Transcriptional mapping of the amplified region encoding the dihydrofolate reductase-thymidylate synthase of Leishmania major reveals a high density of transcripts, including overlapping and antisense RNAs.
We have examined the transcriptional organization of the R region of the protozoan parasite Leishmania major. This region encodes the bifunctional enzyme dihydrofolate reductase-thymidylate synthase (DHFR-TS) and is frequently amplified as a 30-kilobase (kb) extrachromosomal circular DNA in methotrexate-resistant lines. Northern (RNA) blot analysis shows that the R region encodes at least 10 stable cytoplasmic polysomal poly(A)+ RNAs, ranging in size from 1.7 to 13 kb and including the 3.2-kb DHFR-TS mRNA. Transcriptional mapping reveals that these RNAs are closely spaced and collectively cover more than 95% of the 30-kb amplified R region. The organization is complex, including several overlapping RNAs 3' of DHFR-TS and two examples of antisense RNAs 5' of DHFR-TS. The R region RNAs can be grouped into two empirical domains, with eight contiguous RNAs transcribed in the same direction as that of DHFR-TS and two contiguous RNAs transcribed in the orientation opposite to that of DHFR-TS. The two 5'-most RNAs of the DHFR-TS-containing domain overlap the RNAs transcribed from the opposite strand. These data are relevant to models of transcription, including recent studies suggesting polycistronic transcription in trypanosomatids. The abundance of R region RNAs increases uniformly 10- to 15-fold in the amplified R1000-3 line relative to the wild type, and no new RNAs were observed. This suggests that all elements required in cis for DHFR-TS expression are contained within the 30-kb circular DNA. Quantitative analysis reveals that the steady-state DHFR-TS mRNA and protein levels are not growth phase regulated, unlike the monofunctional mouse DHFR. DHFR-TS is developmentally regulated, however, declining about fivefold in lesion amastigotes relative to promastigotes.
Kapler
G M
GM
Department of Biological Chemistry and Molecular Pharmacology, Harvard Medical School, Boston, Massachusetts 02115.
Beverley
S M
SM
D000818:Animals; D002874:Chromosome Mapping; D005784:Gene Amplification; D007895:Leishmania tropica / Q000235:genetics* / Q000254:growth & development / Q000378:metabolism; D011132:Polyribosomes / Q000378:metabolism; D012313:RNA / Q000235:genetics / Q000378:metabolism; D016372:RNA, Antisense; D012333:RNA, Messenger / Q000235:genetics; D012689:Sequence Homology, Nucleic Acid; D013762:Tetrahydrofolate Dehydrogenase / Q000235:genetics*; D013940:Thymidylate Synthase / Q000235:genetics*; D014158:Transcription, Genetic
D016372:RNA, Antisense; D012333:RNA, Messenger; D012313:RNA; D013762:Tetrahydrofolate Dehydrogenase; D013940:Thymidylate Synthase