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    Marathon cDNA Amplification Kit
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    634913
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    Structured Review

    TaKaRa marathon cdna amplification kit
    Expression of CK metabolic and response genes during tomato fruit development. Real-time <t>PCR</t> was performed with <t>cDNA</t> prepared from pollinated ovaries (Poll.) at 1, 3, 5, 10, 15, and 20 days after anthesis (black bars), and unpollinated ovaries (Unpoll.) at –2, 0, 1, and 3 days after anthesis (grey bars). Expression levels are normalized to SAND expression levels. Values are mean ± SE ( n = 3).

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    Images

    1) Product Images from "Roles and regulation of cytokinins in tomato fruit development"

    Article Title: Roles and regulation of cytokinins in tomato fruit development

    Journal: Journal of Experimental Botany

    doi: 10.1093/jxb/ers207

    Expression of CK metabolic and response genes during tomato fruit development. Real-time PCR was performed with cDNA prepared from pollinated ovaries (Poll.) at 1, 3, 5, 10, 15, and 20 days after anthesis (black bars), and unpollinated ovaries (Unpoll.) at –2, 0, 1, and 3 days after anthesis (grey bars). Expression levels are normalized to SAND expression levels. Values are mean ± SE ( n = 3).
    Figure Legend Snippet: Expression of CK metabolic and response genes during tomato fruit development. Real-time PCR was performed with cDNA prepared from pollinated ovaries (Poll.) at 1, 3, 5, 10, 15, and 20 days after anthesis (black bars), and unpollinated ovaries (Unpoll.) at –2, 0, 1, and 3 days after anthesis (grey bars). Expression levels are normalized to SAND expression levels. Values are mean ± SE ( n = 3).

    Techniques Used: Expressing, Real-time Polymerase Chain Reaction

    2) Product Images from "A Novel Zinc Finger Protein 219-like (ZNF219L) is Involved in the Regulation of Collagen Type 2 Alpha 1a (col2a1a) Gene Expression in Zebrafish Notochord"

    Article Title: A Novel Zinc Finger Protein 219-like (ZNF219L) is Involved in the Regulation of Collagen Type 2 Alpha 1a (col2a1a) Gene Expression in Zebrafish Notochord

    Journal: International Journal of Biological Sciences

    doi: 10.7150/ijbs.7126

    Genomic organization and expression profiles of zebrafish znf219L . Genomic organization of zebrafish znf219L , and the mouse and human znf219 genes were shown in panel (A). Coding regions are shown as filled boxes numbered from 3 to 5. The 5'- and 3'-untranslated regions are shown as open boxes, while solid lines indicate introns. RT-PCR was performed with gene-specific primers. β -actin was used as an internal control to normalize the amount of cDNA prepared from different adult zebrafish tissues (B) and from zebrafish embryos at different developmental stages (C). The developmental expression profile of zebrafish znf219L mRNA was examined in embryos from 12 hpf to 144 hpf. Blank PCR was performed using gene-specific primers and β -actin primers without the addition of cDNA template. Whole-mount in situ hybridization with antisense znf219L was performed at the indicated times post-fertilization (D). The images were taken from 22 to 48 hpf in the lateral view (panels a, b) and 48 hpf in dorsal view in panel c. The boxed region is enlarged to show the signal in the notochord (nc) from 22 to 48 hpf of both lateral and dorsal view (panels a', b', and c'). Dorsal view of znf219L mRNA signals in midbrain hinfbrain boundry (mhb), and hindbrain (hb) were detected from 48 to 96 hpf (panel d, e, and f). mhb, midbrain hindbrain boundry; hb, hindbrain; ov, otic vesicle; pf, pectoral fin, nc, notochord. Scale bars=100 μm.
    Figure Legend Snippet: Genomic organization and expression profiles of zebrafish znf219L . Genomic organization of zebrafish znf219L , and the mouse and human znf219 genes were shown in panel (A). Coding regions are shown as filled boxes numbered from 3 to 5. The 5'- and 3'-untranslated regions are shown as open boxes, while solid lines indicate introns. RT-PCR was performed with gene-specific primers. β -actin was used as an internal control to normalize the amount of cDNA prepared from different adult zebrafish tissues (B) and from zebrafish embryos at different developmental stages (C). The developmental expression profile of zebrafish znf219L mRNA was examined in embryos from 12 hpf to 144 hpf. Blank PCR was performed using gene-specific primers and β -actin primers without the addition of cDNA template. Whole-mount in situ hybridization with antisense znf219L was performed at the indicated times post-fertilization (D). The images were taken from 22 to 48 hpf in the lateral view (panels a, b) and 48 hpf in dorsal view in panel c. The boxed region is enlarged to show the signal in the notochord (nc) from 22 to 48 hpf of both lateral and dorsal view (panels a', b', and c'). Dorsal view of znf219L mRNA signals in midbrain hinfbrain boundry (mhb), and hindbrain (hb) were detected from 48 to 96 hpf (panel d, e, and f). mhb, midbrain hindbrain boundry; hb, hindbrain; ov, otic vesicle; pf, pectoral fin, nc, notochord. Scale bars=100 μm.

    Techniques Used: Expressing, Reverse Transcription Polymerase Chain Reaction, Polymerase Chain Reaction, In Situ Hybridization

    3) Product Images from "Molecular and Functional Diversity of Neural Connexins in the Retina"

    Article Title: Molecular and Functional Diversity of Neural Connexins in the Retina

    Journal: The Journal of Neuroscience

    doi: 10.1523/JNEUROSCI.20-22-08331.2000

    Multiple-tissue Northern blots and RT-PCRs of fish connexins. A , Northern blot of carpCx43 mRNA. CarpCx43 mRNA is highest in brain and retina, whereas liver reveals no detectable levels. B , Ethidium bromide-stained agarose gel of A that shows comparative loading of mRNA (18S and 28S band). C , Multiple-tissue RT-PCR of the three zebrafish connexins (ethidium bromide-stained agarose gels). zfCx44.1 is less abundant in brain and retina, with higher levels in lens and heart. zfCx27.5 shows tissue-restricted expression in brain and retina, whereas zfCx55.5 is exclusively expressed in the retina. All cDNA preparations were controlled by PCR using primers specific for β-actin. D , PCR control (with β-actin primers) omitting reverse transcription. No amplification is evident with the exception of the genomic DNA, indicative of lack of genomic DNA contamination in the mRNA samples.
    Figure Legend Snippet: Multiple-tissue Northern blots and RT-PCRs of fish connexins. A , Northern blot of carpCx43 mRNA. CarpCx43 mRNA is highest in brain and retina, whereas liver reveals no detectable levels. B , Ethidium bromide-stained agarose gel of A that shows comparative loading of mRNA (18S and 28S band). C , Multiple-tissue RT-PCR of the three zebrafish connexins (ethidium bromide-stained agarose gels). zfCx44.1 is less abundant in brain and retina, with higher levels in lens and heart. zfCx27.5 shows tissue-restricted expression in brain and retina, whereas zfCx55.5 is exclusively expressed in the retina. All cDNA preparations were controlled by PCR using primers specific for β-actin. D , PCR control (with β-actin primers) omitting reverse transcription. No amplification is evident with the exception of the genomic DNA, indicative of lack of genomic DNA contamination in the mRNA samples.

    Techniques Used: Northern Blot, Fluorescence In Situ Hybridization, Staining, Agarose Gel Electrophoresis, Reverse Transcription Polymerase Chain Reaction, Expressing, Polymerase Chain Reaction, Amplification

    4) Product Images from "A Presumptive Developmental Role for a Sea Urchin Cyclin B Splice Variant "

    Article Title: A Presumptive Developmental Role for a Sea Urchin Cyclin B Splice Variant

    Journal: The Journal of Cell Biology

    doi:

    PCR characterization of alternative splicing in cyclin B mRNA. (Lanes 1 and 9 ) Molecular mass markers (φ X 174/HaeIII and λ/EcoRI + HindIII, respectively). (Lanes 2 and 3 ) RT-PCR was done using poly(A) + mRNA from unfertilized eggs as a template, and CB5-CB64 and CB5– CB62 primer pairs, respectively (see Fig. 2 ). Both pairs amplified fragments of the expected sizes. (Lanes 4 and 5 ) The same primer pairs were used, with genomic DNA as a template. While CB5-CB62 gave rise to the right-sized PCR product (compare lanes 3 and 5 ), CB5-CB64 amplified a 2.5-kbp PCR fragment (compare lanes 2 and 4). This last product was reamplified with CB5-CB62 and gave rise to a right-sized product (compare lanes 3 , 5 , and 6 ), showing part of the clone 2 cDNA sequence comprising primer CB62 was included in the 2.5-kbp genomic CB5-CB64 fragment. (Lanes 7 and 8 ) PCR amplification of genomic DNA with respectively primers CB8-CB64 and CB10-CB64 (see Fig. 2 ). Only CB10-CB64 amplified a fragment (1,300 bp, lane 8 ), whereas amplification with CB8-CB64 was unsuccessful, showing part of the clone 2 cDNA, comprising primer CB8, was not included in the 2.5-kbp genomic CB5-CB64 fragment. All PCR products showed in this figure were confirmed by cloning and partial sequencing (not shown).
    Figure Legend Snippet: PCR characterization of alternative splicing in cyclin B mRNA. (Lanes 1 and 9 ) Molecular mass markers (φ X 174/HaeIII and λ/EcoRI + HindIII, respectively). (Lanes 2 and 3 ) RT-PCR was done using poly(A) + mRNA from unfertilized eggs as a template, and CB5-CB64 and CB5– CB62 primer pairs, respectively (see Fig. 2 ). Both pairs amplified fragments of the expected sizes. (Lanes 4 and 5 ) The same primer pairs were used, with genomic DNA as a template. While CB5-CB62 gave rise to the right-sized PCR product (compare lanes 3 and 5 ), CB5-CB64 amplified a 2.5-kbp PCR fragment (compare lanes 2 and 4). This last product was reamplified with CB5-CB62 and gave rise to a right-sized product (compare lanes 3 , 5 , and 6 ), showing part of the clone 2 cDNA sequence comprising primer CB62 was included in the 2.5-kbp genomic CB5-CB64 fragment. (Lanes 7 and 8 ) PCR amplification of genomic DNA with respectively primers CB8-CB64 and CB10-CB64 (see Fig. 2 ). Only CB10-CB64 amplified a fragment (1,300 bp, lane 8 ), whereas amplification with CB8-CB64 was unsuccessful, showing part of the clone 2 cDNA, comprising primer CB8, was not included in the 2.5-kbp genomic CB5-CB64 fragment. All PCR products showed in this figure were confirmed by cloning and partial sequencing (not shown).

    Techniques Used: Polymerase Chain Reaction, Reverse Transcription Polymerase Chain Reaction, Amplification, Sequencing, Clone Assay

    5) Product Images from "Overexpression of pigeonpea stress-induced cold and drought regulatory gene (CcCDR) confers drought, salt, and cold tolerance in Arabidopsis"

    Article Title: Overexpression of pigeonpea stress-induced cold and drought regulatory gene (CcCDR) confers drought, salt, and cold tolerance in Arabidopsis

    Journal: Journal of Experimental Botany

    doi: 10.1093/jxb/eru224

    Molecular characterization of the Cajanus cajan cold and drought regulatory gene ( CcCDR ) and comparison of its protein product with other plant proteins. (A) Northern blot analysis of CcCDR : 4-week-old plants of pigeonpea were subjected to no stress (1); PEG (20%) stress for 6h (2); NaCl (1M) stress for 6h (3); or cold (4 °C) stress for 6h (4). About 20 μg of total RNA was used for northern blot analysis. The blot was hybridized with the cDNA fragment of CcCDR . Ethidium bromide-stained 28S rRNA is shown for equal RNA loading. (B) Southern blot analysis of CcCDR : lane 1, Eco RI-digested; lane 2, Bam HI-digested; lane 3, Hin dIII-digested; lane 4, Sal I-digested genomic DNA of pigeonpea. Positions of 2.0kb and 10.0kb fragments in the gel are indicated. (C) Comparison of the deduced amino acid sequence of CcCDR with other proteins. Multiple sequence alignment of CcCDR (GU444042) with Carica papaya maturation-associated like Src1 protein (AAL73185), Glycine max cold-induced protein Src1 (BAA19768), G. max low temperature-inducible protein (ABO70349), and G. max KS-type dehydrin (ABQ81887). Identical and conserved amino acids are represented in black and grey, respectively. (This figure is available in colour at JXB online.)
    Figure Legend Snippet: Molecular characterization of the Cajanus cajan cold and drought regulatory gene ( CcCDR ) and comparison of its protein product with other plant proteins. (A) Northern blot analysis of CcCDR : 4-week-old plants of pigeonpea were subjected to no stress (1); PEG (20%) stress for 6h (2); NaCl (1M) stress for 6h (3); or cold (4 °C) stress for 6h (4). About 20 μg of total RNA was used for northern blot analysis. The blot was hybridized with the cDNA fragment of CcCDR . Ethidium bromide-stained 28S rRNA is shown for equal RNA loading. (B) Southern blot analysis of CcCDR : lane 1, Eco RI-digested; lane 2, Bam HI-digested; lane 3, Hin dIII-digested; lane 4, Sal I-digested genomic DNA of pigeonpea. Positions of 2.0kb and 10.0kb fragments in the gel are indicated. (C) Comparison of the deduced amino acid sequence of CcCDR with other proteins. Multiple sequence alignment of CcCDR (GU444042) with Carica papaya maturation-associated like Src1 protein (AAL73185), Glycine max cold-induced protein Src1 (BAA19768), G. max low temperature-inducible protein (ABO70349), and G. max KS-type dehydrin (ABQ81887). Identical and conserved amino acids are represented in black and grey, respectively. (This figure is available in colour at JXB online.)

    Techniques Used: Northern Blot, Staining, Southern Blot, Sequencing

    6) Product Images from "RASPBERRY3 Gene Encodes a Novel Protein Important for Embryo Development"

    Article Title: RASPBERRY3 Gene Encodes a Novel Protein Important for Embryo Development

    Journal: Plant Physiology

    doi: 10.1104/pp.004010

    Gene organization of RSY3 and T-DNA insertion in rsy3 mutant. A, Diagrammatic representation of a portion of the lambda genomic clone containing the RSY3 gene in chromosome 3. The predicted RSY3 gene (annotated for Columbia ecotype as MOB24.14 ) is expanded below the clone to highlight exons represented by solid arrows in orange and numbered accordingly. The genomic fragments tH989, tNH989, and tE989 used in the complementation analysis are outlined above the genomic clone. The cDNA clones are designated below the expanded region of RSY3 gene. Clone pC989–41 represents a partial cDNA isolated from a library, and clone pC989–41 represents the nearly full-length cDNA that were isolated using 5′- and 3′-RACE. Only the areas highlighted in colors within the rectangles represent the cDNA sequences. B, Diagrammatic representation of the T-DNA insertion in the rsy3 embryo mutant. Two T-DNAs that are arranged in concatemer are inserted in exon 9. Some of the Eco RI and SaI fragments, as revealed by plasmid rescue analysis, are highlighted with the approximate sizes written above the lines. Some of the restriction sites relevant to the DNA analysis shown in C are indicated. C, Restriction analysis of genomic DNAs isolated from wild-type (WT) and heterozygous (HZ) rsy3 individual segregants. DNAs were digested with restriction enzymes as indicated and were size separated by electrophoresis in a 1% (w/v) gel. The resulting blots were hybridized with a left or a right border probe as indicated in each panel. Restriction enzymes used are indicated. Note: Diagrams in A and B are not drawn to scale.
    Figure Legend Snippet: Gene organization of RSY3 and T-DNA insertion in rsy3 mutant. A, Diagrammatic representation of a portion of the lambda genomic clone containing the RSY3 gene in chromosome 3. The predicted RSY3 gene (annotated for Columbia ecotype as MOB24.14 ) is expanded below the clone to highlight exons represented by solid arrows in orange and numbered accordingly. The genomic fragments tH989, tNH989, and tE989 used in the complementation analysis are outlined above the genomic clone. The cDNA clones are designated below the expanded region of RSY3 gene. Clone pC989–41 represents a partial cDNA isolated from a library, and clone pC989–41 represents the nearly full-length cDNA that were isolated using 5′- and 3′-RACE. Only the areas highlighted in colors within the rectangles represent the cDNA sequences. B, Diagrammatic representation of the T-DNA insertion in the rsy3 embryo mutant. Two T-DNAs that are arranged in concatemer are inserted in exon 9. Some of the Eco RI and SaI fragments, as revealed by plasmid rescue analysis, are highlighted with the approximate sizes written above the lines. Some of the restriction sites relevant to the DNA analysis shown in C are indicated. C, Restriction analysis of genomic DNAs isolated from wild-type (WT) and heterozygous (HZ) rsy3 individual segregants. DNAs were digested with restriction enzymes as indicated and were size separated by electrophoresis in a 1% (w/v) gel. The resulting blots were hybridized with a left or a right border probe as indicated in each panel. Restriction enzymes used are indicated. Note: Diagrams in A and B are not drawn to scale.

