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Objective: To enhance adipic acid production, a computer-aided approach was employed to engineer the adipyl-CoA synthetase from Thermobifida fusca by combining sequence analysis, protein structure modeling, in silico site-directed mutagenesis, and molecular dynamics simulation., Results: Two single mutants of T. fusca adipyl-CoA synthetase, E210βN and E210βQ, achieved a specific enzyme activity of 1.95 and 1.84 U/mg, respectively, which compared favorably with the 1.48 U/mg for the wild-type. The laboratory-level fermentation experiments showed that E210βN and E210βQ achieved a maximum adipic acid titer of 0.32 and 0.3 g/L. In contrast, the wild-type enzyme yielded a titer of 0.15 g/L under the same conditions. Molecular dynamics (MD) simulations revealed that the mutants (E210βN and E210βQ) could accelerate the dephosphorylation process in catalysis and enhance enzyme activity., Conclusions: The combined computational-experimental approach provides an effective strategy for enhancing enzymatic characteristics, and the mutants may find a useful application for producing adipic acid.

Background: The chromobox (CBX) proteins CBX2, CBX4, CBX6, CBX7, and CBX8, also known as Polycomb (Pc) proteins, are canonical components of the Polycomb repressive complex 1 (PRC1). Abundant evidence indicates that abnormal expression of Pc proteins is associated with a variety of tumors, but their role in the pathogenesis of hepatocellular carcinoma (HCC) has not been fully elucidated. In the present study, we performed a case-control study to investigate the relationship between single nucleotide polymorphisms (SNPs) of CBX genes and HCC., Methods: Nine SNPs on CBX genes (rs7217395, rs2036316 of CBX2 ; rs3764374, rs1285251, rs2289728 of CBX4 ; rs7292074 of CBX6 ; and rs710190, rs139394, rs5750753 of CBX7 ) were screened and genotyped using MassARRAY technology in 334 HCC cases and 321 controls. The association between SNPs and their corresponding gene expressions was analyzed through bioinformatics methods using the Ensembl database and Blood eQTL browser online tools., Results: The results indicated that rs2289728 (G>A) of CBX4 ( P = 0.03, OR = 0.56, 95% CI: 0.33-0.94) and rs139394 (C>A) of CBX7 ( P = 0.02, OR = 0.55, 95% CI: 0.33-0.90) decreased the risk of HCC. Interaction between rs2036316 and HBsAg increased the risk of HCC ( P = 0.02, OR = 6.88, 95% CI: 5.20-9.11), whereas SNP-SNP interaction between rs710190 and rs139394 reduced the risk of HCC ( P = 0.03, OR = 0.33, 95% CI: 0.12-0.91). Gene expression analyses showed that the rs2289728 A allele and the rs139394 A allele significantly reduced CBX4 and CBX7 expression, respectively., Conclusion: Our findings suggest that CBX4 rs2289728 and CBX7 rs139394 are protective SNPs against HCC. The two SNPs may reduce the risk of HCC while suppressing the expression of CBX4 and CBX7 .

Curcuma longa L., accumulates substantial amount of curcumin and essential oil. Little is known about the differential expression of curcumin synthase (CURS) gene and consequent curcumin content variations at different agroclimatic zones. The present study aimed to evaluate the effect of climate, soil and harvesting phase on expression of CURS gene for curcumin yield in two high yielding turmeric cultivars. Expression of CURS gene at different experimental zones as well as at different harvesting phase was studied through transcriptional analysis by qRT-PCR. Curcumin varied from 1.5 to 5% and 1.4-5% in Surama and Roma respectively. The expression of CURS also varied from 0.402 to 5.584 fold in Surama and 0.856-5.217 fold in Roma. Difference in curcumin content at a particular zone varied among different harvesting period from 3.95 to 4.31% in Surama and 3.57-3.83% in Roma. Expression of CURS gene was also effected by harvesting time of the rhizome which varied from 7.389 to 16.882 fold in Surama and 4.41-8.342 fold in Roma. The CURS gene expression was found regardless of variations in curcumin content at different experimental zones. This may be due to the effects of soil and environmental variables. Expression was positively correlated with curcumin content with different harvesting time at a particular zone. This find indicates effect of soil and environment on molecular and biochemical dynamics of curcumin biosynthesis and could be useful in genetic improvement of turmeric., (Copyright © 2017 Elsevier Masson SAS. All rights reserved.)

The increasing prevalence of polymyxin-resistant bacteria has stimulated the search for improved polymyxin lipopeptides. Here we describe the sequence and product profile for polymyxin D nonribosomal peptide synthetase from Paenibacillus polymyxa ATCC 10401. The polymyxin D synthase gene cluster comprised five genes that encoded ABC transporters (pmxC and pmxD) and enzymes responsible for the biosynthesis of polymyxin D (pmxA, pmxB, and pmxE). Unlike polymyxins B and E, polymyxin D contains d-Ser at position 3 as opposed to l-α,γ-diaminobutyric acid and has an l-Thr at position 7 rather than l-Leu. Module 3 of pmxE harbored an auxiliary epimerization domain that catalyzes the conversion of l-Ser to the d-form. Structural modeling suggested that the adenylation domains of module 3 in PmxE and modules 6 and 7 in PmxA could bind amino acids with larger side chains than their preferred substrate. Feeding individual amino acids into the culture media not only affected production of polymyxins D 1 and D 2 but also led to the incorporation of different amino acids at positions 3, 6, and 7 of polymyxin D. Interestingly, the unnatural polymyxin analogues did not show antibiotic activity against a panel of Gram-negative clinical isolates, while the natural polymyxins D 1 and D 2 exhibited excellent in vitro antibacterial activity and were efficacious against Klebsiella pneumoniae and Acinetobacter baumannii in a mouse blood infection model. The results demonstrate the excellent antibacterial activity of these unusual d-Ser 3 polymxyins and underscore the possibility of incorporating alternate amino acids at positions 3, 6, and 7 of polymyxin D via manipulation of the polymyxin nonribosomal biosynthetic machinery.

Little is known about the influence of arsenic (As) exposure on monoamine neurotransmitters and the underlying mechanisms, although arsenic toxicity on the central nervous system has been well documented. In the present study, the levels of norepinephrine (NE), dopamine (DA), and 5-HT were determined by high performance liquid chromatography in the cerebrum and cerebellum of mice exposed to 1, 2 and 4 ppm As2O3 through drinking water for 60 days. The ultra-structural change of vesicles in the synapses of mice brains was observed by transmission electron microscopy; the mRNA expressions of dopamine beta hydroxylase (DBH), tyrosine hydroxylase (TH) and tryptophan hydroxylase (TPH) as NE, DA and 5-HT synthetases were quantitatively assessed by real time reverse transcription-polymerase chain reaction. It was shown that the concentrations of NE, DA and 5-HT in the cerebrum or cerebellum of mice exposed to As were significantly lower than those in the control group. The number of synaptic vesicles significantly decreased in the brain of mice exposed to As. Moreover, the expressions of TH, TPH and DBH genes were significantly lower in the brains of mice exposed to As than those in the controls. These results suggested that subchronic exposure to As might decrease the concentrations of the three monoamine neurotransmitters in the mouse brain and downregulate TH, TPH and DBH gene expressions. It was also indicated that the decreased concentrations of the three monoamine neurotransmitters in the brain might be related to the down-regulated gene expressions of these synthetases by As., (Copyright © 2013 IBRO. Published by Elsevier Ltd. All rights reserved.)

