Gradual compaction of the nascent peptide during cotranslational folding on the ribosome.

Nascent polypeptides begin to fold in the constrained space of the ribosomal peptide exit tunnel. Here we use force-profile analysis (FPA) and photo-induced energy-transfer fluorescence correlation spectroscopy (PET-FCS) to show how a small α-helical domain, the N-terminal domain of HemK, folds cotranslationally. Compaction starts vectorially as soon as the first α-helical segments are synthesized. As nascent chain grows, emerging helical segments dock onto each other and continue to rearrange at the vicinity of the ribosome. Inside or in the proximity of the ribosome, the nascent peptide undergoes structural fluctuations on the µs time scale. The fluctuations slow down as the domain moves away from the ribosome. Mutations that destabilize the packing of the domain's hydrophobic core have little effect on folding within the exit tunnel, but abolish the final domain stabilization. The results show the power of FPA and PET-FCS in solving the trajectory of cotranslational protein folding and in characterizing the dynamic properties of folding intermediates.

Prognostic value of histone marks H3K27me3 and H3K9me3 and modifying enzymes EZH2, SETDB1 and LSD-1 in colorectal cancer.

PURPOSE: Studies on the performance of epigenetic-based biomarkers in colorectal cancer (CRC) are scarce and have shown contradictory results. Thus, we sought to examine the prognostic value of histone-modifying enzymes (EZH2, SETDB1 and LSD-1) and histone post-translational marks (H3K27me3 and H3K9me3) in CRC. METHODS: A retrospective series of 207 CRC patients primarily submitted to surgery in a cancer center was included in this study. Clinicopathological data were retrieved. One representative paraffin block per case was selected for immunohistochemistry, including normal and CRC tissues whenever possible. The percentage of positive nuclear staining (digital image analysis) was used to classify patients into "low" and "high" expression groups for each biomarker. Correlations between immunoexpression levels, clinicopathological features and clinical outcomes [disease-specific (DSS) and disease-free (DFS) survival] were examined. Statistical significance was set at p < 0.05. RESULTS: CRC tissues showed significantly lower expression of SETDB1 and higher expression of the remainder four biomarkers compared to normal mucosa. High EZH2 expression correlated with disease recurrence/progression, whereas low LSD1 expression and high H3K9me3 and H3K27me3 expression were associated with more advanced stage. In multivariable analysis, cases with high LSD1 expression displayed significantly better DSS and DFS (HR 0.477, 95% confidence interval: 0.247-0.923) adjusted for pathological TNM stage. CONCLUSION: EZH2, SETDB1, LSD1, H3K9me3 and H3K27me3 expression are altered in CRC and may play a role in colorectal carcinogenesis. LSD1 immunoexpression levels independently predicted patient outcome in this cohort. Further investigations, using larger series, are warranted to confirm its potential clinical value and unravel underlying molecular mechanisms.

The arginine methyltransferase PRMT5 regulates CIITA-dependent MHC II transcription.

Class II major histocompatibility complex (MHC II) dependent antigen presentation serves as a key step in mammalian adaptive immunity and host defense. In antigen presenting cells (e.g., macrophages), MHC II transcription can be activated by interferon gamma (IFN-γ) and mediated by class II transactivator (CIITA). The underlying epigenetic mechanism, however, is not completely understood. Here we report that following IFN-γ stimulation, symmetrically dimethylated histone H3 arginine 2 (H3R2Me2s) accumulated on the MHC II promoter along with CIITA. IFN-γ augmented expression, nuclear translocation, and promoter binding of the protein arginine methyltransferase PRMT5 in macrophages. Over-expression of PRMT5 potentiated IFN-γ induced activation of MHC II transcription in an enzyme activity-dependent manner. In contrast, PRMT5 silencing or inhibition of PRMT5 activity by methylthioadenosine (MTA) suppressed MHC II transactivation by IFN-γ. CIITA interacted with and recruited PRMT5 to the MHC II promoter and mediated the synergy between PRMT5 and ASH2/WDR5 to activate MHC II transcription. PRMT5 expression was down-regulated in senescent and H2O2-treated macrophages rendering ineffectual induction of MHC II transcription by IFN-γ. Taken together, our data reveal a pathophysiologically relevant role for PRMT5 in MHC II transactivation in macrophages.

