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The mini-exon gene repeats from two isolates of Trypanosoma (Nannomonas) simiae were amplified by polymerase chain reaction (PCR). The products from each isolate differed in size, however they cross-hybridised in Southern blots. The nature of the size variation was revealed by comparison of the DNA sequence of each repeat: relative to the BAN7 strain, the mini-exon gene of KETRI 2431 contained three apparent deletions that were flanked by short (> or = 6-bp) direct repeats. Furthermore, one of the cloned repeats was used as a hybridisation probe against DNA from other closely-related African trypanosomes. The lack of hybridisation of the T. (N.) simiae mini-exon gene to genomic DNA from the Forest, Kilifi and Savannah subgroups of T. (N.) congolense and T. (N.) godfreyi indicate that this PCR-hybridisation assay may be useful for distinguishing T. (N.) simiae from other members of the subgenus Nannomonas.

In Kinetoplastid protozoa, trans-splicing is a central step in the maturation of nuclear mRNAs. In Leishmania, a common 39 nt spliced-leader (SL) is transferred via trans-splicing from the precursor 96 nt SL RNA to the 5' terminus of all known protein-encoding RNAs. In this study, promoter elements of the L. tarentolae SL RNA gene have been identified with respect to transcriptional activity and putative transcription factor binding. We have mapped the essential regions in the SL RNA gene promoter at single nucleotide resolution using both in vivo transcription and in vitro protein/DNA binding approaches. Two regions located upstream of the SL RNA gene were identified: a GN3CCC element at -39 to -33 and a GACN5G element at -66 to -58 were essential for SL RNA gene transcription in stably transfected cells. Consistent with other known bipartite promoter elements, the spacing between the GN3CCC and GACN5G elements was found to be critical for proper promoter function and correct transcription start point selection, as was the distance between the two elements and the wild-type transcription start point. The GACN5G element interacts specifically and in a double-stranded form with a protein(s) in Leishmania nuclear extracts. The degree of this protein DNA interaction in vitro correlated with SL RNA gene transcription efficiency in vivo, consistent with a role of the protein as a transcription factor. The core nucleotides GACN5G fit the consensus PSE promoter structure of pol II-transcribed snRNA genes in metazoa.

First characterized in Trypanosoma brucei, the spliced leader-associated (SLA) RNA gene locus has now been isolated from the kinetoplastids Leishmania tarentolae and Trypanosoma cruzi. In addition to the T. brucei SLA RNA, both L. tarentolae and T. cruzi SLA RNA repeat units also yield RNAs of 75 or 76 nucleotides (nt), 92 or 94 nt, and approximately 450 or approximately 350 nt, respectively, each with significant sequence identity to transcripts previously described from the T. brucei SLA RNA locus. Cell fractionation studies localize the three additional RNAs to the nucleolus; the presence of box C/D-like elements in two of the transcripts suggests that they are members of a class of small nucleolar RNAs (snoRNAs) that guide modification and cleavage of rRNAs. Candidate rRNA-snoRNA interactions can be found for one domain in each of the C/D element-containing RNAs. The putative target site for the 75/76-nt RNA is a highly conserved portion of the small subunit rRNA that contains 2'-O-ribose methylation at a conserved position (Gm1830) in L. tarentolae and in vertebrates. The 92/94-nt RNA has the potential to form base pairs near a conserved methylation site in the large subunit rRNA, which corresponds to position Gm4141 of small rRNA 2 in T. brucei. These data suggest that trypanosomatids do not obey the general 5-bp rule for snoRNA-mediated methylation.

