New p53 target, phosphatase of regenerating liver 1 (PRL-1) downregulates p53.

Most of the p53 target genes, all except MDM2, COP1 and PIRH2, perform functions in apoptosis, differentiation and cell cycle arrest. The aforementioned oncogenes downregulate p53 through a negative feedback mechanism, and thus contribute to tumor development. In this study, we report a new p53 target, PRL-1, which is believed to be a significant regulator in the development and metastasis of a variety of cancer types. Phosphatase of regenerating liver 1 (PRL-1) overexpression reduced the levels of endogenous and exogenous p53 proteins, and inhibited p53-mediated apoptosis. On the other hand, the ablation of PRL-1 by small interfering RNA (siRNA) increased p53 protein levels. The p53 downregulation was mediated by p53 ubiquitination and subsequent proteasomal degradation. Furthermore, p53 ubiquitination by PRL-1 was achieved through two independent pathways, by inducing PIRH2 transcription and by inducing MDM2 phosphorylation through Akt signaling. In addition, we showed that the PRL-1 gene harbors a p53 response element in the first intron, and its transcription is regulated by the p53 protein. These findings imply that the new oncogenic p53 target, PRL-1, may contribute to tumor development by the downregulation of p53 by a negative feedback mechanism.

Taxonomic discrimination of flowering plants by multivariate analysis of Fourier transform infrared spectroscopy data.

Fourier transform infrared spectroscopy (FTIR) provides biochemical profiles containing overlapping signals from a majority of the compounds that are present when whole cells are analyzed. Leaf samples of seven higher plant species and varieties were subjected to FTIR to determine whether plants can be discriminated phylogenetically on the basis of biochemical profiles. A hierarchical dendrogram based on principal component analysis (PCA) of FTIR data showed relationships between plants that were in agreement with known plant taxonomy. Genetic programming (GP) analysis determined the top three to five biomarkers from FTIR data that discriminated plants at each hierarchical level of the dendrogram. Most biomarkers determined by GP analysis at each hierarchical level were specific to the carbohydrate fingerprint region (1,200-800 cm(-1)) of the FTIR spectrum. Our results indicate that differences in cell-wall composition and structure can provide the basis for chemotaxonomy of flowering plants.

Taxonomic discrimination of higher plants by pyrolysis mass spectrometry.

Pyrolysis mass spectrometry (PyMS) is a rapid, simple, high-resolution analytical method based on thermal degradation of complex material in a vacuum and has been widely applied to the discrimination of closely related microbial strains. Leaf samples of six species and one variety of higher plants (Rosa multiflora, R. multiflora var. platyphylla, Sedum kamtschaticum, S. takesimense, S. sarmentosum, Hepatica insularis, and H. asiatica) were subjected to PyMS for spectral fingerprinting. Principal component analysis of PyMS data was not able to discriminate these plants in discrete clusters. However, canonical variate analysis of PyMS data separated these plants from one another. A hierarchical dendrogram based on canonical variate analysis was in agreement with the known taxonomy of the plants at the variety level. These results indicate that PyMS is able to discriminate higher plants based on taxonomic classification at the family, genus, species, and variety level.

Enhanced thermal stability of an alkaline protease, AprP, isolated from a Pseudomonas sp. by mutation at an autoproteolysis site, Ser-331.

The thermal stability of the alkaline protease extracellular subtilisin-type serine protease (AprP) from Pseudomonas sp. KFCC 10818 was improved by altering an amino acid residue at an autoproteolytic cleavage site. N-terminal sequence analysis of the autoproteolytic products of the protein revealed the presence of two cleavage sites, Ser-307 and Ser-331. To increase the thermal stability of the enzyme, serine residues of these sites were replaced with aspartate. The S331D mutant enzyme was successfully purified and characterized whereas the S307D mutant was not. The half-lives of the S331D mutant at 55 degrees C and 60 degrees C were 1.5 and 2.4 times longer than that of the wild-type enzyme, respectively. In addition, the catalytic efficiency was also enhanced.

Lipase and its modulator from Pseudomonas sp. strain KFCC 10818: proline-to-glutamine substitution at position 112 induces formation of enzymatically active lipase in the absence of the modulator.

