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ARTICLE

Transcription Initiation and Termination on Leishmania major Chromosome 3

Santiago Martínez-Calvillo, Dan Nguyen, Kenneth Stuart, Peter J. Myler
Santiago Martínez-Calvillo
1Seattle Biomedical Research Institute, Seattle, Washington 98109-1651
2Departments of Pathobiology
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Dan Nguyen
1Seattle Biomedical Research Institute, Seattle, Washington 98109-1651
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Kenneth Stuart
1Seattle Biomedical Research Institute, Seattle, Washington 98109-1651
2Departments of Pathobiology
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Peter J. Myler
1Seattle Biomedical Research Institute, Seattle, Washington 98109-1651
2Departments of Pathobiology
3Medical Education and Biomedical Informatics, University of Washington, Seattle, Washington 98195
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  • For correspondence: peter.myler@sbri.org
DOI: 10.1128/EC.3.2.506-517.2004
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  • FIG. 1.
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    FIG. 1.

    Effect of UV irradiation on chr3 transcription. Nuclear run-on assays were performed with nuclei isolated from 2 × 108 promastigotes which were irradiated with three different intensities of UV light (1.25, 2.5, and 5 kJ/m2, as indicated below each panel). Run-on RNA was radiolabeled by 6-min incubation, extracted, and hybridized to dot blots of single-stranded M13 DNAs (1 μg) that contain inserts which are complementary to the top (T) or bottom (B) strand of several regions of chr3. A control experiment with nonirradiated cells is shown in the left panel. Control DNAs include genes 27 to 32 and fragments AC and DE from chr1, fragments R-1 to R-4 from the rRNA locus on chr27, and α-tubulin. A map of chr3 is shown below the figure. Arrows indicate the regions of Pol II transcription initiation. The dashed arrow denotes the putative initiation region for gene 68.

  • FIG. 2.
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    FIG. 2.

    Relative rates of transcription as a function of UV intensity. (A to C) The results shown in Fig. 1 and another similar experiment (not shown) were quantified, and the transcription signal for each clone, relative to the nonirradiated control, was plotted against UV dose. The average slope (K) of the curves from each fragment was calculated using the equation K = (ln R0/R)/d, where R0 is nascent RNA synthesis in nuclei made from nonirradiated cells and R is nascent RNA synthesis at UV dose d (13). (D to F) K is plotted versus the distance from the putative promoter to the midpoint of each fragment. (A and D) Gene 1 and fragments I, II, and III; (B and E) genes 2 to 5, 67, and 68 and fragment IV; (C and F) genes 98, 95, and 88 and fragments VII to IX.

  • FIG. 3.
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    FIG. 3.

    Pol II transcription on chr3 initiates in two different regions. (A) Sequence between genes 1 (LmjF3.0010) and 2 (LmjF3.0020). (B) Sequence upstream of gene 98 (LmjF3.0980). The two putative TSSs for gene 1, the putative TSS for gene 2 and the two putative TSSs for gene 98 are indicated with the arrows. The SL acceptor sites (CT for genes 1 and 98, and AG for gene 2) are underlined and highlighted. Protein-coding sequences are shown in capital letters and boldface type. The sequences of fragments III, VI, I and VIII are boxed.

  • FIG. 4.
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    FIG. 4.

    Identification of the RNA polymerases that transcribe chr3. Nuclear run-on RNA was radiolabeled in the presence of α-amanitin (200 μg/ml) or tagetitoxin (160 μM) and hybridized to filters containing single-stranded DNAs from the tRNALys gene and genes 3 and 4 from chr3. A tRNA fragment from chr23 was used as a control, together with α-tubulin, small-subunit rRNA (18S), and mp18 vector with no insert (M13). The left panel shows a control experiment performed in the absence of any transcription inhibitor. Abbreviations: T, DNA complementary to top strand; B, DNA complementary to bottom strand.

  • FIG. 5.
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    FIG. 5.

    Sequence around the tRNALys gene (LmjF3.tRNALys.01). The tRNALys gene fragment used in the nuclear run-on assays and cloned into pNBUC and pLMRIB is highlighted. Also highlighted is the sequence of fragment VI. Coding sequences are shown in capital letters and boldface type. The putative internal control elements for the tRNA gene, boxes A and B, are underlined, as well as the run of four Ts located downstream of the gene and the C located at position 259345. The three putative polyadenylation sites for gene 68 and the two for gene 69 are indicated.

  • FIG. 6.
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    FIG. 6.