4-Hydroxycoumarin (4HC) type anticoagulants (for example, warfarin) are known to have a significant role in the treatment of thromboembolic diseases--a leading cause of patient morbidity and mortality worldwide. 4HC serves as an immediate precursor of these synthetic anticoagulants. Although 4HC was initially identified as a naturally occurring product, its biosynthesis has not been fully elucidated. Here we present the design, validation, in vitro diagnosis and optimization of an artificial biosynthetic mechanism leading to the microbial biosynthesis of 4HC. Remarkably, function-based enzyme bioprospecting leads to the identification of a characteristic FabH-like quinolone synthase from Pseudomonas aeruginosa with high efficiency on the 4HC-forming reaction, which promotes the high-level de novo biosynthesis of 4HC in Escherichia coli (~500 mg l⁻¹ in shake flasks) and further in situ semisynthesis of warfarin. This work has the potential to be scaled-up for microbial production of 4HC and opens up the possibility of biosynthesizing diverse coumarin molecules with pharmaceutical importance.

1-Hydroxy-2-methyl-2-(E)-butenyl-4-diphosphate reductase (HDR) or isopentenyl diphosphate synthase (IDS) is an enzyme at the final step of the MEP pathway. The multi-copy nature of IDS gene in a gymnosperm Ginkgo biloba is known. To evaluate the function of each isogene, the roles of the promoters were examined in Arabidopsis model. Among the promoters of GbIDS series, about 1.3 kb of GbIDS1pro and 1.5 kb of GbIDS2pro were cloned and fused with GUS. The GbIDS1pro::GUS was introduced into Arabidopsis to show GUS expression in most organs except for roots, petals, and stamina, whereas the GbIDS2pro::GUS was expressed only in the young leaves, internodes where the flower and shoot branched, and notably in primary root junction. This pattern of GUS expression correlated with high transcript level of GbIDS2 compared to that of GbIDS1 in Ginkgo roots. Methyl jasmonate (MeJA) treatment resulted in down-regulated GbIDS1pro activity in Arabidopsis leaves and upregulated GbIDS2pro activity in roots. The same pattern of gene regulation in roots was also seen upon treatments of gibberellins, abscisic acid, and indole butyric acid., (Copyright © 2012 Elsevier Masson SAS. All rights reserved.)

Sinorhizobium meliloti Rm2011 responds to sudden shifts to nitrogen or carbon starvation conditions by an accumulation of the stringent response alarmone ppGpp and remodelling of the transcriptome. The gene product of relA, Rel(Sm) , responsible for synthesis of ppGpp, shows functional similarities to E. coli SpoT. Using promoter-egfp gene fusions, we showed that in Rm2011 relA is expressed at a low rate, as a readthrough from the rpoZ promoter and from its own weak promoter. The low level of relA expression is physiologically relevant, since overexpression of Rel(Sm) inhibits ppGpp accumulation. The N-terminal portion of Rel(Sm) is required for ppGpp degradation in nutrient-sufficient cells and might be involved in regulation of the ppGpp synthase and hydrolase activities of the protein. Expression profiling of S. meliloti subjected to sudden nitrogen or carbon downshifts revealed that repression of 'house-keeping' genes is largely dependent on relA whereas activation of gene targets of the stress sigma factor RpoE2 occurred independently of relA. The regulatory genes nifA, ntrB, aniA and sinR, as well as genes related to modulation of protein biosynthesis and nucleotide catabolism, were induced in a relA-dependent manner. dksA was required for the majority of the relA-dependent regulations., (© 2011 Blackwell Publishing Ltd.)

The biologically and commercially important terpenoids are a large and diverse class of natural products that are targets of metabolic engineering. However, in the context of metabolic engineering, the otherwise well-documented spatial subcellular arrangement of metabolic enzyme complexes has been largely overlooked. To boost production of plant sesquiterpenes in yeast, we enhanced flux in the mevalonic acid pathway toward farnesyl diphosphate (FDP) accumulation, and evaluated the possibility of harnessing the mitochondria as an alternative to the cytosol for metabolic engineering. Overall, we achieved 8- and 20-fold improvement in the production of valencene and amorphadiene, respectively, in yeast co-engineered with a truncated and deregulated HMG1, mitochondrion-targeted heterologous FDP synthase and a mitochondrion-targeted sesquiterpene synthase, i.e. valencene or amorphadiene synthase. The prospect of harnessing different subcellular compartments opens new and intriguing possibilities for the metabolic engineering of pathways leading to valuable natural compounds., (Copyright © 2011 Elsevier Inc. All rights reserved.)

Tanshinone is a group of active diterpenes widely used in treatment of cardiovascular diseases. Here, we report the introduction of genes encoding 3-hydroxy-3-methylglutaryl CoA reductase (HMGR), 1-deoxy-D-xylulose-5-phosphate synthase (DXS) and geranylgeranyl diphosphate synthase (GGPPS) involved in tanshinone biosynthesis into Salvia miltiorrhiza hairy roots by Agrobacterium-mediated gene transfer technology. Overexpression of SmGGPPS and/or SmHMGR as well as SmDXS in transgenic hairy root lines can significantly enhance the production of tanshinone to levels higher than that of the control (P<0.05). SmDXS showed much more powerful pushing effect than SmHMGR in tanshinone production, while SmGGPPS plays a more important role in stimulating tanshinone accumulation than the upstream enzyme SmHMGR or SmDXS in S. miltiorrhiza. Co-expression of SmHMGR and SmGGPPS resulted in highest production of tanshinone (about 2.727 mg/g dw) in line HG9, which was about 4.74-fold higher than that of the control (0.475 mg/g dw). All the tested transgenic hairy root lines showed higher antioxidant activity than the control. To our knowledge, this is the first report on enhancement of tanshinone content and antioxidant activity achieved through metabolic engineering of hairy roots by push-pull strategy in S. miltiorrhiza., (Copyright © 2011 Elsevier Inc. All rights reserved.)

