High expression of human carboxypeptidase M in Pichia pastoris: Purification and partial characterization

Carboxypeptidase M (CPM) is an extracellular glycosylphosphatidyl-inositol-anchored membrane glycoprotein, which removes the C-terminal basic residues, lysine and arginine, from peptides and proteins at neutral pH. CPM plays an important role in the control of peptide hormones and growth factor activity on the cell surface. The present study was carried out to clone and express human CPM in the yeast Pichia pastoris in order to evaluate the importance of this enzyme in physiological and pathological processes. The cDNA for the enzyme was amplified from total placental RNA by RT-PCR and cloned in the vector pPIC9, which uses the methanol oxidase promoter and drives the expression of high levels of heterologous proteins in P. pastoris. The cpm gene, after cloning and transfection, was integrated into the yeast genome, which produced the active protein. The recombinant protein was secreted into the medium and the enzymatic activity was measured using the fluorescent substrate dansyl-Ala-Arg. The enzyme was purified by a two-step protocol including gel filtration and ion-exchange chromatography, resulting in a 1753-fold purified active protein (16474 RFU mg protein-1 min-1). This purification protocol permitted us to obtain 410 mg of the purified protein per liter of fermentation medium. SDS-PAGE showed that recombinant CPM migrated as a single band with a molecular mass similar to that of native placental enzyme (62 kDa), suggesting that the expression of a glycosylated protein had occurred. These results demonstrate for the first time the establishment of a method using P. pastoris to express human CPM necessary to the development of specific antibodies and antagonists, and the analysis of the involvement of this peptidase in different physiological and pathological processes

Cloning, sequencing and characterization of a chitin synthase gene fragment from the fungus Phascolomyces articulosus /

Phascolomyces articulosus genomic DNA was isolated from 48 h old hyphae and was used for amplification of a chitin synthase fragment by the polymerase chain reaction method. The primers used in the amplification corresponded to two widely conserved amino acid regions found in chitin synthases of many fimgi. Amphfication resulted in four bands (820, 900, 1000 and 1500 bp, approximately) as visualized in a 1.2% agarose gel. The lowest band (820 bp) was selected as a candidate for chitin synthase because most amplified regions from other fimgi so far exhibited similar sizes (600-750 bp). The selected fragment was extracted from the gel and cloned in the Hinc n site of pUC19. The derived plasmid and insert were designated ^\5C\9'PaCHS and PaCHS respectively. The plasmid pUC19-PaC/fS was digested by several restriction enzymes and was found to contain BamHl and HincU sites. Sequencing of PaCHS revealed two intron sequences and a total open reading frame of 200 amino acids. The derived polypeptide was compared with other related sequences from the EMBL database (Heidelberg, Germany) and was matched to 36 other fiilly or partially sequenced fimgal chitin synthase genes. The closest resemblance was with two genes (74.5% and 73.1% identity) from Rhizopus oligosporus. Southern hybridization with the cloned fragment as a probe to the PCR reaction showed a strong signal at the fragment selected for cloning and weaker signals at the other two fragments. Southern hybridization with partially digested Phascolomyces articulosus genomic DNA showed a single band. The amino acid sequence was compared with sequences from other chitin synthase gene classes using the CLUSTALW program. The chitin synthase fragment from Phascolomyces articulosus was initially grouped in class n along with chitin synthase fragments from Rhizopus oligosporus and Phycomyces blakesleeanus which also belong to the same class, Zygomycetes. Bootstrap analysis using the neighbor-joining method available by CLUSTALW verified such classification. Comparison of PaCHS revealed conservation of intron positions that are characteristic of chitin synthase gene fragments of zygomycetous fungi.

Partial characterization of the cloned dihydrofolate reductase gene of Saccharomyces cerevisiae