    Techniques Used: Mutagenesis, Clone Assay, Isolation, Plasmid Preparation, Electrophoresis

    7) Product Images from "Polyserase-I, a human polyprotease with the ability to generate independent serine protease domains from a single translation product"

    Article Title: Polyserase-I, a human polyprotease with the ability to generate independent serine protease domains from a single translation product

    Journal: Proceedings of the National Academy of Sciences of the United States of America

    doi: 10.1073/pnas.1633392100

    Analysis of polyserase-I expression in human tissues and tumor cell lines. Northern blot analysis of polyserase-I expression in fetal and adult human tissues and a panel of tumor cell lines. Approximately 2 μg of polyadenylated RNA from the indicated tissues or tumor cell lines was hybridized with a 5′ probe of the polyserase-I full-length cDNA. The positions of the RNA markers are shown. The filters were subsequently hybridized with an actin probe to ascertain the differences in RNA loading among the different tissues.
    Figure Legend Snippet: Analysis of polyserase-I expression in human tissues and tumor cell lines. Northern blot analysis of polyserase-I expression in fetal and adult human tissues and a panel of tumor cell lines. Approximately 2 μg of polyadenylated RNA from the indicated tissues or tumor cell lines was hybridized with a 5′ probe of the polyserase-I full-length cDNA. The positions of the RNA markers are shown. The filters were subsequently hybridized with an actin probe to ascertain the differences in RNA loading among the different tissues.

    Techniques Used: Expressing, Northern Blot

    8) Product Images from "Isolation of a cDNA Encoding a Granule-Bound 152-Kilodalton Starch-Branching Enzyme in Wheat 1"

    Article Title: Isolation of a cDNA Encoding a Granule-Bound 152-Kilodalton Starch-Branching Enzyme in Wheat 1

    Journal: Plant Physiology

    doi:

    Isolation of cDNA corresponding to 5′ end of 4.6-kb Sbe1c transcript. A, Schematic illustration of the 4.6-kb Sbe1c transcript and product obtained from 5′-RACE analysis. Start of pRN60 sequence and location of PCR primers used in the 5′-RACE and RT-PCR reactions are indicated. B, Gel analysis of 5′-RACE products obtained in reactions with primers indicated and poly(A + ) RNA prepared from 12-d-old wheat kernels. Arrow indicates migration of product carrying the 5′ end of the 4.6-kb Sbe1c cDNA. Migration of standard DNA fragments are indicated to the right. C, Gel analysis of RT-PCR products obtained from reactions with PCR primers BE65 and BE38.
    Figure Legend Snippet: Isolation of cDNA corresponding to 5′ end of 4.6-kb Sbe1c transcript. A, Schematic illustration of the 4.6-kb Sbe1c transcript and product obtained from 5′-RACE analysis. Start of pRN60 sequence and location of PCR primers used in the 5′-RACE and RT-PCR reactions are indicated. B, Gel analysis of 5′-RACE products obtained in reactions with primers indicated and poly(A + ) RNA prepared from 12-d-old wheat kernels. Arrow indicates migration of product carrying the 5′ end of the 4.6-kb Sbe1c cDNA. Migration of standard DNA fragments are indicated to the right. C, Gel analysis of RT-PCR products obtained from reactions with PCR primers BE65 and BE38.

    Techniques Used: Isolation, Sequencing, Polymerase Chain Reaction, Reverse Transcription Polymerase Chain Reaction, Migration

    9) Product Images from "Hepatitis B Virus X-Associated Protein 2 Is a Subunit of the Unliganded Aryl Hydrocarbon Receptor Core Complex and Exhibits Transcriptional Enhancer Activity"

    Article Title: Hepatitis B Virus X-Associated Protein 2 Is a Subunit of the Unliganded Aryl Hydrocarbon Receptor Core Complex and Exhibits Transcriptional Enhancer Activity

    Journal: Molecular and Cellular Biology

    doi:

    Presence of XAP2 in COS-1 cells. (A) Northern blot analysis of XAP2 mRNA. Poly(A) + mRNA (4 μg) from COS-1 cells was probed with simian XAP2 cDNA. (B) Immunoblot analysis of XAP2. Lanes: 1, XAP2 translated in vitro; 2, 100 μg of COS-1 cytosol.
    Figure Legend Snippet: Presence of XAP2 in COS-1 cells. (A) Northern blot analysis of XAP2 mRNA. Poly(A) + mRNA (4 μg) from COS-1 cells was probed with simian XAP2 cDNA. (B) Immunoblot analysis of XAP2. Lanes: 1, XAP2 translated in vitro; 2, 100 μg of COS-1 cytosol.

    Techniques Used: Northern Blot, In Vitro

    10) Product Images from "Molecular Cloning and Characterization of the Estrogen Receptor from the Striped Bitterling (Acheilognathus yamatsutae)"

    Article Title: Molecular Cloning and Characterization of the Estrogen Receptor from the Striped Bitterling (Acheilognathus yamatsutae)

    Journal: Environmental Health and Toxicology

    doi: 10.5620/eht.2011.26.e2011005

    Cloning strategy and schematic view of SB ER cDNAs. The open reading frame is represented by a thick black line. The positions of the start codon (ATG) and the stop codon (TGA) are indicated. The binding sites of two degenerated primers (NF and NR) and four gene-specific primers (R1, F1, ER-F1, and ER-R1) are shown. The AP1 primer was provided in the RACE kit (Clontech Lab, CA, USA). The complete cDNA sequence was determined by overlapping the PCR products using the Chromas 3.2 analysis program. SB ER: striped bitterling estrogen receptor, AP1 primer: adaptor primer, RACE kit: Marathon cDNA Amplification kit.
    Figure Legend Snippet: Cloning strategy and schematic view of SB ER cDNAs. The open reading frame is represented by a thick black line. The positions of the start codon (ATG) and the stop codon (TGA) are indicated. The binding sites of two degenerated primers (NF and NR) and four gene-specific primers (R1, F1, ER-F1, and ER-R1) are shown. The AP1 primer was provided in the RACE kit (Clontech Lab, CA, USA). The complete cDNA sequence was determined by overlapping the PCR products using the Chromas 3.2 analysis program. SB ER: striped bitterling estrogen receptor, AP1 primer: adaptor primer, RACE kit: Marathon cDNA Amplification kit.

    Techniques Used: Clone Assay, Binding Assay, Sequencing, Polymerase Chain Reaction, Amplification

    11) Product Images from "CMS: An adapter molecule involved in cytoskeletal rearrangements"

    Article Title: CMS: An adapter molecule involved in cytoskeletal rearrangements

    Journal: Proceedings of the National Academy of Sciences of the United States of America

    doi:

    Tissue distribution of CMS mRNA. ( A ) Northern blot analysis of poly(A) + RNA from adult human tissues by using the 32 P-labeled CMS cDNA clone isolated in the two-hybrid screen. Hybridization analysis of the same Northern blot by using a 32 P-labeled β-actin cDNA probe ( Lower ). ( B ) Relative expression levels of CMS in different human tissues and developmental stages. Adult tissue: A1, whole brain; A2, amygdala; A3, caudate nucleus; A4, cerebellum; A5, cerebral cortex; A6, frontal lobe; A7, hippocampus; A8, medulla oblongata; B1, occipital lobe; B2, putamen; B3, substantia nigra; B4, temporal lobe; B5, thalamus; B6, nucleus accumbeus; B7, spinal cord; C1, heart; C2, aorta; C3, skeletal muscle; C4, colon; C5, bladder; C6, uterus; C7, prostate; C8, stomach; D1, testis; D2, ovary, D3, pancreas, D4, pituitary gland; D5, adrenal gland; D6, thyroid gland; D7, salivary gland; D8, mammary gland; E1, kidney; E2, liver; E3, small intestine; E4, spleen; E5, thymus; E6, peripheral leukocyte; E7, lymph node; E8, bone marrow; F1, appendix; F2, lung; F3, trachea; F4, placenta. Fetal tissue: G1, brain; G2, heart; G3, kidney; G4, liver; G5, spleen; G6, thymus; G7, lung. Controls (100 ng each): H1, yeast total RNA; H2, yeast tRNA; H3, Escherichia coli rRNA; H4, E. coli DNA; H5, poly(rA); H6, human C 0 t 1 DNA; H7, human DNA; H8, human DNA (500 ng).
    Figure Legend Snippet: Tissue distribution of CMS mRNA. ( A ) Northern blot analysis of poly(A) + RNA from adult human tissues by using the 32 P-labeled CMS cDNA clone isolated in the two-hybrid screen. Hybridization analysis of the same Northern blot by using a 32 P-labeled β-actin cDNA probe ( Lower ). ( B ) Relative expression levels of CMS in different human tissues and developmental stages. Adult tissue: A1, whole brain; A2, amygdala; A3, caudate nucleus; A4, cerebellum; A5, cerebral cortex; A6, frontal lobe; A7, hippocampus; A8, medulla oblongata; B1, occipital lobe; B2, putamen; B3, substantia nigra; B4, temporal lobe; B5, thalamus; B6, nucleus accumbeus; B7, spinal cord; C1, heart; C2, aorta; C3, skeletal muscle; C4, colon; C5, bladder; C6, uterus; C7, prostate; C8, stomach; D1, testis; D2, ovary, D3, pancreas, D4, pituitary gland; D5, adrenal gland; D6, thyroid gland; D7, salivary gland; D8, mammary gland; E1, kidney; E2, liver; E3, small intestine; E4, spleen; E5, thymus; E6, peripheral leukocyte; E7, lymph node; E8, bone marrow; F1, appendix; F2, lung; F3, trachea; F4, placenta. Fetal tissue: G1, brain; G2, heart; G3, kidney; G4, liver; G5, spleen; G6, thymus; G7, lung. Controls (100 ng each): H1, yeast total RNA; H2, yeast tRNA; H3, Escherichia coli rRNA; H4, E. coli DNA; H5, poly(rA); H6, human C 0 t 1 DNA; H7, human DNA; H8, human DNA (500 ng).

    Techniques Used: Northern Blot, Labeling, Isolation, Two Hybrid Screening, Hybridization, Expressing

    Schematic representation of the human CMS and protein sequence. ( A ) Alignment of human cDNA clones isolated in the yeast two-hybrid screen, 5′ rapid amplification of complementary DNA ends (RACE) and λgt11 cDNA library screen with respect to the full-length CMS cDNA. PR, proline-rich region. ( B ) Predicted protein sequence of CMS. Sequence numbers are shown on the left. The SH3 domains are underlined. Proline-rich sequences are marked in bold, the CC domain is marked in italics, and the putative actin binding sites are bold underlined. IP, immunoprecipitation. ( C ) In vitro interaction of p130 Cas and CMS. 293T cells were transiently transfected with the flag-tagged CMS yeast TH clone (CMS-TH) together with the CasSH3 domain or full-length Cas (both GST tagged) or the vector alone (GST control). Cell lysates were immunoprecipitated with anti-flag antibody or glutathione Sepharose beads, and precipitates were subjected to SDS/PAGE and probed with anti-GST antibody and anti-flag antibody, respectively. The upper blot was striped and reprobed with anti-flag antibody. ( D ) Direct interaction of CMS with p130 Cas . Five hundred micrograms of protein from 293T cells expressing the various CMS peptides or C3G used as a positive control was immunoprecipitated with the antibodies indicated above. Blots were probed for binding with 32 P-labeled GST-p130 Cas SH3 domain.
    Figure Legend Snippet: Schematic representation of the human CMS and protein sequence. ( A ) Alignment of human cDNA clones isolated in the yeast two-hybrid screen, 5′ rapid amplification of complementary DNA ends (RACE) and λgt11 cDNA library screen with respect to the full-length CMS cDNA. PR, proline-rich region. ( B ) Predicted protein sequence of CMS. Sequence numbers are shown on the left. The SH3 domains are underlined. Proline-rich sequences are marked in bold, the CC domain is marked in italics, and the putative actin binding sites are bold underlined. IP, immunoprecipitation. ( C ) In vitro interaction of p130 Cas and CMS. 293T cells were transiently transfected with the flag-tagged CMS yeast TH clone (CMS-TH) together with the CasSH3 domain or full-length Cas (both GST tagged) or the vector alone (GST control). Cell lysates were immunoprecipitated with anti-flag antibody or glutathione Sepharose beads, and precipitates were subjected to SDS/PAGE and probed with anti-GST antibody and anti-flag antibody, respectively. The upper blot was striped and reprobed with anti-flag antibody. ( D ) Direct interaction of CMS with p130 Cas . Five hundred micrograms of protein from 293T cells expressing the various CMS peptides or C3G used as a positive control was immunoprecipitated with the antibodies indicated above. Blots were probed for binding with 32 P-labeled GST-p130 Cas SH3 domain.

    Techniques Used: Sequencing, Clone Assay, Isolation, Two Hybrid Screening, Amplification, cDNA Library Assay, Binding Assay, Immunoprecipitation, In Vitro, Transfection, Plasmid Preparation, SDS Page, Expressing, Positive Control, Labeling

    12) Product Images from "Molecular cloning, gene structure and expression profile of two mouse peroxisomal 3-ketoacyl-CoA thiolase genes"

    Article Title: Molecular cloning, gene structure and expression profile of two mouse peroxisomal 3-ketoacyl-CoA thiolase genes

    Journal: BMC Biochemistry

    doi: 10.1186/1471-2091-5-3

    Nucleotide sequence and deduced aa sequence of the mThB cDNA and comparison with mThA cDNA. The complete nucleotide sequence for mThB cDNA and its deduced aa sequence are shown in full. Differences with mThA cDNA are indicated above the mThB sequence, whereas differences in the deduced aa sequence are indicated below. The positions of nucleotides and aa residues are given on the right. Nucleotide 1 (for each cDNA) corresponds to the major transcription initiation site determined by ribonuclease protection assay (figure 1c ). The start codon (ATG) is indicated in bold and underlined, and the stop codon (TGA) is in bold and denoted by End. The coding regions are written in capital letters, whereas the untranslated regions are in lower case letters. Dashes (-) correspond to the absence of 5' and 3'-untranslated extension in each cDNA. The 5'-extension of mThA cDNA (124 nt) appears in italics. The underlined sequences correspond to specific probes (used for Northern blot) of thiolase A and B cDNAs, respectively. The putative polyadenylation signal AGTAAA is double-framed in the two sequences. The putative Peroxisomal Target Signal (PTS 2) is framed and shaded. The three thiolase signature patterns are framed in dotted lanes. The putative catalytic residues (Cys123, His377 and Cys408) are framed. Arrows indicate the putative cleavage site for the mature thiolase proteins. The thiolase A and B cDNAs have been deposited in the GenBank database: accession nos AY273811 and AY273812, respectively.
    Figure Legend Snippet: Nucleotide sequence and deduced aa sequence of the mThB cDNA and comparison with mThA cDNA. The complete nucleotide sequence for mThB cDNA and its deduced aa sequence are shown in full. Differences with mThA cDNA are indicated above the mThB sequence, whereas differences in the deduced aa sequence are indicated below. The positions of nucleotides and aa residues are given on the right. Nucleotide 1 (for each cDNA) corresponds to the major transcription initiation site determined by ribonuclease protection assay (figure 1c ). The start codon (ATG) is indicated in bold and underlined, and the stop codon (TGA) is in bold and denoted by End. The coding regions are written in capital letters, whereas the untranslated regions are in lower case letters. Dashes (-) correspond to the absence of 5' and 3'-untranslated extension in each cDNA. The 5'-extension of mThA cDNA (124 nt) appears in italics. The underlined sequences correspond to specific probes (used for Northern blot) of thiolase A and B cDNAs, respectively. The putative polyadenylation signal AGTAAA is double-framed in the two sequences. The putative Peroxisomal Target Signal (PTS 2) is framed and shaded. The three thiolase signature patterns are framed in dotted lanes. The putative catalytic residues (Cys123, His377 and Cys408) are framed. Arrows indicate the putative cleavage site for the mature thiolase proteins. The thiolase A and B cDNAs have been deposited in the GenBank database: accession nos AY273811 and AY273812, respectively.

    Techniques Used: Sequencing, Northern Blot

    Cloning of the two mouse peroxisomal thiolase genes and determination of the transcription initiation sites of each gene . a) Southern blot of a 129SV mouse genomic DNA digested with different restriction enzymes. Membrane was hybridized with an exon 9 rat thiolase 32 P-labelled cDNA probe. A molecular weight marker DNA is indicated on the left in kb. b) Structural organizations of the mouse thiolase A and B genes: exon-intron distribution. Exons are numbered from I to XII. Translated sequences are shown as black boxes and untranslated sequences are open boxes. c) Identification of the transcription initiation sites of mouse thiolase A and B genes by ribonuclease protection assay. RNA probes corresponding to the genomic sequence extending upstream from the exon 1-intron 1 border (probes size: 678 nt and 521 nt for mThA and mThB, respectively) were hybridized to mouse liver RNA (lanes 1 and 6) and digested with RNAses. The protected fragments were analysed on a denaturing polyacrylamide gel (for details, see Methods). Lanes 2–5 and 7–10, DNA sequencing ladder from plasmids containing +1 mThA and +1 mThB probes, respectively. Arrows and dots indicate the different bands corresponding to multiple transcription initiation sites.
    Figure Legend Snippet: Cloning of the two mouse peroxisomal thiolase genes and determination of the transcription initiation sites of each gene . a) Southern blot of a 129SV mouse genomic DNA digested with different restriction enzymes. Membrane was hybridized with an exon 9 rat thiolase 32 P-labelled cDNA probe. A molecular weight marker DNA is indicated on the left in kb. b) Structural organizations of the mouse thiolase A and B genes: exon-intron distribution. Exons are numbered from I to XII. Translated sequences are shown as black boxes and untranslated sequences are open boxes. c) Identification of the transcription initiation sites of mouse thiolase A and B genes by ribonuclease protection assay. RNA probes corresponding to the genomic sequence extending upstream from the exon 1-intron 1 border (probes size: 678 nt and 521 nt for mThA and mThB, respectively) were hybridized to mouse liver RNA (lanes 1 and 6) and digested with RNAses. The protected fragments were analysed on a denaturing polyacrylamide gel (for details, see Methods). Lanes 2–5 and 7–10, DNA sequencing ladder from plasmids containing +1 mThA and +1 mThB probes, respectively. Arrows and dots indicate the different bands corresponding to multiple transcription initiation sites.

