Notes: The authors identify PARTING DANCERS as a gene involved in male meiosis in Arabidopsis using a microarray generated from meiotic-stage anthers. To confirm the sequence obtained by microarray analysis, RT-PCR was performed and the resulting cDNA was cloned into the pGEM^®-T Vector and sequenced. To generate probes for in situ hybridization, fragments of ptd were amplified, cloned into the pGEM^®-T Vector, then labeled with digoxygenin through in vitro transcription. (3468)

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: Constructs of Rabex-5, a guanine nucleotide exchange factor, and Rabaptin-5, a multivalent adaptor protein, were created for transfection. The plasmid pGEM-Myc-human Rabaptin-5 was digested using NcoI/PstI, the fragment filled in using T4 DNA polymerase and ligated into SmaI-cut pCI-neo Mammalian Expression Vector. This new construct was digested with ApaI and NotI to remove Rabaptin-5 while Rabex-5 was digested using SacII/SpeI. Both digestion products were subjected to T4 DNA polymerase and the blunt-ended fragments ligated to create pCI-neo-Myc-Rabex-5. Furthermore, the Rabex-5 construct was subjected to site-directed mutatgenesis. HeLa cells were transiently transfected using the various pCI-neo-Myc-Rabex-5 vectors and the cells extracts immunoprecipitated to determine if ubiquitination of Myc-Rabex-5 had occurred. (3513)

Notes: To characterize sequence variability of the catalytic center of the hepatitis delta virus (HDV) ribozyme, which includes a self-cleaving motif, the authors developed an in vitro selection process that allowed them to select self-cleaving sequence variants from a pool of HDV ribozymes. The selection process started with a library of DNA oligonucleotides corresponding to the HDV ribozyme sequence that had been randomized at a 25-nucleotide sequence. The oligos contained known sequences at the 5´ and 3´ ends to allow amplification. The library was amplified by PCR, then transcribed. The resulting RNA was separated by polyacylamide gel electrophoresis, and the self-cleaved transcripts were excised, purified and reverse transcribed. A 3´ extension using terminal deoxynucleotidyl transferase added a known sequence to the 3´ end so that the resulting cDNA could by amplified by PCR. Thus another cycle of selection could begin. The PCR products generated at the start of the process and at each cycle were cloned into the pGEM^®-T Vector. (3467)

Notes: The authors used 5´ and 3´RACE to amplify the gene mimitin, and the resulting cDNA was cloned into the pGEM^®-T Vector. A genomic DNA fragment containing the mimitin promoter sequence was amplified by PCR and cloned into the pGEM^®-T Vector. The promoter was then cloned into the pGL3-Basic Vector. The activity of the wildtype and mutated promoters was determined using a luciferase assay. The pRL CMV Vector was used to normalize for differences in transfection efficiency. (3466)

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 authors investigated common variants of the APOA5 gene that have been associated with differences in plasma triglyceride (TG) levels. PCR fragments containing either the –1131T --> C promoter variant or containing both the –1131T --> C and –3G --> A promoter variants were cloned into the pGEM^®-T Vector System. The fragments were subsequently cloned into the pGL3 Basic Vector and transiently transfected into Huh7 and HepG2 cells along with the luciferase control vector, pRL-TK. The cells were lysed 48 hours after transfection and Luciferase activity was measured with the Dual-Luciferase^® Reporter Assay System. The function of the 1891T --> C variant in the 3´ UTR was tested the same way; with the exception that site-directed mutagenesis was performed to introduce the T --> C at position 1891 before the fragment was cloned into the pGL3 Basic Vector. The functionality of the Kozak sequence –3A --> G variant was determined by cloning cDNAs into the pGEM^®-7Zf Vector. Transcription/translation experiments were performed using the TNT^® Quick Coupled Transcription/Translation System and the proteins were labeled using the FluorTect™ Green_Lys System. In addition, a primer extension inhibition assay was performed using capped mRNAs generated with the Riboprobe^® System –T7 and the Ribo m⁷G Cap Analog. Ribosome binding reactions were performed using the Rabbit Reticulocyte Lysate System, Nuclease Treated. (3460)

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: In this report, satellite RNA from the full-length RPV serotype (satRPV RNA) of Cereal yellow dwarf virus was transcribed in vitro using the RiboMAX™ Large Scale RNA Production System. Self-cleavage of the synthesized RNA was then induced by incubation with a cleavage buffer. Serial deletions were created in the wild-type satRPV cloned in pGEM^®-3Zf(–) Vector. The mutants generated were tested to see how well they replicated with internal sections of satRPV sequence removed. (3080)