The chip and quantitative real-time PCR (qPCR) assays were optimized to study the expression of microcystin biosynthesis genes (mcy) with RNA samples extracted from cyanobacterial strains and environmental water samples. Both microcystin-producing Anabaena and Microcystis were identified in Lake Tuusulanjärvi samples. Microcystis transcribed the mcyE genes throughout the summer of 2006, while expression by Anabaena became evident later in August and September. Active mcyE gene expression was also detectable when microcystin concentrations were very low. Detection of Anabaena mcyE transcripts by qPCR, as well as certain cyanobacterial 16S rRNAs with the chip assay, showed slightly reduced sensitivity compared with the DNA analyses. In contrast, even groups undetectable or present in low quantities as determined by microscopy could be identified with the chip assay from DNA samples. The methods introduced add to the previously scarce repertoire of applications for mcy expression profiling in environmental samples and enable in situ studies of regulation of microcystin synthesis in response to environmental factors.

The Escherichia coli entD gene, which encodes an Sfp-type phosphopantetheinyl transferase (PPTase) that is involved in the biosynthesis of siderophore, is available as a high-expression ASKA clone (pCA24N::entD) constructed from the E. coli K-12 strain AG1. In E. coli DH5alpha, pCA24N::entD complemented a pfaE-deficient clone that comprised pfaA, pfaB, pfaC and pfaD, which are four of the five pfa genes that are responsible for the biosynthesis of eicosapentaenoic acid derived from Shewanella pneumatophori SCRC-2738. Sfp-type PPTases are classified into the EntD and PfaE groups, based on differences between their N-terminal-domain structures. Here, we showed that all Sfp-type PPTases may have the potential to promote the biosynthesis of long-chain n-3 polyunsaturated fatty acids.

The fungal entomopathogen Metarhizium robertsii (formerly known as M. anisopliae var. anisopliae) is a prolific producer of secondary metabolites of which very little is known at the genetic level. To establish the genetic bases for the biosynthesis of the mutagenic compound NG- 391, we identified a 19,818 kb genomic region harboring the predicted hybrid polyketide synthase-nonribosomal peptide synthetase NGS1, plus five additional ORFs. NGS1 knockouts generated by Agrobacterium-mediated transformation failed to produce detectable levels of NG-391, indicating the involvement of this locus in its biosynthesis. NGS1 deletion mutants had no significant changes in virulence levels against larvae of Spodoptera exigua and in resistance to hydrogen peroxide-generated oxidative stress compared to the wild-type strain. All 6 ORFs were expressed in medium supporting production of NG-391, and NGS1 was expressed during the interaction with the S. exigua host. The use of an NGS1 promoter-GFP reporter fusion showed that during in vitro growth in still broth cultures, NGS1 expression is restricted to the early exponential phase and is affected by M. robertsii cell density.

Polyamines are ubiquitously present in all organisms. In addition to the common polyamines, thermophilic archaea synthesize long-chain polyamines. In the present study polyamine synthases from Hyperthermus butylicus and Pyrobaculum aerophilum were cloned and their substrate specificity was analyzed. The polyamine synthase HbSpeE II from H. butylicus synthesized long-chain polyamines with high activity using the same mechanism that is used by a wide range of organisms to synthesize common polyamines, in which the aminopropyl residue derives from decarboxylated S-adenosylmethionine. This is the first polyamine synthase described that synthesizes a polyamine longer than a tetramine with high activity.

Linoleate (10R)-dioxygenase (10R-DOX) of Aspergillus fumigatus was cloned and expressed in insect cells. Recombinant 10R-DOX oxidized 18:2n-6 to (10R)-hydroperoxy-8(E),12(Z)-octadecadienoic acid (10R-HPODE; approximately 90%), (8R)-hydroperoxylinoleic acid (8R-HPODE; approximately 10%), and small amounts of 12S(13R)-epoxy-(10R)-hydroxy-(8E)-octadecenoic acid. We investigated the oxygenation of 18:2n-6 at C-10 and C-8 by site-directed mutagenesis of 10R-DOX and 7,8-linoleate diol synthase (7,8-LDS), which forms approximately 98% 8R-HPODE and approximately 2% 10R-HPODE. The 10R-DOX and 7,8-LDS sequences differ in homologous positions of the presumed dioxygenation sites (Leu-384/Val-330 and Val-388/Leu-334, respectively) and at the distal site of the heme (Leu-306/Val-256). Leu-384/Val-330 influenced oxygenation, as L384V and L384A of 10R-DOX elevated the biosynthesis of 8-HPODE to 22 and 54%, respectively, as measured by liquid chromatography-tandem mass spectrometry analysis. The stereospecificity was also decreased, as L384A formed the R and S isomers of 10-HPODE and 8-HPODE in a 3:2 ratio. Residues in this position also influenced oxygenation by 7,8-LDS, as its V330L mutant augmented the formation of 10R-HPODE 3-fold. Replacement of Val-388 in 10R-DOX with leucine and phenylalanine increased the formation of 8R-HPODE to 16 and 36%, respectively, whereas L334V of 7,8-LDS was inactive. Mutation of Leu-306 with valine or alanine had little influence on the epoxyalcohol synthase activity. Our results suggest that Leu-384 and Val-388 of 10R-DOX control oxygenation of 18:2n-6 at C-10 and C-8, respectively. The two homologous positions of prostaglandin H synthase-1, Val-349 and Ser-353, are also critical for the position and stereospecificity of the cyclooxygenase reaction.

The heterologous production of fungal polyketides was investigated using 6-methylsalicylic acid synthase (6-MSAS) as a model polyketide synthase and Saccharomyces cerevisiae as a host. In order to improve the production of 6-MSA by enhancing the supply of precursors, the promoter of the gene (ACC1) encoding acetyl-CoA carboxylase, which catalyzes the conversion of acetyl-CoA to malonyl-CoA, was replaced with a strong, constitutive promoter (TEF1p) in a strain harboring two plasmids carrying the genes encoding 6-MSAS from Penicillium patulum and PPTase from Aspergillus nidulans, respectively. The strain was characterized in batch cultivations with a glucose minimal media (20 g/L), and a 60% increase in 6-MSA titer was observed compared to a strain having the native promoter in front of ACC1. The production of 6-MSA was scaled up by the cultivation in minimal media containing 50 g/L of glucose, and hereby a final titer of 554+/-26 mg/L of 6-MSA was obtained.

The rpoZ gene for the omega subunit of Escherichia coli RNA polymerase constitutes single operon with the spoT gene, which is responsible for the maintenance of stringent response under nutrient starvation conditions. To identify the physiological role of the omega subunit, we compared the gene expression profile of wild-type Escherichia coli with that of an rpoZ deleted strain by microarray analysis using an E. coli DNA chip. Here we report on a set of genes which show changes in expression profile following the removal of rpoZ. We have seen that relA, which is responsible for the synthesis of the stringent factor ppGpp and many ribosomal proteins, exhibited noticeable changes in mRNA levels and were therefore further analyzed for their expression using a GFP/RFP two-fluorescent protein promoter assay vector. In the absence of rpoZ, the promoter for the relA gene was severely impaired, but the promoters from the ribosomal protein genes were not affected as much. Taking these results together we propose that the omega subunit is involved in regulation of the relA gene, but induction of the stringently controlled genes in the absence of rpoZ is, at least in part, attributable to a decrease in ppGpp level.