A lipase gene, lipK, and a lipase modulator gene, limK, of Pseudomonas sp. strain KFCC 10818 have been cloned, sequenced, and expressed in Escherichia coli. The limK gene is located immediately downstream of the lipK gene. Enzymatically active lipase was produced only in the presence of the limK gene. The effect of the lipase modulator LimK on the expression of active lipase was similar to those of the Pseudomonas subfamily I.1 and I.2 lipase-specific foldases (Lifs). The deduced amino acid sequence of LimK shares low homology (17 to 19%) with the known Pseudomonas Lifs, suggesting that Pseudomonas sp. strain KFCC 10818 is only distantly related to the subfamily I.1 and I.2 Pseudomonas species. Surprisingly, a lipase variant that does not require LimK for its correct folding was isolated in the study to investigate the functional interaction between LipK and LimK. When expressed in the absence of LimK, the P112Q variant of LipK formed an active enzyme and displayed 63% of the activity of wild-type LipK expressed in the presence of LimK. These results suggest that the Pro(112) residue of LipK is involved in a key step of lipase folding. We expect that the novel finding of this study may contribute to future research on efficient expression or refolding of industrially important lipases and on the mechanism of lipase folding.

Effect of metal ions on the stability of metallothionein in the degradation by cellular fractions in vitro.

Metallothioneins (MT), small molecular weight metal binding proteins are known to play an important protective role against heavy metal toxicity, either as antioxidants or pre-oxidants. However, the mode of metabolic fate of MTs in various metal complexes is not clearly understood. This study was carried out to better understand the mode of selective turnover rate of various form of MT in complexes with different metals. The degradation of in vitro translated mouse 35S-cysteine-MT was examined in lysosomal or cytosolic fractions from mouse liver by gel electrophoresis and autoradiography. Overnight incubations of MT showed extensive proteolysis in the lysosomal fraction but not in cytosolic fractions. However, Cu2+-MT was found to be stable under the same experimental condition. In contrast, Zn did not interfere with MT degradation. These results suggest that lysosomes are chiefly responsible for MT removal and appears to be selective on the metals involved in the MT complex. In vitro, translated, radiolabeled MT provides a suitable substrate for investigating the characteristics of MT degradation.

Methylation in the p53 promoter is a supplementary route to breast carcinogenesis: correlation between CpG methylation in the p53 promoter and the mutation of the p53 gene in the progression from ductal carcinoma in situ to invasive ductal carcinoma.

Aberrant methylation in the CpG sites located in the promoter region of several tumor suppressor genes has been reported in various types of cancers. However, the methylation status of the p53 promoter has not been clearly determined and no information is available on its role in breast cancer. The aim of the study was to determine the presence and timing of the methylation of CpG sites in the p53 promoter, in the progression from ductal carcinoma in situ to invasive cancer. We also explored the correlation between the CpG methylation of the p53 promoter and p53 mutation during the progression of breast cancer. The corresponding lesions of both the invasive and noninvasive types were microdissected in paraffin-embedded tissue of 26 breast carcinomas. Bisulfite-modified DNA sequencing for methylation status in the p53 promoter was carried out, and double-strand DNA sequencing was performed in the promoter region and exons 4 to 9 of the p53 gene. CpG site methylation in the p53 promoter was detected in three cases (11.5%). Two noninvasive and three invasive lesions harbored CpG methylation in the p53 promoter. Methylations in more than one site were observed in three lesions, all of which contained methylation in two sites. The methylated CpG sites were located near the AP1 and YY-1 binding sites and at the YY-1 binding site. The p53 mutation was not found in the lesions where methylation in p53 promoter region was evident. In 16 cases (61.5%), neither methylation nor p53 mutation was detected. We conclude that the methylation in the p53 promoter region is found in the breast cancer irrespective of the status of invasion, and that the hypermethylation in the p53 promoter region is an alternative pathway to tumorigenesis where there is no p53 gene mutation.

Correlation between genotype and phenotype in Korean patients with spinal muscular atrophy.

The goal of this study was to define the correlation between genotype and phenotype in Korean patients with spinal muscular atrophy (SMA). The SMA can be classified into three groups based on the age of onset and the clinical course. The candidate genes, survival motor neuron (SMN) gene, neuronal apoptosis inhibitory protein (NAIP) gene, and p44 gene were mapped and duplicated with telomeric and centromeric. The loss of the telomeric SMN occurs by a different mechanism. That is the deletion or conversion of telomeric SMN to centromeric SMN, in which case the conversion could produce a mild phenotype and deletion could produce a severe one. It has been known that there may be a balance between the numbers of copies expressed by the centromeric and telomeric SMN genes. In our study, ten patients with type I SMA and two type II patients were identified by their clinical findings and DNA studies. The major deletion of SMA candidate genes, deletion of the SMN gene, NAIP gene, and p44 gene were identified in six patients with type I SMA, while the rest of type I and all the type II patients showed the deletion of the SMN gene only. Allele numbers of the C212 marker were compared in patients and normal controls in order to find the correlation between the copy numbers and the clinical severity. The result was that type I patients had 2-5 alleles and the normal controls had 4-6. This suggests that the deletion is a major determining factor in the clinical phenotype. However, two type I patients with telomeric NAIP gene deletion notably had 4-5 alleles, as in the normal controls. This result implies that the correlation between the copy numbers and the severity is uncertain as opposed to the previous hypothesis. One type I patient showed the conversion of the centromeric SMN gene to the telomeric, which supports the conclusion that gene conversion is an important molecular mechanism for SMA. In the study of one hundred normal newborns, two physically normal newborns showed deletion of the centromeric SMN gene, suggesting frequent rearrangement in the locus.