The cloned dihydrofolate reductase gene of Saccharomyces cerevisiae (DFR 1) is expressed in Escherichia coli. Bacterial strain JF1754 transformed with plasmids containing DFR 1 is at least 5X more resistant to inhibition by the folate antagonist trimethoprim. Expression of yeast DFR 1 in E. coli suggests it is likely that the gene lacks intervening sequences. The 1.8 kbp DNA fragment encoding yeast dhfr activity probably has its own promotor, as the gene is expressed in both orientations in E. coli. Expression of the yeast dhfr gene cloned into M13 viral vectors allowed positive selection of DFR 1 - M13 bacterial transfectants in medium supplemented with trimethoprim. A series of nested deletions generated by nuclease Bal 31 digestion and by restriction endonuclease cleavage of plasmids containing DFR 1 physically mapped the gene to a 930 bp region between the Pst 1 and Sal 1 cut sites. This is consistent with the 21,000 molecular weight attributed to yeast dhfr in previous reports. From preliminary DNA sequence analysis of the dhfr DNA fragment the 3' terminus of DFR 1 was assigned to a position 27 nucleotides from the Eco Rl cut site on the Bam Hi - Eco Rl DNA segment. Several putative yeast transcription termination consensus sequences were identified 3' to the opal stop codon. DFR 1 is expressed in yeast and it confers resistance to the antifolate methotrexate when the gene is present in 2 - 10 copies per cell. Plasmid-dependent resistance to methotrexate is also observed in a rad 6 background although the effect is somewhat less than that conferred to wild-type or rad 18 cells. Integration of DFR 1 into the yeast genome showed an intermediate sensitivity to folate antagonists. This may suggest a gene dosage effect. No change in petite induction in these yeast strains was observed in transformed cells containing yeast dhfr plasmids. The sensitivity of rad 6 , rad 18 and wild-type cell populations to trimethoprim were unaffected by the presence of DFR 1 in transformants. Moreover, trimethoprim did not induce petites in any strain tested, which normally results if dhfr is inhibited by other antifolates such as methotrexate. This may suggest that the dhfr enzyme is not the only possible target of trimethoprim in yeast. rad 6 mutants showed a very low level of spontaneous petite formation. Methotrexate failed to induce respiratory deficient mutants in this strain which suggested that rad 6 might be an obligate grande. However, ethidium bromide induced petites to a level approximately 50% of that exhibited by wild-type and rad 18 strains.

Cloning and Characterisation of the human gene gremlin promoter

L’ostéoarthrose (OA) est une maladie articulaire invalidante caractérisée par la perte de l’intégrité du cartilage articulaire. Les recherches tentent de comprendre les mécanismes moléculaires de la maladie afin de trouver des inhibiteurs efficaces pouvant prévenir la dégradation du cartilage articulaire. Les BMPs (bone morphogenic proteins) jouent un rôle dans le processus pathophysiologique de cette maladie. Cette étude cible le rôle d’un antagoniste des BMPs, le gremlin. Nous avons étudié la régulation de l’expression de gremlin par le clonage et la caractérisation de son promoteur et en déterminant si gremlin pouvait jouer un rôle autre qu’antagoniste des BMP, en affectant l’expression d’autres gènes par l’activation d’une cascade de signalisation dans la cellule. Les résultats ont identifié une région importante dans le promoteur de gremlin qui affecte son activité basale et induite, et ont montré que le gremlin ne pouvait pas affecter l’expression génique et l’activation de signalisation intracellulaire indépendamment des BMPs. Cette étude démontre que le rôle de gremlin dans l’OA en est un essentiellement d’antagoniste des BMPs.

Molecular cloning of alkaline protease gene from marine fungus Engyodontium album

Department of Biotechnology, Cochin University of Science and Technology

Partial identification of spirochaetes from two dairy cows with digital dermatitis by polymerase chain reaction analysis of the 16S ribosomal RNA gene

Specimens taken postmortem from typical lesions of digital dermatitis in two dairy cows were tested by the polymerase chain reaction (PCR) for the presence of a spirochaetal 16S rRNA gene. Seven different assays detected the gene in the samples from both cows. Two of the PCR products were sequenced and a comparison of the nucleotide sequences revealed that the spirochaete belonged to the genus Treponema and was closely related to Treponema denticola. A PCR specific for the detection of the digital dermatitis-associated treponeme was developed.

Cloning and overexpression of the xylose isomerase gene from Burkholderia sacchari and production of polyhydroxybutyrate from xylose

A different organization for the xyl operon was found in different genomes of Burkholderia and Pseudomomas species. Degenerated primers were designed based on Burkholderia genomes and used to amplify the xylose isomerase gene (xylA) from Burkholderia sacchari IPT101 The gene encoded a protein of 329 amino acids, which showed the highest similarity (90%) to the homologous gene of Burkholderia dolosa. It was cloned in the broad host range plasmid pBBR1MCS-2, which partially restored growth and polyhydroxybutyrate production capability in xylose to a B. sacchari xyl(-) mutant. When xylA was overexpressed in the wild-type strain, it was not able to increase growth and polyhydroxybutyrate production, suggesting that XylA activity is not limiting for xylose utilization in B. sacchari.