Panax ginseng produces triterpene saponins called ginsenosides, which are classified into two groups by the skeleton of aglycones, namely dammarane type and oleanane type. Dammarane-type ginsenosides dominate over oleanane type not only in amount but also in structural varieties. However, their sapogenin structure is restricted to two aglycones, protopanaxadiol and protopanaxatriol. So far, the genes encoding oxidosqualene cyclase (OSC) responsible for formation of dammarane skeleton have not been cloned, although OSC yielding oleanane skeleton (beta-amyrin synthase) has been successfully cloned from this plant. In this study, cDNA cloning of OSC producing dammmarane triterpene was attempted from hairy root cultures of P. ginseng by homology based PCR method. A new OSC gene (named as PNA) obtained was expressed in a lanosterol synthase deficient (erg7) Saccharomyces cerevisiae strain GIL77. LC-MS and NMR analyses identified the accumulated product in the yeast transformant to be dammarenediol-II, demonstrating PNA to encode dammarenediol-II synthase.

A cryptic plasmid of Methylobacterium extorquens AM1 was found to encode tslI, a truncated luxI homolog. tslI was shown to be expressed and to control transcription of the acyl-homoserine lactone (HSL) synthase gene msaI and thus, indirectly, acyl-HSL production. In addition, tslI was found to positively regulate extracellular polysaccharide production.

High-yield production of HLA-A*0201 heavy chain is a prerequisite to the preparation of HLA-A2 tetramer. The present study was aimed to construct the expression vector of recombinant HLA-A*0201-BSP fusion gene for preparing HLA-A2 tetramers. The extracellular region HLA*0201 was cloned by RT-PCR from HLA-A2(+) donor, and a 15-amino acid biotin-protein ligase (BirA) substrate peptide (BSP) for BirA-dependent biotinylation was added to the COOH-terminus of HLA-A*0201 heavy chain. Then the fusion gene was cloned into pBV220 vector at EcoRI and Bam HI sites and its sequence was confirmed by DNA sequence analysis. The recombinant plasmid pBV220-HLA-A*0201-BSP was transformed to the competent cells of E.coli DH5alpha. The results showed that the HLA-A*0201-BSP fusion protein was successfully expressed in the form of inclusion body and amounted to over 28% of total cell proteins via induction at 42 degrees C. After washed with triton X-100 and urea, the inclusion body was dissolved with 8 mol/L urea and then purified with Sepharcyl S-300 HR, and the final purity reached above 90%. It is concluded that the HLA-A*0201-BSP fusion gene was cloned successfully and expressed efficiently in E.coli DH5alpha. This work establishes a convenient approach for purification of large quantity of recombinant HLA-A*0201-BSP. This provides the basis for the preparation of HLA-A2 tetramers.

The alarmone ppGpp is an important signal molecule for the stringent response. Escherichia coli relA encodes a ppGpp synthetase, and although the regulation of RelA protein activity has been studied extensively, the regulation of relA transcription remains unclear. Here, we describe a novel relA promoter, relAP2. According to quantitative measurement of mRNA by primer extension analysis, the previously reported promoter relAP1 is constitutively active throughout growth, while relAP2 is induced temporarily at the transition state between the exponential growth and stationary phases. A chromosomal transcriptional lacZ fusion (relAP2-lacZ) showed that relAP2 is positively regulated by H-NS and CRP. Furthermore, the reduced activity of relAP2-lacZ in an hns mutant could be rescued by an rpoS mutation, which is sufficient to derepress the relAP2-lacZ activity. These data suggest that transient expression from the relAP2 promoter is controlled by several global regulators. This may account for the complex regulation of relA expression in Escherichia coli.

In the attempt to establish a reliable real-time PCR protocol for transcriptional analysis of secondary metabolism in Sorangium cellulosum strain So ce56, a RNA extraction method and a reverse transcription protocol was developed. In order to validate chivosazol or etnangien gene cluster transcripts as good candidates to develop the real-time PCR protocol, stability measurements of the transcripts were performed proving both transcripts to be very stable. The chivosazol biosynthetic gene cluster was taken as the test case to evaluate the special problems arising from the large size of the transcripts and the high G/C-content of the encoding DNA. A set of primer pairs targeting the presumed 90 kbp chivosazol transcript at different positions was employed. The production rate of chivosazol was compared to the transcription of the operon in time course experiments revealing that during the logarithmic growth phase transcription is maximally induced and levels out during the stationary phase. Some deviations in transcript numbers could be measured depending on the primer pair used, but cross-evaluation strengthened the notion that the measured numbers reflect the whole transcript quantities and the in vivo level. Finally, a putative promoter located between chiA and chiB was examined by using the developed real-time PCR protocol.

The dual-function Rel(Mtb) protein from Mycobacterium tuberculosis catalyzes both the synthesis and hydrolysis of (p)ppGpp, the effector of the stringent response. In our previous work [Avarbock, D., Avarbock, A., and Rubin, H. (2000) Biochemistry 39, 11640], we presented evidence that the Rel(Mtb) protein might catalyze its two opposing reactions at distinct active sites. In the study presented here, we purified and characterized fragments of the 738-amino acid Rel(Mtb) protein and confirmed the hypothesis that amino acid fragment 1-394 contains both synthesis and hydrolysis activities, amino acid fragment 87-394 contains only (p)ppGpp synthesis activity, and amino acid fragment 1-181 contains only (p)ppGpp hydrolysis activity. Mutation of specific residues within fragment 1-394 results in the loss of synthetic activity and retention of hydrolysis (G241E and H344Y) or loss of hydrolytic activity with retention of synthesis (H80A and D81A). The C-terminally cleaved Rel(Mtb) fragment proteins have basal activities similar to that of full-length Rel(Mtb), but are no longer regulated by the previously described Rel(Mtb) activating complex (RAC). Residues within the C-terminus of Rel(Mtb) (D632A and C633A) are shown to have a role in interaction with the RAC. Additionally, size exclusion chromatography indicates Rel(Mtb) forms trimers and removal of the C-terminus results in monomers. The C-terminal deletion, 1-394, which exists as a mixture of monomers and trimers, will dissociate from the trimer state upon the addition of substrate. Furthermore, the trimer state of fragment 1-394 appears to be a catalytically less efficient state than the monomer state.

Smad ubiquitination regulatory factor 2 (Smurf2), a ubiquitin ligase for Smads, plays critical roles in the regulation of transforming growth factor-beta (TGF-beta)-Smad signaling via ubiquitin-dependent degradation of Smad2 and Smad7. We found that TGF-beta stimulates Smurf2 expression. TGF-beta activated the Smurf2 promoter in a TGF-beta responsive cell lines, whereas IL-1alpha, PDGF and epidermal growth factor did not. TGF-beta-mediated Smurf2 promoter activation was inhibited by Smad7 or an activin receptor-like kinase 5 inhibitor but not by dominant negative Smad or disruption of Smad-binding elements in the promoter. Moreover, inhibition of the phosphatidil inositol 3 kinase (PI3K)/Akt pathway suppressed TGF-beta-mediated Smurf2 induction. These results suggest that TGF-beta stimulates Smurf2 expression by Smad-independent pathway such as PI3K/Akt pathway via TGF-beta receptor.