Translocation and accumulation of exogeneous hepatitis B virus preS surface proteins in the cell nucleus.

Recurrent reports about protease-sensitive sites in the junction of the preS and S region of the hepatitis B virus large surface protein have raised the question about a possible biological role of S protein-depleted, independent preS protein fragments in the virus life cycle. In the present study, this question was addressed by exogenous introduction of fluorescence-labeled recombinant preS proteins into permeabilized HepG2 cells. While maltose-binding proteins (MBP) were evenly distributed throughout the cytoplasm, MBP-preS fusion proteins selectively accumulated in the nucleus. Using truncated preS proteins, the effective domain for this nuclear accumulation was localized around the preS2 region. The mode of this action differs from conventional nuclear translocation mechanism in its energy- and mediator-independency and in that it is not saturated regardless of the increase of preS protein concentration. The biological meaning of this phenomenon has to be further studied. However, in regard to hepatitis B virus infection, this observation might provide a clue for unveiling the still poorly characterized events after initial internalization of the virus, which might make use of the nuclear translocation effect of the preS2 region to facilitate the infection.

Production of biologically active human granulocyte colony stimulating factor in the milk of transgenic goat.

We have developed a transgenic female goat harboring goat beta-casein promoter/human granulocyte colony stimulating factor (G-CSF) fusion gene by microinjection into fertilized one-cell goat zygotes. Human G-CSF was produced at levels of up to 50 microg/ml in transgenic goat milk. Its biological activity was equivalent to recombinant human G-CSF expressed from Chinese hamster ovary (CHO) cell when assayed using in vitro HL-60 cell proliferation. Human G-CSF from transgenic goat milk increased the total number of white blood cells in C57BL/6N mice with leucopenia induced by cyclophosphamide (CPA). The secreted human G-CSF was glycosylated although the degree of O-glycosylation was lower compared to CHO cell-derived human G-CSF.

The first successful prenatal diagnosis on a Korean family with citrullinemia.

DNA prenatal diagnosis was successfully performed on a family with citrullinemia. The father carried the G324S mutation and the mother carried the IVS6-2A > G mutation in the argininosuccinate synthase gene. They had a previous child with citrullinemia who died in the week after birth owing to complicated hyperammonemia. The lost child turned out to be a compound heterozygote. DNA was extracted from the cultured amniotic cells after amniocentesis done at 18-week gestation. For the detection of the G324S mutation, the PCR and restriction fragment length polymorphism method was used, and for the IVS6-2A > G mutation, allele-specific PCR was performed. The fetus was found to carry G324S but not IVS6-2A > G, suggesting a heterozygote carrier. Pregnancy was continued and a healthy boy was born. Plasma amino acid analysis performed on the third day after birth was normal and the serial ammonia level was in the normal range. A molecular study on his genomic DNA after birth also agreed with the previous fetal DNA analysis. He is now 2-months old with normal growth and development.

Molecular cloning and characterization of a human cDNA and gene encoding a novel acid ceramidase-like protein.