    Techniques Used: Clone Assay, Southern Blot, Molecular Weight, Marker, Sequencing, DNA Sequencing

    Mouse peroxisomal 3-ketoacyl-CoA thiolase A and B mRNA levels in different tissues and effect of fenofibrate on their expression in liver. a) Total RNAs (15 μg per lane from 3 mice) extracted from different tissues were hybridized with 32 P-labelled cDNA probes: upper panel, mouse peroxisomal 3-ketoacyl-CoA thiolase A (mThA); middle panel, mouse peroxisomal 3-ketoacyl-CoA thiolase B (mThB); lower panel, 36B4 (acidic ribosomal phosphoprotein (P0)) as loading control. Peroxisomal 3-ketoacyl-CoA thiolase A ( mThA ) or thiolase B ( mThB ) mRNA signal was quantified, standardized with 36B4 mRNA signal for the same tissue and expressed as a percentage in comparison with the signal observed in liver for each isoform. b) Relative proportions of thiolase A and B mRNAs in mouse liver by Slot blot analysis. 1, 2, 5 and 10 μg total RNAs extracted from mouse liver were deposed onto nitrocellulose membrane and then hybridized with 32 P-labelled cDNA probes: upper panel, mouse peroxisomal 3-ketoacyl-CoA thiolase A (mThA), and lower panel, mouse peroxisomal 3-ketoacyl-CoA thiolase B (mThB). The total signals of thiolase A and thiolase B were fixed to 100%. Relative proportions of thiolase A and B mRNAs were calculated and expressed as a percentage. c) Total RNAs (15 μg per lane from 3 independent animals for each condition) extracted from mouse liver of control and fenofibrate-treated mice were loaded. The membrane was hybridized with 32 P-labelled cDNA probes: upper panel, mouse peroxisomal 3-ketoacyl-CoA thiolase A (mThA); middle panel, mouse peroxisomal 3-ketoacyl-CoA thiolase B (mThB); lower panel, 36B4 (acidic ribosomal phosphoprotein (P0)) as loading control. Fold variation represents mThA (or mThB ) mRNA / 36B4 mRNA variation. These values were fixed to 1 for liver of untreated mice.
    Figure Legend Snippet: Mouse peroxisomal 3-ketoacyl-CoA thiolase A and B mRNA levels in different tissues and effect of fenofibrate on their expression in liver. a) Total RNAs (15 μg per lane from 3 mice) extracted from different tissues were hybridized with 32 P-labelled cDNA probes: upper panel, mouse peroxisomal 3-ketoacyl-CoA thiolase A (mThA); middle panel, mouse peroxisomal 3-ketoacyl-CoA thiolase B (mThB); lower panel, 36B4 (acidic ribosomal phosphoprotein (P0)) as loading control. Peroxisomal 3-ketoacyl-CoA thiolase A ( mThA ) or thiolase B ( mThB ) mRNA signal was quantified, standardized with 36B4 mRNA signal for the same tissue and expressed as a percentage in comparison with the signal observed in liver for each isoform. b) Relative proportions of thiolase A and B mRNAs in mouse liver by Slot blot analysis. 1, 2, 5 and 10 μg total RNAs extracted from mouse liver were deposed onto nitrocellulose membrane and then hybridized with 32 P-labelled cDNA probes: upper panel, mouse peroxisomal 3-ketoacyl-CoA thiolase A (mThA), and lower panel, mouse peroxisomal 3-ketoacyl-CoA thiolase B (mThB). The total signals of thiolase A and thiolase B were fixed to 100%. Relative proportions of thiolase A and B mRNAs were calculated and expressed as a percentage. c) Total RNAs (15 μg per lane from 3 independent animals for each condition) extracted from mouse liver of control and fenofibrate-treated mice were loaded. The membrane was hybridized with 32 P-labelled cDNA probes: upper panel, mouse peroxisomal 3-ketoacyl-CoA thiolase A (mThA); middle panel, mouse peroxisomal 3-ketoacyl-CoA thiolase B (mThB); lower panel, 36B4 (acidic ribosomal phosphoprotein (P0)) as loading control. Fold variation represents mThA (or mThB ) mRNA / 36B4 mRNA variation. These values were fixed to 1 for liver of untreated mice.

    Techniques Used: Expressing, Mouse Assay, Dot Blot

    13) Product Images from "The human formin-binding protein 17 (FBP17) interacts with sorting nexin, SNX2, and is an MLL-fusion partner in acute myelogeneous leukemia"

    Article Title: The human formin-binding protein 17 (FBP17) interacts with sorting nexin, SNX2, and is an MLL-fusion partner in acute myelogeneous leukemia

    Journal: Proceedings of the National Academy of Sciences of the United States of America

    doi: 10.1073/pnas.121433898

    ( A ) Schematic representation of the in-frame MLL / FBP17 gene fusion. In the predicted chimeric MLL/FBP17 fusion protein, the MLL zinc finger domain is disrupted and the MLL SET [Su(var) 3-9, Enhancer-of-zeste, Thritorax] domain is replaced by the FBP17 Src homology 3 domain. ( B ) Southern blot analysis revealed a rearrangement of the MLL gene in the index patient. Lane M, size standards; lane 1, the normal 8.3-kb MLL germ-line fragment (cell line HL60); lane 2, the rearranged MLL fragments of the leukemia sample. ( C ) Lane M, size marker VI (Roche Diagnostics). Lane 1, RT-PCR analysis with an MLL exon 5 sense primer and an FBP17 antisense primer detects a chimeric MLL/FBP17 transcript. The smaller faint PCR product is caused by alternative splicing of MLL exon 6. Lanes 2 and 3, negative controls. Lanes 4–6, sequence of the ABL gene amplified to ensure the integrity of the RNA from the patient and the two cell lines that were used for control (HL60 and THP1). ( D ) Western blot after transfection of 293T cells with chimeric MLL/FBP17 cDNA. Almost all MLL/FBP17 protein is localized into the nucleus (lane 3), whereas only a small amount is retained in the cytoplasm (lane 1). Lanes 2 and 4 are negative controls.
    Figure Legend Snippet: ( A ) Schematic representation of the in-frame MLL / FBP17 gene fusion. In the predicted chimeric MLL/FBP17 fusion protein, the MLL zinc finger domain is disrupted and the MLL SET [Su(var) 3-9, Enhancer-of-zeste, Thritorax] domain is replaced by the FBP17 Src homology 3 domain. ( B ) Southern blot analysis revealed a rearrangement of the MLL gene in the index patient. Lane M, size standards; lane 1, the normal 8.3-kb MLL germ-line fragment (cell line HL60); lane 2, the rearranged MLL fragments of the leukemia sample. ( C ) Lane M, size marker VI (Roche Diagnostics). Lane 1, RT-PCR analysis with an MLL exon 5 sense primer and an FBP17 antisense primer detects a chimeric MLL/FBP17 transcript. The smaller faint PCR product is caused by alternative splicing of MLL exon 6. Lanes 2 and 3, negative controls. Lanes 4–6, sequence of the ABL gene amplified to ensure the integrity of the RNA from the patient and the two cell lines that were used for control (HL60 and THP1). ( D ) Western blot after transfection of 293T cells with chimeric MLL/FBP17 cDNA. Almost all MLL/FBP17 protein is localized into the nucleus (lane 3), whereas only a small amount is retained in the cytoplasm (lane 1). Lanes 2 and 4 are negative controls.

    Techniques Used: Southern Blot, Marker, Reverse Transcription Polymerase Chain Reaction, Polymerase Chain Reaction, Sequencing, Amplification, Western Blot, Transfection

    14) Product Images from "A Rac Homolog Functions Downstream of Ras1 To Control Hyphal Differentiation and High-Temperature Growth in the Pathogenic Fungus Cryptococcus neoformans"

    Article Title: A Rac Homolog Functions Downstream of Ras1 To Control Hyphal Differentiation and High-Temperature Growth in the Pathogenic Fungus Cryptococcus neoformans

    Journal: Eukaryotic Cell

    doi: 10.1128/EC.4.6.1066-1078.2005

    Physical interaction of Rac1 and Ste20α assessed using the yeast two-hybrid system. (A) The cDNA sequences of the C. neoformans STE20α , RAC1 , CDC42 , and RAC1-G15V alleles were cloned into the two-hybrid vectors pGBT9 and pGAD424 and cotransformed
    Figure Legend Snippet: Physical interaction of Rac1 and Ste20α assessed using the yeast two-hybrid system. (A) The cDNA sequences of the C. neoformans STE20α , RAC1 , CDC42 , and RAC1-G15V alleles were cloned into the two-hybrid vectors pGBT9 and pGAD424 and cotransformed

    Techniques Used: Clone Assay

    15) Product Images from "characterization of the full length mRNA coding for Lucina pectinata HbIII revealed an alternative polyadenylation site"

    Article Title: characterization of the full length mRNA coding for Lucina pectinata HbIII revealed an alternative polyadenylation site

    Journal: Gene

    doi: 10.1016/j.gene.2007.12.005

    1% Agarose Gel Electrophoresis of Degenerative RT-PCR and PCR-RACE products The reverse transcriptase-polymerase chain reaction (RT-PCR and Rapid amplification of cDNA ends (RACE) methods were employed to synthesize the cDNA fragments as described in section 2.2. A) Degenerative RT-PCR product. A 342nt fragment was obtained. Lane 1: Marker, Lane 2: RT-PCR product. B) 5′-RACE Product. A 318 nt fragment was obtained. Lane 1 Marker, Lane 2 5′-RACE Product (C) 3′-RACE products. Two DNA fragments of 640 nt and 455nt were obtained. Lane 1 Marker, Lane 2 3′-RACE Product
    Figure Legend Snippet: 1% Agarose Gel Electrophoresis of Degenerative RT-PCR and PCR-RACE products The reverse transcriptase-polymerase chain reaction (RT-PCR and Rapid amplification of cDNA ends (RACE) methods were employed to synthesize the cDNA fragments as described in section 2.2. A) Degenerative RT-PCR product. A 342nt fragment was obtained. Lane 1: Marker, Lane 2: RT-PCR product. B) 5′-RACE Product. A 318 nt fragment was obtained. Lane 1 Marker, Lane 2 5′-RACE Product (C) 3′-RACE products. Two DNA fragments of 640 nt and 455nt were obtained. Lane 1 Marker, Lane 2 3′-RACE Product

    Techniques Used: Agarose Gel Electrophoresis, Reverse Transcription Polymerase Chain Reaction, Polymerase Chain Reaction, Rapid Amplification of cDNA Ends, Marker

    Complete nucleotide sequence for the HbIII cDNA The overlapping sequences of degenerate RT-PCR products with the 5′ RACE and 3′ RACE products were used to obtain the complete nucleotide sequence for the HbIII cDNA. Most of the HbIII cDNA sequence (97%) was confirmed by end to end RT-PCR. A. Schematic Organization of cDNA Fragments. An initial 342-nt cDNA clone encoding 114 amino acid residues of HbIII (amino acid 26 to 139) was amplified from total RNA using degenerate oligonucleotides. The 5′ RACE product contained a cDNA sequence encoding 43 amino acid residues of HbIII (amino acids 1 to 43) and 62 nt of the 5′ UTR. The 3′ RACE products contained a cDNA sequence encoding 71 amino acid residues of HbIII (amino acid 82 to 152) and two 3′UTR regions (168 and 355 nt long). The numbers at each end of the boxes identify the amino acid numbers of the HbIII aminoacid sequence included in the amplification product. Numbers in parentheses indicates the length in nucleotides of the cDNA regions indicated. Black boxes represent the coding regions and white boxes the UTRs. B . Schematic diagram of the HbIII cDNA. The untranslated region (5′UTR) is composed of 62 nt whereas two 3′UTR regions were isolated revealing the presence of alternative polyadenylation site. The shortest 3′UTR is composed of 168nt out of 689 nucleotides-long cDNA. The longest 3′UTR (355 nt long) is found in 876 nt-long cDNA. The PA indicates the poly-adenylation signal. Numbers in parentheses indicate the length in nucleotides of the cDNA regions indicated. C. Full length cDNA Sequence and Derived Amino Acid Sequence of Hemoglobin III from Lucina pectinata. A single amino acid difference (Asn72Asp) with the reported Lucina pectinata HbIII amino sequence is shown in bold. The two polyadenylation signals are underlined. The ▽ symbol represents the site where the poly (A)+ tail is added in the smaller cDNA. Primers used in RT-PCR, RACE and Primer Extension experiments are shown.
    Figure Legend Snippet: Complete nucleotide sequence for the HbIII cDNA The overlapping sequences of degenerate RT-PCR products with the 5′ RACE and 3′ RACE products were used to obtain the complete nucleotide sequence for the HbIII cDNA. Most of the HbIII cDNA sequence (97%) was confirmed by end to end RT-PCR. A. Schematic Organization of cDNA Fragments. An initial 342-nt cDNA clone encoding 114 amino acid residues of HbIII (amino acid 26 to 139) was amplified from total RNA using degenerate oligonucleotides. The 5′ RACE product contained a cDNA sequence encoding 43 amino acid residues of HbIII (amino acids 1 to 43) and 62 nt of the 5′ UTR. The 3′ RACE products contained a cDNA sequence encoding 71 amino acid residues of HbIII (amino acid 82 to 152) and two 3′UTR regions (168 and 355 nt long). The numbers at each end of the boxes identify the amino acid numbers of the HbIII aminoacid sequence included in the amplification product. Numbers in parentheses indicates the length in nucleotides of the cDNA regions indicated. Black boxes represent the coding regions and white boxes the UTRs. B . Schematic diagram of the HbIII cDNA. The untranslated region (5′UTR) is composed of 62 nt whereas two 3′UTR regions were isolated revealing the presence of alternative polyadenylation site. The shortest 3′UTR is composed of 168nt out of 689 nucleotides-long cDNA. The longest 3′UTR (355 nt long) is found in 876 nt-long cDNA. The PA indicates the poly-adenylation signal. Numbers in parentheses indicate the length in nucleotides of the cDNA regions indicated. C. Full length cDNA Sequence and Derived Amino Acid Sequence of Hemoglobin III from Lucina pectinata. A single amino acid difference (Asn72Asp) with the reported Lucina pectinata HbIII amino sequence is shown in bold. The two polyadenylation signals are underlined. The ▽ symbol represents the site where the poly (A)+ tail is added in the smaller cDNA. Primers used in RT-PCR, RACE and Primer Extension experiments are shown.

    Techniques Used: Sequencing, Reverse Transcription Polymerase Chain Reaction, Amplification, Isolation, Derivative Assay

    L pectinata HbIII mRNA tissue distribution A. RT-PCR for 28S rRNA . Internal loading control detected similar levels of starting RNA in each lanes B. RT-PCR products. The tissue distribution of the Lucina pectinata HbIII mRNA was determined using RT-PCR using the HbIIImRNAF1 and HbIIImRNAR2 primers to amplify a 673 bp cDNA fragment. Lane L: 123 bp DNA ladder, Lane 1: mantle, Lane 2: adductor muscle, Lane 3: foot, Lane 4: pericardial sac, Lane 5: ctenidia, Lane 6: gonad and intestine, Lane C+: Positive Control (ctenidia RNA), Lane 7: digestive gland, Lane M: Mock (human mRNA) C. 1% Agarose Gel Electrophoresis of 3′ PCR-RACE products. RACE reactions were carried out as described in section 2.2. Lane L:1 kb bp DNA Ladder, Lane C+: Positive control, Lane C-: Negative control (no RNA), Lane 1: mantle, Lane 2: adductor muscle, Lane 3: foot, Lane 4: ctenidia
    Figure Legend Snippet: L pectinata HbIII mRNA tissue distribution A. RT-PCR for 28S rRNA . Internal loading control detected similar levels of starting RNA in each lanes B. RT-PCR products. The tissue distribution of the Lucina pectinata HbIII mRNA was determined using RT-PCR using the HbIIImRNAF1 and HbIIImRNAR2 primers to amplify a 673 bp cDNA fragment. Lane L: 123 bp DNA ladder, Lane 1: mantle, Lane 2: adductor muscle, Lane 3: foot, Lane 4: pericardial sac, Lane 5: ctenidia, Lane 6: gonad and intestine, Lane C+: Positive Control (ctenidia RNA), Lane 7: digestive gland, Lane M: Mock (human mRNA) C. 1% Agarose Gel Electrophoresis of 3′ PCR-RACE products. RACE reactions were carried out as described in section 2.2. Lane L:1 kb bp DNA Ladder, Lane C+: Positive control, Lane C-: Negative control (no RNA), Lane 1: mantle, Lane 2: adductor muscle, Lane 3: foot, Lane 4: ctenidia

    Techniques Used: Reverse Transcription Polymerase Chain Reaction, Positive Control, Agarose Gel Electrophoresis, Polymerase Chain Reaction, Negative Control

    16) Product Images from "Renal agenesis in mice homozygous for a gene trap mutation in the gene encoding heparan sulfate 2-sulfotransferase"

    Article Title: Renal agenesis in mice homozygous for a gene trap mutation in the gene encoding heparan sulfate 2-sulfotransferase

    Journal: Genes & Development

    doi:

    Hs2st transcripts are disrupted by the gene trap insertion. ( A ) Schematic of mouse Hs2st cDNA showing position of gene trap vector insertion site. The shaded box represents a 1068-bp ORF; the integration site corresponds to position 588 (start of translation = position 1). (SA) Splice acceptor; (TM) transmembrane-encoding region. ( B ) Northern hybridization of 10 μg of total RNA isolated from a wild-type (+/+), a heterozygote (+/−), and a homozygous mutant (−/−) 15.5-d.p.c. embryo using a probe specific to the ST125 5′ RACE product. The weak 4-kb signal (asterisk) detected in the wild-type RNA may represent an endogenous unprocessed Hs2st RNA or splice variant that is disrupted in the mutants and is represented too poorly in a heterozygote RNA population to be visualized. β-Actin was used as a loading control.
    Figure Legend Snippet: Hs2st transcripts are disrupted by the gene trap insertion. ( A ) Schematic of mouse Hs2st cDNA showing position of gene trap vector insertion site. The shaded box represents a 1068-bp ORF; the integration site corresponds to position 588 (start of translation = position 1). (SA) Splice acceptor; (TM) transmembrane-encoding region. ( B ) Northern hybridization of 10 μg of total RNA isolated from a wild-type (+/+), a heterozygote (+/−), and a homozygous mutant (−/−) 15.5-d.p.c. embryo using a probe specific to the ST125 5′ RACE product. The weak 4-kb signal (asterisk) detected in the wild-type RNA may represent an endogenous unprocessed Hs2st RNA or splice variant that is disrupted in the mutants and is represented too poorly in a heterozygote RNA population to be visualized. β-Actin was used as a loading control.

