Maturation of mRNA precursors in trypanosomes involves an apparent trans splicing event in which a 39-nucleotide miniexon sequence, common to all trypanosome mRNAs, is joined to the 5' end of a protein-coding exon. We demonstrate that the processing machinery responsible for the maturation of tubulin mRNA precursors in Trypanosoma brucei can be disrupted by heat shock. This results in an accumulation of polycistronic RNA species and a decrease in the abundance of branched splicing intermediates. At normal temperatures, tubulin polycistronic transcripts were also detected and were shown in pulse-chase experiments to be abundantly synthesized and very rapidly turned over. These results, combined with results of the heat shock experiments, suggest that these polycistronic transcripts are the precursors of the (monocistronic) tubulin mRNAs of trypanosomes.Formation of the 5' end of trypanosome mRNAs entails the addition of a 39-nucleotide miniexon or spliced-leader sequence to an mRNA precursor (reviewed in references 2 and 4). In Trypanosoma brucei this miniexon sequence initially exists as the first 39 nucleotides of an approximately 140-nucleotide capped RNA (13,45,53) termed the miniexon-derived RNA (medRNA; 6, 24, 34). Equivalent RNAs, ranging in size down to the 84-nucleotide medRNA of Crithidiafasciculata (38), have been described for a number of kinetoplastid species (34,35). In every species examined, medRNA is encoded by a segment of the nuclear genome that is tandemly repeated approximately 200 times (10,35,38,42). The function of the miniexon sequence (per se) remains unknown.Although conventional introns are apparently absent from the trypanosome genome, the involvement of splicing in the synthesis of mRNA in trypanosomes is clearly indicated by the presence of a consensus 5' splice site (36) on the medRNA immediately downstream of the miniexon sequence (6,24,34) and by consensus 3' splice sites (36) directly preceding the protein-coding exons of all genes examined (3,15,54,57,58).Several models describing the mechanism of attachment of the miniexon sequence to the acceptor pre-mRNA have been proposed (6,25,34). Recently evidence supporting a trans splicing mechanism has been provided by the discovery that the 102-nucleotide intron portion of medRNA (termed minRNA, for mini-intron RNA) can be released as a discrete product from high-molecular-weight poly(A)+ RNA by treatment with HeLa cell debranching activity (26,40,52). This suggests that medRNA participates in a trans splicing reaction in which the intron portion of medRNA becomes transiently associated with pre-mRNA through a 2'-5' phosphodiester linkage (53a Little is known about the structure of transcription units in trypanosomes. Many of the protein-coding genes of trypanosomes are found to be organized as tandem repeats (7,27,33,37,43). The protein-coding regions of all genes examined to date are continuous, lacking internal introns.The tubulin locus of T. brucei consists of approximately 15 head-to-tail repeats of alternating a and a coding se...

A 2.76 kb segment of the 12 kb divergent region of the Leishmania tarentolae kinetoplast maxicircle DNA consists almost entirely of repeated sequences. The repeats can be grouped into six families, some of which are present throughout the remainder of the divergent region. The repeats are oriented in a head-to-tail fashion with the three simplest repeats clustered into large arrays. A 47 bp palindrome and two copies of a "supercluster" of three different types of repeats are also present in the sequenced region. A sequence change in the divergent region is described for a clonal strain of L. tarentolae which was passaged continuously for several years. The repetitive sequences found in the divergent region appear to be appropriate substrates for the presumed deletion/insertion/recombination events occurring in this rapidly evolving portion of the maxicircle.