Every kinetoplastid mRNA receives a common, conserved leader sequence via the process of trans-splicing. In Leishmania tarentolae the precursor spliced leader RNA is 96 nucleotides, with a 39-nucleotide exon that is 7meG-capped and methylated on the first 4 nucleotides. trans-Splicing was inferred from the presence of tagged leader in the high molecular weight RNA population and confirmed for accuracy by cDNA cloning. Linker scan substitutions within the exon between positions 10 and 39 did not affect trans-splicing. The trans-splicing efficiency for three of the scan exons was proportional to the tagged:wild type ratio in the spliced leader precursor population. Two scan leader RNAs that were efficiently spliced showed reduced methylation. Longer exons showed reduced splicing, whereas 10- or 20-base pair deletions abolished splicing. These results indicate that size, but not content, of the exon is a constraint on the splicing process. These results, in combination with previous data eliminating a role in transcription initiation, suggest that translation may be the selective pressure on the leader content.

We have designed and synthesized eight compounds 2-9 which incorporate various amino acid residues in positions 17, 18, and 21 of the glucagon molecule: 2, [Lys17]glucagon amide; 3, [Lys18]glucagon amide; 4, [Nle17,Lys18,Glu21]glucagon amide; 5, [Orn17,18, Glu21]glucagon amide; 6, [d-Arg17]glucagon; 7, [d-Arg18]glucagon; 8, [d-Phe17]glucagon; and 9, [d-Phe18]glucagon. Compared to glucagon (IC50 = 1.5 nM), analogues 2-9 were found to have binding affinity IC50 values (in nM) of 0.7, 4.1, 1.0, 2.0, 5.0, 25.0, 43.0, and 32.0, respectively. When these compounds were tested for their ability to stimulate adenylate cyclase (AC) activity, they were found to be full or partial agonists having maximum stimulation values of 100, 100, 100, 100, 87, 78, 94, and 100%, respectively. On the basis of the X-ray crystal structure of [Lys17,18,Glu21]glucagon amide reported here, the ability to form a salt bridge between Lys18 and Glu21 is probably key to their increased binding and second messenger activities. Among the eight analogues synthesized here, only analogue 4 preserves the ability to form a salt bridge between Lys18 and Glu21. However, since these modifications are minor they do not seem to change the amphiphilic character of the C-terminus, allowing these analogues to reach 78-100% stimulation in the adenylate cyclase assay. Biological data from analogues 6-9 supports the idea that position 18 of glucagon may influence binding only, while position 17 may influence both receptor recognition and transduction.

To develop molecular markers for lower trypanosmatids, we have examined the mini-exon gene repeats of 17 isolates that were classified as Crithidia by traditional methods. Representative repeats were amplified by polymerase chain reaction and the amplification products were cloned and used as hybridization probes against genomic DNA. Six hybridization groups of Crithidia were defined on the basis of the DNA blotting experiments. The three endosymbiont-bearing species (C. deanei, C. desouzai and C. oncopelti) and C. acanthocephali each belonged to single-member hybridization groups, while the C. fasciculata group contained additional named and undesignated species. The Crithidia lucilae thermophila probe hybridized to multiple undesignated isolates. The DNA sequence of the cloned products revealed that the specificity of the hybridization probes was due to substantial differences in the intron and the nontranscribed spacer regions. These data indicate substantial heterogeneity within the mini-exon gene locus of the taxon Crithidia.

We have designed and synthesized eight compounds 2-9 which incorporate neutral, hydrophobic amino acid residues in positions 9, 11 and 16 of the glucagon molecule: (2) [desHis1, Val9. Ile11,16] glucagon amide, (3) [desHis1, Val9,11,16] glucagon amide, (4) [desHis1, Val9, Leu11,16]glucagon amide, (5) [desHis1, Nle9, Ile11,16]glucagon amide, (6) [desHis1, Nle9, Val11,16] glucagon amide, (7) [desHis1,-Nle9, Leu11,16] glucagon amide, (8) [desHis1, Val9, Leu11,16, Lys17,18, Glu21] glucagon amide and (9) [desHis1, Nle9, Leu11,16, Lys17,18, Glu21] glucagon amide. The effect of neutral, hydrophobic residues at positions 9, 11 and 16 led to good binding to the glucagon receptor. Compared to glucagon (IC50 = 1.5 nM), analogues 2-9 were found to have IC50 values of 6.0, 6.0, 11.0, 9.0, 2.5, 2.8, 6.5 and 7.0 nM, respectively. When these compounds were tested for their ability to block adenylate cyclase (AC) activity, they were found to be antagonists having no stimulation of adenyl cyclase, with pA2 values of 6.15, 6.20, 6.30, 7.25, 6.10, 7.30, 6.25 and 7.25, respectively.

