hela jvm  (Nissui Pharmaceutical)

Journal: PLoS Biology

Article Title: Spliced integrated retrotransposed element (SpIRE) formation in the human genome

doi: 10.1371/journal.pbio.2003067

Figure Lengend Snippet: (A) Schematic of the luciferase constructs and the relative position of northern blot probes. The L1.3 5′UTR (gray rectangle) was used to drive the transcription of the firefly luciferase reporter gene (green rectangle) present in plasmid pGL4.11. The following plasmids were created: pPL WT LUC contains the full-length L1.3 5′UTR; pPL 97/622 LUC contains the SpIRE 97/622 5′UTR; pPL SDm LUC contains a U 99 C SD mutation (red asterisk) in the L1.3 5′UTR; pPL SAm LUC contains an A 620 C SA mutation (light blue asterisk) in the L1.3 5′UTR. The relative positions of complementary riboprobes used in the northern blot experiments (ribonucleotides 7–99 [purple line], ribonucleotides 103–336 [red line], and the 3′ end of the luciferase gene [blue line]) are indicated below the schematic. (B) Representative northern blots. The black arrowhead indicates the predicted size of full-length L1/luciferase mRNA (about 2.7 kb). Construct names are indicated above the gel lanes; UTF = untransfected HeLa-JVM cells. The probe used in the northern blot experiment is indicated below the autoradiograph. Actin served as an mRNA loading control (2.1 kb). RNA size standards (kb) (Millenium RNA Markers) are indicated to the left of the autoradiograph panels. (C) Results from the luciferase assays. The x-axis indicates the name of the luciferase expression plasmid. The y-axis indicates the relative firefly luciferase units normalized to a co-transfected Renilla luciferase internal control. These data represent the averages of three biological replicates . Each biological replicate contained six technical replicates. Error bars indicate the standard deviation between three biological replicates. P -values were determined using a Student one-tailed t test. (D) Results from RT-PCR assays: A 1.2% agarose gel depicting the results from a representative qualitative RT-PCR experiment. DNA size markers (1 kb Plus DNA Ladder) are indicated at the left of the gel. Plasmid names are indicated above the gel; UTF = untransfected HeLa-JVM cells, H2O = water control for PCR reactions. The inset to below the gel indicates the major (* and #) and minor (+) cDNA products detected in the experiments. FL, full-length; H2O, water control for PCR reactions; kb, kilobase; L1, Long interspersed element-1; M, marker; RT-PCR, reverse transcription PCR; SA, splice acceptor; SD, splice donor; SpIRE, spliced integrated retrotransposed element; UTF, untransfected HeLa-JVM cells; UTR; untranslated region; WT, wild-type.

Article Snippet: Briefly, 2×10 5 HeLa-JVM cells were plated into 60-mm dishes (BD Biosciences).

Techniques: Luciferase, Construct, Northern Blot, Plasmid Preparation, Mutagenesis, Autoradiography, Expressing, Transfection, Standard Deviation, One-tailed Test, Reverse Transcription Polymerase Chain Reaction, Agarose Gel Electrophoresis, Marker

Journal: PLoS Biology

Article Title: Spliced integrated retrotransposed element (SpIRE) formation in the human genome

