Abstract. Null mutants of the Trypanosoma cruzi insect stage-specific glycoprotein GP72 were created by targeted gene replacement. Targeting plasmids were constructed in which the neomycin phosphotransferase and hygromycin phosphotransferase genes were flanked by GP72 sequences. These plasmids were sequentially transfected into T. cruzi epimastigotes by electroporation. Southern blot analyzes indicated that precise replacement of the two genes had occurred. No abetrant rearrangements occurred at the GP72 locus and no GP72 gene sequences had been translocated elsewhere in the genome. Western blots confirmed that GP72 is not expressed in these null mutants. The morphology of the mutants is dramatically different from wild-type. In both mutant and wild-type parasites, the flagellum emerges from the flagellar pocket. In the null mutant the normal attachment of the flagellum to the cell membrane of the parasite is lost.

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Mature mRNAs of trypanosomatid protozoa result from the joining of at least two exons, which are initially transcribed as separate RNAs. In all trypanosomatids examined to date, the first exon (mini-exon) is encoded by approximately 200 tandemly reiterated genes. In characterizing the mini-exon genes of Leptomonas seymouri, we identified two predominant size classes of repetitive sequences that hybridized strongly to the L. seymouri mini-exon sequence. These two sequences are arranged as interspersed clusters. DNA sequence analysis of a clone representing the smaller size class demonstrated that these sequences have the capacity to encode a mini-exon donor (med)RNA corresponding to the 86 nt component seen in Northern blots of L. seymouri RNA. The larger size class comprises a family of related sequences, some of which contain DNA inserted into the mini-exon portion of the medRNA gene. The specific insert identified here (LINS 1) is exclusively associated with medRNA sequences, and is present in approximately 20% of the larger size class of L. seymouri medRNA genes. Disregarding the insertion, the sequences of the smaller bona fide mini-exon genes and the gene copy containing the insert were almost identical. The insert sequence is transcribed in the same direction as medRNA to yield at least four small non-polyadenylated RNAs, which appeared not to be linked to medRNA sequences.

In a previous paper we presented evidence for the existence of at least two alpha 1-acid glycoprotein (AGP) genes in the mouse. One of the cDNA clones characterized in those studies was used to isolate several unique AGP genomic clones. In these studies we present the complete sequence of one of the mouse AGP genes. The sequence analysis includes 595 base pairs (bp) 5' to the site of initiation of transcription and 135 bp 3' to the polyadenylation signal. This mouse AGP gene, designated AGP-1, has six exons, a structure similar to those of the AGP genes in rats and humans. Analysis of the sequence has revealed a number of potential regulatory sites. These include a run of alternating purine-pyrimidine bases [(GT)N] at +2890 to +2945, flanked by three potential glucocorticoid receptor binding sites within intron 5. Two of these TGTTCT at +3069 to +3074 and +3082 to +3087 flank the (GT)n track at its 3' end, and one, which is oriented in the opposite direction (AGAACA), at +2771 to +2776 flanks the track at its 5' end. A longer version of the glucocorticoid receptor site, GGGTACAATGTGTCCT, has been located in the 5' flanking region of the gene (-94 to -79); the sequence AGAACA is another potential glucocorticoid receptor site oriented in the opposite direction and located at -127 to -122. This entire region, from -146 to -42, in the mouse has a strong homology (approximately 85%) to the 5' flanking region of the rat AGP gene, which contains a 78-bp fragment (-120 to -42) that represents the minimal sequence required for glucocorticoid regulation.(ABSTRACT TRUNCATED AT 250 WORDS)

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