Computer-assisted database analysis of sequences homologous to human acid ceramidase (ASAH) revealed a 1233-bp cDNA (previously designated cPj-LTR) whose 266-amino-acid open reading frame had approximately 36% identity with the ASAH polypeptide. Based on this high degree of homology, we undertook further molecular characterization of cPj-LTR and now report the full-length cDNA sequence, complete gene structure (renamed human ASAHL since it is a human acid ceramidase-like sequence), chromosomal location, primer extension and promoter analysis, and transient expression results. The full-length human ASAHL cDNA was 1825 bp and contained an open-reading frame encoding a 359-amino-acid polypeptide that was 33% identical and 69% similar to the ASAH polypeptide over its entire length. Numerous short regions of complete identity were observed between these two sequences and two sequences obtained from the Caenorhabditis elegans genome database. The 30-kb human ASAHL genomic sequence contained 11 exons, which ranged in size from 26 to 671 bp, and 10 introns, which ranged from 150 bp to 6.4 kb. The gene was localized to the chromosomal region 4q21.1 by fluorescence in situ hybridization analysis. Northern blotting experiments revealed a major 2.0-kb ASAHL transcript that was expressed at high levels in the liver and kidney, but at relatively low levels in other tissues such as the lung, heart, and brain. Sequence analysis of the 5'-flanking region of the human ASAHL gene revealed a putative promoter region that lacked a TATA box and was GC rich, typical features of a housekeeping gene promoter, as well as several tissue-specific and/or hormone-induced transcription regulatory sites. 5'-Deletion analysis localized the promoter activity to a 1. 1-kb fragment within this region. A major transcription start site also was located 72 bp upstream from the ATG translation initiation site by primer extension analysis. Expression analysis of a green fluorescence protein/ASAHL fusion protein in COS-1 cells revealed a punctate, perinuclear distribution, although no acid ceramidase activity was detected in the transfected cells using a fluorescence-based in vitro assay system.

Cloning and characterization of the promoter region of the Wilson disease gene.

Wilson disease (WD), an autosomal recessive disorder of copper transport, is marked by impaired biliary excretion and incorporation of copper into ceruloplasmin. Molecular mechanism regulating the expression of the WD gene was studied. We isolated, sequenced, and characterized approximately 1.3 kb of the 5'-flanking region of the WD gene from the human genomic library. The approximately 1.3 kb of the WD sequence directed high level of luciferase activity in HepG2 cells. Interestingly, the 5'-flanking region contained four metal response elements (MREs) and six MRE-like sequences (MLSs), usually found in the metallothionein genes. It also contained a number of putative regulatory elements such as Sp1, AP-1, AP-2, and E-box, but lacked TATA box. The transcription start site was located at 335 base pairs upstream of the translation initiation site. Successive 5'-deletion analyses suggested that the 159-base pair region from -811 to -653, which includes MLS2 (-802 to -796) and MLS3 (-785 to -779), contained one or more positive regulatory element(s). A negative element was also identified at region -1038 to -812. A protein-MLS complex was identified through electrophoretic mobility shift and competition assay using MLS2/MLS3 and HepG2 cell nuclear proteins.

A MADS box gene homologous to AG is expressed in seedlings as well as in flowers of ginseng.

To study the flower development of ginseng, a MADS box cDNA (GAG2) was isolated and characterized. A comparison of the deduced amino acid sequence of GAG2 with the sequences of other MADS box proteins showed higher amino acid identities with AG (71%) from Arabidopsis thaliana, which is specifically expressed in stamens and carpels of flowers than with AGL genes (30 to 60%), suggesting that GAG2 is a ginseng homologue of AG. However northern blot analysis showed that GAG2 was expressed in seedlings. As the ginseng plant grew, the expression of GAG2 was confined to flowers. In situ hybridization experiments showed that GAG2 transcripts accumulated in the three inner whorls of flowers and in the cells surrounding the developing embryo sac. Temporal and spatial differences between GAG2 expression and AG imply that GAG2 alone is not sufficient to determine the identities of sexual organs of ginseng flowers and has additional or unique functions which differ from those of AG.

Molecular cloning of gaf1, a Schizosaccharomyces pombe GATA factor, which can function as a transcriptional activator.

As a first step to elucidate the functions of Schizosaccharomyces pombe (S. pombe) GATA factors, we have isolated the gaf1+ gene (GATA-factor like gene) in S. pombe. The predicted amino acid (aa) sequence of Gaf1 reveals a single zinc finger domain typical of fungal GATA factors, and the zinc finger exhibits 60% aa identity to that of human GATA-1. The open reading frame of Gaf1 predicts a protein of Mr 32 kDa consisting of 290 intronless amino acids. Disruption of this gene has no effect on cell viability and growth rate. The GST-Gaf1 fusion protein binds specifically to GATA motifs of its own promoter as well as DAL7 UAS, a canonical GATA motif of Saccharomyces cerevisiae (S. cerevisiae) The specific DNA-binding activity resides within the N-terminal half of Gaf1 (Gaf1N; aa 1-120) containing the zinc finger, whereas the C-terminal half (Gaf1C; aa 121-290) contains transactivation sequences that induce the expression of the lacZ reporter when fused to the GAL4 DNA binding domain. These results demonstrate that Gaf1 may function as a transcriptional activator consisting of DNA-binding and transactivation domains.

Identification of three novel mutations and a high frequency of the Arg778Leu mutation in Korean patients with Wilson disease.