    Techniques Used: Plasmid Preparation, Northern Blot, Hybridization, Isolation, Mutagenesis, Variant Assay

    17) Product Images from "A Medicago truncatula Homoglutathione Synthetase Is Derived from Glutathione Synthetase by Gene Duplication 1"

    Article Title: A Medicago truncatula Homoglutathione Synthetase Is Derived from Glutathione Synthetase by Gene Duplication 1

    Journal: Plant Physiology

    doi:

    Genomic southern analysis of M. truncatula gshs1 and gshs2 . Total genomic M. truncatula DNA was digested with Eco RI, Eco RV, and Bam HI. The filters were probed with gshs1 cDNA or gshs2 cDNA as described in the experimental procedures. Size markers are indicated on the left.
    Figure Legend Snippet: Genomic southern analysis of M. truncatula gshs1 and gshs2 . Total genomic M. truncatula DNA was digested with Eco RI, Eco RV, and Bam HI. The filters were probed with gshs1 cDNA or gshs2 cDNA as described in the experimental procedures. Size markers are indicated on the left.

    Techniques Used:

    18) Product Images from "CCAAT/enhancer binding protein ? is preferentially up-regulated during granulocytic differentiation and its functional versatility is determined by alternative use of promoters and differential splicing"

    Article Title: CCAAT/enhancer binding protein ? is preferentially up-regulated during granulocytic differentiation and its functional versatility is determined by alternative use of promoters and differential splicing

    Journal: Proceedings of the National Academy of Sciences of the United States of America

    doi:

    Sequence of the two putative human C/EBPɛ promoter regions. The Pβ region is the major promoter used in HL60 cells, because the majority of the clones ( > 60%) identified by RACE–PCR were initiated at this site. The transcriptional start sites of the C/EBPɛ gene were determined by 5′-RACE–PCR and sequence analysis of several independent clones. Large arrows indicate sequence ends of the majority of cDNA clones for each promoter region. Small arrows show ends of less frequent RACE–PCR clones. The translation initiation codon is shown in uppercase and bold characters. Purine stretches are boxed.
    Figure Legend Snippet: Sequence of the two putative human C/EBPɛ promoter regions. The Pβ region is the major promoter used in HL60 cells, because the majority of the clones ( > 60%) identified by RACE–PCR were initiated at this site. The transcriptional start sites of the C/EBPɛ gene were determined by 5′-RACE–PCR and sequence analysis of several independent clones. Large arrows indicate sequence ends of the majority of cDNA clones for each promoter region. Small arrows show ends of less frequent RACE–PCR clones. The translation initiation codon is shown in uppercase and bold characters. Purine stretches are boxed.

    Techniques Used: Sequencing, Clone Assay, Polymerase Chain Reaction

    19) Product Images from "Sequence and Expression Characteristics of Long Noncoding RNAs in Honey Bee Caste Development - Potential Novel Regulators for Transgressive Ovary Size"

    Article Title: Sequence and Expression Characteristics of Long Noncoding RNAs in Honey Bee Caste Development - Potential Novel Regulators for Transgressive Ovary Size

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0078915

    Full length cDNAs of honey bee lncov1 and lncov2 and their genomic mapping. (A) Southern Blot showing probe hybridizations with full-length cDNAs of lncov1 and lncov2 produced by 3′5′RACE reactions. The lncov1 probe labelled a cDNA of 1367 bp, the one for lncov2 hybridized to a 684 bp transcript. (B) Full length mRNA sequence of lncov1 . Coverage with the genome sequence is shown on grey background, the tandem repeat sequence lacking in the genome is shown on white background. (C) Genomic mapping of lncov1 ; lncov1 (arrow) is located in the sense strand to the fifth intron of the predicted protein LOC726407. (D) lncov2 (arrow) maps into the fourth intron of fringe , also in sense direction.
    Figure Legend Snippet: Full length cDNAs of honey bee lncov1 and lncov2 and their genomic mapping. (A) Southern Blot showing probe hybridizations with full-length cDNAs of lncov1 and lncov2 produced by 3′5′RACE reactions. The lncov1 probe labelled a cDNA of 1367 bp, the one for lncov2 hybridized to a 684 bp transcript. (B) Full length mRNA sequence of lncov1 . Coverage with the genome sequence is shown on grey background, the tandem repeat sequence lacking in the genome is shown on white background. (C) Genomic mapping of lncov1 ; lncov1 (arrow) is located in the sense strand to the fifth intron of the predicted protein LOC726407. (D) lncov2 (arrow) maps into the fourth intron of fringe , also in sense direction.

    Techniques Used: Southern Blot, Produced, Sequencing

    20) Product Images from "A Mutation of the Mitochondrial ABC Transporter Sta1 Leads to Dwarfism and Chlorosis in the Arabidopsis Mutant starik"

    Article Title: A Mutation of the Mitochondrial ABC Transporter Sta1 Leads to Dwarfism and Chlorosis in the Arabidopsis Mutant starik

    Journal: The Plant Cell

    doi:

    Effects of STA1 Expression on the Growth of Δatm1 Yeast. (A) Cells grown on agar plates with minimal medium containing glucose for 3 days at 30°C. The full-length sta1 cDNA or the yeast ATM1 ). Δatm1 cells were transformed with the plasmids or the vector without DNA insert. To apply selective pressure for maintenance of the vectors in yeast cells, minimal medium was depleted of either tryptophan (-Trp) or uracil (-Ura). (B) Cells grown on YPD medium (1% yeast extract, 2% bactopeptone, 2% glucose, and 1.5% agar) under the same conditions as given for (A) .
    Figure Legend Snippet: Effects of STA1 Expression on the Growth of Δatm1 Yeast. (A) Cells grown on agar plates with minimal medium containing glucose for 3 days at 30°C. The full-length sta1 cDNA or the yeast ATM1 ). Δatm1 cells were transformed with the plasmids or the vector without DNA insert. To apply selective pressure for maintenance of the vectors in yeast cells, minimal medium was depleted of either tryptophan (-Trp) or uracil (-Ura). (B) Cells grown on YPD medium (1% yeast extract, 2% bactopeptone, 2% glucose, and 1.5% agar) under the same conditions as given for (A) .

    Techniques Used: Expressing, Transformation Assay, Plasmid Preparation

    21) Product Images from "A tumor host range selection procedure identifies p150sal2 as a target of polyoma virus large T antigen"

    Article Title: A tumor host range selection procedure identifies p150sal2 as a target of polyoma virus large T antigen

    Journal: Proceedings of the National Academy of Sciences of the United States of America

    doi: 10.1073/pnas.251447198

    The t-hr mutant has an altered large T C terminus that prevents interaction with host factor mSal2. ( A ) Sequencing of the original mutant shows a 20-bp duplication of coding sequences at the large T C terminus causing a reading frame shift shown in red. ( B ) The coding region of mSal2 cDNA is shown schematically in blue with the zinc fingers in orange. Overlapping clones from the C-terminal region of mSal2 interact with the wild-type large T C-terminal fragment (amino acids 333–781) but not with that of the mutant in yeast two-hybrid assays. +, growth on His − plates; His+ colonies were also lac Z positive (not shown). ( C ) Deletion analysis of the wild-type large T C terminus indicating that a minimal deletion of amino acids 774–776 abolishes the binding of mSal2 in yeast.
    Figure Legend Snippet: The t-hr mutant has an altered large T C terminus that prevents interaction with host factor mSal2. ( A ) Sequencing of the original mutant shows a 20-bp duplication of coding sequences at the large T C terminus causing a reading frame shift shown in red. ( B ) The coding region of mSal2 cDNA is shown schematically in blue with the zinc fingers in orange. Overlapping clones from the C-terminal region of mSal2 interact with the wild-type large T C-terminal fragment (amino acids 333–781) but not with that of the mutant in yeast two-hybrid assays. +, growth on His − plates; His+ colonies were also lac Z positive (not shown). ( C ) Deletion analysis of the wild-type large T C terminus indicating that a minimal deletion of amino acids 774–776 abolishes the binding of mSal2 in yeast.

    Techniques Used: Mutagenesis, Sequencing, Zinc-Fingers, Clone Assay, Binding Assay

    Interaction of wild-type but not mutant large T with mSal2. ( A ) In vitro binding. GST alone and GST-fusion protein containing the large T binding domain of mSal2 (amino acids 839–969) are used to pull down large T from cell extracts. The filter is blotted with F4 anti-T monoclonal antibody. *, crossreactive cellular band. Lane a, input extract from wild-type infected BMK cells; lane b, wild-type extract from lane a pulled down with GST alone; lane c, wild-type extract from lane a pulled down with GST-mSal2 fusion protein; lane d, input extract from 3T3 transfected with wild-type large T cDNA; lane e, input extract from 3T3 cells transfected with TMD-25 large T cDNA; lane f, wild-type extract from lane d pulled down with GST alone; lane g, wild-type extract from lane d pulled down with GST-mSal2 fusion protein; lane h, mutant extract from lane e pulled down with GST-mSal2 fusion protein. ( B ) In vivo binding. ( Left ) 3T3 cells were cotransfected with full-length GST-mSal2 fusion construct and either wild-type or TMD 25 large T. Following GST pull-down, blots were developed with anti-T antibody ( Upper ) and with monoclonal anti-mSal2 ( Lower ) to show similar amount of GST-mSal2 was present in both pull-down lanes. Input lanes contain 3% of the extract used for pull-down. ( Right ) A 24-h wild-type virus-infected BMK cell extract was immunoprecipitated with normal rabbit IgG or purified rabbit polyclonal antibody against the N terminus of mSal2. The blot was probed with anti-T monoclonal antibody and reprobed with anti-mSal2 monoclonal antibody.
    Figure Legend Snippet: Interaction of wild-type but not mutant large T with mSal2. ( A ) In vitro binding. GST alone and GST-fusion protein containing the large T binding domain of mSal2 (amino acids 839–969) are used to pull down large T from cell extracts. The filter is blotted with F4 anti-T monoclonal antibody. *, crossreactive cellular band. Lane a, input extract from wild-type infected BMK cells; lane b, wild-type extract from lane a pulled down with GST alone; lane c, wild-type extract from lane a pulled down with GST-mSal2 fusion protein; lane d, input extract from 3T3 transfected with wild-type large T cDNA; lane e, input extract from 3T3 cells transfected with TMD-25 large T cDNA; lane f, wild-type extract from lane d pulled down with GST alone; lane g, wild-type extract from lane d pulled down with GST-mSal2 fusion protein; lane h, mutant extract from lane e pulled down with GST-mSal2 fusion protein. ( B ) In vivo binding. ( Left ) 3T3 cells were cotransfected with full-length GST-mSal2 fusion construct and either wild-type or TMD 25 large T. Following GST pull-down, blots were developed with anti-T antibody ( Upper ) and with monoclonal anti-mSal2 ( Lower ) to show similar amount of GST-mSal2 was present in both pull-down lanes. Input lanes contain 3% of the extract used for pull-down. ( Right ) A 24-h wild-type virus-infected BMK cell extract was immunoprecipitated with normal rabbit IgG or purified rabbit polyclonal antibody against the N terminus of mSal2. The blot was probed with anti-T monoclonal antibody and reprobed with anti-mSal2 monoclonal antibody.

    Techniques Used: Mutagenesis, In Vitro, Binding Assay, Infection, Transfection, In Vivo, Construct, Immunoprecipitation, Purification

    22) Product Images from "Auxin Influx Activity Is Associated with Frankia Infection during Actinorhizal Nodule Formation in Casuarina glauca 1 1 [C] 1 [C] [W] 1 [C] [W] [OA]"

    Article Title: Auxin Influx Activity Is Associated with Frankia Infection during Actinorhizal Nodule Formation in Casuarina glauca 1 1 [C] 1 [C] [W] 1 [C] [W] [OA]

    Journal: Plant Physiology

    doi: 10.1104/pp.107.101337

    CgAUX1 and CgLAX3 are expressed in Casuarina root, shoot, and nodule. Nonquantitative RT-PCR analysis in mature nodule, shoot, and root using tubulin ( CgTUB ) as a control. A control without cDNA and a genomic DNA control were also included. The extra
    Figure Legend Snippet: CgAUX1 and CgLAX3 are expressed in Casuarina root, shoot, and nodule. Nonquantitative RT-PCR analysis in mature nodule, shoot, and root using tubulin ( CgTUB ) as a control. A control without cDNA and a genomic DNA control were also included. The extra

    Techniques Used: Reverse Transcription Polymerase Chain Reaction

    23) Product Images from "Protein purification and cloning of diacylglycerol lipase from rat brain"

    Article Title: Protein purification and cloning of diacylglycerol lipase from rat brain

    Journal: Journal of Biochemistry

    doi: 10.1093/jb/mvw002

    Nucleic acid sequence of rat DDHD2 cDNA. The arrow indicates the nucleotides for 5′-RACE. The region of the antigen peptide used for the generation of the anti-DDHD2 antibody is enclosed by a square. The lipase motif is also enclosed by a square.
    Figure Legend Snippet: Nucleic acid sequence of rat DDHD2 cDNA. The arrow indicates the nucleotides for 5′-RACE. The region of the antigen peptide used for the generation of the anti-DDHD2 antibody is enclosed by a square. The lipase motif is also enclosed by a square.

    Techniques Used: Sequencing

    24) Product Images from "The human GRAF gene is fused to MLL in a unique t(5;11)(q31;q23) and both alleles are disrupted in three cases of myelodysplastic syndrome/acute myeloid leukemia with a deletion 5q"

    Article Title: The human GRAF gene is fused to MLL in a unique t(5;11)(q31;q23) and both alleles are disrupted in three cases of myelodysplastic syndrome/acute myeloid leukemia with a deletion 5q

    Journal: Proceedings of the National Academy of Sciences of the United States of America

    doi:

    ( A ) Results of RT-PCR analyses. Lane M contains a size marker VI (Boehringer Mannheim). Lanes 1, 4, 7, and 12 are negative controls in which the cDNA was replaced by sterile water. Normal fragments are obtained from the patient's unaffected GRAF allele (lane 2) and from the cell line Mono-Mac6 (lane 3). A MLL / GRAF fusion mRNA is detected in the sample with a t(5;11)(q31;q23) (lane 5) but not in the cell line lacking this translocation (lane 6). Normal fragments are obtained from the patient's unaffected MLL allele (lane 8) and the cell line Mono-Mac6 (lane 9). The additional fragments in lanes 5, 8, and 9 are generated by MLL splice variants. Further analysis reveals that the reciprocal GRAF / MLL fragment is neither present in the patient's sample (lane 10) nor in the cell line (lane 11). Control amplifications with primers specific for the ABL gene are shown in lanes 15 (patient sample) and 16 (cell line). ( B ) Long-range PCR results of genomic DNA. Lanes M contain the size markers III and VI (Boehringer Mannheim). Lane 1 is a negative control. A MLL / GRAF fusion product is detected in the patient with the t(5;11)(q31;23) (lane 2) but not in the control cell line Mono-Mac6 (lane 3). Lanes 4, 5, 9, and 17 are negative controls. A normal 8-kb fragment that covers the breakpoint cluster region of the unaffected MLL alleles in the patient with the t(5;11)(q31;q23) (lane 6), in a healthy individual (lane 7), and in the Mono-Mac cell line (lane 8) is seen. No reciprocal GRAF / MLL gene fragment is detected in any of these samples (lanes 10–13), whereas in all of them an approximately 13-kb long intron of GRAF becomes evident (lanes 14–16). ( C ) Sequence and schematic representation of the inverted duplication of MLL within the genomic MLL / GRAF fusion. Numbering of nucleotides within the breakpoint region of MLL . The horizontal arrows indicate the positions of the primers used for amplification of the genomic MLL / GRAF fusion seen in lane 2 of B .
    Figure Legend Snippet: ( A ) Results of RT-PCR analyses. Lane M contains a size marker VI (Boehringer Mannheim). Lanes 1, 4, 7, and 12 are negative controls in which the cDNA was replaced by sterile water. Normal fragments are obtained from the patient's unaffected GRAF allele (lane 2) and from the cell line Mono-Mac6 (lane 3). A MLL / GRAF fusion mRNA is detected in the sample with a t(5;11)(q31;q23) (lane 5) but not in the cell line lacking this translocation (lane 6). Normal fragments are obtained from the patient's unaffected MLL allele (lane 8) and the cell line Mono-Mac6 (lane 9). The additional fragments in lanes 5, 8, and 9 are generated by MLL splice variants. Further analysis reveals that the reciprocal GRAF / MLL fragment is neither present in the patient's sample (lane 10) nor in the cell line (lane 11). Control amplifications with primers specific for the ABL gene are shown in lanes 15 (patient sample) and 16 (cell line). ( B ) Long-range PCR results of genomic DNA. Lanes M contain the size markers III and VI (Boehringer Mannheim). Lane 1 is a negative control. A MLL / GRAF fusion product is detected in the patient with the t(5;11)(q31;23) (lane 2) but not in the control cell line Mono-Mac6 (lane 3). Lanes 4, 5, 9, and 17 are negative controls. A normal 8-kb fragment that covers the breakpoint cluster region of the unaffected MLL alleles in the patient with the t(5;11)(q31;q23) (lane 6), in a healthy individual (lane 7), and in the Mono-Mac cell line (lane 8) is seen. No reciprocal GRAF / MLL gene fragment is detected in any of these samples (lanes 10–13), whereas in all of them an approximately 13-kb long intron of GRAF becomes evident (lanes 14–16). ( C ) Sequence and schematic representation of the inverted duplication of MLL within the genomic MLL / GRAF fusion. Numbering of nucleotides within the breakpoint region of MLL . The horizontal arrows indicate the positions of the primers used for amplification of the genomic MLL / GRAF fusion seen in lane 2 of B .