By screening etiolated Arabidopsis seedlings for mutants with aberrant ethylene-related phenotypes, we identified a mutant that displays features of the ethylene-mediated triple response even in the absence of ethylene. Further characterization showed that the phenotype observed for the dark-grown seedlings of this mutant is reversible by prevention of ethylene perception and is dependent on a modest increase in ethylene production correlated with an increase in 1-aminocyclopropane-1-carboxylic acid (ACC) oxidase (ACO) activity in the hypocotyl. Molecular characterization of leaves of the mutant revealed severely impaired induction of basic chitinase (chiB) and plant defensin (PDF)1.2 following treatment with jasmonic acid and/or ethylene. Positional cloning of the mutation resulted in identification of a 49-bp deletion in RCE1 (related to ubiquitin 1 (RUB1)-conjugating enzyme), which has been demonstrated to be responsible for covalent attachment of RUB1 to the SCF (Skpl Cdc 53 F-box) ubiquitin ligase complex to modify its activity. Our analyses with rce1-2 demonstrate a previously unknown requirement for RUB1 modification for regulation of ethylene biosynthesis and proper induction of defense-related genes in Arabidopsis.

The timely destruction of key regulators through ubiquitin-mediated proteolysis ensures the orderly progression of the cell cycle. The APC (anaphase-promoting complex) is a major component of this degradation machinery and its activation is required for the execution of critical events. Recent studies have just begun to reveal the complex control of the APC through a regulatory network involving WD40 repeat proteins CDC20 and CDH1. In the present paper, we report on the identification and characterization of human CDH1beta, a novel alternatively spliced isoform of CDH1. Both CDH1alpha and CDH1beta can bind to the APC and stimulate the degradation of cyclin B1, but they are differentially expressed in human tissues and cells. CDH1alpha contains a nuclear localization signal which is absent in CDH1beta. Intracellularly, CDH1alpha appears in the nucleus whereas CDH1beta is a predominantly cytoplasmic protein. The forced overexpression of CDH1alpha in cultured cells correlates with the reduction of nuclear cyclin A, but the steady-state amount of cyclin A does not change noticeably in CDH1beta-overexpressed cells. In Xenopus embryos, ectopic overexpression of human CDH1alpha, but not of CDH1beta, induces cell-cycle arrest during the first G(1) phase at the mid-blastula transition. Taken together, our findings document the differential expression, subcellular localization and cell-cycle-regulatory activity of human CDH1 isoforms.

Ubiquitin-dependent proteolysis by the 26S proteasome plays a pivotal role in cell cycle progression as well as in tumorigenesis. In this pathway, ubiquitin-conjugating enzyme (E2), together with ubiquitin ligase (E3), transfers ubiquitin to the specific substrate protein(s); however, little is known about the potential contribution of E2 to tumorigenesis. In this study, we examined the expression levels of 17 E2 genes in 25 different human normal tissues and 24 human cancerous cell lines by using a quantitative real-time reverse transcription-PCR. Among the E2 gene family, the expression level of UbcH10 was extremely low in many of the normal tissues but prominent in the majority of cancerous cell lines. Intriguingly, UbcH10 was expressed at high levels in primary tumors derived from the lung, stomach, uterus, and bladder as compared with their corresponding normal tissues, suggesting that UbcH10 is involved in tumorigenesis or progression of the tumor. To further investigate a possible contribution of UbcH10 to malignant transformation and tumor cell proliferation, NIH3T3 cells were transfected with the expression plasmid encoding UbcH10, and stable transfectants were subsequently established. UbcH10-overexpressing cells exhibited an increased incorporation of bromodeoxyuridine, an enhanced growth rate, an increase in saturation density, and a promotion of colony formation in soft agar medium as compared with parental NIH3T3 cells and the control transfectants. Collectively, our present results provide the first evidence that UbcH10 is highly expressed in various human primary tumors and that UbcH10 has an ability to promote cell growth and malignant transformation.

SFT, a stimulator of iron (Fe) transport, has been described as a transmembrane protein that facilitates the uptake of ferrous and ferric iron in mammalian cells. This study was initiated to investigate the 5' regulatory region of SFT and its role in the etiology of hereditary hemochromatosis. Sequence analyses of the putative 5' regulatory region revealed that the SFT cDNA sequence corresponds to intron 6/exon 7 of UbcH5A, a member of E2 ubiquitin-conjugating enzymes, which is involved in the iron-dependent ubiquitination of the hypoxia-inducible factor (HIF) by the von Hippel-Lindau tumor suppressor (pVHL) E3 ligase complex. Further mRNA expression studies using a sequence-specific reverse transcriptase-polymerase chain reaction (RT-PCR) assay showed that UbcH5A is significantly up-regulated in the liver of iron-overloaded patients with hereditary hemochromatosis, as previously published for SFT. However, in vitro studies on HepG2 cells failed to demonstrate any significant UbcH5A regulation in response to iron loading or iron chelation. In conclusion, in vivo mRNA expression data previously obtained for SFT might be attributed to UbcH5A. The role of UbcH5A and the ubiquitination pathway in the etiology of hereditary hemochromatosis remains to be elucidated further.

Mutations in parkin, which encodes a RING domain protein associated with ubiquitin ligase activity, lead to autosomal recessive Parkinson's disease characterized by midbrain dopamine neuron loss. Here we show that parkin functions in a multiprotein ubiquitin ligase complex that includes the F-box/WD repeat protein hSel-10 and Cullin-1. HSel-10 serves to target the parkin ubiquitin ligase activity to cyclin E, an hSel-10-interacting protein previously implicated in the regulation of neuronal apoptosis. Consistent with the notion that cyclin E is a substrate of the parkin ubiquitin ligase complex, parkin deficiency potentiates the accumulation of cyclin E in cultured postmitotic neurons exposed to the glutamatergic excitotoxin kainate and promotes their apoptosis. Furthermore, parkin overexpression attenuates the accumulation of cyclin E in toxin-treated primary neurons, including midbrain dopamine neurons, and protects them from apoptosis.

M7.1 is a class IV ubiquitin-conjugation enzyme (UBC) that belongs to the ubiquitination cascade in Caenorhabditis elegans. The clone for this UBC has been overexpressed in Escherichia coli and the 16.7 kDa protein was purified from the soluble fraction. M7.1 was crystallized by sitting-drop vapor diffusion in 10% ethanol, 1.5 M NaCl at 277.5 K. Crystals diffracted to 1.75 A and belong to the orthorhombic space group P2(1)2(1)2(1), with unit-cell parameters a = 44.3, b = 54.3, c = 60.2 A. The asymmetric unit contains a single monomer. A molecular-replacement model has been determined and refinement is in progress.