Quantitative proteomic study of human prostate cancer cells with different metastatic potentials.

Metastatic dissemination is a feature of most cancers including prostate cancer (PCa), and is the main cause of treatment failure and mortality. The aim of the study is to explore the mechanisms of PCa metastasis and to search for potential prognostic markers using proteomics. Two-dimensional fluorescent differential gel electrophoresis (2D-DIGE) was used to quantify proteins in normal prostate epithelial cells, bone metastasis-derived PC-3 cells, and visceral metastasis-derived PC-3M cells. Metastatic potential was confirmed by flow cytometry, electron microscopy, proliferating cell nuclear antigen assay, and wound healing assay. Differential protein expression was compared between PCa cells with different metastatic potentials (LNcap, DU145, PC-3 and PC-3M) and normal prostate epithelial cells (RWPE-1). Selected candidate proteins in human prostate tissues were analyzed using GOA, UniProt and GeneCards analyses. Eighty-six proteins were differentially expressed between cell lines (>1.5-fold, P<0.05). Among them, twelve proteins were identified by MALDI-TOF-MS. One protein was upregulated in normal prostate epithelial cells, nine proteins were upregulated in PC-3, and two proteins were upregulated in PC-3M. Proteins were divided into five groups according to their functions. The SETDB1 protein was closely associated with the prognosis of PCa. Bioinformatics suggested that SETDB1 might promote PCa bone metastasis through the WNT pathway. In conclusion, SETDB1 might be associated with the development of bone metastases from PCa. Further study is necessary to assess its exact role in PCa.

Prognostic value of ERCC1, RRM1, BRCA1 and SETDB1 in early stage of non-small cell lung cancer.

INTRODUCTION: Nowadays, 40 % of early-stage NSCLC patients relapse in the 2 years following resection, suggesting a mis-staging in this group of patients who are not receiving adjuvant chemotherapy. Although different biomarkers, such as ERCC1, RRM1 and BRCA1 have been found to present prognostic value in advanced NSCLC patients, in early-stage NSCLC patients its relevance remains unclear. Moreover, SETDB1 has been recently proposed as a bona fide oncogene in lung tumourigenesis and related with metastasis. The aim of the present study was to analyze the prognostic value of ERCC1, RRM1, BRCA1 and SETDB1 expression levels in NSCLC patients at stage I. PATIENTS AND METHODS: ERCC1, RRM1, BRCA1 and SETDB1 expression at mRNA level was analyzed by real-time quantitative RT-PCR in fresh-frozen tumor and normal adjacent lung tissue samples from 64 stage I NSCLC patients. Later, significant association between gene expression levels, clinicopathological characteristics and patient's disease-free survival was assessed. RESULTS: We did not find any statistically significant correlation between gene expression and gender, age, histological type or smoking status. Univariate followed by multivariate Cox analysis showed that higher levels of BRCA1 and SETDB1 expression were significantly associated with shorter disease-free survival in stage I NSCLC patients. CONCLUSION: Our study finds that ERCC1 and RRM1 are not independent prognostic factors of recurrence in stage I NSCLC patients. By contrast, BRCA1 and SETDB1 stand out as the most significant prognostic markers in this group of patients, appearing as promising tools to predict tumor recurrence in early-stage NSCLC patients.

Cotranslational protein folding on the ribosome monitored in real time.

Protein domains can fold into stable tertiary structures while they are synthesized on the ribosome. We used a high-performance, reconstituted in vitro translation system to investigate the folding of a small five-helix protein domain-the N-terminal domain of Escherichia coli N5-glutamine methyltransferase HemK-in real time. Our observations show that cotranslational folding of the protein, which folds autonomously and rapidly in solution, proceeds through a compact, non-native conformation that forms within the peptide tunnel of the ribosome. The compact state rearranges into a native-like structure immediately after the full domain sequence has emerged from the ribosome. Both folding transitions are rate-limited by translation, allowing for quasi-equilibrium sampling of the conformational space restricted by the ribosome. Cotranslational folding may be typical of small, intrinsically rapidly folding protein domains.