doi: 10.1371/journal.pbio.2003067

Figure Lengend Snippet: (A) Schematic of the L1PA1 and L1PA3 5′UTRs. Top schematic, the relative positions of the SD (red lettering), SA (green lettering), and putative branch point sequence (ACCTCAC, black lettering) in the L1PA1 5′UTR that led to the formation of SpIRE 97/790 are indicated in the schematic. Superscript numbers indicate the first and last nucleotide of the indicated sequence. Note that nucleotide positions are indicated in the context of L1.3 (accession #L19088). Numbers below the branch point (underlined A; 95.75) and above the SA A 788 G 789 (84.95) indicate the predicted score of those sequences for utilization in a splicing reaction, as determined using Human Splicing Finder v.3.0 ( http://www.umd.be/HSF3/ ) . Note that predicted scores above 80 are considered “strong” . Bottom schematic, the relative positions of the SD (red lettering), SAs A 851 G 852 (purple lettering), A 916 G 917 (green lettering), and putative branch point sequence (TCCAGAG, black lettering) in the L1PA3 5′UTR are indicated in the schematic. Superscript numbers indicate the first and last nucleotide of the indicated sequence. Numbers below the branch point (underlined A; 75.73) and SAs A 851 G 852 (83.75) and A 916 G 917 (79.66) indicate the predicted strength of those sequences for utilization in a splicing reaction, as determined using Human Splicing Finder v.3.0 ( http://www.umd.be/HSF3/ ) . The L1PA3 5′UTR contains a 129-bp sequence (gray triangle) containing the SA A 851 G 852 that was lost in the transition from the L1PA3 to L1PA2/L1PA1 subfamilies. The 129-bp deletion results in repositioning the SA A 916 G 917 in L1PA3 to closer proximity of a putative branch point in the L1PA2/L1PA1 subfamilies 5′UTR (now noted as A 788 G 789 in the top schematic), leading to a higher predicted score (84.95 in PA1 compared to 79.66 in PA3). (B) Schematic of luciferase constructs and results from luciferase assays. Top panel: the L1 RP 5′UTR (gray rectangle) was used to drive the transcription of the firefly luciferase reporter gene (green rectangle) present in plasmid pGL4.11. The following plasmids were created: pJBM WT LUC contains the full-length L1 RP 5′UTR; pJBM WT 129 PA4 LUC contains the 129-bp (black box in 5′UTR) sequence derived from L1PA4 within the L1 RP 5′UTR; pJBM WT 129 SCR LUC contains a scrambled version of the 129-bp sequence (black and white striped box) within the 5′UTR. Bottom panel: luciferase assay. The x-axis indicates the name of the luciferase expression plasmid. The y-axis indicates the relative firefly luciferase units normalized to a co-transfected Renilla luciferase internal control. These data represent the averages of three biological replicates . Each biological replicate contained six technical replicates. Error bars indicate the standard deviation between three biological replicates. P -values were determined using a Student one-tailed t test and “n.s.” indicates that there was no statistical difference. (C) Results from the EGFP retrotransposition assay: the x-axis indicates the construct names. The y-axis indicates the relative retrotransposition efficiency (%). The relative retrotransposition efficiencies are normalized to pL1 RP -EGFP (set at 100%). The data are from one representative experiment . Error bars represent the standard deviation of technical triplicates for the depicted assay. Each assay was repeated four times, yielding similar results. (D) Results from RT-PCR assays: a 2.0% agarose gel depicting the results from a representative qualitative RT-PCR experiment. DNA size markers (1 kb Plus DNA Ladder) are shown at the left of the gel. Plasmid names are indicated above the gel; UTF = untransfected HeLa-JVM cells, H2O = water control for PCR reactions. The right half of the agarose gel (“NO RT”) indicates the results from a representative experiment conducted without the addition of reverse transcriptase. The inset to the right of the gel indicates the major (*, **, and ***) and minor (+, @, and $) cDNA products detected in the experiments. The assay was repeated four times, yielding similar results. H2O, water control for PCR reactions; L1, Long interspersed element-1; RT-PCR, reverse transcription PCR; SA, splice acceptor; SD, splice donor; SpIRE, spliced integrated retrotransposed element; UTF, untransfected HeLa-JVM cells; UTR, untranslated region.

Article Snippet: Briefly, 2×10 5 HeLa-JVM cells were plated into 60-mm dishes (BD Biosciences).

Techniques: Sequencing, Luciferase, Construct, Plasmid Preparation, Derivative Assay, Expressing, Transfection, Standard Deviation, One-tailed Test, Reverse Transcription Polymerase Chain Reaction, Agarose Gel Electrophoresis