    Techniques Used: Reverse Transcription Polymerase Chain Reaction, Marker, Translocation Assay, Generated, Polymerase Chain Reaction, Negative Control, Sequencing, Amplification

    ( A ) Insertion of 52-bp (capital letters) derived from intron 13 into the final cDNA found in patient #7. The surrounding intronic sequences are shown in lowercase letters. This leads to a reading frame shift followed by a premature stop codon. The GAP domain of Graf is substantially shortened. The intronic regions that were sequenced in patient #7 and 12 healthy controls are indicated by arrows. The splice branch site consensus sequence is shown as follows: Y represents T or C, R either A or G. ( B ) Schematic representation of both cDNA fragments that were coamplified by universal primers I and IV for assessment of their relative amount. Primers II and III amplify the aberrantly spliced fragment only. ( C ) Nested PCR analysis using the first-round primers I and IV and the second-round primers II and III. M, molecular weight marker. Lane 1, negative control. Four of 15 healthy blood donors expressed the aberrantly spliced fragment in their mononuclear cells (lanes 2 and 4–6) because a faint PCR product was seen. Lane 12, positive control. ( D ) Single-round PCR analysis using primers I and IV. Lane 6, two differently sized PCR products are seen from the cDNA of patient #7 even after only one round of PCR. Positive plasmid controls containing the 52-bp insertion (lane 12) or not (lane 13). In each RT-PCR, 2 μg of total RNA was subjected to cDNA synthesis and processed in parallel.
    Figure Legend Snippet: ( A ) Insertion of 52-bp (capital letters) derived from intron 13 into the final cDNA found in patient #7. The surrounding intronic sequences are shown in lowercase letters. This leads to a reading frame shift followed by a premature stop codon. The GAP domain of Graf is substantially shortened. The intronic regions that were sequenced in patient #7 and 12 healthy controls are indicated by arrows. The splice branch site consensus sequence is shown as follows: Y represents T or C, R either A or G. ( B ) Schematic representation of both cDNA fragments that were coamplified by universal primers I and IV for assessment of their relative amount. Primers II and III amplify the aberrantly spliced fragment only. ( C ) Nested PCR analysis using the first-round primers I and IV and the second-round primers II and III. M, molecular weight marker. Lane 1, negative control. Four of 15 healthy blood donors expressed the aberrantly spliced fragment in their mononuclear cells (lanes 2 and 4–6) because a faint PCR product was seen. Lane 12, positive control. ( D ) Single-round PCR analysis using primers I and IV. Lane 6, two differently sized PCR products are seen from the cDNA of patient #7 even after only one round of PCR. Positive plasmid controls containing the 52-bp insertion (lane 12) or not (lane 13). In each RT-PCR, 2 μg of total RNA was subjected to cDNA synthesis and processed in parallel.

    Techniques Used: Derivative Assay, Sequencing, Nested PCR, Molecular Weight, Marker, Negative Control, Polymerase Chain Reaction, Positive Control, Plasmid Preparation, Reverse Transcription Polymerase Chain Reaction

    25) Product Images from "A Single Amino Acid Change in the Hemagglutinin Protein of Measles Virus Determines Its Ability To Bind CD46 and Reveals Another Receptor on Marmoset B Cells"

    Article Title: A Single Amino Acid Change in the Hemagglutinin Protein of Measles Virus Determines Its Ability To Bind CD46 and Reveals Another Receptor on Marmoset B Cells

    Journal: Journal of Virology

    doi:

    Growth of the Edmonston strain of measles virus in New World monkey cell lines is impaired when SCR1 is deleted. (A) Human cervical carcinoma (HeLa), African green monkey kidney (Vero), owl monkey kidney (OMK), marmoset kidney (NZP-60), marmoset B (B95-8), and squirrel monkey lung (SML) cells were infected with the Edmonston strain of measles virus which had previously been adapted for growth in Vero cells. The cells were inoculated with 5 PFU of virus per cell, and infections were allowed to proceed for 72 h, after which the infected cells were subjected to immunoblot analysis with monoclonal antibodies to measles virus H protein. Viral protein synthesis was not observed in the OMK and NZP-60 cell lines, but measles virus H protein was detected in B95-8 and SML cells. (B) FACScan analysis was performed on B95-8, OMK, SML, and NZP-60 cells with an antibody to the SCR1 domain of the moustached tamarin ( Saguinus mystax ) and detected with goat anti-rabbit antibodies which had been conjugated to fluorescein isothiocyanate (solid line). The cells were also tested with rabbit preimmune antisera (dotted line). Shifts in fluorescence were observed in B95-8 and SML cells but not in OMK and NZP-60 cells. (C) mRNA was extracted from B95-8 and SML cells, cDNA was prepared, and PCR products spanning the signal peptide, SCR1, SCR2, and SCR3 domains were prepared and sequenced. The predicted amino acid sequence is shown and was derived from three independent amplification reactions for each sequence. Both deleted and nondeleted forms of mRNA were present in the two cell lines.
    Figure Legend Snippet: Growth of the Edmonston strain of measles virus in New World monkey cell lines is impaired when SCR1 is deleted. (A) Human cervical carcinoma (HeLa), African green monkey kidney (Vero), owl monkey kidney (OMK), marmoset kidney (NZP-60), marmoset B (B95-8), and squirrel monkey lung (SML) cells were infected with the Edmonston strain of measles virus which had previously been adapted for growth in Vero cells. The cells were inoculated with 5 PFU of virus per cell, and infections were allowed to proceed for 72 h, after which the infected cells were subjected to immunoblot analysis with monoclonal antibodies to measles virus H protein. Viral protein synthesis was not observed in the OMK and NZP-60 cell lines, but measles virus H protein was detected in B95-8 and SML cells. (B) FACScan analysis was performed on B95-8, OMK, SML, and NZP-60 cells with an antibody to the SCR1 domain of the moustached tamarin ( Saguinus mystax ) and detected with goat anti-rabbit antibodies which had been conjugated to fluorescein isothiocyanate (solid line). The cells were also tested with rabbit preimmune antisera (dotted line). Shifts in fluorescence were observed in B95-8 and SML cells but not in OMK and NZP-60 cells. (C) mRNA was extracted from B95-8 and SML cells, cDNA was prepared, and PCR products spanning the signal peptide, SCR1, SCR2, and SCR3 domains were prepared and sequenced. The predicted amino acid sequence is shown and was derived from three independent amplification reactions for each sequence. Both deleted and nondeleted forms of mRNA were present in the two cell lines.

    Techniques Used: Infection, Fluorescence, Polymerase Chain Reaction, Sequencing, Derivative Assay, Amplification

    26) Product Images from "Identification of mRNAs differentially-expressed between benign and malignant breast tumour cells"

    Article Title: Identification of mRNAs differentially-expressed between benign and malignant breast tumour cells

    Journal: British Journal of Cancer

    doi: 10.1038/sj.bjc.6600456

    Alignment of the M41 gene and M41 transcripts. The gene region of chromosome 21q22.1 is shown at the top with the exons of the DS-CAM gene shown as vertical bars. The location of the M41 gene is shown as a square to scale beneath the genomic DNA and expanded beneath. The location of response elements for oestrogen (ERE) and progesterone (PRE) and Alu sequences are shown. The region of the original M41 cDNA isolated from the MCF-7 subtracted library, and processing variants of M41 mRNA (A1–A7) obtained from the RACE reactions are shown as horizontal bars with gaps indicating the intronic regions. Numbering refers to the base number of contig NT_011512.3. The 5′ intron exon boundaries revealed by the RACE reactions at 27328651, 27328698, 27328827, 27329381, and the 3′ intron exon boundaries at 27328749, 27328894, 27329821 (enumeration of contig NT_011512.3) all contained conserved GT / AG sequences and exhibited 62.5, 75, 87.5, 100, 75, 93.75, and 81.25% identity, respectively, to the consensus sequences for 5′ or 3′ intron exon boundaries ( Mount, 1982 ). The symbols (A) at the ends of the lines indicate the poly(A) addition sites. Variants contained one of two alternative poly(A) addition sites (at 27330601 and 27330228 of NT_011512.3), with a consensus AATAAA poly(A)-addition signal, 29 and 19 nucleotides upstream of the poly(A)-addition sites, respectively. The lengths of the proposed mRNA variants arising from the 7 alternatively-spliced exons correspond broadly to the sizes of two of the bands observed in the Northern blot for the mammary tumour cell line MCF-7 ( Figure 1 ), strongly suggesting that the RACE products are defining near full length mRNAs. Horizontal arrows indicate the direction of transcription of the genes.
    Figure Legend Snippet: Alignment of the M41 gene and M41 transcripts. The gene region of chromosome 21q22.1 is shown at the top with the exons of the DS-CAM gene shown as vertical bars. The location of the M41 gene is shown as a square to scale beneath the genomic DNA and expanded beneath. The location of response elements for oestrogen (ERE) and progesterone (PRE) and Alu sequences are shown. The region of the original M41 cDNA isolated from the MCF-7 subtracted library, and processing variants of M41 mRNA (A1–A7) obtained from the RACE reactions are shown as horizontal bars with gaps indicating the intronic regions. Numbering refers to the base number of contig NT_011512.3. The 5′ intron exon boundaries revealed by the RACE reactions at 27328651, 27328698, 27328827, 27329381, and the 3′ intron exon boundaries at 27328749, 27328894, 27329821 (enumeration of contig NT_011512.3) all contained conserved GT / AG sequences and exhibited 62.5, 75, 87.5, 100, 75, 93.75, and 81.25% identity, respectively, to the consensus sequences for 5′ or 3′ intron exon boundaries ( Mount, 1982 ). The symbols (A) at the ends of the lines indicate the poly(A) addition sites. Variants contained one of two alternative poly(A) addition sites (at 27330601 and 27330228 of NT_011512.3), with a consensus AATAAA poly(A)-addition signal, 29 and 19 nucleotides upstream of the poly(A)-addition sites, respectively. The lengths of the proposed mRNA variants arising from the 7 alternatively-spliced exons correspond broadly to the sizes of two of the bands observed in the Northern blot for the mammary tumour cell line MCF-7 ( Figure 1 ), strongly suggesting that the RACE products are defining near full length mRNAs. Horizontal arrows indicate the direction of transcription of the genes.

    Techniques Used: Chick Chorioallantoic Membrane Assay, Isolation, Northern Blot

    27) Product Images from "Characterization of Transcripts Expressed from Human Herpesvirus 6A Strain GS Immediate-Early Region B U16-U17 Open Reading Frames"

    Article Title: Characterization of Transcripts Expressed from Human Herpesvirus 6A Strain GS Immediate-Early Region B U16-U17 Open Reading Frames

    Journal: Journal of Virology

    doi:

    ). The locations of the 22.2-kb Bam HI genomic fragment pZVB70 and the 3.8-kb Sal ) are indicated, as are the seven TATA sequences and the two potential polyadenylation signal sequences P. (B and C) Relationships of the ORFs identified in HHV-6A(U1102) (B) and HHV-6A(GS) (C) to comparable regions, as well as their designations, are shown. The deduced ORFs A to C in the 3.8-kb DNA sequence are in the leftward reading frame (arrow). The first methionine codon in the HHV-6A(GS) ORF B, located at nucleotide 1377, and the location of the transactivating B701 ORF, used to screen the cDNA library, are shown in the hatched box. (D) Schematic representation of the virus-specific portion (E1E2) of the 1.9-kb cDNA and the included ORF. (E) Schematic representation of the 1.8-kb cDNA and the deduced ORFs. ∧, intron locations. The polyadenylation signal used in each transcript is indicated by AAAAA. The arrow indicates the 5′-to-3′ orientation of the cDNA ORFs; the nucleotide positions are numbered from right to left. The small hatched box indicates a 96-aa ORF.
    Figure Legend Snippet: ). The locations of the 22.2-kb Bam HI genomic fragment pZVB70 and the 3.8-kb Sal ) are indicated, as are the seven TATA sequences and the two potential polyadenylation signal sequences P. (B and C) Relationships of the ORFs identified in HHV-6A(U1102) (B) and HHV-6A(GS) (C) to comparable regions, as well as their designations, are shown. The deduced ORFs A to C in the 3.8-kb DNA sequence are in the leftward reading frame (arrow). The first methionine codon in the HHV-6A(GS) ORF B, located at nucleotide 1377, and the location of the transactivating B701 ORF, used to screen the cDNA library, are shown in the hatched box. (D) Schematic representation of the virus-specific portion (E1E2) of the 1.9-kb cDNA and the included ORF. (E) Schematic representation of the 1.8-kb cDNA and the deduced ORFs. ∧, intron locations. The polyadenylation signal used in each transcript is indicated by AAAAA. The arrow indicates the 5′-to-3′ orientation of the cDNA ORFs; the nucleotide positions are numbered from right to left. The small hatched box indicates a 96-aa ORF.

    Techniques Used: Sequencing, cDNA Library Assay

    (A) Reactivity of monoclonal antibody against the B701 (U16) protein. Lane 1, reactivities of antibodies in the Western blot reaction with the B701 protein expressed in the pET-3b vector. The 20K protein and the higher-molecular-weight dimeric and/or multimeric forms of B701 protein recognized are indicated by the arrowheads. (B) SDS-PAGE analysis of in vitro-expressed polypeptides from the 1.8-kb cDNA insert. In vitro-synthesized mRNA, transcribed from the cDNA insert in the pGEM-T plasmid, was translated in vitro using rabbit reticulocyte lysate. Lanes 1, 4, and 7, translation reaction components without the addition of RNA. Lanes 2, 6, and 9, translation from RNA transcribed with SP6 RNA polymerase. Lanes 3, 5, and 8, translation from RNA transcribed with T7 RNA polymerase. Lanes 1 to 3, total translated products. Lanes 4 to 6, immunoprecipitations of translated products using a monoclonal antibody against the B701 (U16) protein. Lanes 7 to 9, immunoprecipitations of translation products using polyclonal rabbit antisera raised against the B701 protein. Samples were analyzed on 12% acrylamide cross-linked with bisacrylamide, and standard molecular mass markers were included in parallel lanes. The numbers indicate approximate molecular masses (in kilodaltons) of the prominent polypeptides identified.
    Figure Legend Snippet: (A) Reactivity of monoclonal antibody against the B701 (U16) protein. Lane 1, reactivities of antibodies in the Western blot reaction with the B701 protein expressed in the pET-3b vector. The 20K protein and the higher-molecular-weight dimeric and/or multimeric forms of B701 protein recognized are indicated by the arrowheads. (B) SDS-PAGE analysis of in vitro-expressed polypeptides from the 1.8-kb cDNA insert. In vitro-synthesized mRNA, transcribed from the cDNA insert in the pGEM-T plasmid, was translated in vitro using rabbit reticulocyte lysate. Lanes 1, 4, and 7, translation reaction components without the addition of RNA. Lanes 2, 6, and 9, translation from RNA transcribed with SP6 RNA polymerase. Lanes 3, 5, and 8, translation from RNA transcribed with T7 RNA polymerase. Lanes 1 to 3, total translated products. Lanes 4 to 6, immunoprecipitations of translated products using a monoclonal antibody against the B701 (U16) protein. Lanes 7 to 9, immunoprecipitations of translation products using polyclonal rabbit antisera raised against the B701 protein. Samples were analyzed on 12% acrylamide cross-linked with bisacrylamide, and standard molecular mass markers were included in parallel lanes. The numbers indicate approximate molecular masses (in kilodaltons) of the prominent polypeptides identified.

