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.

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.Trypanosomatid protozoan parasites comprise a group of eucaryotic organisms which use a number of unconventional strategies for gene expression. These include obligatory trans-splicing of a common nucleotide sequence, the miniexon, onto the 5' ends of all mRNAs (17, 57, 63, 70, 74; reviewed in 11), as well as editing of mitochondrial mRNAs (4,26). An increasing body of data suggests that some protein-coding mRNAs may be synthesized as larger polycistronic precursors, up to 60 kilobases (kb) in length, which are subsequently cleaved and processed into mature mRNAs. Evidence supporting this view includes the following: (i) determination of the size of a variant surface glycoprotein gene transcriptional unit by UV inactivation (38); (ii) demonstration of comparable transcription rates for flanking, mRNA-coding, and intergenic regions (30,46,72); and (iii) the identification of a putative polycistronic mRNA precursor (56). Whether this process is ubiquitous and what DNA sequences are involved in transcription initiation and mRNA processing remain to be determined because of the lack of striking sequence homology with consensus eucaryotic elements and the lack of in vitro or in vivo functional assays such as DNA transfection.We have examined the gene encoding ...

Stable transfection of the human parasite Leishmania major delineates a 30-kilobase region sufficient for extrachromosomal replication and expression.

“…As the DHFR-TS mRNA is not abundant (27,28,42) and transfected genes frequently are expressed less well than endogenous genes, we anticipated low levels of expression of the Neo-DHFR-TS hybrid mRNA. We employed a gradual stepwise selection, beginning at 2 ,ug of G418 per ml and progressing through three 1:10 dilutions of cells into 4 ,ug/ml and then 8 ,ug/ml (protocol A).…”

“…The locations of BgIII (B) and KpnI (K) restriction sites are shown, and the map positions (in kilobases) relative to the start codon of the DHFR-TS gene are shown at the top of the figure. Above the restriction map, the locations of poly(A)+ RNAs transcribed from this region are shown by wavy lines, with arrows denoting the direction of transcription (27). Below the restriction map are shown the segments amplified in the R1000-3 and other amplified lines (R1000 [6, 8, 21; unpublished data]) and the D7B-R1000 line (D7B-R1000 [8,23]).…”

“…that the circular DNAs contain all elements required in cis for DNA replication and expression (6,8,16,17,27). However, it is possible that the amplified DNAs are transcriptionally silent but in some manner potentiate the expression of the chromosomal locus, perhaps by titration of trans-acting factors.…”

“…and Leishmania enrietti were capable of transiently taking up and expressing exogenous DNA constructs containing genomic sequences fused to the coding region of the chloramphenicol acetyltransferase gene (3,29). We used the amplified dihydrofolate reductase-thymidylate synthase (DHFR-TS) gene contained within the extrachromosomal circular amplified R region (6) as the starting point for the construction of a DNA transfection vector, since a great deal is known about the protein-coding and transcriptional properties of this amplified DNA (7, 27; T. E. Ellenberger (27). Moreover, the locations of cis-acting elements required for transcription, processing, trans splicing, or replication have not been genetically defined for this or any other trypanosomatid gene.…”

“…Poly(A)+ RNA was prepared by chromatography on oligo(dT)-cellulose (1). Northern (RNA) blotting was performed as previously described (27). Double-stranded DNA probes were radioactively labeled with [a-32P]dCTP by random priming (20).…”