    The tRNALys-gene region is involved in termination of transcription. (A) Maps of plasmids used in the transfection experiments. Ble-Luc represents the phleomycin-luciferase fusion gene, which is flanked by 5′- and 3′-dihydrofolate reductase intergenic regions from L. major. The tRNA box represents the tRNALys gene from chr3. The arrow on the rRNA box indicates the initiation site of the LmjF rRNA genes. A fragment from open reading frame 72 from chr1 (Chr1_0650) was cloned into pLM72-5′ and -3′. (B and C) Transient-transfection assays. Luciferase activity was tested 24 h after LmjF promastigotes were transfected by electroporation with the constructs indicated. The results shown were obtained from three independent experiments, each performed in triplicate. The numbers above each column represent a relative percentage of activity, with the results obtained with the parental plasmids pNBUC (no promoter) and pLMRIB (with the rRNA promoter) set at 100%. Error bars, standard deviations.

Tables

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  • TABLE 1.

    Primers used in this study

    PrimerSequence (5′ to 3′)
    501-5′ATGTAAGCTTAGGACGCTCTCGATCCAGTAAG
    501-3′ATGTGAGCTCCCAGTTGTCTCTTCCTAACGGC
    502-5′ATGTAAGCTTAAGAGAACGGAAACCGATGACG
    502-3′ATGTGAGCTCTTGCTTTCTCTCGATCACCACG
    LRRP1-1ATTCGAGAGAGGATGCGACTGGCAC
    LRRP1-2TCTGGAGGTGCTGGACATTGGAGGA
    1000-5′ATGTAAGCTTACCGCGAATGATAGAGAGGAAC
    1000-3′ATGTGAGCTCCTTGCGACTGTTCCACAGAGAC
    1001-5′ATGTAAGCTTTGCGATTTGTATCGGAACGAGG
    1001-3′ATGTGAGCTCCGCTATGTTCTTTGGCACGTGG
    1002-5′ATGTAAGCTTCGTATTTCTCCGTTCGGGTATC
    1002-3′ATGTGAGCTCAAGCGAGAGCTGATGCAAAGTG
    D3PGDH-5′ATGTAAGCTTTGGGATCGGCTGTTTCTGCATC
    D3PGDH-3′ATGTGAGCTCTGCAGCTCCTTGTTCGACCCAG
    2AEPAT-5′ATGTAAGCTTATCAGTGCCTTTGGCGGTATCC
    2AEPAT-3′ATGTGAGCTCGTCAATGCCCATGGATTTGAGC
    L952.4-5′ATGTAAGCTTGAGGCCAACCGTCGTGTATGAC
    L952.4-3′ATGTGAGCTCAAGATCAGCAATGCGGACGAAC
    U2AF23-5′ATGTAAGCTTCATTGACTGTGCGACACTTCAC
    U2AF23-3′ATGTGAGCTCACTGCTCCTTCTCCAGCTTCTC
    L6202.3-5′ATGTAAGCTTGCGCCCTTCAAGTATTATACGC
    L6202.3-3′ATGTGAGCTCCTCCACAGAGAGCAGACAAGGC
    L6910.7-5′ATGTAAGCTTACTCCACTACAATGCACTGTGC
    L6910.7-3′ATGTGAGCTCTAGAACGGTGATAATTCAACGG
    2-int.L6910.7/L6290.1-5′ATGTAAGCTTTGTTGTTGGTTTCGCTTTGCTC
    2-int.L6910.7/L6290.1-3′ATGTGAGCTCGATCGGCATTTGTGTAGGCATG
    L6290.1-5′ATGTAAGCTTTAACCGCGCCTGAGGTGTACTC
    L6290.1-5′ATGTGAGCTCTTCCATTCTTCAGCGCCGTAAC
    int.L6290.1/tRNA-5′ATGTAAGCTTGGTGTGGACACGCTGACGAAAG
    int.L6290.1/tRNA-3′ATGTGAGCTCCGGCGTAATGTCAACGAAGACC
    tRNA-Lys-5′ATGTAAGCTTTGCCTACGGCTTTGTCCAGGAG
    tRNA-Lys-3′ATGTGAGCTCTTCAACACCCTCCCACCCACAG
    HEL2-5′ATGTAAGCTTGTGGCGGAAGAAGACGATGTGG
