,C., Lew-Smith,J.E., Lingner,C. et al. (2000) Nucleic Acids Res., 28, 73–76], Unigene [Wheeler,D.L., Chappey,C., Lash,A.E., Leipe,D.D., Madden,T.L., Schuler,G.D., Tatusova,T.A. and Rapp,B.A. (2000) Nucleic Acids Res., 28, 10–14], dbEST [Boguski,M.S., Lowe,T.M. and Tolstoshev,C.M. (1993) Nature Genet., 4, 332–333] and SWISS-PROT [Bairoch,A. and Apweiler,R. (2000) Nucleic Acids Res., 28, 45–48] and can be accessed at http://genome-www.stanford.edu/microarray.

Delineating developmental and metabolic pathways in vivo by expression profiling using the RIKEN set of 18,816 full-length enriched mouse cDNA arrays

We have systematically characterized gene expression patterns in 49 adult and embryonic mouse tissues by using cDNA microarrays with 18,816 mouse cDNAs. Cluster analysis defined sets of genes that were expressed ubiquitously or in similar groups of tissues such as digestive organs and muscle. Clustering of expression profiles was observed in embryonic brain, postnatal cerebellum, and adult olfactory bulb, reflecting similarities in neurogenesis and remodeling. Finally, clustering genes coding for known enzymes into 78 metabolic pathways revealed a surprising coordination of expression within each pathway among different tissues. On the other hand, a more detailed examination of glycolysis revealed tissue-specific differences in profiles of key regulatory enzymes. Thus, by surveying global gene expression by using microarrays with a large number of elements, we provide insights into the commonality and diversity of pathways responsible for the development and maintenance of the mammalian body plan.

An orchestrated gene expression component of neuronal programmed cell death revealed by cDNA array analysis

Programmed cell death (PCD) during neuronal development and disease has been shown to require de novo RNA synthesis. However, the time course and regulation of target genes is poorly understood. By using a brain-biased array of over 7,500 cDNAs, we profiled this gene expression component of PCD in cerebellar granule neurons challenged separately by potassium withdrawal, combined potassium and serum withdrawal, and kainic acid administration. We found that hundreds of genes were significantly regulated in discreet waves including known genes whose protein products are involved in PCD. A restricted set of genes was regulated by all models, providing evidence that signals inducing PCD can regulate large assemblages of genes (of which a restricted subset may be shared in multiple pathways).

GenEST, a powerful bidirectional link between cDNA sequence data and gene expression profiles generated by cDNA-AFLP

The release of vast quantities of DNA sequence data by large-scale genome and expressed sequence tag (EST) projects underlines the necessity for the development of efficient and inexpensive ways to link sequence databases with temporal and spatial expression profiles. Here we demonstrate the power of linking cDNA sequence data (including EST sequences) with transcript profiles revealed by cDNA-AFLP, a highly reproducible differential display method based on restriction enzyme digests and selective amplification under high stringency conditions. We have developed a computer program (GenEST) that predicts the sizes of virtual transcript-derived fragments (TDFs) of in silico-digested cDNA sequences retrieved from databases. The vast majority of the resulting virtual TDFs could be traced back among the thousands of TDFs displayed on cDNA-AFLP gels. Sequencing of the corresponding bands excised from cDNA-AFLP gels revealed no inconsistencies. As a consequence, cDNA sequence databases can be screened very efficiently to identify genes with relevant expression profiles. The other way round, it is possible to switch from cDNA-AFLP gels to sequences in the databases. Using the restriction enzyme recognition sites, the primer extensions and the estimated TDF size as identifiers, the DNA sequence(s) corresponding to a TDF with an interesting expression pattern can be identified. In this paper we show examples in both directions by analyzing the plant parasitic nematode Globodera rostochiensis. Various novel pathogenicity factors were identified by combining ESTs from the infective stage juveniles with expression profiles of ∼4000 genes in five developmental stages produced by cDNA-AFLP.

Characterization of Two Divergent Endo-β-1,4-Glucanase cDNA Clones Highly Expressed in the Nonclimacteric Strawberry Fruit

Two cDNAs clones (Cel1 and Cel2) encoding divergent endo-β-1,4-glucanases (EGases) have been isolated from a cDNA library obtained from ripe strawberry (Fragaria x ananassa Duch) fruit. The analysis of the amino acid sequence suggests that Cel1 and Cel2 EGases have different secondary and tertiary structures and that they differ in the presence of potential N-glycosylation sites. By in vitro translation we show that Cel1 and Cel2 bear a functional signal peptide, the cleavage of which yields mature proteins of 52 and 60 kD, respectively. Phylogenetic analysis revealed that the Cel2 EGase diverged early in evolution from other plant EGases. Northern analysis showed that both EGases are highly expressed in fruit and that they have different temporal patterns of accumulation. The Cel2 EGase was expressed in green fruit, accumulating as the fruit turned from green to white and remaining at an elevated, constant level throughout fruit ripening. In contrast, the Cel1 transcript was not detected in green fruit and only a low level of expression was observed in white fruit. The level of Cel1 mRNA increased gradually during ripening, reaching a maximum in fully ripe fruit. The high levels of Cel1 and Cel2 mRNA in ripe fruit and their overlapping patterns of expression suggest that these EGases play an important role in softening during ripening. In addition, the early expression of Cel2 in green fruit, well before significant softening begins, suggests that the product of this gene may also be involved in processes other than fruit softening, e.g. cell wall expansion.