Notes: Fungi of the genus Cercospora are plant pathogens that cause leaf spot and blight diseases, and produce the polyketide toxin cercosporin. The bacterium Xanthomonas campestris is able to rapidly degrade cercosporin. In this study, X. campestris mutants unable to degrade cercosporin were created by chemical mutagenesis. Complementation studies with a plasmid-based library of X. campestris DNA showed that the ability to degrade cercosporin was restored upon transformation with plasmids containing an oxidoreductase gene and a putative transcriptional regulator. These genes were then amplified from the mutant strains by high-fidelity PCR. The PCR products were separated by agarose gel electrophoresis, purified using the Wizard^® SV Gel and PCR Clean-Up System, and subcloned into the pGEM^®-T Easy Vector. The mutant genes were then sequenced to identify the nature of the mutations. (3531)

Notes: The outer membrane protein A (ompA) gene of Enterobacter sakazakii was amplified using PCR primers based on E. coli ompA sequences. The resulting PCR product was ligated into the pGEM®-T Easy Vector, and the sequence was confirmed. The ompA sequence was used to develop a PCR for detection of Enterobacter sakazakii in reconstituted infant formula. (3464)

Notes: This paper highlights the first use of the HaloTag™ Interchangeable Protein Labeling Technology in plant cells. The cDNA for the HaloTag™ protein was amplified by PCR from the pHT2 Vector and cloned into the pGEM^®-T Easy^® Vector, from which it was excised and transferred to a second vector where its expression was under the control of the cauliflower mosaic virus (CaMV)-³⁵S promoter. The construct was transformed into tobacco protoplasts and bioballistically transformed into tobacco leaf cells. Localization was followed using the HaloTag™ TMR and diAcFAM Ligands. (3503)

Notes: In this study, insertional mutagenesis with the transposon TN5367 was used to generate a library of M. paratuberculosis mutants. Sequences containing transposons were then amplified, gel purified using the Wizard^® SV Gel and PCR Clean-Up System, and cloned into the pGEM^®-T Easy Vector prior to sequencing. Bioinformatic screening was then used to identify potential virulence determinants for further study in a mouse model of M. paratuberculosis infection. (3534)

Notes: Overexpression of IGF-1 can delay or prevent aging problems in motor neurons and skeletal muscle. The authors of this paper were able to target IGF-1 to motor neurons using a fusion protein containing tetanus toxin fragment C (TTC). Motor neurons will bind, take up and transport the TTC fragment with no toxicity to the neurons. Full-length human IGF-1 cDNA was generated by PCR and inserted into the pGEM^®-T Easy Vector. TTC amplified from Clostridium tetani CN655 genomic DNA was inserted into the vector. The new IFG-1-TTC insert was used for PCR to eventually produce the fusion protein for the studies. (3635)

Notes: This study compared detection of Pythium species in soil samples by DNA array hybridization and PCR cloning. Three Pythium species were amplified from soil samples, a single 3´ A was added to the resulting PCR product, and the DNA was ligated into the pGEM^®-T Easy Vector at 4°C overnight. After the ligation was transformed into JM109 Competent Cells, and 100 colonies were chosen and grown overnight in LB broth. The plasmid DNA was isolated using the Wizard^® SV 96 Plasmid DNA Purification System and then sequenced. (3437)

Notes: The coding sequence of the Human Papilloma Virus (HPV16) E7 oncogene was isolated following total RNA purification from CaSki cells, RT-PCR and PCR and then cloning into the pGEM^®-T Easy vector. In order to test the effectiveness of antisense HPV16 E7 therapy against cervical cancer, an adeno-associated virus vector was used to transfer the antisense construct of the E7 coding sequence into CaSki cervical cancer cells. (3395)

Notes: The authors of this study investigated the role of apoptosis signal-regulating kinase 1 (ASK1) in neural cell apoptosis during retinal development and ischemic injury. Nucleotides 283 to 713 of the ASK1 cDNA were amplified by PCR and cloned into the pGEM^®-T Easy Vector, and sense and antisense probes for in situ hybridization experiments were generated. Anti-ACTIVE^® p38 polyclonal antibody was used for immunohistochemistry analyses to investigate the localization of phosphorylated p38 in mouse retina. (3530)

Notes: To purify stolbur phytoplasma DNA from total DNA of infected periwinkle plants, two rounds of suppression subtractive hybridization (SSH) were performed, followed by amplification with Taq DNA polymerase. The resultant PCR products (1µl) were ligated into 50ng of pGEM^®-T Easy Vector using 3 units T4 DNA Ligase. After transformation of DH10B cells, ampicillin-resistant colonies were grown and the plasmids purified using the Wizard^® Plus SV Minipreps DNA Purification System. The insert lengths were estimated after EcoR I digestion and agarose gel electrophoresis prior to amplification and labeling with digoxigenin. These probes were used for dot hybridization with denatured healthy or infected plant DNA (10µg) and the corresponding plasmid as a positive control (100 ng). (3436)