We previously reported that haloperidol, a dopamine-D(2) receptor antagonist, induced striatal expression of parkin gene, which mutations cause autosomal recessive juvenile parkinsonism. Because of an involvement of the parkin gene defect in selective degeneration of dopaminergic neurons, we herein examined the effect of the neurotoxic dose of methamphetamine (METH; 40 mg/kg, i.p.) on gene expression of parkin and its substrate Pael-receptor (R) in the dopamine-rich areas of the rat brain, using reverse transcription-polymerase chain reaction. parkin mRNA levels in the striatum, but not in other regions, decreased at 1 and 2 h and returned to the pre-drug basal levels at 4 h after METH administration. METH also decreased Pael-R mRNA levels in the striatum and substantia nigra within 2 h after METH, while haloperidol (2 mg/kg, s.c.) increased Pael-R mRNA levels in the substantia nigra at 2 h after administration. These results suggest that temporary suppression of gene expression of parkin and Pael-R may be associated with the METH-induced dopaminergic neurotoxicity. Taken together with our previous report, dopaminergic modulation of the expression of parkin and Pael-R genes in the nigro-striatal pathway may have significant implication for pathophysiology and treatment of parkinson disease., (Copyright 2003 Elsevier Science Ltd.)

Two cDNAs and their corresponding genes (GhUBC1 and GhUBC2) encoding ubiquitin-conjugating enzymes (E2s) have been cloned and characterized from allotetraploid cotton Gossypium hirsutum ((AD)(1) genome). Three additional E2 genes (GaUBC1, GtUBC2, and GrUBC2) have also been identified from diploid cottons Gossypium arboreum (A(2) genome), Gossypium thurberi (D(1) genome), and Gossypium raimondii (D(5) genome), respectively. The derived amino acid sequences of the five closely related cotton E2s are 77-79% identical to yeast ScUBC4 and ScUBC5. The GhUBC1/2 gene family is composed of two members, and genomic origin analysis indicates that GhUBC1 and 2 are individually present in the A and D subgenomes of G. hirsutum. The transcript levels of GhUBC1/2 increased significantly in leaves and flowers at senescence, suggesting that GhUBC1/2 may play a role in the degradation of target proteins that function in the delay of the senescence program. Correlated with high auxin content and auxin-associated effects, GhUBC1/2 are also highly expressed in the youngest leaves, the apical part of lateral roots, and elongating fibers. Genetic complementation experiments revealed that GhUBC1 and 2 can substitute for the function of ScUBC4 and 5 required for the selective degradation of abnormal and short-lived proteins in a yeast ubc4ubc5 double mutant.

Recently in yeast and animal cells, one particular class of ubiquitin ligase (E3), called the SCF, was demonstrated to regulate diverse processes including cell cycle and development. In plants SCF-dependent proteolysis is also involved in different developmental and hormonal regulations. To further investigate the function of SCF, we characterized at the molecular level the Arabidopsis RING-H2 finger protein AtRbx1. We demonstrated that the plant gene is able to functionally complement a yeast knockout mutant strain and showed that AtRbx1 protein interacts physically with at least two members of the Arabidopsis cullin family (AtCul1 and AtCul4). AtRbx1 also associates with AtCul1 and the Arabidopsis SKP1-related proteins in planta, indicating that it is part of plant SCF complexes. AtRbx1 mRNAs accumulate in various tissues of the plant, but at higher levels in tissues containing actively dividing cells. Finally to study the function of the gene in planta, we either overexpressed AtRbx1 or reduced its expression by a dsRNA strategy. Down-regulation of AtRbx1 impaired seedling growth and development, indicating that the gene is essential in plants. Furthermore, the AtRbx1-silenced plants showed a reduced level of AtCul1 protein, but accumulated higher level of cyclin D3.

Transforming growth factor beta (TGF-beta) regulates growth of various cells, and inactivation of the TGF-beta signaling pathway contributes to tumor progression. Smad2 is phosphorylated and activated by TGF-beta, resulting in the antiproliferative effects of TGF-beta signaling. Smurf2 (Smad ubiquitination regulatory factor 2) was identified as the Smad ubiquitin ligase that induces the ubiquitination and degradation of Smad2. This study was undertaken to elucidate the relationships between Smurf2 expression and the clinicopathological characteristics of patients with esophageal squamous cell carcinoma (SCC) and the correlation between Smurf2 and Smad2 expression. Surgical specimens obtained from 80 patients with esophageal SCC were subjected to immunohistochemical staining. Our data indicated that high-level expression of Smurf2 correlated with depth of invasion, lymph node metastasis, and a poor survival rate. We also found an inverse correlation between the expression of Smurf2 and Smad2. Western blotting analysis of esophageal SCC-derived cell lines revealed similar inverse correlations. We demonstrated that high-level expression of Smurf2 appears to correlate with tumor development and poor prognosis in patients with esophageal SCC and that alteration of Smad2 expression in the TGF-beta signaling pathway may be induced by enhancement of Smad2 degradation mediated by high-level expression of Smurf2.

Death-associated protein kinase (DAPK) is a multi-domain Ser/Thr protein kinase with an important role in apoptosis regulation. In these studies we have identified a DAPK-interacting protein called DIP-1, which is a novel multi-RING finger protein. The RING finger motifs of DIP-1 have E3 ligase activity that can auto-ubiquitinate DIP-1 in vitro. In vivo, DIP-1 is detected as a polyubiquitinated protein, suggesting that the intracellular levels of DIP-1 are regulated by the ubiquitin-proteasome system. Transient expression of DIP-1 in HeLa cells antagonizes the anti-apoptotic function of DAPK to promote a caspase-dependent apoptosis. These studies also demonstrate that DAPK is an in vitro and in vivo target for ubiquitination by DIP-1, thereby providing a mechanism by which DAPK activities can be regulated through proteasomal degradation.

Adenovirus penton base protein is involved in virus internalization. Searching for the cellular partners of this protein, we used dodecahedra, adenovirus subviral particles composed of 12 bases, for screening a human lung expression library. This screen yielded three ubiquitin-protein ligases, WWP1, WWP2, and AIP4, all of which belong to the HECT family and contain multiple WW domains. The xPPxY motif, known to interact with WW domains in partner proteins occurs twice in the N-terminal part of the base polypeptide chain. The recruitment of three ubiquitin-protein ligases was shown for two distinct virus serotypes, Ad2 and Ad3. The first N-terminal xPPxY motif in the base protein sequence is indispensable for the interaction. The association in vitro was shown by the protein overlay technique and in vivo by cotransfection followed by immunoprecipitation. The binding parameters studied by surface plasmon resonance confirmed the interaction of base protein with three ubiquitin-protein ligases. In case of WWP1 when the saturation of binding was achieved, the apparent dissociation constant of 65nM was calculated. This is the first demonstration of the interaction of nonenveloped viruses with ubiquitin-protein ligases of host cells.

