Article Figures & Data
Figures
- 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.
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.
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.
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.
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.
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
- TABLE 1.
Primers used in this study
Primer Sequence (5′ to 3′) 501-5′ ATGTAAGCTTAGGACGCTCTCGATCCAGTAAG 501-3′ ATGTGAGCTCCCAGTTGTCTCTTCCTAACGGC 502-5′ ATGTAAGCTTAAGAGAACGGAAACCGATGACG 502-3′ ATGTGAGCTCTTGCTTTCTCTCGATCACCACG LRRP1-1 ATTCGAGAGAGGATGCGACTGGCAC LRRP1-2 TCTGGAGGTGCTGGACATTGGAGGA 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-nested GCACGTGTATCGGTCGTCAAGATT LRRP1-nested 2 TCTGGTTGTGCTTGCAATCGTC LRRP1-nested 3 TTGTCACAGTCCTGTGTGCGAGGT L952.3-nested 1 AAGCGATTGAAACGGATCAAA L952.3-nested 2 AGGACAGAAGGATACACCAGATGC L952.3-nested 3 AACAGTGCCTGCGCACCACATCT L952.3-nested 4 GGATAGTAGTGGCACGACCTTT Miniexon AACGCTATATAAGTATCAGTT D3PGDH-nested 3 CTGTCAGGTGACAGAGATGATG D3PGDH-nested 4 GCGACACCTTTCGTAAGTACTT D3PGDH-nested 5 ACTTGAGCCAACAATCACCTCC EIF-2a-RT TGCGGCCTACCTTGATTAGCTTTC EIF-2a-nested TCACCTCCGTGTACGGAATAATGC EIF-2a-21 CAGAGCAGAAGCGACACACCATT Nested(dT) CCTCTGAAGGTTCACGGATCCACATCTAGA(T)18VN B1 CCTCTGAAGGTTCACGGAT B2 CACGGATCCACATCTAGAT L6290.1-PA-1 GTTACGGCGCTGAAGAATGGAA L6290.1-PA-2 CAGTGCCTGATGAACATCGATG HEL2-PA-1 CGGCTGCTGTCGAACATATCA HEL2-PA-2 GGAGGAGGACCTCAAATTCTAC 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′ end TTGCGAAGCATTCCTAGCTCAGT