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: Researchers cloned the Photinus and Renilla luciferase ORFs into the pSP64 Poly(A) Vector to create a dual-reporter vector named SP6P. A similar vector, SP6R.4G(-508/-3).P, was created in which a 5´ untranslated region from the Saccharomyces cerevisiae TIF4631 gene was cloned between the two reporter genes. These two vectors were used to transform yeast strains. The resultant transformants were lysed using Passive Lysis Buffer and a modified lysis procedure.   Lysates were analyzed for luciferase activities using the Dual-Luciferase^® Reporter Assay System and a TD20/20 luminometer. The researchers also cloned and sequenced the 5´  untranslated region of TIF4631 by using a RACE-PCR technique followed by cloning the PCR amplimers into the pGEM^®-T Vector. (2845)

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 immunohistochmical 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 counter stained 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: The entire sti gene (~7kb) of Drosophila was PCR cloned into the pGEM^®-T Vector. The cloned sequences were then sequenced and analyzed for mutations. The researchers also used the T7 RiboMAX™ Large Scale RNA Production System to create dsRNAs from PCR-amplified regions of the sti and GFP coding regions. TransFast™ Transfection Reagent was used to transfect 2 x 10⁶ Schneider S2 cells with 10 μg of dsRNA in a 35mm Petri dish. The cells were fixed after transfection and examined for multinucleate cells, or immunocytochemically stained for actin and anillin. (3259)

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 authors used Xenopus laevis oocytes to show that unliganded thyroid hormone receptor (TR) recruits N-CoR (nuclear receptor corepressor) to modulate metamorphosis.  To study this influence, the cytoplasm of stage VI oocytes from X. laevis was injected with the indicated mRNAs [TR, retinoic acid receptor (RXR) and FLAG-tagged N-CoR].  The reporter plasmid TRE-Luc (0.33 ng/oocyte; thyroid hormone response elements from a Xenopus promoter driving expression of the firefly luciferase gene) and the control vector phRG-TK (0.03 ng/oocyte) were co-injected into the germinal vesicle (nucleus) after mRNA injection. After overnight incubation at 18°C, oocyte lysates were prepared by lysing six oocytes in 90µL 1X Passive Lysis Buffer. The Dual-Luciferase^® Reporter Assay System was then used to assay 7µl of the lysate. The researchers also used an expression vector (based on the pGEM^®-4Z Vector) containing the 5’ and 3’ untranslated regions of the X. laevis beta-globin gene flanking the multiple cloning site. (3088)

Notes: GoTaq^® DNA Polymerase was used in a multiplexed genotyping reaction with three primers to differentiate wild-type, heterozygous, and mutant transgenic mouse alleles simultaneously. The wild-type target was 670bp and the mutant or knockout target was 1,030bp. Various other amplimers (1.4kb, 471bp, & 311bp) were subcloned with the aid of the pGEM^®-T Easy Vector System. (3360)

Notes: In this study, the SV Total RNA Isolation System was used to isolate total RNA from venom sacs from Naja Atra (Asian cobra). The Access RT-PCR System was used to reverse transcribe atratoxin and atratoxin-b cDNA from the isolated RNA. The amplified cDNAs were then cloned into the pGEM®-T vector. (3126)

Notes: The authors of this study investigated the function of the daf-15 gene in C. elegans. The daf-15 gene encodes the C. elegans ortholog of raptor, a protein involved in the TOR signaling pathway. In C. elegans, daf-15 transcription is regulated by DAF-16, a FOXO transcription factor which is itself regulated by the insulin/IGF-1 signaling pathway. This work links regulation of the TOR pathway, which controls cell growth, to the insulin/IGF-1 pathway, known to affect lifespan, development and metabolism. Three candidate daf-15 genes were amplified by PCR and cloned into the pGEM^®-T Vector. The Riboprobe^® SP6/T7 transcription system was used to transcribe RNA from the candidate clones. Semiquantitative RT-PCR was performed to compare daf-15 mRNA levels in daf-2 and daf-16; daf-2 mutant animals. The mRNA was purified from total worm RNA using the PolyATtract^® mRNA Isolation System. (3633)