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1.  Generating In Vivo Cloning Vectors for Parallel Cloning of Large Gene Clusters by Homologous Recombination 
PLoS ONE  2013;8(11):e79979.
A robust method for the in vivo cloning of large gene clusters was developed based on homologous recombination (HR), requiring only the transformation of PCR products into Escherichia coli cells harboring a receiver plasmid. Positive clones were selected by an acquired antibiotic resistance, which was activated by the recruitment of a short ribosome-binding site plus start codon sequence from the PCR products to the upstream position of a silent antibiotic resistance gene in receiver plasmids. This selection was highly stringent and thus the cloning efficiency of the GFPuv gene (size: 0.7 kb) was comparable to that of the conventional restriction-ligation method, reaching up to 4.3 × 104 positive clones per μg of DNA. When we attempted parallel cloning of GFPuv fusion genes (size: 2.0 kb) and carotenoid biosynthesis pathway clusters (sizes: 4 kb, 6 kb, and 10 kb), the cloning efficiency was similarly high regardless of the DNA size, demonstrating that this would be useful for the cloning of large DNA sequences carrying multiple open reading frames. However, restriction analyses of the obtained plasmids showed that the selected cells may contain significant amounts of receiver plasmids without the inserts. To minimize the amount of empty plasmid in the positive selections, the sacB gene encoding a levansucrase was introduced as a counter selection marker in receiver plasmid as it converts sucrose to a toxic levan in the E. coli cells. Consequently, this method yielded completely homogeneous plasmids containing the inserts via the direct transformation of PCR products into E. coli cells.
doi:10.1371/journal.pone.0079979
PMCID: PMC3823602  PMID: 24244585
2.  Phosphate-Responsive Promoter of a Pichia pastoris Sodium Phosphate Symporter▿ † 
Applied and Environmental Microbiology  2009;75(11):3528-3534.
To develop a functional phosphate-regulated promoter in Pichia pastoris, a phosphate-responsive gene, PHO89, which encodes a putative sodium (Na+)-coupled phosphate symporter, was isolated. Sequencing analyses revealed a 1,731-bp open reading frame encoding a 576-amino-acid polypeptide with 12 putative transmembrane domains. The properties of the PHO89 promoter (PPHO89) were investigated using a bacterial lipase gene as a reporter in 5-liter jar fermentation experiments. PPHO89 was tightly regulated by phosphate and was highly activated when the cells were grown in a phosphate-limited external environment. Compared to translation elongation factor 1α and the glyceraldehyde-3-phosphate dehydrogenase promoter, PPHO89 exhibited strong transcriptional activity with higher specific productivity (amount of lipase produced/cell/h). Furthermore, a cost-effective and simple PPHO89-based fermentation process was developed for industrial application. These results demonstrate the potential for efficient use of PPHO89 for controlled production of recombinant proteins in P. pastoris.
doi:10.1128/AEM.02913-08
PMCID: PMC2687300  PMID: 19329662
3.  Mini-open Muscle Resection Procedure under Local Anesthesia for Lateral and Medial Epicondylitis 
Clinics in Orthopedic Surgery  2009;1(3):123-127.
Background
This study examined the clinical results of surgical treatment using a mini-open muscle resection procedure under local anesthesia for intractable lateral or medial epicondylitis.
Methods
Forty two elbows (41 patients) were treated surgically for lateral or medial epicondylitis. The indication for surgery was refractory pain after six months of conservative treatment, or a history of more than three local injections of steroid, or severe functional impairment in the occupational activities. The treatment results were assessed in terms of the pain using the visual analogue scale (VAS), Roles & Maudsley score, and Nirschl & Pettrone grade.
Results
The preoperative VAS scores of pain were an average of 5.36 at rest, 6.44 at daily activities, and 8.2 at sports or occupational activities. After surgery, the VAS scores improved significantly (p < 0.01): 0.3 at rest, 1.46 at daily activities, and 2.21 at sports or occupational activities. The preoperative Roles & Maudsley score was acceptable in 6 cases, and poor in 36 cases, which was changed to excellent in 23 cases, good in 16 cases, acceptable in 3 cases after surgery. According to the grading system by Nirschl & Pettrone, 23 cases were excellent, 18 cases were good, and the remaining 1 case was fair. Overall, 41 cases (97.6%) achieved satisfactory results. Postoperative complications were encountered in three cases. Subcutaneous seroma due to the leakage of joint fluid in two patients was managed by additional surgery and suction drainage, and resulted in a satisfactory outcome. One patient complained of continuous pain on occupational activity, but her pain at rest was improved greatly.
