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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.

Department of Biological Chemistry and Molecular Pharmacology, Harvard Medical School, Boston, Massachusetts 02115.

ABSTRACT

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.

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