Transcriptional regulators TRIM28, SETDB1, and TP53 are aberrantly expressed in porcine embryos produced by in vitro fertilization in comparison to in vivo- and somatic-cell nuclear transfer-derived embryos.

In vitro embryo production is important for research in animal reproduction, embryo transfer, transgenics, and cloning. Yet, in vitro-fertilized (IVF) embryos are generally developmentally delayed and are inferior to in vivo-derived (IVV) embryos; this discrepancy is likely a result of aberrant gene expression. Transcription of three genes implicated to be important in normal preimplantation embryo development, TRIM28, SETDB1, and TP53, was determined by quanitative PCR in IVF, somatic-cell nuclear transfer (SCNT), parthenogenetic, and IVV porcine oocytes and embryos. There was no difference in TRIM28 or SETDB1 abundance between oocytes matured in vitro versus in vivo (P > 0.05), whereas TP53 levels were higher in in vitro-matured oocytes. TRIM28 increased from metaphase-II oocytes to the 4-cell and blastocyst stages in IVF embryos, whereas IVV embryos showed a reduction in TRIM28 abundance from maturation throughout development. The relative abundance of TP53 increased by the blastocyst stage in all treatment groups, but was higher in IVF embryos compared to IVV and SCNT embryos. In contrast, SETDB1 transcript levels decreased from the 2-cell to blastocyst stage in all treatments. For each gene analyzed, SCNT embryos of both hard-to-clone and easy-to-clone cell lines were more comparable to IVV than IVF embryos. Knockdown of TRIM28 also had no effect on blastocyst development or expression of SETDB1 or TP53. Thus, TRIM28, SETDB1, and TP53 are dynamically expressed in porcine oocytes and embryos. Furthermore, TRIM28 and TP53 abundances in IVV and SCNT embryos are similar, but different from quantities in IVF embryos.

Immunohistochemistry for histone h3 lysine 9 methyltransferase and demethylase proteins in human melanomas.

Methylation and demethylation of histone H3 lysine 9 (H3K9) play a role in the transcriptional regulation of several cancer-related genes and are closely associated with malignant tumor behavior. A novel study has recently demonstrated that SETDB1, a member of the H3K9 methyltransferases, accelerates tumor formation significantly in a zebrafish melanoma model. However, the expression of H3K9 methyltransferases including SETDB1 and demethylases has not been systematically examined in samples of human melanoma. Here, we used immunohistochemistry to examine the expression of the H3K9 methyltransferases, EHMT2 and SETDB1, and a H3K9 demethylase, LSD1, in 67 patients with melanoma. Overexpression of EHMT2, SETDB1, and LSD1 was observed in 14 (21%), 38 (57%), and 53 (79%) of the 67 patients, respectively. A significant relationship was observed between overexpression of EHMT2 or SETDB1 and aggressive tumor behavior such as lymph node metastasis and/or distant metastasis (P < 0.05), whereas no significant relationship was evident for LSD1 immunoreactivity. Univariate log-rank tests demonstrated that patients with melanoma overexpressing EHMT2 had a poorer outcome (P < 0.001), whereas overexpression of SETDB1 or LSD1 had no prognostic impact. These results suggest that overexpression of EHMT2 might be a prognostic marker in patients with melanoma.

Anthrax SET protein: a potential virulence determinant that epigenetically represses NF-κB activation in infected macrophages.