Cloning, sequencing and expression analysis of the equine hepcidin gene by real-time PCR

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Genetic variability of porcine parvovirus isolates revealed by analysis of partial sequences of the structural coding gene VP2

The 3'-terminal 853 nt (and the putative 283 aa) sequence of the VP2-encoding gene from 29 field strains of porcine parvovirus (PPV) were determined and compared both to each other and with other published sequences. Sequences were examined using maximum-parsimony and statistical analyses for nucleotide diversity and sequence variability. Among the nucleotide sequences of the PPV field strains, 26 polymorphic sites were encountered; 22 polymorphic sites were detected in the putative amino acid sequence. Mapping polymorphic sites of protein data onto the three-dimensional (3D) structure of PPV VP2 revealed that almost all substitutions were located on the external surface of the viral capsid. Mapping amino acid substitutions to the alignment between PPV VP2 sequences and the 3D structure of canine parvovirus (CPV) capsid, many PPV substitutions were observed to map to regions of recognized antigenicity and/or to contain phenotypically important residues for CPV and other parvoviruses. In spite of the high sequence similarity, genetic analysis has shown the existence of at least two virus lineages among the samples. In conclusion, these results highlight the need for close surveillance on PPV genetic drift, with an assessment of its potential ability to modify the antigenic make-up of the virus.

Partial molecular characterization of the Nile tilapia (Oreochromis niloticus) alpha-cardiac muscle actin gene and its relationship to actin isoforms of other fish species

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Identification of unannotated exons of low abundance transcripts in Drosophila melanogaster and cloning of a new serine protease gene upregulated upon injury

Abstract Background The sequencing of the D.melanogaster genome revealed an unexpected small number of genes (~ 14,000) indicating that mechanisms acting on generation of transcript diversity must have played a major role in the evolution of complex metazoans. Among the most extensively used mechanisms that accounts for this diversity is alternative splicing. It is estimated that over 40% of Drosophila protein-coding genes contain one or more alternative exons. A recent transcription map of the Drosophila embryogenesis indicates that 30% of the transcribed regions are unannotated, and that 1/3 of this is estimated as missed or alternative exons of previously characterized protein-coding genes. Therefore, the identification of the variety of expressed transcripts depends on experimental data for its final validation and is continuously being performed using different approaches. We applied the Open Reading Frame Expressed Sequence Tags (ORESTES) methodology, which is capable of generating cDNA data from the central portion of rare transcripts, in order to investigate the presence of hitherto unnanotated regions of Drosophila transcriptome. Results Bioinformatic analysis of 1,303 Drosophila ORESTES clusters identified 68 sequences derived from unannotated regions in the current Drosophila genome version (4.3). Of these, a set of 38 was analysed by polyA+ northern blot hybridization, validating 17 (50%) new exons of low abundance transcripts. For one of these ESTs, we obtained the cDNA encompassing the complete coding sequence of a new serine protease, named SP212. The SP212 gene is part of a serine protease gene cluster located in the chromosome region 88A12-B1. This cluster includes the predicted genes CG9631, CG9649 and CG31326, which were previously identified as up-regulated after immune challenges in genomic-scale microarray analysis. In agreement with the proposal that this locus is co-regulated in response to microorganisms infection, we show here that SP212 is also up-regulated upon injury. Conclusion Using the ORESTES methodology we identified 17 novel exons from low abundance Drosophila transcripts, and through a PCR approach the complete CDS of one of these transcripts was defined. Our results show that the computational identification and manual inspection are not sufficient to annotate a genome in the absence of experimentally derived data.

A novel GH-1 gene mutation (GH-P59L) causes partial GH deficiency type II combined with bioinactive GH syndrome

Despite the differences in the main characteristics between the autosomal dominant form of GH deficiency (IGHD II) and the bioinactive GH syndrome, a common feature of both is their impact on linear growth leading to short stature in all affected patients.

Cloning and expression of the gene encoding the major surface protein 5 (MSP5) of Anaplasma phagocytophilum and potential application for serodiagnosis

BACKGROUND: Anaplasma phagocytophilum (formerly known as the human granulocytic ehrlichia, Ehrlichia equi and Ehrlichia phagocytophila) is an obligate intracellular organism causing clinical disease in humans and various species of domestic animals. OBJECTIVES: The objectives of this investigation were to sequence and clone the major surface protein 5 (MSP5) of A phagocytophilum and to evaluate the suitability of this antigen in the serologic diagnosis of anaplasmosis in humans and dogs. METHODS: The msp5 gene of A phagocytophilum was sequenced, cloned, and expressed in Escherichia coli. The predicted amino acid sequence homology of the various MSP5/major antigenic protein 2 orthologs was compared among various Anaplasma and Ehrlichia species. Recombinant MSP5 of A phagocytophilum was used in an ELISA to detect antibodies in serum samples from humans and dogs infected with the organism. RESULTS: Serum samples from 104 individuals previously diagnosed with A phagocytophilum infection, as well as samples from clinically healthy humans, were tested. In addition, multiple samples from 4 dogs experimentally infected with 2 different geographic isolates of A phagocytophilum and 5 dogs naturally infected with a Swiss isolate were tested using ELISA. Using this group of immunofluorescent antibody test-positive and immunofluorescent antibody test-negative samples, we found the overall agreement between assays to be >90%. CONCLUSIONS: These results indicate that recombinant MSP5 has potential for use as a diagnostic test antigen to detect infection with A phagocytophilum in both dogs and humans. However, sequence similarities among orthologs of MSP5 in related species of anaplasma and ehrlichia suggest that cross-reactivity among these pathogens is likely if the entire peptide is used as a test antigen.