    Techniques Used: Western Blot, Positron Emission Tomography, Plasmid Preparation, Molecular Weight, SDS Page, In Vitro, Synthesized

    (A) SDS-PAGE analysis of in vitro-expressed polypeptides from the 1.1-kb E1E2 (U17/U16) cDNA insert. In vitro-synthesized mRNA, transcribed from the cDNA insert in the pGEMEX plasmid, was translated in vitro using rabbit reticulocyte lysate. Lanes 1, 3, 5, and 7, translation from RNA transcribed with SP6 RNA polymerase. Lanes 2, 4, 6, and 8, translation from RNA transcribed with T3 RNA polymerase. Lanes 7 and 8, total translated products. Lanes 1, 2, 5, and 6, immunoprecipitations of translated products with two different polyclonal rabbit antisera raised against the B701 protein. Lanes 3 and 4, immunoprecipitations of translation products with a monoclonal antibody against the B701 (U16) protein. Samples were analyzed on a 12% acrylamide gel cross-linked with bisacrylamide, and standard molecular mass markers were included in parallel lanes. The numbers indicate the approximate molecular masses (in kilodaltons) of the prominent polypeptides identified. (B) Expression of U17/U16 from the 1.1-kb cDNA in pGEMEX-E1E2. Bacteria transformed with either pGEMEX or pGEMEX-E1E2 were induced with IPTG for various lengths of time, and the lysate pellets and soluble supernatants were collected. The proteins were analyzed on SDS–12% PAGE, transferred to nitrocellulose membranes, and reacted with monoclonal antibody against B701 (U16) protein. Lanes 1 and 2, pellet and supernatant from bacteria with control pGEMEX plasmid induced with IPTG for 6 h. Lanes 3 and 4, pellet and supernatant from bacteria with pGEMEX-E1E2 plasmid induced with IPTG for 2 h. Lanes 5 and 6, pellet and supernatant from bacteria with pGEMEX-E1E2 plasmid induced with IPTG for 4 h. The fusion protein recognized is indicated by the arrowhead on the right.
    Figure Legend Snippet: (A) SDS-PAGE analysis of in vitro-expressed polypeptides from the 1.1-kb E1E2 (U17/U16) cDNA insert. In vitro-synthesized mRNA, transcribed from the cDNA insert in the pGEMEX plasmid, was translated in vitro using rabbit reticulocyte lysate. Lanes 1, 3, 5, and 7, translation from RNA transcribed with SP6 RNA polymerase. Lanes 2, 4, 6, and 8, translation from RNA transcribed with T3 RNA polymerase. Lanes 7 and 8, total translated products. Lanes 1, 2, 5, and 6, immunoprecipitations of translated products with two different polyclonal rabbit antisera raised against the B701 protein. Lanes 3 and 4, immunoprecipitations of translation products with a monoclonal antibody against the B701 (U16) protein. Samples were analyzed on a 12% acrylamide gel cross-linked with bisacrylamide, and standard molecular mass markers were included in parallel lanes. The numbers indicate the approximate molecular masses (in kilodaltons) of the prominent polypeptides identified. (B) Expression of U17/U16 from the 1.1-kb cDNA in pGEMEX-E1E2. Bacteria transformed with either pGEMEX or pGEMEX-E1E2 were induced with IPTG for various lengths of time, and the lysate pellets and soluble supernatants were collected. The proteins were analyzed on SDS–12% PAGE, transferred to nitrocellulose membranes, and reacted with monoclonal antibody against B701 (U16) protein. Lanes 1 and 2, pellet and supernatant from bacteria with control pGEMEX plasmid induced with IPTG for 6 h. Lanes 3 and 4, pellet and supernatant from bacteria with pGEMEX-E1E2 plasmid induced with IPTG for 2 h. Lanes 5 and 6, pellet and supernatant from bacteria with pGEMEX-E1E2 plasmid induced with IPTG for 4 h. The fusion protein recognized is indicated by the arrowhead on the right.

    Techniques Used: SDS Page, In Vitro, Synthesized, Plasmid Preparation, Acrylamide Gel Assay, Expressing, Transformation Assay, Polyacrylamide Gel Electrophoresis

    Northern blot analysis of the transcripts encoded by U16 and U17/U16. (A) Transcripts detected by radiolabeled antisense riboprobe generated from the U16 ORF of HHV-6A(GS). The approximate sizes (in kilobases) of the transcripts detected are indicated next to the arrows. (B) Transcripts detected by radiolabeled antisense riboprobe generated from the HHV-6A(GS) U17/U16 spliced cDNA. (C) Transcripts detected by radiolabeled antisense riboprobe generated from the HHV-6A(GS) U27 early gene. (A, B, and C) Each blot was stripped and reprobed with an antisense riboprobe generated from the GAPDH host cell gene. The GAPDH-specific Northern blot is shown below its respective viral-gene blot. Lanes 1, poly(A) + -selected RNA from HHV-6A(GS)-infected J-Jhan cells expressing viral antigens in more than 50% of the cells. Lanes 2, RNA isolated from uninfected J-Jhan cells. Lanes 3 to 7, kinetics of RNA expression from the U17/U16 region. J-Jhan cells were infected with HHV-6A(GS) as described in Materials and Methods, and samples were collected at various times after the initiation of infection. Lane 3, 8-h infections in the presence of cycloheximide. Lane 4, 8-h infections. Lane 5, 24-h infections. Lane 6, 24-h infections in the presence of PAA. Lane 7, 48-h infections.
    Figure Legend Snippet: Northern blot analysis of the transcripts encoded by U16 and U17/U16. (A) Transcripts detected by radiolabeled antisense riboprobe generated from the U16 ORF of HHV-6A(GS). The approximate sizes (in kilobases) of the transcripts detected are indicated next to the arrows. (B) Transcripts detected by radiolabeled antisense riboprobe generated from the HHV-6A(GS) U17/U16 spliced cDNA. (C) Transcripts detected by radiolabeled antisense riboprobe generated from the HHV-6A(GS) U27 early gene. (A, B, and C) Each blot was stripped and reprobed with an antisense riboprobe generated from the GAPDH host cell gene. The GAPDH-specific Northern blot is shown below its respective viral-gene blot. Lanes 1, poly(A) + -selected RNA from HHV-6A(GS)-infected J-Jhan cells expressing viral antigens in more than 50% of the cells. Lanes 2, RNA isolated from uninfected J-Jhan cells. Lanes 3 to 7, kinetics of RNA expression from the U17/U16 region. J-Jhan cells were infected with HHV-6A(GS) as described in Materials and Methods, and samples were collected at various times after the initiation of infection. Lane 3, 8-h infections in the presence of cycloheximide. Lane 4, 8-h infections. Lane 5, 24-h infections. Lane 6, 24-h infections in the presence of PAA. Lane 7, 48-h infections.

    Techniques Used: Northern Blot, Generated, Infection, Expressing, Isolation, RNA Expression

    28) Product Images from "Pheromone gland-specific fatty-acyl reductase of the silkmoth, Bombyx mori"

    Article Title: Pheromone gland-specific fatty-acyl reductase of the silkmoth, Bombyx mori

    Journal: Proceedings of the National Academy of Sciences of the United States of America

    doi: 10.1073/pnas.1531993100

    cDNA and deduced amino acid sequence of B. mori pgFAR. The region corresponding to the primer sequence used for 5′ RACE is underlined. The C-terminal part obtained by the first PCR is boxed. The proposed NAD(P)H binding motif is double underlined.
    Figure Legend Snippet: cDNA and deduced amino acid sequence of B. mori pgFAR. The region corresponding to the primer sequence used for 5′ RACE is underlined. The C-terminal part obtained by the first PCR is boxed. The proposed NAD(P)H binding motif is double underlined.

    Techniques Used: Sequencing, Polymerase Chain Reaction, Binding Assay

    29) Product Images from "Mutations in the Human Orthologue of the Mouse underwhite Gene (uw) Underlie a New Form of Oculocutaneous Albinism, OCA4"

    Article Title: Mutations in the Human Orthologue of the Mouse underwhite Gene (uw) Underlie a New Form of Oculocutaneous Albinism, OCA4

    Journal: American Journal of Human Genetics

    doi:

    Expression of the Matp gene in weanling eyes. For northern analysis, two identical blots containing 5 μg of total eye RNA from each of the strains indicated were hybridized with probes from either the mouse Matp cDNA (nucleotides 118–1004 [ upper panel ]) or the mouse p cDNA (nucleotides 375–2773 [ lower panel ]).
    Figure Legend Snippet: Expression of the Matp gene in weanling eyes. For northern analysis, two identical blots containing 5 μg of total eye RNA from each of the strains indicated were hybridized with probes from either the mouse Matp cDNA (nucleotides 118–1004 [ upper panel ]) or the mouse p cDNA (nucleotides 375–2773 [ lower panel ]).

    Techniques Used: Expressing, Northern Blot

    30) Product Images from "A small nuclear RNA, hdm365, is the major processing product of the human mdm2 gene"

    Article Title: A small nuclear RNA, hdm365, is the major processing product of the human mdm2 gene

    Journal: Nucleic Acids Research

    doi:

    hdm365 precedes S-hdm2 expression. Note that already at the time of first hdm365 appearance the intron 5 probe detects a long precursor RNA and a band corresponding to the excised intron 5. Hybridisations with probes from distinct regions of the hdm2 cDNA and with an intron 5 probe are presented. 28S rRNA as visualised by ethidium bromide (EtBr) staining of the formaldehyde gel is shown as a loading control.
    Figure Legend Snippet: hdm365 precedes S-hdm2 expression. Note that already at the time of first hdm365 appearance the intron 5 probe detects a long precursor RNA and a band corresponding to the excised intron 5. Hybridisations with probes from distinct regions of the hdm2 cDNA and with an intron 5 probe are presented. 28S rRNA as visualised by ethidium bromide (EtBr) staining of the formaldehyde gel is shown as a loading control.

    Techniques Used: Expressing, Staining

    31) Product Images from "Molecular Cloning and Functional Expression of Chitinase-Encoding cDNA from the Cabbage Moth, Mamestra brassicae"

    Article Title: Molecular Cloning and Functional Expression of Chitinase-Encoding cDNA from the Cabbage Moth, Mamestra brassicae

    Journal: Molecules and Cells

    doi: 10.1007/s10059-012-2133-4

    Amplified conserved partial domain and RACE products of chitinase-encoding cDNA isolated from M. brassicae. CPD, Conserved partial domain; 5′ and 3′ RP, 5′ and 3′ RACE products; M, Marker DNAs.
    Figure Legend Snippet: Amplified conserved partial domain and RACE products of chitinase-encoding cDNA isolated from M. brassicae. CPD, Conserved partial domain; 5′ and 3′ RP, 5′ and 3′ RACE products; M, Marker DNAs.

    Techniques Used: Amplification, Isolation, Marker

    32) Product Images from "Molecular cloning and characterization of ZFF29: a protein containing a unique Cys2His2 zinc-finger motif"

    Article Title: Molecular cloning and characterization of ZFF29: a protein containing a unique Cys2His2 zinc-finger motif

    Journal: Biochemical Journal

    doi: 10.1042/BJ20040394

    Expression of ZFF29 mRNA ( A ) Transcripts of ZFF29a and ZFF29b were detected by Northern blotting. The riboprobes indicated above the lanes were hybridized to 4 μg of poly(A) + RNA extracted from K562 cells. The positions of 18 S and 28 S rRNA species are shown. Arrows indicate bands at the authentic positions of ZFF29a and ZFF29b. Bands marked with asterisks seem to be cross-hybridized to residual 28 S rRNA; the significance of bands marked by arrowheads is obscure. ( B , C ) ZFF29 expression in adult mouse tissues analysed by RT-PCR. Amplified cDNAs and numbers of PCR cycles are indicated on the left, and cDNA source tissues are shown above the lanes. Primers were derived from sequence common to ZFF29a and ZFF29b cDNAs. Note that intense bands were formed from the bone marrow and ovary cDNAs among those diluted to give rise to similar band intensities on amplification of the 28 S rRNA gene. ( D ) ZFF29 expression in mouse haematopoietic tissues analysed by RT-PCR. Amplified cDNAs and the numbers of PCR cycles are indicated on the left, and cDNA source tissues are shown above the lanes. The cDNA samples had been diluted to give rise to similar band intensities on amplification of the GATA-1 gene.
    Figure Legend Snippet: Expression of ZFF29 mRNA ( A ) Transcripts of ZFF29a and ZFF29b were detected by Northern blotting. The riboprobes indicated above the lanes were hybridized to 4 μg of poly(A) + RNA extracted from K562 cells. The positions of 18 S and 28 S rRNA species are shown. Arrows indicate bands at the authentic positions of ZFF29a and ZFF29b. Bands marked with asterisks seem to be cross-hybridized to residual 28 S rRNA; the significance of bands marked by arrowheads is obscure. ( B , C ) ZFF29 expression in adult mouse tissues analysed by RT-PCR. Amplified cDNAs and numbers of PCR cycles are indicated on the left, and cDNA source tissues are shown above the lanes. Primers were derived from sequence common to ZFF29a and ZFF29b cDNAs. Note that intense bands were formed from the bone marrow and ovary cDNAs among those diluted to give rise to similar band intensities on amplification of the 28 S rRNA gene. ( D ) ZFF29 expression in mouse haematopoietic tissues analysed by RT-PCR. Amplified cDNAs and the numbers of PCR cycles are indicated on the left, and cDNA source tissues are shown above the lanes. The cDNA samples had been diluted to give rise to similar band intensities on amplification of the GATA-1 gene.

    Techniques Used: Expressing, Northern Blot, Reverse Transcription Polymerase Chain Reaction, Amplification, Polymerase Chain Reaction, Derivative Assay, Sequencing

    33) Product Images from "DNA Supercoiling Factor Localizes to Puffs on Polytene Chromosomes in Drosophila melanogaster"

    Article Title: DNA Supercoiling Factor Localizes to Puffs on Polytene Chromosomes in Drosophila melanogaster

    Journal: Molecular and Cellular Biology

    doi:

    Transcripts from the SCF locus. (A) Northern blot hybridization with region-specific probes. A blot of total cellular RNA from prepupae was successively probed with the indicated DNA fragments (probes 1-3) and a full-length cDNA (probe 4). Size marker RNAs were run in a parallel lane on the same gel, and their positions are indicated. N and C represent the N and C termini of the full-length protein. Signal peptide denotes the signal-peptide-like sequence, and I to V represent the loops of the EF-Hand domains. (B) Mapping of the 5′ end of each mRNA by 5′ RACE. Underlining represents sequences of the longest PCR products from 1.6 and 1.8 kb mRNAs. INIT consensus, initiator consensus sequence; DPE consensus, downstream promoter element consensus. The filled triangles show the presumptive initiation sites for each mRNA.
    Figure Legend Snippet: Transcripts from the SCF locus. (A) Northern blot hybridization with region-specific probes. A blot of total cellular RNA from prepupae was successively probed with the indicated DNA fragments (probes 1-3) and a full-length cDNA (probe 4). Size marker RNAs were run in a parallel lane on the same gel, and their positions are indicated. N and C represent the N and C termini of the full-length protein. Signal peptide denotes the signal-peptide-like sequence, and I to V represent the loops of the EF-Hand domains. (B) Mapping of the 5′ end of each mRNA by 5′ RACE. Underlining represents sequences of the longest PCR products from 1.6 and 1.8 kb mRNAs. INIT consensus, initiator consensus sequence; DPE consensus, downstream promoter element consensus. The filled triangles show the presumptive initiation sites for each mRNA.

    Techniques Used: Northern Blot, Hybridization, Marker, Sequencing, Polymerase Chain Reaction

    34) Product Images from "Mouse RC/BTB2, a Member of the RCC1 Superfamily, Localizes to Spermatid Acrosomal Vesicles"

    Article Title: Mouse RC/BTB2, a Member of the RCC1 Superfamily, Localizes to Spermatid Acrosomal Vesicles

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0039846

    The Rc/btb2 gene encodes two major messages. A. Analysis of Rc/btb2 mRNA expression in the indicated tissues by Northern blot analysis. A multiple tissue blot was hybridized to a 32 P-α-dCTP labeled Rc/btb2 cDNA probe, the blot was exposure to an X-ray film overnight. Notice that the 2.3 kb message is present in most of the somatic tissues, the 2.5 kb message is only present in the testis. B. Identification of two transcripts ( Rc/btb2-s and Rc/btb2-t ) by 5′- and 3′- RACE experiments using mouse heart and testis cDNA libraries; C: Schematic representation of the two transcripts. Rc/btb2-t contains an extra exon (1c) compared to Rc/btb2-s ; D: Examination of tissue distribution of the two transcripts by RT-PCR using primer sets indicated in (C). Rc/btb2-t is only present in the testis.
    Figure Legend Snippet: The Rc/btb2 gene encodes two major messages. A. Analysis of Rc/btb2 mRNA expression in the indicated tissues by Northern blot analysis. A multiple tissue blot was hybridized to a 32 P-α-dCTP labeled Rc/btb2 cDNA probe, the blot was exposure to an X-ray film overnight. Notice that the 2.3 kb message is present in most of the somatic tissues, the 2.5 kb message is only present in the testis. B. Identification of two transcripts ( Rc/btb2-s and Rc/btb2-t ) by 5′- and 3′- RACE experiments using mouse heart and testis cDNA libraries; C: Schematic representation of the two transcripts. Rc/btb2-t contains an extra exon (1c) compared to Rc/btb2-s ; D: Examination of tissue distribution of the two transcripts by RT-PCR using primer sets indicated in (C). Rc/btb2-t is only present in the testis.