Proper initiation of transcription by RNA polymerase II requires the TATA-consensus-binding transcription factor TFIID. A cDNA clone encoding the Drosophila TFIID protein has been isolated and characterized. The deduced amino acid sequence reveals an open reading frame of 353 residues. The carboxyl-terminal 180 amino acids are approximately 80% identical to yeast TFID and 88% identical to human TFIIID. The amino-terminal portions of the yeast and Drosophila TFIID proteins lack appreciable homology, whereas the Drosophila and human amino termini appear qualitatively similar. In addition, the amino-terminal region of the Drosophila TFID contains several sequence motifs that are found in other Drosophia proteins which appear to regulate transcription. (6)(7)(8). One key general transcription factor is TFIID, which binds to the TATA element in Drosophila (9), human (10, 11), and yeast (12)(13)(14). TFIID has been shown to be required for preinitiation complex formation, suggesting that it functions at an early critical step in the initiation process (15, 16). The yeast and human TFIID proteins function interchangeably to support a basal level oftranscription in vitro, implying significant structural conservation between these molecules (12)(13)(14).The recent cloning ofthe yeast (Saccharomyces cerevisiae) TFIID gene has allowed an examination of the functional and putative structural properties of this transcription factor (17-21 A Drosophila melanogaster embryonic (0-24 hr) cDNA library in AZAP II (Stratagene) was screened by hybridization with a cloned PCR probe. Hybridizations were performed at 400C in 6x SSPE/0.25% nonfat dried milk/50%o deionized formamide. Filters were washed at 650C in 2x SSC (lx = 0.15 M NaCl/15 mM sodium citrate, pH 7.0)/0.1% NaDodSO4. After plaque purification, individual cDNA inserts were recovered in the form of chimeric pBluescript SK(+) phagemids by in vivo excision from the A vector as described by the library supplier.Overproduction of Recombinant Drosophila TFVD. The conserved region ofDrosophila TFIID was subcloned into an Escherichia coli expression system as follows. The Apa I-EcoRI fragment of the Drosophila TFIID cDNA insert encoding the carboxyl-terminal 1% amino acid residues was inserted into the pET-8c (T7 expression) vector (27) at the Nco I-BamHI restriction sites. A BamHI site was added to the EcoRI site of the TFIID insert by ligation of an EcoRIBamHI (duplex oligonucleotide) adaptor (New England Biolabs). The modified TFIID insert was then ligated into pET-8c cut with BamHI and Nco I. The 3' overhang of the TFIID Apa I site was joined to the 5' overhang of the Nco I site of pET-8c by use of an eight-base "bridging oligonucleotide" with sequence complementarity to both the Nco I and Apa I overhangs (5'-CATGGGCC-3'). The trimolecular ligation joined the initiating methionine codon of the pET-8c vector in-frame to the Gly-158 codon of the TFIID open reading frame. Plasmid DNA samples prepared from XL1-Abbreviations: PCR, polymerase chain reaction; IPTG, isopropy...

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A repeated sequence from the Crithidia fasciculata nuclear genome has been isolated which is homologous to the mini-exon genes of other kinetoplastid protozoa. Sequence analysis of the 417 bp monomeric unit confirmed the presence of a 35 nt sequence within the repeat that is 77% homologous with the Trypanosoma brucei 35-mer mini-exon or spliced leader sequence. The repeat is present at approximately 250 copies per cell and is organized into one, or a few, large head to tail tandem clusters predominantly on a single chromosome. The mini-exon repeat unit hybridizes to a major 84 nt and a minor 87 nt poly (A)- steady state transcript, the first 35 nts of which comprise the mini-exon sequence found at the 5' end of mRNAs in several other kinetoplastid species. The 3'-termini of the transcripts map to positions on the DNA sense strand directly preceeding a stretch of 8 thymidine residues. Crithidia represents the most primitive kinetoplastid species which apparently possesses a discontinuous type of mRNA processing, implying that this represents a conserved feature in possibly all genera of kinetoplastid protozoa.

Transcripts for six Leishmania tarentolae maxicircle structural genes (cytochrome oxidase subunits I, II and III, cytochrome b, human mitochondrial unidentified reading frames 4 and 5) and several unidentified open reading frames were mapped, and the locations of the 5' ends determined by primer runoff analysis. All genes studied here are transcribed from the same strand as the 12S and 9S ribosomal RNAs except for the cytochrome oxidase subunit I gene. In two cases (ORF3 and ORF4, ORF5 and ORF6), a single transcript covers two contiguous overlapping reading frames. The 5' ends of the RNAs are located 20-64 nt from the putative translation initiation codons. Primary transcripts from a mitochondrial RNA preparation were 5' end-labeled with guanylyltransferase and alpha -32P-GTP; the major labeled species comigrated with the 12S and 9S mitochondrial rRNAs, and in addition there were at least four higher molecular weight labeled species.