Toxins play a major role in the pathogenesis of Bacillus anthracis by subverting the host defenses. However, besides toxins, B. anthracis expresses effector proteins, whose role in pathogenesis are yet to be investigated. Here we present that suppressor-of-variegation, enhancer-of-zeste, trithorax protein from B. anthracis (BaSET) methylates human histone H1, resulting in repression of NF-κB functions. Notably, BaSET is secreted and undergoes nuclear translocation to enhance H1 methylation in B. anthracis-infected macrophages. Compared with wild type Sterne, delayed growth kinetics and altered septum formation were observed in the BaSET knock-out (BaΔSET) bacilli. Uncontrolled BaSET expression during complementation of the BaSET gene in BaΔSET partially restored growth during stationary phase but resulted in substantially shorter bacilli throughout the growth cycle. Importantly, in contrast to Sterne, the BaΔSET B. anthracis is avirulent in a lethal murine bacteremia model of infection. Collectively, BaSET is required for repression of host transcription as well as proper B. anthracis growth, making it a potentially unique virulence determinant.

Protein arginine methyltransferase 5 (PRMT5) signaling suppresses protein kinase Cδ- and p38δ-dependent signaling and keratinocyte differentiation.

PKCδ is a key regulator of keratinocyte differentiation that activates p38δ phosphorylation leading to increased differentiation as measured by an increased expression of the structural protein involucrin. Our previous studies suggest that p38δ exists in association with protein partners. A major goal is to identify these partners and understand their role in regulating keratinocyte differentiation. In this study we use affinity purification and mass spectrometry to identify protein arginine methyltransferase 5 (PRMT5) as part of the p38δ signaling complex. PRMT5 is an arginine methyltransferase that symmetrically dimethylates arginine residues on target proteins to alter target protein function. We show that PRMT5 knockdown is associated with increased p38δ phosphorylation, suggesting that PRMT5 impacts the p38δ signaling complex. At a functional level we show that PRMT5 inhibits the PKCδ- or 12-O-tetradecanoylphorbol-13-acetate-dependent increase in human involucrin expression, and PRMT5 dimethylates proteins in the p38δ complex. Moreover, PKCδ expression reduces the PRMT5 level, suggesting that PKCδ activates differentiation in part by reducing PRMT5 level. These studies indicate antagonism between the PKCδ and PRMT5 signaling in control of keratinocyte differentiation.

A germ cell-specific gene, Prmt5, works in somatic cell reprogramming.

Germ cells possess the unique ability to acquire totipotency during development in vivo as well as give rise to pluripotent stem cells under the appropriate conditions in vitro. Recent studies in which somatic cells were experimentally converted into pluripotent stem cells revealed that genes expressed in primordial germ cells (PGCs), such as Oct3/4, Sox2, and Lin28, are involved in this reprogramming. These findings suggest that PGCs may be useful for identifying factors that successfully and efficiently reprogram somatic cells into toti- and/or pluripotent stem cells. Here, we show that Blimp-1, Prdm14, and Prmt5, each of which is crucial for PGC development, have the potential to reprogram somatic cells into pluripotent stem cells. Among them, Prmt5 exhibited remarkable reprogramming of mouse embryonic fibroblasts into which Prmt5, Klf4, and Oct3/4 were introduced. The resulting cells exhibited pluripotent gene expression, teratoma formation, and germline transmission in chimeric mice, all of which were indistinguishable from those induced with embryonic stem cells. These data indicate that some of the factors that play essential roles in germ cell development are also active in somatic cell reprogramming.

Cloning and primarily function study of two novel putative N5-glutamine methyltransferase (Hemk) splice variants from mouse stem cells.

Methylation is one of epigenetic mechanisms regulating gene expression. The methylation pattern is determined during embryogenesis and passed over to differentiating cells and tissues. Beginning with the ESTs which were highly expressed in undifferentiated human ES cells and using homology research in mouse dbEST database, we cloned two novel putative (N (5))-glutamine methyltransferase (Hemk) splice variants termed mHemk1 and mHemk2 (Genbank accession number AY456393 and AY583759). Sequence analysis revealed that mHemk1 and mHemk2 cDNAs are 1,792 bp and 1,696 bp in length respectively. The deduced proteins have 214 amino acid residues (mHemk1) and 138 residues (mHemk2) in length and both share significant homology with (N (5))-glutamine methyltransferase (Hemk proteins) in database. Northern blot and RT-PCR analysis showed that mHemk mRNAs were abundantly expressed in undifferentiated ES cells, testis and brain, weakly expressed in differentiated ES cells and kidney, and not expressed in muscle, heart, placenta, pancreas, lung and stomach. Immunohistochemical analysis further revealed that the protein was most abundant in undifferentiated ES cells. The green fluorescent protein produced by pEGFP-C3/mHemk1 was detected mainly in the nucleus of COS7 cell lines after 24 h post-transfection. RNA interference (RNAi)-mediated knock-down method was established. Cell cycle analysis suggests that the cell proliferation decreases after RNAi with mHemk1. In vitro bioactivity assay showed that no evidence for a DNA adenine-methyltransferase activity was detected. The accumulating functional information from Hemk homology proteins in bacteria and yeast suggests that it may be an uncharacterized new mammalian N(5)-glutamine methyltransferase.