    HEL2-3′ATGTGAGCTCCTTCGACGCCACCGTTGAGATC
    L505.2-5′ATGTAAGCTTTGTCTGACGGCACCGTCGATTG
    L505.2-3′ATGTGAGCTCGGGCGCATCACTGGATGATTGG
    L7234.2-5′ATGTAAGCTTTCCATCGACGACGAGCTTGTAC
    L7234.2-3′ATGTGAGCTCGTGGACGCCCTTCTCACCTATG
    MCO1-5′ATGTAAGCTTAGCGACATGTTTATGTAGCACG
    MCO1-3′ATGTGAGCTCTGTCTTGACGCTAGTGAACGAC
    EIF-2a-5′TCCTTCAGCTCTGTATTAGTCCG
    EIF-2a-3′CAATAACGAGCGTCCAGAGTGGA
    2001-5′ATGTGAATTCTTATTGCTACCCTTCGTCTCAC
    2001-3′ATGTGCATGCGCCAATTTCAACTGTTCAAGTC
    2002-5′ATGTGAATTCACCGTGGCACGAACAATAAACG
    2002-3′ATGTGCATGCGACAAGCTTCCGCTCAGCAGAC
    2003-5′ATGTGAATTCGTCGTCGCAGTTTGTTCTCCTG
    2003-3′ATGTGCATGCCGCACTCCTATGGGTTAACGTG
    tRNA cluster-5′GAAGGACAGACAGTTGGGACCA
    tRNA cluster-3′TACCAGGTTCAGGTGGGAGGAA
    LRRP1-RT′CAGATGTAGTGGTCGCAGTGATTG
    LRRP1-nestedGCACGTGTATCGGTCGTCAAGATT
    LRRP1-nested 2TCTGGTTGTGCTTGCAATCGTC
    LRRP1-nested 3TTGTCACAGTCCTGTGTGCGAGGT
    L952.3-nested 1AAGCGATTGAAACGGATCAAA
    L952.3-nested 2AGGACAGAAGGATACACCAGATGC
    L952.3-nested 3AACAGTGCCTGCGCACCACATCT
    L952.3-nested 4GGATAGTAGTGGCACGACCTTT
    MiniexonAACGCTATATAAGTATCAGTT
    D3PGDH-nested 3CTGTCAGGTGACAGAGATGATG
    D3PGDH-nested 4GCGACACCTTTCGTAAGTACTT
    D3PGDH-nested 5ACTTGAGCCAACAATCACCTCC
    EIF-2a-RTTGCGGCCTACCTTGATTAGCTTTC
    EIF-2a-nestedTCACCTCCGTGTACGGAATAATGC
    EIF-2a-21CAGAGCAGAAGCGACACACCATT
    Nested(dT)CCTCTGAAGGTTCACGGATCCACATCTAGA(T)18VN
    B1CCTCTGAAGGTTCACGGAT
    B2CACGGATCCACATCTAGAT
    L6290.1-PA-1GTTACGGCGCTGAAGAATGGAA
    L6290.1-PA-2CAGTGCCTGATGAACATCGATG
    HEL2-PA-1CGGCTGCTGTCGAACATATCA
    HEL2-PA-2GGAGGAGGACCTCAAATTCTAC
    tRNA-Bam-5′ATGTGGATCCTGCCTACGGCTTTGTCCAGGAG
    tRNA-Bam-3′ATGTGGATCCTTCAACACCCTCCCACCCACAG
    72-Bam-5′ATGTGGATCCCAACGTCGTCACAACGTACAGC
    72-Bam-3′ATGTGGATCCTGGTGAATGTAGCAGCGACACG
    tRNA-dTTTT-5′CGATCCCCACGGAGTGCGCCCCCCTTGGCTGCGCCAAGCC
    tRNA-dTTTT-3′GGCTTGGCGCAGCCAAGGGGGGCGCACTCCGTGGGGATCG
    chr3-tss2-5′ACACAAGCACGGGAGCTGCGGTGA
    tRNA-5′ endTTGCGAAGCATTCCTAGCTCAGT
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Transcription Initiation and Termination on Leishmania major Chromosome 3
Santiago Martínez-Calvillo, Dan Nguyen, Kenneth Stuart, Peter J. Myler
Eukaryotic Cell Apr 2004, 3 (2) 506-517; DOI: 10.1128/EC.3.2.506-517.2004

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Transcription Initiation and Termination on Leishmania major Chromosome 3
Santiago Martínez-Calvillo, Dan Nguyen, Kenneth Stuart, Peter J. Myler
Eukaryotic Cell Apr 2004, 3 (2) 506-517; DOI: 10.1128/EC.3.2.506-517.2004
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KEYWORDS

Chromosomes
Leishmania
Transcription, Genetic

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