Photoreceptor differentiation in the Drosophila eye disc progresses from posterior to anterior in a wave driven by the Hedgehog and Decapentaplegic signals. Cells mutant for the hyperplastic discs gene misexpress both of these signaling molecules in anterior regions of the disc, leading to premature photoreceptor differentiation and overgrowth of surrounding tissue. The two genes are independently regulated by hyperplastic discs; decapentaplegic can still be misexpressed in cells mutant for both hyperplastic discs and hedgehog, and a repressor form of the transcription factor Cubitus interruptus can block decapentaplegic misexpression but not hedgehog misexpression. Loss of hyperplastic discs causes the accumulation of full-length Cubitus interruptus protein, but not of Smoothened, in both the eye and wing discs. hyperplastic discs encodes a HECT domain E3 ubiquitin ligase that is likely to act by targeting Cubitus interruptus and an unknown activator of hedgehog expression for proteolysis.

An RBCC (RING finger, B-box, and coiled-coil) protein was identified that belongs to the superfamily of zinc-binding proteins and is specifically expressed in the gill of eel, Anguilla japonica. Euryhaline fishes such as eels can migrate between freshwater and seawater, which is considered to be accomplished by efficient remodeling of the architecture and function of the gill, a major osmoregulatory organ. To identify molecules involved in such adaptive changes, we performed differential display using mRNA preparations from freshwater and seawater eel gills and obtained an RBCC clone among several differentially expressed clones. The clone encoded a protein of 514 amino acid residues with structural features characteristic of the RBCC protein; we therefore named it eRBCC (e for eel). eRBCC mRNA was specifically expressed in the gills with a greater extent in the gills of freshwater eels. Immunohistochemistry revealed that the expression of eRBCC is confined to particular epithelial cells of the gills including freshwater-specific lamellar chloride cells. The RING finger of eRBCC was found to have a ubiquitin ligase activity, suggesting an important regulatory role of eRBCC in the remodeling of branchial cells.

F-Box protein p45(SKP2) is the substrate-specific receptor of ubiquitin-protein ligase SCF/p45(SKP2) and is involved in the degradation of p27(Kip1) through the ubiquitin/proteasome pathway. In addition, p45(SKP2) facilitates proteolysis of other molecules related to the cell cycle, is frequently over-expressed in transformed cells, and induces S phase in quiescent cells. The aim of this study was to determine whether p45(SKP2) expression is altered in aggressive lesions of Kaposi's sarcoma and its relation to p27(KIP1)down-regulation. We performed immunohistochemistry using antibodies directed to p45(SKP2), p27(KIP1), and Ki67 on paraffin blocks corresponding to 47 cases of Kaposi's sarcoma (8 macules, 10 plaques, 12 tumors, and 15 extracutaneous lesions). p45(SKP2) nuclear over-expression was present in all Kaposi's sarcoma stages, being significantly increased in skin tumors (mean +/- 95% confidence interval: 39.2 +/- 18.8) and extracutaneous lesions (25.8 +/- 17.3) as compared with macules (18.9 +/- 8.2) and plaques (29.2 +/- 12.0; P =.0199). On the other hand, Kaposi's sarcoma progression was associated with a decrease in p27(KIP1) expression and Ki67 immunoreactivity was independent of disease stage. No statistically significant differences were found in regard to patients' sex and human immunodeficiency virus status and regression analysis failed to show a correlation among p45(SKP2), p27(KIP1) and Ki67 immunostaining scores. These findings suggest that p45(SKP2) is involved in Kaposi's sarcoma progression, not only by promoting the degradation of p27(KIP1) but also through other mechanisms still unknown.

A mutation in the parkin gene has been identified as the cause for an autosomal recessively inherited form of Parkinson's disease (PD). The authors have recently isolated the mRNA coding for the rat homolog of parkin and showed its widespread expression in the central nervous system (CNS) by in situ hybridization. In the present study, we investigated the distribution of parkin in the rat CNS with a polyclonal antibody that reacts with a approximately 52-kDa protein, mainly localized in the cytoplasm and corresponding to the predicted molecular mass of parkin. Immunohistochemistry on adult rat brain sections showed a widespread distribution of parkin. This included labeling of cell bodies, nuclei as well as processes in the hippocampus, cerebral cortex, cerebellum, and several nuclei in the brainstem. The regional expression of parkin-immunoreactivity (IR) correlated well with the parkin-mRNA levels assessed by real-time quantitative reverse transcription-polymerase chain reaction (RT-PCR). This study provides the detailed analysis of the regional and cellular distribution of parkin in the rat brain and may be useful in elucidating its pathophysiological role.

A search was made for genes that confer resistance to methylmercury in yeast using a genomic DNA library derived from Saccharomyces cerevisiae. The genomic library was introduced into yeast and transformants that grew in the presence of a normally toxic concentration of methylmercury were selected. We sequenced the genomic DNA fragment in the plasmid from the clone with the highest resistance to methylmercury and analyzed the sequence for presence of an open reading frame that might confer resistance to methylmercury. We identified a gene, CDC34 (also known as UBC3), that increased resistance to methylmercury when overexpressed in yeast. CDC34 encodes a ubiquitin-conjugating enzyme; such proteins play important roles in the selective targeting of proteins for degradation. Overexpression of UBC4 and of UBC7, two other genes for ubiquitin-conjugating enzymes, also conferred resistance to methylmercury. Yeast strains transformed with the CDC34 gene were resistant not only to methylmercury but also to mercuric chloride and p-chloromercuribenzoate. To our knowledge, this is the first demonstration that overexpression of genes for ubiquitin-conjugating enzymes confers resistance to xenobiotics. Our results suggest that ubiquitination system might be involved in protection against the toxicity of mercury compounds, such as methylmercury, in eukaryotic cells.

We have isolated by differential RNA display a cDNA that is up-regulated in metastatic mammary tumor lines. This cDNA corresponds to HR6B, the yeast homologue of Rad6, a ubiquitin-conjugating enzyme, and a key player in postreplication repair and induced mutagenesis in the yeast. We show that Rad6 protein expressed in metastatic tumor lines is wild type and functional, because it is able to catalyze the transfer of ubiquitin to histone H2b and is predominantly localized in the nucleus as compared with cytoplasmic localization in normal or nonmetastatic mammary cells. This pattern of Rad6 protein expression/localization is not restricted to breast cancer cell lines, because human breast carcinomas display similar patterns of Rad6 up-regulation and nuclear localization suggesting that deregulation in expression of Rad6 may be an important step in transformation to malignant phenotype. Constitutive overexpression of exogenous human HR6B cDNA into normal-behaving MCF10A human breast epithelial cells induced cell-cell fusion that resulted in generation of multinucleated cells, centrosome amplification, multipolar mitotic spindles, aneuploidy, and ability for anchorage-independent growth. Double immunofluorescence labeling experiments demonstrated the colocalization of Rad6 protein with gamma-tubulin on centrosomes. This physical association of Rad6 with centrosomes is maintained throughout the interphase and mitotic phases of the cell cycle. The Rad6 protein exhibits notable alterations in distribution during interphase and mitotic stages of the cell cycle that are compatible with its function as a transcription factor. These findings suggest that Rad6 is an important ubiquitin-conjugating enzyme that may play a significant role in the maintenance of genomic integrity of mammalian cells and that an imbalance in the levels and activity of Rad6 could lead to chromosomal instability and transformation in vitro.