Four mutations--R778L, A874V, L1083F, and 2304delC--in the copper-transporting enzyme, P-type ATPase (ATP7B), were identified in Korean Patients with Wilson disease. Arg778Leu, the most frequently reported mutation of this enzyme, was found in six of eight unrelated patients studied, an allele frequency of 37.5%, which is considerably higher than those in other Asian populations. The novel single nucleotide deletion, 2304delC, was found in one patient. Since a mutation at cDNA nucleotide 2302 (2302insC) had been previously described, this region of the ATP7B gene may be susceptible to gene rearrangements causing Wilson disease.

Frequent occurrence of transgene deletion in transgenic plants.

The status of the transgene in tobacco plants transformed by Agrobacterium was analyzed with PCR. Twelve percent of the transgenic plants with the nptII gene showed different levels of transgene deletion, which was also found in transgenic watermelon (10-30%) and carrot (40-60%). It appeared that the percentage of transgenic plants carrying deleted transgenes depended on both the transgene and the plant. It is suggested that the transgene should be inserted between a right border and a selection marker to reduce the number of transgenic plants containing deleted transgenes after selection.

Molecular assembly of the extracellular domain of P2X2, an ATP-gated ion channel.

We have produced the putative extracellular domain (ECD) of the ATP-gated ion channel, P2X2, in a bacterial expression system. The hexahistidine-tagged protein was purified by immobilized metal affinity chromatography and refolded by sulfitolysis and dialysis. We demonstrate that P2X2-ECD forms a stable tetramer in solution by gel filtration chromatography, dynamic light scattering and analytical sedimentation centrifugation. [alpha-32P]ATP has been covalently cross-linked by UV irradiation to the P2X2-ECD and this binding is specific and competable by antagonists suramin and cibacron blue. These results indicate that the binding affinity among P2X2-ECD subunits is appreciably stronger than 3.4 microM (0.1 mg/ml), implying that the extracellular domain of P2X2 is primarly responsible for tetramerization of whole P2X2 and thus probably plays a role in determining homo- and heteromerization specificity of P2X channel subunits.

Cloning of novel trinucleotide-repeat (CAG) containing genes in mouse brain.

CAG trinucleotide repeat (CTR) sequence often appears in mammalian genome including transcription-regulatory protein and homeobox genes. Its expansion is associated with six genetic disorders in human. To identify novel CTR-containing genes expressed in mouse brain, a brain cDNA library was screened using an oligonucleotide, (CTG)10. Eight clones were novel mouse genes and they were sequenced on both strands. The size of the cloned DNA ranged from 0.5 to 2.1 kb. The number of the CAG repeats in the clones ranged from 6 to 25. The inserts of the clones were analyzed for open reading frames and the peptide sequences were used for a GenBank homology search. Of the clones, one (CAG-6) shared 13 consecutive identical amino acid residues with the OB-cadherin gene, a member of cadherin family. CAG-14 showed high homology (657 nucleotides identity in 1022 nucleotides; 64%) with the 3'-untranslated region of rat leukocyte common antigen-related (LAR) tyrosine phosphatase receptor. All the 8 clones were originated from mouse DNA as judged by Southern blot analysis of mouse genomic DNA. The expression of the clones in mouse brain was addressed by RT-PCR and 4 clones showed specific expression.

Identification of the transcription termination site of the mouse nkx-1.2 gene: involvement of sequence-specific factors.

We have identified a transcription termination site in the 3' flanking region of the mouse nkx-1.2 gene. A downstream transcription regulatory element in the mouse nkx-1.2 gene was characterized by transferring its 3'-fragment into a chloramphenicol acetyl transferase (CAT) expression vector. Analysis of recombinant plasmids transfected into mouse NIH3T3 cells by CAT assay showed the possible region of regulation. There were two direct repeat structures containing poly(dG-dT) x poly(dC-dA) sequences (GT repeats) in this region. The precise location of transcription termination was mapped by nuclease S1 analysis of the transcripts from recombinant plasmids transfected into COSM6 cells. It was approximately 20 nucleotides upstream of the first GT repeat within the 5' sequences of the first element of the two direct repeats. Gel mobility shift assay and footprinting analysis demonstrated that nuclear DNA binding proteins bound specifically to the sequences where the termination occurred as well as the other sequences in the second element of the direct repeats. Southwestern analysis showed that 90-, 54-, 36- and 15-kDa nuclear proteins bound to the region of the termination. It is possible that one or more of those proteins are involved in blocking the elongation of the mouse nkx-1.2 gene transcript and then result in termination.