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.

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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...

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.