Regulation of epithelial Na+ channels (ENaC) by methylation: a novel methyltransferase stimulates ENaC activity.

Aldosterone acts to increase apical membrane permeability by activation of epithelial Na(+) channels (ENaC). We have previously shown that aldosterone activates ENaC early in the course of its action by stimulating the methylation of the beta subunit of this heteromeric channel in A6 cells. Aldosterone also stimulates the expression and methylation of k-ras in A6 cells. To determine whether aldosterone-stimulated methylations are seen in mammalian cells, we examined the effect of aldosterone on methylation and ras activation in a continuous line of cultured epithelial cells derived from mouse cortical collecting duct (CCD) and determined that beta mENaC is a substrate for methylation by an enzyme contained in CCD cells. Aldosterone stimulated protein base labile methylation in CCD cells. Aldosterone stimulated Na(+) transport in CCD cells within 1 h of addition and without an increase in cellular amount of any ENaC subunits over the first 4 h. Inhibition of methylation, using the inhibitor 3-deaza-adenosine, blocked the stimulation of Na(+) transport induced by aldosterone at early time points (1-4 h) without affecting cellular amounts of any ENaC subunits. In contrast to 3-deaza-adenosine (3-DZA), which inhibits all methylation reactions, specific inhibitors of small G-protein methylation or prenylation had no effect on the early aldosterone-induced current. Overexpression of isoprenylcysteine carboxylmethyltransferase (PCMTase), the enzyme that methylates ras, had little effect on basal transport but enhanced aldosterone-stimulated transport in A6 cells. Overexpression of PCMTase in CCD cells had no effect on either basal or aldosterone-stimulated transport. Moreover PCMTase had no effect on ENaC activity when co-expressed in Xenopus oocytes. Aldosterone had no effect on either message or protein levels of k-ras in CCD cells. Searching a mouse kidney library, we identified a methyltransferase that stimulates ENaC activity in Xenopus oocytes without affecting surface expression of ENaC. Our results demonstrate that aldosterone stimulates protein methylation in CCD cells, and this is required for expression of the early transport response. In CCD cells this effect is not mediated via methylation of ras, which is not induced by aldosterone in these cells, and the enzyme that methylates ras has no direct effect on ENaC activity. beta ENaC is a substrate for methylation in CCD cells. A novel methyltransferase that stimulates ENaC directly has been identified in CCD cells.

Human PRMT5 expression is enhanced during in vitro tubule formation and after in vivo ischemic injury in renal epithelial cells.

BACKGROUND: The interactions between cells and the extracellular matrix (ECM) are important in the regulation of cell growth and differentiation. Cells cultured in ECM differentiate and develop tubular structures. The kidney has the ability to partially recover function after an ischemic insult through repairing its tubular epithelium. The factors that contribute to tubule formation in vitro may mediate tubule regeneration in the recovery stage of acute tubular necrosis. METHODS: RNA purified from cells grown on plastic, on Matrigel and in Matrigel matrix were subjected to differential display analysis to identify the transcripts that are differentially expressed during in vitro tubulogenesis. RESULTS: Protein arginine methyltransferase 5 (PRMT5) expression increased in renal epithelial cells undergoing tubule formation. PRMT5 expression is developmentally regulated and ubiquitously expressed in a variety of adult tissues. We also demonstrated that expression of PRMT5 is enhanced in the renal tubular epithelium of animals subjected to ischemic reperfusion injury (IRI). CONCLUSION: The role of PRMT5 in the regulation of mitosis, its induction in renal epithelial cells undergoing tubule formation in vitro and its expression in the tubules of the kidneys subjected to IRI suggest that it functions in the regulation of cell growth and differentiation during tubule formation and regeneration.