    Techniques Used: Expressing, Northern Blot, Labeling, Reverse Transcription Polymerase Chain Reaction

    35) Product Images from "Cloning and Functional Characterization of Roaz, a Zinc Finger Protein that Interacts with O/E-1 to Regulate Gene Expression: Implications for Olfactory Neuronal Development"

    Article Title: Cloning and Functional Characterization of Roaz, a Zinc Finger Protein that Interacts with O/E-1 to Regulate Gene Expression: Implications for Olfactory Neuronal Development

    Journal: The Journal of Neuroscience

    doi: 10.1523/JNEUROSCI.17-11-04159.1997

    Structure and sequence of Roaz cDNA clones. A , Schematic representation of Roaz and various cDNA clones isolated from cDNA library screens and RACE-PCR. Each shaded box represents one zinc finger structure. RoazD86 is the original clone isolated from the yeast two-hybrid screen. The other clones represent isolates from subsequent cDNA library screens and RACE-PCR, as indicated in the text. The 3′-UTR of Roaz is not shown in this diagram. B , cDNA sequence and predicted open reading frame of Roaz. The amino acid sequences of zinc finger structures are underlined . Figure continues.
    Figure Legend Snippet: Structure and sequence of Roaz cDNA clones. A , Schematic representation of Roaz and various cDNA clones isolated from cDNA library screens and RACE-PCR. Each shaded box represents one zinc finger structure. RoazD86 is the original clone isolated from the yeast two-hybrid screen. The other clones represent isolates from subsequent cDNA library screens and RACE-PCR, as indicated in the text. The 3′-UTR of Roaz is not shown in this diagram. B , cDNA sequence and predicted open reading frame of Roaz. The amino acid sequences of zinc finger structures are underlined . Figure continues.

    Techniques Used: Sequencing, Clone Assay, Isolation, cDNA Library Assay, Polymerase Chain Reaction, Two Hybrid Screening

    36) Product Images from "ELFN1-AS1: A Novel Primate Gene with Possible MicroRNA Function Expressed Predominantly in Human Tumors"

    Article Title: ELFN1-AS1: A Novel Primate Gene with Possible MicroRNA Function Expressed Predominantly in Human Tumors

    Journal: BioMed Research International

    doi: 10.1155/2014/398097

    Identification of the primary structure of the Hs.633957-specific RNA. (a) Scheme of the transcript variants for the locus Hs.633957. Splice sites and polyadenylation sites are marked and their positions are given according to the leftmost TSS. Arrows indicate position of the primers which were used for identification of the major splice form. BX119057-like variant is filled in dark grey. (b) Results of cDNA PCR amplification with primers bordering the 39 to 3642 gene region. (c) Results of cDNA PCR amplification with primers bordering the 39–3289 gene region. cDNA samples: 1: gallbladder, adenocarcinoma; 2: rectum, well differentiated adenocarcinoma; 3: ureter, papillary transitional cell carcinoma; 4: T-cell Hodgkin's lymphoma; 5: kidney; 6: liver; NTC: no template control.
    Figure Legend Snippet: Identification of the primary structure of the Hs.633957-specific RNA. (a) Scheme of the transcript variants for the locus Hs.633957. Splice sites and polyadenylation sites are marked and their positions are given according to the leftmost TSS. Arrows indicate position of the primers which were used for identification of the major splice form. BX119057-like variant is filled in dark grey. (b) Results of cDNA PCR amplification with primers bordering the 39 to 3642 gene region. (c) Results of cDNA PCR amplification with primers bordering the 39–3289 gene region. cDNA samples: 1: gallbladder, adenocarcinoma; 2: rectum, well differentiated adenocarcinoma; 3: ureter, papillary transitional cell carcinoma; 4: T-cell Hodgkin's lymphoma; 5: kidney; 6: liver; NTC: no template control.

    Techniques Used: Variant Assay, Polymerase Chain Reaction, Amplification

    37) Product Images from "Molecular Cloning and Functional Expression of the Rat 175-kDa Hyaluronan Receptor for Endocytosis"

    Article Title: Molecular Cloning and Functional Expression of the Rat 175-kDa Hyaluronan Receptor for Endocytosis

    Journal: Molecular Biology of the Cell

    doi: 10.1091/mbc.02-03-0048

    Expression of a Functional HA Receptor for Endocytosis from the 175-kDa HARE cDNA
    Figure Legend Snippet: Expression of a Functional HA Receptor for Endocytosis from the 175-kDa HARE cDNA

    Techniques Used: Expressing, Functional Assay

    Nucleic acid and deduced amino acid sequences of the 4.7-kb cDNA encoding the rat 175-kDa HARE. The artificial cDNA containing 4708 nucleotides encodes a 1431-amino acid recombinant 175-kDa HARE protein, whose deduced amino acid sequence begins with a serine. Amino acid sequences verified by peptide sequence analysis of the purified HARE are underlined, and the two N-terminal peptides found in the purified protein are underlined and in italics. Putative N -glycosylation sites are in boldface, and Cys residues are highlighted in boldface and italics. Three alternative N -glycosylation sites of the type N-X-C are located at N 135 , N 218 , and N 930 . The predicted transmembrane domain of the type I membrane protein is underlined and in boldface. The three shaded regions in the cytoplasmic domain are potential motifs for targeting the receptor to clathrin-coated pits. Potential HA-binding motifs of the type B-X 7 -B, which are in the predicted extracellular domain, are enclosed in boldface brackets.
    Figure Legend Snippet: Nucleic acid and deduced amino acid sequences of the 4.7-kb cDNA encoding the rat 175-kDa HARE. The artificial cDNA containing 4708 nucleotides encodes a 1431-amino acid recombinant 175-kDa HARE protein, whose deduced amino acid sequence begins with a serine. Amino acid sequences verified by peptide sequence analysis of the purified HARE are underlined, and the two N-terminal peptides found in the purified protein are underlined and in italics. Putative N -glycosylation sites are in boldface, and Cys residues are highlighted in boldface and italics. Three alternative N -glycosylation sites of the type N-X-C are located at N 135 , N 218 , and N 930 . The predicted transmembrane domain of the type I membrane protein is underlined and in boldface. The three shaded regions in the cytoplasmic domain are potential motifs for targeting the receptor to clathrin-coated pits. Potential HA-binding motifs of the type B-X 7 -B, which are in the predicted extracellular domain, are enclosed in boldface brackets.

    Techniques Used: Recombinant, Sequencing, Purification, Binding Assay

    Confocal microscopy of the 175-kDa HARE in SK-175HARE cells. The cellular distributions of the recombinant HARE, fl-HA, clathrin, and lysosomes were determined in SK-175HARE-34 cells as described in MATERIALS AND METHODS. (A–C) Colocalization of clathrin (A) and HARE (B) in the overlay picture (C). The different distribution patterns of HARE (D) and Lysotracker (E) in cells incubated with unlabeled HA are shown in the overlay picture (F). (I) Colocalization pattern of fl-HA (G) and Lysotracker (H). The effect of excess unlabeled HA on the uptake of fl-HA is shown in J. The background staining of SK-175HARE cells with rabbit IgG is shown in K. (L) Anti-HARE staining of SK-Hep-1 cells stably transfected with the backbone plasmid (containing no cDNA insert). The bar in A (20 μm) applies to A–C, and the bar in D (50 μm) applies to D–L.
    Figure Legend Snippet: Confocal microscopy of the 175-kDa HARE in SK-175HARE cells. The cellular distributions of the recombinant HARE, fl-HA, clathrin, and lysosomes were determined in SK-175HARE-34 cells as described in MATERIALS AND METHODS. (A–C) Colocalization of clathrin (A) and HARE (B) in the overlay picture (C). The different distribution patterns of HARE (D) and Lysotracker (E) in cells incubated with unlabeled HA are shown in the overlay picture (F). (I) Colocalization pattern of fl-HA (G) and Lysotracker (H). The effect of excess unlabeled HA on the uptake of fl-HA is shown in J. The background staining of SK-175HARE cells with rabbit IgG is shown in K. (L) Anti-HARE staining of SK-Hep-1 cells stably transfected with the backbone plasmid (containing no cDNA insert). The bar in A (20 μm) applies to A–C, and the bar in D (50 μm) applies to D–L.

    Techniques Used: Confocal Microscopy, Recombinant, Incubation, Staining, Stable Transfection, Transfection, Plasmid Preparation

    Expression of a Functional HA Receptor for Endocytosis from the 175-kDa HARE cDNA
    Figure Legend Snippet: Expression of a Functional HA Receptor for Endocytosis from the 175-kDa HARE cDNA

    Techniques Used: Expressing, Functional Assay

    Northern blot analysis of rat LEC RNA with 175-kDa HARE cDNA probes. Total RNA (lanes 1) or mRNA (lanes 2) samples prepared from isolated rat LECs were subjected to formamide gel electrophoresis, transferred to nylon membranes, and processed as described in MATERIALS AND METHODS. The membranes were allowed to hybridize separately with three different 32 P-labeled DNA probes, 5′RACE#11 (A), ZAP1P3 (B), or ZAP9P3 (C), which are located at the 5′ end, middle, or 3′ end of the 175-kDa HARE cDNA sequence, respectively.
    Figure Legend Snippet: Northern blot analysis of rat LEC RNA with 175-kDa HARE cDNA probes. Total RNA (lanes 1) or mRNA (lanes 2) samples prepared from isolated rat LECs were subjected to formamide gel electrophoresis, transferred to nylon membranes, and processed as described in MATERIALS AND METHODS. The membranes were allowed to hybridize separately with three different 32 P-labeled DNA probes, 5′RACE#11 (A), ZAP1P3 (B), or ZAP9P3 (C), which are located at the 5′ end, middle, or 3′ end of the 175-kDa HARE cDNA sequence, respectively.

    Techniques Used: Northern Blot, Isolation, Nucleic Acid Electrophoresis, Labeling, Sequencing

    Schematic map of the recombinant 175-kDa HARE construct. A 5′-end fragment (1.1 kb) of the 175-kDa HARE open reading frame was amplified by RT-PCR with primers containing Eco RI sites, and cloned in-frame with the Ig κ-chain leader sequence of the pSecTag2 vector. The DNA insert was cut with Nhe I and Asn I and then cloned into pcDNA3.1 together with two other 175-kDa HARE cDNA fragments of 2.2 and 1.4 kb, which were derived from the RT-PCR and cDNA library screenings, respectively, and which encode the remainder of the 175-kDa HARE protein. The fragments were digested with different restriction enzymes as indicated and assembled as described in MATERIALS AND METHODS.
    Figure Legend Snippet: Schematic map of the recombinant 175-kDa HARE construct. A 5′-end fragment (1.1 kb) of the 175-kDa HARE open reading frame was amplified by RT-PCR with primers containing Eco RI sites, and cloned in-frame with the Ig κ-chain leader sequence of the pSecTag2 vector. The DNA insert was cut with Nhe I and Asn I and then cloned into pcDNA3.1 together with two other 175-kDa HARE cDNA fragments of 2.2 and 1.4 kb, which were derived from the RT-PCR and cDNA library screenings, respectively, and which encode the remainder of the 175-kDa HARE protein. The fragments were digested with different restriction enzymes as indicated and assembled as described in MATERIALS AND METHODS.

    Techniques Used: Recombinant, Construct, Amplification, Reverse Transcription Polymerase Chain Reaction, Clone Assay, Sequencing, Plasmid Preparation, Derivative Assay, cDNA Library Assay

    38) Product Images from "Control of Growth Cone Motility and Morphology by LIM Kinase and Slingshot via Phosphorylation and Dephosphorylation of Cofilin"

    Article Title: Control of Growth Cone Motility and Morphology by LIM Kinase and Slingshot via Phosphorylation and Dephosphorylation of Cofilin

    Journal: The Journal of Neuroscience

    doi: 10.1523/JNEUROSCI.23-07-02527.2003

    Subcellular localization of chLIMK1 in chick DRG neurons. A , Specificity of anti-chLIMK1 antibody. COS-7 cells were transfected with chLIMK1 cDNA expression plasmid ( lanes 1, 3, 4 ) or mock-transfected with pMYC-C1 vector alone ( lane 2 ). Lysates were immunoprecipitated ( IP ) with C20 anti-chLIMK1 antibody ( C20 , lanes 1, 2, 4 ) or preimmune serum ( Pre , lane 3 ), run on SDS-PAGE, and then immunoblotted with C20 anti-chLIMK1 antibody. In lane 4 , anti-chLIMK1 antibody was pretreated with excess amounts of antigenic peptide ( pep ). The positions of molecular weight marker proteins are indicated on the left . IgH , Ig heavy chain. B , Subcellular localization of chLIMK1 in chick DRG neurons. Chick E7 DRG neurons were costained with rhodamine-conjugated phalloidin ( bottom ) and C20 anti-chLIMK1 antibody ( top ) in the absence ( left ) or presence ( right ) of excess amounts of antigenic peptide. Dot-like staining of endogenous chLIMK1 was specifically detected in growth cones and axonal shafts at the top left . Scale bar, 20 μm. C , Axonal transport of LIMK1(WT)-YFP. Chick E7 DRG neurons were infected with HSV coding for YFP-fused chLIMK1(WT) and recorded 12 hr later by video fluorescence microscopy. Each frame shows the fluorescence image of YFP at 10 sec intervals. Arrowheads ), in which retrograde movements can be seen. Scale bars: white, 10 μm; black, 5 μm.
    Figure Legend Snippet: Subcellular localization of chLIMK1 in chick DRG neurons. A , Specificity of anti-chLIMK1 antibody. COS-7 cells were transfected with chLIMK1 cDNA expression plasmid ( lanes 1, 3, 4 ) or mock-transfected with pMYC-C1 vector alone ( lane 2 ). Lysates were immunoprecipitated ( IP ) with C20 anti-chLIMK1 antibody ( C20 , lanes 1, 2, 4 ) or preimmune serum ( Pre , lane 3 ), run on SDS-PAGE, and then immunoblotted with C20 anti-chLIMK1 antibody. In lane 4 , anti-chLIMK1 antibody was pretreated with excess amounts of antigenic peptide ( pep ). The positions of molecular weight marker proteins are indicated on the left . IgH , Ig heavy chain. B , Subcellular localization of chLIMK1 in chick DRG neurons. Chick E7 DRG neurons were costained with rhodamine-conjugated phalloidin ( bottom ) and C20 anti-chLIMK1 antibody ( top ) in the absence ( left ) or presence ( right ) of excess amounts of antigenic peptide. Dot-like staining of endogenous chLIMK1 was specifically detected in growth cones and axonal shafts at the top left . Scale bar, 20 μm. C , Axonal transport of LIMK1(WT)-YFP. Chick E7 DRG neurons were infected with HSV coding for YFP-fused chLIMK1(WT) and recorded 12 hr later by video fluorescence microscopy. Each frame shows the fluorescence image of YFP at 10 sec intervals. Arrowheads ), in which retrograde movements can be seen. Scale bars: white, 10 μm; black, 5 μm.

    Techniques Used: Transfection, Expressing, Plasmid Preparation, Immunoprecipitation, SDS Page, Molecular Weight, Marker, Staining, Infection, Fluorescence, Microscopy, Size-exclusion Chromatography

    39) Product Images from "Arabidopsis STERILE APETALA, a multifunctional gene regulating inflorescence, flower, and ovule development"

    Article Title: Arabidopsis STERILE APETALA, a multifunctional gene regulating inflorescence, flower, and ovule development

    Journal: Genes & Development

    doi:

    SAP genomic structure and sequences of SAP cDNA and mutant alleles. ( A ) Genomic structure of the SAP gene. Solid bars represent exons and the single intron of approximately 3 kb in length is indicated by an interrupted line. The locations of the start (ATG) and stop codon (GTA) are shown. The thin line represents genomic DNA outside the SAP gene. The position of the I/dSpm insertion is indicated by the large open triangle. Primer 4 was used for 5′ RACE. (E) Eco RI; (H) Hin fI restriction sites. ( B ) Nucleotide sequence of SAP cDNA and the deduced amino acid sequence. The position of the intron is shown (▾); the position of the I/dSpm insertion is indicated ( ). The serine–glysine-rich domain is underlined. (*) The stop codon of the ORF. ( C ) Footprint alleles generated after excision of the I/dSpm element from the sap:I/dSpm allele. The phenotype is either as wild-type or sap mutant plants. The sequence of the duplications is underlined. In allele SAP-5.510 a duplication of four nucleotides results in the generation of a stopcodon indicated by an asterisks. In allele SAP-4.42 a duplication of two nucleotides (TA) is accompanied by a GC deletion, restoring the ORF.
    Figure Legend Snippet: SAP genomic structure and sequences of SAP cDNA and mutant alleles. ( A ) Genomic structure of the SAP gene. Solid bars represent exons and the single intron of approximately 3 kb in length is indicated by an interrupted line. The locations of the start (ATG) and stop codon (GTA) are shown. The thin line represents genomic DNA outside the SAP gene. The position of the I/dSpm insertion is indicated by the large open triangle. Primer 4 was used for 5′ RACE. (E) Eco RI; (H) Hin fI restriction sites. ( B ) Nucleotide sequence of SAP cDNA and the deduced amino acid sequence. The position of the intron is shown (▾); the position of the I/dSpm insertion is indicated ( ). The serine–glysine-rich domain is underlined. (*) The stop codon of the ORF. ( C ) Footprint alleles generated after excision of the I/dSpm element from the sap:I/dSpm allele. The phenotype is either as wild-type or sap mutant plants. The sequence of the duplications is underlined. In allele SAP-5.510 a duplication of four nucleotides results in the generation of a stopcodon indicated by an asterisks. In allele SAP-4.42 a duplication of two nucleotides (TA) is accompanied by a GC deletion, restoring the ORF.