Notes: Monocarboxylate transporters (MCT) transport lactic acid across the cell membrane. The promoters of 4 MCT family members (MCT1, MCT2, MCT3 and MCT4), were amplified by PCR and cloned into the pGEM^®-T Easy Vector. The sequences were confirmed, and the promoters were cloned into the pGL3-Basic Vector. The Dual-Glo™ Luciferase Assay System was used to quantitate promoter activity under basal and hypoxic conditions in HeLa cells. The pRL-SV40 Vector was used to normalize for differences in transfection efficiency. (3463)

Notes: A transposase protein (Tnp) open reading frame (amino acids 1 to 345) was amplified from the Drosophila mauritiana Mos1 mariner transposable element using GoTaq^® DNA polymerase. The Tnp open reading frame was then cloned with into the pGEM^®-T Easy Vector and sequenced before further subloning. Tnp protein was purified and used in gel-shift assays to demonstrate Tnp binding regions in the Mos1 transposable element. (3344)

Notes: An open reading frame encoding a putative DNA polymerase, SpPol4, was amplified from S. pombe genomic DNA by PCR. The resulting PCR product was cloned into the pGEM^®-T Easy Vector, and the sequence was verified. Based on sequence analysis, the authors hypothesize that SpPol4 has deoxyribose phosphate (dRP) lyase activity, suggesting that the enzyme plays a role in base excision repair. The authors perform a dRP lyase activity assay with an oligonucleotide substrate labeled using [³²P]ddATP and terminal deoxynucleotidyl transferase. (3465)

Notes: The investigators cloned three β-defensins, which are antimicrobial peptides, from canine testes. A canine expressed sequence tag (EST) was identified based on similarity to human β-defensins. Full-length cDNAs were obtained using 5´- and 3´RACE, then amplified by PCR and cloned into the pGEM^®-T Easy Vector. cDNA sequences were confirmed using the SP6 and T7 Promoter Primers. The tissue-specific expression of canine β-defensins (cBDs) was characterized using the AccessQuick™ RT-PCR System to amplify β-defensin RNA from a variety of tissues. RNA was treated with RQ1 RNase-Free DNase prior to RT-PCR. The identity of the RT-PCR products was confirmed by electrophoresis, transfer to nylon membranes and hybridization to probes derived from sequence-confirmed β-defensin clones; the probes were synthesized using the Prime-a-Gene^® Labeling System. To localize expression of the three β-defensin isoforms in canine testes, in situ hybridization (ISH) was performed. The 3´-RACE products were cloned into the pGEM^®-T Vector, which was then linearized and treated with exonuclease III to delete an approximately 80-bp region shared by the three cBD isoforms. The resulting product was used to synthesize sense and antisense ISH probes. (3453)

Notes: To study the transcription levels of several genes involved in cysteine and methionine metabolism, Lactococcus lactis strains deficient in these genes were created by double-crossover homologous integration. To do so, PCR was performed to amplify sequences upstream and downstream of the target gene, and the PCR products were ligated and cloned into the pGEM^®-T Easy Vector. The resulting vectors were fused to pGhost9, a vector designed for homologous recombination in L. lactis. Once the gene deletions were verified, quantitative real-time RT-PCR was performed to quantitate expression levels of a variety of genes involved in Cys and Met metabolism. (3461)

Notes: In this study, the binding of the response regulator RR06 to the promoter region of cbpA was investigated. DNA encoding the rr06 gene was cloned into the pGEM^®-T Easy vector and transformed into E. coli strain DH5α. Cell lysates were prepared and incubated with a labeled cbpA promoter fragment, and binding was evaluated in an electrophoretic mobility shift assay (EMSA). To further investigate the RR06/cbpA interaction, the cbpA promoter region, or a control rRNA sequence, was biotin labeled and attached to Streptavidin MagneSphere^® Paramagnetic Particles. The beads were incubated with E. coli lysates expressing RR06, or with control lysates. After washing, bound proteins were eluted by boiling. Eluted samples were then probed with specific anti-RR06 antisera. (3397)

Notes: Researchers used GoTaq^® DNA polymerase to amplify 139bp and 815bp regions of hOST-PTP cDNA for detection and probe synthesis. The full-length 4006bp cDNA was amplified with Pfu DNA Polymerase. Fragments were gel purified using the Wizard^® SV Gel and PCR Clean-Up System prior to cloning into the pGEM^®-T Easy Vector.  The Prime-a-Gene^® Labeling System was used to make ³²P-dCTP labeled probes, which were used to screen cDNA clones. (3111)

Notes: The researchers used pGEM®-T Easy Vectors (Cat.# A1360) to clone Zika virus PCR products. (4651)