[Constitutive activation and clinical significance of Stat3 in human gastric cancer tissues and cell lines].

OBJECTIVE: To investigate the activation of signal transducers and activators of transcription 3 (Stat3) in different types of gastric cancer cell lines and tissues and evaluate the relationship with their clinicopathological parameters. METHODS: Western blotting and electrophoretic mobility shift assay (EMSA) were used to detected the expression of Stat3 protein and Stat3 DNA-binding activity in normal human gastric epithelial cell line 3T3 and five gastric cancer cell lines with different differentiation: MKN28, SGC7901, MKN45, AGS and NCI-SNU-1, respectively. The localization of phospho-Stat3 was determined by immunocytochemistry. The expressive intensity of phospho-Stat3 protein in 50 cases of gastric cancer tissues and adjacent normal mucosa were measured by immunohistochemistry. RESULTS: Compared with normal gastric epithelial cell line 3T3, elevated activities of Stat3 were found in five different human gastric cancer cell lines. The Stat3 DNA-binding activity in moderately and poorly differentiated stomach adenocarcinoma cell lines (SGC7901, MKN45 and AGS) was higher than that of other cell lines (MKN28 and NCI-SNU-1). Phospho-Stat3 was detected primarily in the nuclei of AGS cells. The expressive intensity of phospho-Stat3 protein was significantly increased in gastric cancer tissues as compared with the adjacent normal gastric mucosa, especially in moderately and poorly differentiated cancers (both P < 0.05). The expressive intensity of phospho-Stat3 protein in stage II and stage III tumors was higher than that in stage I tumors (P < 0.05). No statistic difference of phospho-Stat3 expression was found between stage IV and stage I tumors (P > 0.05). The expression of phospho-Stat3 was closely correlated with the differentiation of gastric cancer. CONCLUSION: Elevated activity of Stat3 can be found in different types of human gastric cancer cell lines and gastric cancer. JAK/STAT signal transduction pathway may play an important role in the development of human gastric cancer.

Protein phosphatase methyltransferase 1 (Ppm1p) is the sole activity responsible for modification of the major forms of protein phosphatase 2A in yeast.

Protein phosphatase 2A (PP2A) is a major threonine/serine phosphatase that is involved in regulating a variety of cellular processes. It has been shown in both yeast and mammals that the PP2A catalytic subunit (PP2Ac) is methyl-esterified at the conserved C-terminal Leu residue. The recent characterization of a mammalian PP2A carboxyl methyltransferase has led to the identification of two ORFs in Saccharomyces cerevisiae as potential orthologues of the mammalian PP2A methyltransferase: protein phosphatase methyltransferase 1 (PPM1) and protein phosphatase methyltransferase 2 (PPM2). To experimentally identify the PP2A methyltransferase in yeast, we obtained deletion mutants of PPM1 and PPM2 and then constructed double mutants. Using in vivo-labeling techniques, we demonstrate that only the PPM1 gene is required for PP2Ac methylation at the C-terminus. Because yeast has at least three homologues of PP2Ac (PPH21, PPH22, and PPH3), we then asked whether all of these catalytic subunits are methylated by the PPM1 and/or PPM2 putative methyltransferases. We modified the segment corresponding to the N-terminal coding region of all three PP2Ac genomic genes with a hemagglutinin (HA) tag in the parent, ppm1, ppm2, and ppm1ppm2 mutant genetic backgrounds. Using immuoprecipitation with anti-HA antibodies followed by methyl ester analysis, we showed that only in the ppm1 mutant were both Pph21p and Pph22p not methylated. We did not detect any methylesterification of Pph3p under our conditions. Our results indicate that PPM1 is the sole methyltransferase responsible for methylating the two major homologues of PP2Ac in yeast. The function of the PPM2 gene product remains unclear.