We have isolated and characterized a developmentally regulated gene in Trypanosoma brucei, arbitrarily termed BS2. BS2 mRNA is substantially more abundant in bloodstream-form trypanosomes than in procyclic culture forms. Its nucleotide sequence reveals a single contiguous open-reading frame of 497 codons and is predicted to encode a protein of approximately 55.5 kilodaltons. A search of the NBRF protein data base revealed that within the predicted amino acid sequence are two of the evolutionarily conserved redox sites typified by thioredoxin of bacteria. Of this family of proteins, the recently sequenced rat genes encoding protein-disulfide isomerase (PDI) and form I phosphoinositide-specific phospholipase C (PIPLC) showed homology extending over the length of all three proteins (i.e., between BS2, PDI, and PIPLC). Although this homology includes the acidic C-terminus characteristic of proteins localized to the lumen of the endoplasmic reticulum, the BS2 product is predicted to possess multiple sites for N-linked glycosylation while PDI and PIPLC have none. Possible roles of the BS2 gene product in trypanosome physiology are discussed.

The conserved portions of the maxicircle DNAs of Leishmania tarentolae and Trypanosoma brucei are organized in a basically colinear manner over a 15-to 17-kilobase region that is interrupted by two small less-homologous sequences. The most highly conserved regions are those encoding the 9S and 12S genes. An approximately 12-kilobase region directly upstream of the 12S gene in the L. tarentolae maxicircle showed no sequence homology with the T. brucei maxicircle and also was not transcribed. An approximately 6-kilobase region in the T. brucei maxicircle in the same relative location also showed no sequence homology with the L. tarentolae maxicircle. We propose that evolution of maxicircle DNA occurs mainly within.this "divergent region."The mtDNA or kinetoplast DNA (kDNA) of the kinetoplastid protozoa (1, 2) consists of a catenated network of minicircles and maxicircles. The minicircles comprise approximately 95% of the mass of the network, are usually heterogeneous in sequence, and are probably not transcribed (3-5). The maxicircles are present at 20-50 copies per network, are homogeneous in sequence, and are extensively transcribed (6-9). Maxicircle DNA contains the genes for the putative mitochondrial miniribosomal RNAs and also contains sequences homologous to several structural genes from yeast (10) and Zea mays (11). In the African trypanosome Trypanosoma brucei, the maxicircle appears to be evolving in a complex fashion involving both base substitutions and insertions/deletions of up to 1.5 kilobases (kb) in a region of the molecule termed the "silent" or "variable" region (12).Interspecific size variation of maxicircle DNA is much more pronounced, ranging from 3 to 17 kb (2). Conservation of the 9S and 12S gene sequences has been shown for several species (13)(14)(15)(16), and conservation of other maxicircle sequences has also been reported for T. brucei and Trypanosoma cruzi (17) (19). The purified maxicircle DNA was digested with Msp I and the fragments were separated in agarose and isolated by electroelution into dialysis bags. T. brucei kDNA was digested with HindIII and the 9.3-kb maxicircle A fragment (see Fig. 2) was isolated by electroelution after separation in agarose. The 6.5-kb B and the 6.1-kb C maxicircle HindIII fragments were obtained from the pBR322 clones, pTbM-2 and pTbM-1, described previously (16). HindIII-excised inserts were recovered by electroelution following electrophoresis through agarose. The Hha I subfragments, a and b, of the L. tarentolae maxicircle Msp I fragment 3 were cloned into the EcoRI site of pBR322 after treatment with the Klenow fragment of Escherichia coli DNA polymerase I and EcoRI linker addition. Insert DNA was isolated from EcoRI-digested plasmid by agarose electrophoresis and electroelution. The 6.6-kb EcoRI/BamHII fragment (see Fig. 6) of the L. tarentolae maxicircle was obtained from the pBR322 clone, pLtl20 (6). Purified 120 insert DNA was digested with Sau3A and the fragments were isolated by electroelution after separation in agarose. Control ...