Notes: The authors identified a TERMINAL FLOWER homolog, CsTFL, in Washington navel oranges (Citrus sinensis) and investigated its role and the role of other genes in juvenility and flower production. The CsTFL gene was amplified from genomic DNA using PCR and degenerate primers, and amplification products were cloned into the pGEM^®-T Easy Vector. The resulting clones were sequenced using the fmol^® DNA Cycle Sequencing System. The CsTFL cDNA was amplified by RT-PCR, using 4 µg of total RNA from whole flowers and ImProm-II™ Reverse Transcriptase. The amplified cDNA was then cloned into the pGEM^®-T Easy Vector. To evaluate CsTFL gene copy number and allele origins, the authors performed a Southern blot with 10 µg of genomic DNA and a CsTFL probe labeled with the Prime-a-Gene^® Labeling System. To characterize CsTFL expression in various citrus tissues, RT-PCR was performed with 2 µg of total RNA and ImProm-II™ Reverse Transcriptase. The levels of CsTFL RNA and other RNAs were determined by cDNA synthesis using ImProm-II™ Reverse Transcriptase, followed by real-time PCR. Amplification products were quantitated by SYBR^® Green fluorescence. Standard curves for each real-time PCR target were generated using known amounts of in vitro transcribed RNA. Prior to reverse transcription and real-time PCR, total RNA samples were treated with RQ1 RNase-Free DNase to remove DNA contaminants. (3650)

Notes: In this article, a 2.5- and a 1.7-kb fragment of the myostatin promoter were amplified by PCR and cloned into the pGEM®-T Easy vector. These clones were then subcloned into the pGL3-Basic Vector.  Deletion mutants of the constructs were made and used to transient transfect mouse muscle C₂C₁₂ cells. The Luciferase Assay System was then used to analyze transfectants. The researchers also injected luciferase-reporter constructs into mouse muscle. Luciferase activity in the mouse muscle was examined by grinding isolated muscles in liquid nitrogen and resuspending them in Cell Culture Lysis Buffer. Ten micron cryosections of the muscles were also used in immunohistochemical staining experiments.  For these experiments, the researchers used a 1:50 dilution of Promega’s polyclonal anti-luciferase antibody, a secondary antibody and tertiary fluorescein-labeled conjugate. The slides were counterstained with DAPI. (3147)

Notes: The authors cloned and sequenced the dsRNA from Penicillium chrysogenum virus (PcV) and analyzed the gene products of the segmented virus. After reverse transcription followed by PCR to confirm the 5’ and 3’ ends of the dsRNA segments, RT-PCR was used to amplify the complete cDNAs for the four viral segments. The products were cloned into pGEM®-T Vector after A-tailing. To confirm the ORFs predicted for each of the four segments, the cDNAs were added to TNT® T7 Quick Coupled Transcription/Translation System reactions that included radiolabeled methionine. The resulting proteins ranged in size from 94-128 kDa and were separated on an 8% SDS-PAGE gel and analyzed by autoradiography. Gradient-purified PcV virions were digested with Sequencing Grade Modified Trypsin at 37°C for 18 hours and the digestion products were separated by reverse-phase HPLC on a C18 column. Two highly resolved peptides were selected for amino acid sequencing by automated Edman degradation. (3089)

Notes: HeLa and Saos-2 were transiently transfected with luciferase reporter vectors made from the pGL3-Promoter vector containing various intron sequences from the human integrinα3 gene ITGA3. The first intron sequence was originally cloned by PCR cloning into the pGEM®-T Easy vector. Cell cultures were assessed for luciferase activity by making lysates with the Glo Lysis Buffer and assaying with the Steady-Glo® Assay kit. (3225)

Notes: Nuclear transfer (NT) embryos (macaque fibroblasts introduced into matured metaphase II stage rabbit oocytes) were placed into PCR tubes containing 10µl of lysis solution, ReadyAmp™ Genomic DNA Purification System resin and 200µg/ml proteinase K. To break open the cells and access the genomic DNA, the embryos were incubated for 30 minutes at 55°C, boiled for 10 minutes, centrifuged for 1 minute then used for PCR. The amplified DNA was then cloned into the pGEM^®-T Easy Vector and sequenced to determine if the product was macaque or rabbit. (3423)

Notes: The Wizard^® Genomic DNA Purification Kit was used to purify Bacillus anthracis chromosomal DNA, which was then used in PCR to amplify a luxN ortholog (named open reading frame BA5047). Amplified DNA containing the luxN ortholog region was then cloned into the pGEM^®-T Easy Vector. This construct, when transformed into Vibrio harveyi (AI-1^-, and AI-2⁺), allowed the detection of an upregulated signaling system by a bioluminescent assay. (3092)

Notes: Total RNA was obtained from non-small cell lung cancer cell lines and the breast cancer resistance protein message was amplified using real-time quantitative RT-PCR. RT-PCR products were subcloned into the pGEM^®-T Easy Vector. (2714)

Notes: The pGEM^®-T Easy Vector was used to subclone products of a 5´ RACE reaction. A promoter construct, assembled in the pGL3 Basic Vector, was co-transfected with a zebrafish interferon expression vector in the ZF4 zebrafish embryo fibroblast cell line using the TransFast™ Reagent (details provided). Luciferase levels were examined with the BrightGlo™ Luciferase Assay Reagent. Induction of zebrafish mRNA was also examined in zebrafish liver cells (ZFL) following treatment with the known interferon inducer, poly(I)-poly(C). RNA was extracted and reverse transcribed using ImProm-II™ Reverse Transcriptase. The resulting cDNA was used for quantitative, real-time RT-PCR with a SYBR green-based assay. (2627)