Positional cloning and characterization of the human aniridia and murine small eye genes

Aniridia (AN) is a congenital, panocular disorder of the eye characterized by the complete or partial absence of the iris. The disease can occur in both the sporadic and familial forms which, in the latter case, is inherited as an autosomal dominant trait with high penetrance. The objective of this study was to isolate and characterize the genes involved in AN and Sey, and thereby to gain a better understanding of the molecular basis of the two disorders.^ Using a positional cloning strategy, I have approached and cloned from the AN locus in human chromosomal band 11p13 a cDNA that is deleted in two patients with AN. The deletions in these patients overlap by about 70 kb and encompass the 3$\sp\prime$ end of the cDNA. This cDNA detects a 2.7 kb mRNA encoded by a transcription unit estimated to span approximately 50 kb of genomic DNA. The message is specifically expressed in all tissues affected in all forms of AN, namely within the presumptive iris, lens, neuroretina, the superficial layers of the cornea, the olfactory bulbs, and the cerebellum. Sequence analysis of the AN cDNA revealed a number of motifs characteristic of certain transcription factors. Chief among these are the presence of the paired domain, the homeodomain, and a carboxy-terminal domain rich in serine, threonine and proline residues. The overall structure shows high homology to the Drosophila segmentation gene paired and members of the murine Pax family of developmental control genes.^ Utilizing a conserved human genomic DNA sequence as probe, I was able to isolate an embryonic murine cDNA which is over 92% homologous in nucleotide sequence and virtually identical at the amino acid level to the human AN cDNA. The expression pattern of the murine gene is the same as that in man, supporting the conclusion that it probably corresponds to the Sey gene. Its specific expression in the neuroectodermal component of the eye, in glioblastomas, but not in the neural crest-derived PC12 pheochromocytoma cell line, suggests that a defect in neuroectodermal rather mesodermal development might be the common etiological factor underlying AN and Sey. ^

The cloning and expression of myogenin, a gene regulating myogenesis

Expression of the differentiated skeletal muscle phenotype is a process that appears to occur in at least two stages. First, pluripotent stem cells become committed to the myogenic lineage. Although undifferentiated and capable of continued proliferation, determined myoblasts are restricted to a single developmental fate. Upon receiving the appropriate environmental signals, these determined myoblasts withdraw from the cell cycle, fuse to form multi-nucleated myotubes, and begin to express a battery of muscle-specific gene products that make up the functional and contractile apparatus of the muscle. This project is aimed at the identification and characterization of factors that control the determination and differentiation of myogenic cells. We have cloned a cDNA, called myogenin, that plays an important role in these processes. Myogenin is expressed exclusively in skeletal muscle in vivo and myogenic cell lines in vitro. Its expression is sharply upregulated during differentiation. When constitutively expressed in fibroblasts, myogenin converts these cells to the myogenic lineage. Transfected cells behave as myogenic tissue culture cells with respect to the genes they express, the way they respond to environmental cues, and are capable of fusing to form multinucleated myotubes. Sequence analysis showed that this cDNA has homology to a family of transcription factors in a region of 72 amino acids known as the basic helix-loop-helix motif. This domain appears to mediate binding to a DNA sequence element known as an E-box (CANNTG) essential for the activity of the enhancers of many muscle-specific genes.^ Analysis of myogenin in tissue culture cells showed that its expression is responsive to many of the environmental cues, such as the presence of growth factors and oncogenes, that modulate myogenesis. In an attempt to identify the cis- and trans-elements that control myogenin expression and thereby understand what factors are responsible for the establishment of the myogenic lineage, we have cloned the myogenin gene. After analysis of the gene structure, we constructed a series of reporter constructs from the 5$\prime$ upstream sequence of the myogenin gene to determine which cis-acting sequences might be important in myogenin regulation. We found that 184 nucleotides of the 5$\prime$ sequence was sufficient to direct high-level muscle-specific expression of the reporter gene. Two sequence elements present in the 184 fragment, an E-box and a MEF-2 site, have been shown previously to be important in muscle-specific transcription. Mutagenesis of these sites revealed that both sites are necessary for full activity of the myogenin promoter, and suggests that a complex hierarchy of transcription factors control myogenic differentiation. ^