Conclusions
The mini-open muscle resection procedure under local anesthesia appears to be one of effective methods for intractable lateral or medial epicondylitis.
doi:10.4055/cios.2009.1.3.123
PMCID: PMC2766749  PMID: 19885046
Lateral epicondylitis; Medial epicondylitis; Local anesthesia; Mini-open; Muscle resection
4.  Efficient Library Construction by In Vivo Recombination with a Telomere-Originated Autonomously Replicating Sequence of Hansenula polymorpha 
A high frequency of transformation and an equal gene dosage between transformants are generally required for activity-based selection of mutants from a library obtained by directed evolution. An efficient library construction method was developed by using in vivo recombination in Hansenula polymorpha. Various linear sets of vectors and insert fragments were transformed and analyzed to optimize the in vivo recombination system. A telomere-originated autonomously replicating sequence (ARS) of H. polymorpha, reported as a recombination hot spot, facilitates in vivo recombination between the linear transforming DNA and chromosomes. In vivo recombination of two linear DNA fragments containing the telomeric ARS drastically increases the transforming frequency, up to 10-fold, compared to the frequency of circular plasmids. Direct integration of the one-end-recombined linear fragment into chromosomes produced transformants with single-copy gene integration, resulting in the same expression level for the reporter protein between transformants. This newly developed in vivo recombination system of H. polymorpha provides a suitable library for activity-based selection of mutants after directed evolution.
doi:10.1128/AEM.69.8.4448-4454.2003
PMCID: PMC169078  PMID: 12902228
5.  Integrative Transformation System for the Metabolic Engineering of the Sphingoid Base-Producing Yeast Pichia ciferrii 
We have developed an integrative transformation system for metabolic engineering of the tetraacetyl phytosphingosine (TAPS)-secreting yeast Pichia ciferrii. The system uses (i) a mutagenized ribosomal protein L41 gene of P. ciferrii as a dominant selection marker that confer resistance to the antibiotic cycloheximide and (ii) a ribosomal DNA (rDNA) fragment of P. ciferrii as a target for multicopy gene integration into the chromosome. A locus within the nontranscribed region located between 5S and 26S rDNAs was selected as the integration site. A maximum frequency of integrative transformation of approximately 1,350 transformants/μg of DNA was observed. To improve the de novo synthesis of sphingolipid, the LCB2 gene, encoding a subunit of serine palmitoyltransferase, which catalyzes the first committed step of sphingolipid synthesis, was cloned from P. ciferrii and overexpressed under the control of the P. ciferrii glyceraldehyde-3-phosphate dehydrogenase promoter. After transformation of an LCB2 gene expression cassette, several transformants that contained approximately five to seven copies of transforming DNA in the chromosome and exhibited about 50-fold increase in LCB2 mRNA relative to the wild type were identified. These transformants were observed to produce approximately two times more TAPS than the wild type.
doi:10.1128/AEM.69.2.812-819.2003
PMCID: PMC143681  PMID: 12570999
6.  A Family of Telomere-Associated Autonomously Replicating Sequences and Their Functions in Targeted Recombination in Hansenula polymorpha DL-1 
Journal of Bacteriology  1999;181(3):1005-1013.
A family of multiple autonomously replicating sequences (ARSs) which are located at several chromosomal ends of Hansenula polymorpha DL-1 has been identified and characterized. Genomic Southern blotting with an ARS, HARS36, originating from the end of a chromosome, as a probe showed several homologues in the genome of H. polymorpha. Nucleotide sequences of the three fragments obtained by a selective cloning for chromosomal ends were nearly identical to that of HARS36. All three fragments harbored an ARS motif and ended with 18 to 23 identical repetitions of 5′-GGGTGGCG-3′ which resemble the telomeric repeat sequence in other eukaryotes. Transformation of H. polymorpha with nonlinearized plasmids containing the newly obtained telomeric ARSs almost exclusively resulted in the targeted integration of a single copy or multiple tandem copies of the plasmid into the chromosomes. The sensitivity to exonuclease Bal31 digestion of the common DNA fragment in all integrants confirmed the telomeric origin of HARS36 homologues, suggesting that several chromosomal ends, if not all of them, consisted of the same ARS motif and highly conserved sequences observed in HARS36. Even though the frequencies of targeted recombination were varied among the ends of the chromosomes, the overall frequency was over 96%. The results suggested that the integration of the plasmids containing telemeric ARSs occurred largely through homologous recombination at the telomeric repeats, which serve as high-frequency recombination targets.
PMCID: PMC93470  PMID: 9922267

Results 1-6 (6)