Tight junctions create a highly selective diffusion barrier between epithelial and endothelial cells by preventing the free passage of molecules and ions across the paracellular pathway. Although the regulation of this barrier is still enigmatic, there is evidence that junctional transmembrane proteins are critically involved. Recent evidence confirms the notion that occludin, a four-pass integral plasma-membrane protein, is a functional component of the paracellular barrier. The overall hydrophilicity of occludin predicts two extracellular loops bounded by NH(2)- and COOH-terminal cytoplasmic domains. To date, the binding of the COOH terminus of occludin to intracellular proteins is well documented, but information concerning the function of the cytoplasmic NH(2) terminus is still lacking. Using yeast two-hybrid screening we have identified a novel interaction between occludin and the E3 ubiquitin-protein ligase Itch, a member of the HECT domain-containing ubiquitin-protein ligases. We have found that the NH(2)-terminal portion of occludin binds specifically to a multidomain of Itch, consisting of four WW motifs. This interaction has been confirmed by our results from in vivo and in vitro co-immunoprecipitation experiments. In addition, we provide evidence that Itch is specifically involved in the ubiquitination of occludin in vivo, and that the degradation of occludin is sensitive to proteasome inhibition.

The autosomal recessive juvenile parkinsonism is caused by the mutations of the gene encoding a novel protein called parkin. It has been reported that parkin is expressed in the central nervous system and functions as a ubiquitin-protein ligase (E3) which suppresses neuronal cell degeneration by ubiquitinating misfolded proteins. Thus far, however, it remains unknown if parkin is expressed and functions in the peripheral nervous system. In order to begin to address to this question, we investigated the expression of parkin in bovine peripheral nerve. Reverse transcription polymerase chain reaction analysis demonstrated the presence of parkin transcript in bovine peripheral nerve. The obtained bovine parkin cDNA sequence was identical to that of human except a single nucleotide. Immunoblot analysis demonstrated the expression of parkin protein in bovine peripheral nerve. Immunohistochemical analysis demonstrated the localization of parkin in the axoplasm of myelinated nerve fibers, the Schwann cell cytoplasm and the Schwann cell outer membrane. Furthermore, fractionation analysis indicated the presence of two fractions of parkin in bovine peripheral nerve, the cytosolic fraction and the cell membrane-bound fraction. All together, these results point to diverse roles of parkin in not only the central but also the peripheral nervous system.

Using an in silico (electronic database) subtraction, we identified a new member of the Ret Finger Protein-Like gene family, Rfpl4. Rfpl4 encodes a 287 amino acid putative E3 ubiquitin-protein ligase with a RING finger-like domain and a B30.2 motif. Reverse transcriptase polymerase chain reaction and Northern blot analyses reveal that Rfpl4 encodes a 1.7kb mRNA detectable exclusively in the gonads of adult mice. In situ hybridization localizes Rfpl4 transcripts within the ovary to oocytes of primary and later stage follicles and in the testis to elongating spermatids. The Rfpl4 gene comprises three exons and maps to mouse chromosome 7. We have identified the human ortholog, which maps to 19q13.4. These studies suggest that RFPL4 mediates protein degradation pathways important for gametogenesis or early embryonic development.

Objective: To clone and express 3'-terminal of Parkin gene in E. coli, and prepare its antiserum for further study., Methods: The glutathion-sulfate-transferase (GST) fusion expression plasmid of 3'-terminal of Parkin gene (937-1959 bp) was constructed and transferred to JM 105. After being treated with Triton-100 (1%) and Tween-20 (1%) and purified with affinity chromatograph, GST-Parkin C was used to immunize New Zealand rabbits to acquire antiserum. Antiserum was analysed with immunoblot., Results: The GST-Parkin C protein was expressed in JM 105, existing in the form of inclusion body with a molecular weight of around 42 kD; The purity of GST-Parkin C was up to 95%; the titer of antiserum was 1:64; Immunoblotting showed that the prepared antiserum could react specifically with 51.6 kD protein extracted from the mouse brain., Conclusion: A high level of expression of GST-Parkin C is obtained in JM 105, and its antiserum can be prepared successfully.

Mutations of the parkin gene are a cause of autosomal recessive juvenile parkinsonism. Although the parkin gene has been isolated from mouse, rat, and human, little is known about its expression in neural and nonneural tissues during development. In this study, we used a polyclonal antibody to a peptide downstream of the parkin ubiquitin domain to investigate (1) the differential expression of parkin isoforms in protein extracts from fetal and adult mouse tissues, and (2) the distribution of parkin in mouse fetal tissues at different developmental stages and in adult CNS tissues. By Western blot analyses, at least three isoforms of parkin of 22, 50, and 55 kDa were differentially expressed in mouse tissues. The p22 and p50 isoforms were found in fetal and adult mouse CNS tissues, while the p55 isoform was found only in adult tissues. The p50 isoform is the predominant form in both fetal and adult tissues. Immunolocalization in mouse fetuses showed that parkin was expressed only after neuronal differentiation. Although parkin was localized throughout the cytoplasm, the highest level of parkin was found in the neurites of both fetal and adult neurons.

Previous work has established that compound mutations and homozygous loss of function of the parkin gene cause early-onset, autosomal recessive parkinsonism. Classically, this disease has been associated with loss of dopaminergic neurons in the substantia nigra pars compacta and locus ceruleus, without Lewy body pathology. We have sequenced the parkin gene of 38 patients with early-onset Parkinson's disease (<41 years). Two probands with mutations were followed up. Clinical evaluation of their families was performed, blinded to both genetic and pathological findings. Chromosome 6q25.2-27 haplotype analysis was carried out independently of the trait; parkin gene expression was examined at both the RNA and protein levels. Haplotype analysis of these families revealed a common chromosome 6, with a novel 40 bp exon 3 deletion that cosegregated with disease. In the proband of the smaller kindred, an exon 7 R275W substitution was identified in addition to the exon 3 deletion; RNA analysis demonstrated that the mutations were on alternate transcripts. However, Lewy body pathology typical of idiopathic Parkinson's disease was found at autopsy in the proband from the smaller kindred. These data suggest that compound heterozygous parkin mutations and loss of parkin protein may lead to early-onset parkinsonism with Lewy body pathology, while a hemizygous mutation may confer increased susceptibility to typical Parkinson's disease.