    Techniques Used: Mutagenesis, Sequencing, Generated

    40) Product Images from "Subpopulations of chloroplast ribosomes change during photoregulated development of Zea mays leaves: Ribosomal proteins L2, L21, and L29"

    Article Title: Subpopulations of chloroplast ribosomes change during photoregulated development of Zea mays leaves: Ribosomal proteins L2, L21, and L29

    Journal: Proceedings of the National Academy of Sciences of the United States of America

    doi:

    L29 is a maize homologue of B. stearothermophilus ribosomal protein L29. ( A ) The nucleotide sequence and the deduced amino acid sequences are numbered at the left. The two primers used in the DDA are labeled with arrows. Four bases in the AP12 primer did not match the cDNA sequence. The gene-specific primer used in a 5′-RACE reaction is underlined. Three N-myristylation sites are boxed, and four potential protein kinase C phosphorylation sites are marked with asterisks. The boldfaced letters (amino acids 133–136) designate the potential cAMP/cGMP-dependent protein kinase phosphorylation site (accession no. AF 147725). ( B ) The amino acid alignment of the homologous regions of maize L29, B. stearothermophilus (BACST) and Bacillus subtilis (BACSLI) is shown. Double and single dots indicate amino acid identity and similarity, respectively. Gaps represented by dashes were inserted to maximize the sequence identity. Consensus amino acids are residues that are identical in the three proteins. Numbers in parentheses indicate the position of the start of an amino acid sequence with respect to the initiator methionine of the corresponding protein. B. stearothermophilus L29 exhibited the highest identity with maize L29 of all sequences in the NCBI protein databases at the time of submission. The region of L29 that can be crosslinked to 23S rRNA in ribosomes of B. stearothermophilus ) are underlined, and the crosslinked lysyl residues are in boldface type (K). Accession numbers for the bacterial L29 proteins shown: B. stearothermophilus (SPIP04457) and B. subtilis (SPIP12873).
    Figure Legend Snippet: L29 is a maize homologue of B. stearothermophilus ribosomal protein L29. ( A ) The nucleotide sequence and the deduced amino acid sequences are numbered at the left. The two primers used in the DDA are labeled with arrows. Four bases in the AP12 primer did not match the cDNA sequence. The gene-specific primer used in a 5′-RACE reaction is underlined. Three N-myristylation sites are boxed, and four potential protein kinase C phosphorylation sites are marked with asterisks. The boldfaced letters (amino acids 133–136) designate the potential cAMP/cGMP-dependent protein kinase phosphorylation site (accession no. AF 147725). ( B ) The amino acid alignment of the homologous regions of maize L29, B. stearothermophilus (BACST) and Bacillus subtilis (BACSLI) is shown. Double and single dots indicate amino acid identity and similarity, respectively. Gaps represented by dashes were inserted to maximize the sequence identity. Consensus amino acids are residues that are identical in the three proteins. Numbers in parentheses indicate the position of the start of an amino acid sequence with respect to the initiator methionine of the corresponding protein. B. stearothermophilus L29 exhibited the highest identity with maize L29 of all sequences in the NCBI protein databases at the time of submission. The region of L29 that can be crosslinked to 23S rRNA in ribosomes of B. stearothermophilus ) are underlined, and the crosslinked lysyl residues are in boldface type (K). Accession numbers for the bacterial L29 proteins shown: B. stearothermophilus (SPIP04457) and B. subtilis (SPIP12873).

    Techniques Used: Sequencing, Labeling

    DDA of cDNAs derived from mRNAs from etiolated and illuminated maize leaves and Northern blot analyses of these mRNAs. ( A ) Part of a gel from a DDA done with primers T12MG and AP12 (GATCTAACCG) is shown. Total RNA preparations from 10-day-old etiolated maize leaves (D) and greening leaves illuminated with white light (W) or red light (R) for 8 hr or 24 hr were subjected to differential display reverse transcription-PCR. Band No. 40 represents the light-regulated cDNA segment of L29. ( B ) Confirmation of the differential expression pattern of L29. Twenty-five μg of total RNA from etiolated leaves (D) or greening leaves illuminated with blue light (B) or red light (R) for 8 hr or 24 hr was fractionated electrophoretically in a 1% formaldehyde agarose gel, transferred, and probed with [α- 32 P]dCTP-labeled cDNA of reamplified fragment No. 40. A single band of about 0.8 kbp was detected in both blue light- and red light-treated samples but not in the etiolated samples. Equal loading was confirmed by 23S rRNA hybridization.
    Figure Legend Snippet: DDA of cDNAs derived from mRNAs from etiolated and illuminated maize leaves and Northern blot analyses of these mRNAs. ( A ) Part of a gel from a DDA done with primers T12MG and AP12 (GATCTAACCG) is shown. Total RNA preparations from 10-day-old etiolated maize leaves (D) and greening leaves illuminated with white light (W) or red light (R) for 8 hr or 24 hr were subjected to differential display reverse transcription-PCR. Band No. 40 represents the light-regulated cDNA segment of L29. ( B ) Confirmation of the differential expression pattern of L29. Twenty-five μg of total RNA from etiolated leaves (D) or greening leaves illuminated with blue light (B) or red light (R) for 8 hr or 24 hr was fractionated electrophoretically in a 1% formaldehyde agarose gel, transferred, and probed with [α- 32 P]dCTP-labeled cDNA of reamplified fragment No. 40. A single band of about 0.8 kbp was detected in both blue light- and red light-treated samples but not in the etiolated samples. Equal loading was confirmed by 23S rRNA hybridization.

    Techniques Used: Derivative Assay, Northern Blot, Polymerase Chain Reaction, Expressing, Agarose Gel Electrophoresis, Labeling, Hybridization

    Time course of L29 mRNA accumulation in leaves on illumination of etiolated maize seedlings. Total RNA was extracted from leaves of 10-day-old etiolated (D) or greening plants illuminated with white light for the number of hours indicated and fractionated electrophoretically in a 1% formaldehyde agarose gel (25 μg/lane). After transfer to Gene Screen filter (DuPont/NEN), the filter was hybridized with [α- 32 P] dCTP-labeled maize L29 full-length cDNA, and 23S rRNA probe was used to assess loading differences.
    Figure Legend Snippet: Time course of L29 mRNA accumulation in leaves on illumination of etiolated maize seedlings. Total RNA was extracted from leaves of 10-day-old etiolated (D) or greening plants illuminated with white light for the number of hours indicated and fractionated electrophoretically in a 1% formaldehyde agarose gel (25 μg/lane). After transfer to Gene Screen filter (DuPont/NEN), the filter was hybridized with [α- 32 P] dCTP-labeled maize L29 full-length cDNA, and 23S rRNA probe was used to assess loading differences.

    Techniques Used: Agarose Gel Electrophoresis, Labeling

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    TaKaRa marathon cdna amplification kit
    Expression of CK metabolic and response genes during tomato fruit development. Real-time <t>PCR</t> was performed with <t>cDNA</t> prepared from pollinated ovaries (Poll.) at 1, 3, 5, 10, 15, and 20 days after anthesis (black bars), and unpollinated ovaries (Unpoll.) at –2, 0, 1, and 3 days after anthesis (grey bars). Expression levels are normalized to SAND expression levels. Values are mean ± SE ( n = 3).
    Marathon Cdna Amplification Kit, supplied by TaKaRa, used in various techniques. Bioz Stars score: 99/100, based on 28 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Expression of CK metabolic and response genes during tomato fruit development. Real-time PCR was performed with cDNA prepared from pollinated ovaries (Poll.) at 1, 3, 5, 10, 15, and 20 days after anthesis (black bars), and unpollinated ovaries (Unpoll.) at –2, 0, 1, and 3 days after anthesis (grey bars). Expression levels are normalized to SAND expression levels. Values are mean ± SE ( n = 3).

    Journal: Journal of Experimental Botany

    Article Title: Roles and regulation of cytokinins in tomato fruit development

    doi: 10.1093/jxb/ers207

    Figure Lengend Snippet: Expression of CK metabolic and response genes during tomato fruit development. Real-time PCR was performed with cDNA prepared from pollinated ovaries (Poll.) at 1, 3, 5, 10, 15, and 20 days after anthesis (black bars), and unpollinated ovaries (Unpoll.) at –2, 0, 1, and 3 days after anthesis (grey bars). Expression levels are normalized to SAND expression levels. Values are mean ± SE ( n = 3).

    Article Snippet: RACE (Rapid Amplification of cDNA Ends) was performed to identify the sequences of 5’ and 3’ regions of the genes using a Marathon cDNA amplification kit and an Advantage 2 PCR kit (Clontech).

    Techniques: Expressing, Real-time Polymerase Chain Reaction

    Genomic organization and expression profiles of zebrafish znf219L . Genomic organization of zebrafish znf219L , and the mouse and human znf219 genes were shown in panel (A). Coding regions are shown as filled boxes numbered from 3 to 5. The 5'- and 3'-untranslated regions are shown as open boxes, while solid lines indicate introns. RT-PCR was performed with gene-specific primers. β -actin was used as an internal control to normalize the amount of cDNA prepared from different adult zebrafish tissues (B) and from zebrafish embryos at different developmental stages (C). The developmental expression profile of zebrafish znf219L mRNA was examined in embryos from 12 hpf to 144 hpf. Blank PCR was performed using gene-specific primers and β -actin primers without the addition of cDNA template. Whole-mount in situ hybridization with antisense znf219L was performed at the indicated times post-fertilization (D). The images were taken from 22 to 48 hpf in the lateral view (panels a, b) and 48 hpf in dorsal view in panel c. The boxed region is enlarged to show the signal in the notochord (nc) from 22 to 48 hpf of both lateral and dorsal view (panels a', b', and c'). Dorsal view of znf219L mRNA signals in midbrain hinfbrain boundry (mhb), and hindbrain (hb) were detected from 48 to 96 hpf (panel d, e, and f). mhb, midbrain hindbrain boundry; hb, hindbrain; ov, otic vesicle; pf, pectoral fin, nc, notochord. Scale bars=100 μm.

    Journal: International Journal of Biological Sciences

    Article Title: A Novel Zinc Finger Protein 219-like (ZNF219L) is Involved in the Regulation of Collagen Type 2 Alpha 1a (col2a1a) Gene Expression in Zebrafish Notochord

    doi: 10.7150/ijbs.7126

    Figure Lengend Snippet: Genomic organization and expression profiles of zebrafish znf219L . Genomic organization of zebrafish znf219L , and the mouse and human znf219 genes were shown in panel (A). Coding regions are shown as filled boxes numbered from 3 to 5. The 5'- and 3'-untranslated regions are shown as open boxes, while solid lines indicate introns. RT-PCR was performed with gene-specific primers. β -actin was used as an internal control to normalize the amount of cDNA prepared from different adult zebrafish tissues (B) and from zebrafish embryos at different developmental stages (C). The developmental expression profile of zebrafish znf219L mRNA was examined in embryos from 12 hpf to 144 hpf. Blank PCR was performed using gene-specific primers and β -actin primers without the addition of cDNA template. Whole-mount in situ hybridization with antisense znf219L was performed at the indicated times post-fertilization (D). The images were taken from 22 to 48 hpf in the lateral view (panels a, b) and 48 hpf in dorsal view in panel c. The boxed region is enlarged to show the signal in the notochord (nc) from 22 to 48 hpf of both lateral and dorsal view (panels a', b', and c'). Dorsal view of znf219L mRNA signals in midbrain hinfbrain boundry (mhb), and hindbrain (hb) were detected from 48 to 96 hpf (panel d, e, and f). mhb, midbrain hindbrain boundry; hb, hindbrain; ov, otic vesicle; pf, pectoral fin, nc, notochord. Scale bars=100 μm.

    Article Snippet: Rapid Amplification of cDNA Ends (RACE) In order to obtain full-length cDNA, the 5' ends of zebrafish znf219L mRNA were amplified by random amplification of cDNA 5' ends (RACE) PCR using a Marathon cDNA amplification kit (Clontech, Palo Alto, CA, USA) according to the supplier's instructions.

    Techniques: Expressing, Reverse Transcription Polymerase Chain Reaction, Polymerase Chain Reaction, In Situ Hybridization

    Multiple-tissue Northern blots and RT-PCRs of fish connexins. A , Northern blot of carpCx43 mRNA. CarpCx43 mRNA is highest in brain and retina, whereas liver reveals no detectable levels. B , Ethidium bromide-stained agarose gel of A that shows comparative loading of mRNA (18S and 28S band). C , Multiple-tissue RT-PCR of the three zebrafish connexins (ethidium bromide-stained agarose gels). zfCx44.1 is less abundant in brain and retina, with higher levels in lens and heart. zfCx27.5 shows tissue-restricted expression in brain and retina, whereas zfCx55.5 is exclusively expressed in the retina. All cDNA preparations were controlled by PCR using primers specific for β-actin. D , PCR control (with β-actin primers) omitting reverse transcription. No amplification is evident with the exception of the genomic DNA, indicative of lack of genomic DNA contamination in the mRNA samples.

    Journal: The Journal of Neuroscience

    Article Title: Molecular and Functional Diversity of Neural Connexins in the Retina

    doi: 10.1523/JNEUROSCI.20-22-08331.2000

    Figure Lengend Snippet: Multiple-tissue Northern blots and RT-PCRs of fish connexins. A , Northern blot of carpCx43 mRNA. CarpCx43 mRNA is highest in brain and retina, whereas liver reveals no detectable levels. B , Ethidium bromide-stained agarose gel of A that shows comparative loading of mRNA (18S and 28S band). C , Multiple-tissue RT-PCR of the three zebrafish connexins (ethidium bromide-stained agarose gels). zfCx44.1 is less abundant in brain and retina, with higher levels in lens and heart. zfCx27.5 shows tissue-restricted expression in brain and retina, whereas zfCx55.5 is exclusively expressed in the retina. All cDNA preparations were controlled by PCR using primers specific for β-actin. D , PCR control (with β-actin primers) omitting reverse transcription. No amplification is evident with the exception of the genomic DNA, indicative of lack of genomic DNA contamination in the mRNA samples.

    Article Snippet: In addition to the screening of the genomic zebrafish library, we performed RACE extension using carp mRNA as a template for the Marathon cDNA Amplification Kit (Clontech Laboratories, Heidelberg, Germany).

    Techniques: Northern Blot, Fluorescence In Situ Hybridization, Staining, Agarose Gel Electrophoresis, Reverse Transcription Polymerase Chain Reaction, Expressing, Polymerase Chain Reaction, Amplification

    PCR characterization of alternative splicing in cyclin B mRNA. (Lanes 1 and 9 ) Molecular mass markers (φ X 174/HaeIII and λ/EcoRI + HindIII, respectively). (Lanes 2 and 3 ) RT-PCR was done using poly(A) + mRNA from unfertilized eggs as a template, and CB5-CB64 and CB5– CB62 primer pairs, respectively (see Fig. 2 ). Both pairs amplified fragments of the expected sizes. (Lanes 4 and 5 ) The same primer pairs were used, with genomic DNA as a template. While CB5-CB62 gave rise to the right-sized PCR product (compare lanes 3 and 5 ), CB5-CB64 amplified a 2.5-kbp PCR fragment (compare lanes 2 and 4). This last product was reamplified with CB5-CB62 and gave rise to a right-sized product (compare lanes 3 , 5 , and 6 ), showing part of the clone 2 cDNA sequence comprising primer CB62 was included in the 2.5-kbp genomic CB5-CB64 fragment. (Lanes 7 and 8 ) PCR amplification of genomic DNA with respectively primers CB8-CB64 and CB10-CB64 (see Fig. 2 ). Only CB10-CB64 amplified a fragment (1,300 bp, lane 8 ), whereas amplification with CB8-CB64 was unsuccessful, showing part of the clone 2 cDNA, comprising primer CB8, was not included in the 2.5-kbp genomic CB5-CB64 fragment. All PCR products showed in this figure were confirmed by cloning and partial sequencing (not shown).

    Journal: The Journal of Cell Biology

    Article Title: A Presumptive Developmental Role for a Sea Urchin Cyclin B Splice Variant

    doi:

    Figure Lengend Snippet: PCR characterization of alternative splicing in cyclin B mRNA. (Lanes 1 and 9 ) Molecular mass markers (φ X 174/HaeIII and λ/EcoRI + HindIII, respectively). (Lanes 2 and 3 ) RT-PCR was done using poly(A) + mRNA from unfertilized eggs as a template, and CB5-CB64 and CB5– CB62 primer pairs, respectively (see Fig. 2 ). Both pairs amplified fragments of the expected sizes. (Lanes 4 and 5 ) The same primer pairs were used, with genomic DNA as a template. While CB5-CB62 gave rise to the right-sized PCR product (compare lanes 3 and 5 ), CB5-CB64 amplified a 2.5-kbp PCR fragment (compare lanes 2 and 4). This last product was reamplified with CB5-CB62 and gave rise to a right-sized product (compare lanes 3 , 5 , and 6 ), showing part of the clone 2 cDNA sequence comprising primer CB62 was included in the 2.5-kbp genomic CB5-CB64 fragment. (Lanes 7 and 8 ) PCR amplification of genomic DNA with respectively primers CB8-CB64 and CB10-CB64 (see Fig. 2 ). Only CB10-CB64 amplified a fragment (1,300 bp, lane 8 ), whereas amplification with CB8-CB64 was unsuccessful, showing part of the clone 2 cDNA, comprising primer CB8, was not included in the 2.5-kbp genomic CB5-CB64 fragment. All PCR products showed in this figure were confirmed by cloning and partial sequencing (not shown).

    Article Snippet: Race PCR To isolate 5′ ends of incomplete cDNA we used 5′RACE-PCR method with the Marathon™ cDNA Amplification Kit ( CLONTECH Laboratories, Inc., Palo Alto, CA) according to the protocol of manufacturer.

    Techniques: Polymerase Chain Reaction, Reverse Transcription Polymerase Chain Reaction, Amplification, Sequencing, Clone Assay