Polyglutamine expansion down-regulates specific neuronal genes before pathologic changes in SCA1.

The expansion of an unstable CAG repeat causes spinocerebellar ataxia type 1 (SCA1) and several other neurodegenerative diseases. How polyglutamine expansions render the resulting proteins toxic to neurons, however, remains elusive. Hypothesizing that long polyglutamine tracts alter gene expression, we found certain neuronal genes involved in signal transduction and calcium homeostasis sequentially downregulated in SCA1 mice. These genes were abundant in Purkinje cells, the primary site of SCA1 pathogenesis; moreover, their downregulation was mediated by expanded ataxin-1 and occurred before detectable pathology. Similar downregulation occurred in SCA1 human tissues. Altered gene expression may be the earliest mediator of polyglutamine toxicity.

Rapid mapping of genomic P1 clones: the mouse L-isoaspartyl/D-aspartyl methyltransferase gene.

We report the mapping of the gene for the murine protein-L-isoaspartate (D-aspartate) O-methyltransferase (EC 2.1.1. 77) from a 129 mouse strain. This gene encodes an enzyme present in all tissues that can catalyze the first step of a repair reaction in which age-damaged proteins containing abnormal l-isoaspartyl (or d-aspartyl) residues can be converted to forms containing normal l-aspartyl residues. We first mapped the restriction sites from a genomic P1 clone using a rapid method generally applicable to all bacteriophage P1 clones containing large DNA inserts. We show that a single pulsed-field electrophoresis blot can be used to map an entire 89-kb P1 clone insert for eight restriction endonucleases with an error of no more than 2% of the length of the fragment, or 1 kb at the middle of the insert. In this method, we combine complete restriction endonuclease digestion at rare sites within the P1 vector with partial restriction endonuclease digestion within the insert. After size separation by pulsed-field gel electrophoresis and blotting, the fragments are detected by Southern hybridization with probes to the vector. This method is potentially useful for restriction mapping other large DNA clones such as artificial chromosomes. We then determined the positions of the exons of the methyltransferase gene by restriction mapping of long PCR fragments. The previously unidentified exon 8, which encodes the -DEL C-terminus of the more acidic isozyme II, was sequenced and mapped 5. 3 kb from the end of exon 7.

Cloning, expression, and purification of rat brain protein L-isoaspartyl methyltransferase.

Protein L-isoaspartyl methyltransferase (PIMT) methylates isoaspartyl residues in peptides and proteins using S-adenosyl-L-methionine as the methyl donor. A cloned source of this enzyme should be useful in the identification of cellular substrates and for quantitation and localization of isoaspartyl sites in purified proteins, including recombinant proteins used as pharmaceuticals. Rat brain PIMT cDNA was amplified using the polymerase chain reaction. The reaction product was directionally cloned into the expression vector p delta blue (M. E. Brandt and L. E. Vickery, Arch. Biochem. 294, 735-740, 1992). The vector contains the strong promoter lambda pL and allows for the direct expression of cloned genes. After transformation, Escherichia coli cells containing the plasmid constitutively produced recombinant rat brain PIMT (rrPIMT) at levels between 2 and 3% of total soluble protein. Recombinant enzyme was purified to homogeneity by ammonium sulfate precipitation of the crude extract followed by anion-exchange chromatography. The specific activity was 14,000 pmol methyl groups transferred/min/mg protein at 30 degrees C using bovine gamma-globulin as the methyl acceptor. A typical yield was 12 mg of purified rrPIMT per liter of bacterial culture. Subsequent dye ligand chromatography increased the specific activity of the preparation to 16,800 pmol methyl groups transferred/min/mg protein with an overall yield of 5 mg per liter of bacterial culture. Using isoaspartyl delta sleep-inducing peptide as the methyl acceptor, rrPIMT exhibited normal Michaelis-Menten kinetics that yielded the following constants: Km (S-adenosyl-L-methionine) = 1.1 microM, Km (peptide) = 16 microM, Vmax = 60,000 pmol/min/mg.