Interleukin-1 beta (IL-1 beta) significantly inhibits insulin secretion from glucose stimulated islet cells. The mechanism for this inhibition has been hypothesized to be due to stimulation of the inducible form of nitric oxide synthase and a resulting increase in nitric oxide (NO) concentration. Ways to block the effect of IL-1 beta have focused on blocking the binding of IL-1 beta to the IL-1 receptor and the use of antioxidants to neutralize increases in NO. This report focuses on a 33 residue peptide synthesized based on the C-terminal region of the IL-1 beta molecule, a reported binding site of the IL-1 beta molecule, and the redoxcycling antioxidant pyrroloquinoline quinone (PQQ). The 33 residue peptide did not function as an antagonist, but as a weak agonist. High concentrations of PQQ itself inhibited glucose-dependent insulin release while low concentrations did not. PQQ had no effect on the actions of IL-1 beta. Three isosteric and isomeric analogues of PQQ were also investigated. One of the PQQ isomers had an inhibitory effect on insulin secretion at low concentrations where PQQ had no effect. These results reflect the sensitivity of islets to oxidative stress.

The role of position 10 in the beta-turn region of glucagon was investigated by substituting chiral constrained amino acids and other modifications in the N-terminal region. A series of glucagon analogues have been designed and synthesized by incorporating beta-methylphenylalanine isomers (2S,3S, 2S,3R, 2R,3R, and 2R,3S) at position 10 in order to explore the structural and topographical requirements of the glucagon receptor, and, in addition, utilizing previous studies which indicated that antagonism could be enhanced by modifications (des-His1, Glu9) and a bulky group at position 5. The structures of the new analogues are as follows: [des-His1,-Tyr5,Glu9]glucagon-NH2 (II), [des-His1,Tyr5,Glu9,Phe10]glucagon-NH2 (III), [des-His1,Tyr5,Glu9,-Ala10]glucagon-NH2 (IV), [des-His1,Tyr5,Glu9,(2S,3R)-beta-MePhe10]glucagon-NH2 (V), [des-His1,-Tyr5,Glu9,(2S,3S)-beta-MePhe10]glucagon-NH2 (VI), [des-His1,Tyr5,Glu9,D-Tyr10]glucagon-NH2 (VII), [des-His1,Tyr5,Glu9,D-Phe10]glucagon-NH2 (VIII), [des-His1,Tyr5,Glu9,D-Ala10]glucagon-NH2 (IX), [des-His1,Tyr5,Glu9,(2R,3R)-beta-MePhe10]glucagon-NH2 (X), and [des-His1,Tyr5,Glu9,(2R,3S)-beta-MePhe10]glucagon-NH2 (XI). These analogues led to dramatically different changes in in vitro binding affinities for glucagon receptors. Their receptor binding potencies IC50 values (nM) are 2.3 (II), 4.1 (III), 395.0 (IV), 10.0 (V), 170.0 (VI), 74.0 (VII), 34.5 (VIII), 510.0 (IX), 120.0 (X), and 180.0 (XI). Analogues II, III, V, VI, and XI were found to be weak partial agonists/partial antagonists with maximum stimulation between 5%-9%, while the other compounds (IV and VII-X) were antagonists unable to activate the adenylate cyclase system even at concentrations as high as 10(-5) M. In competition experiments, all of the analogues caused a right shift of the glucagon-stimulated adenylate cyclase dose-response curve. The pA2 values were 6.60 (II), 6.85 (III), 6.20 (IV), 6.20 (V), 6.10 (VI), 6.50 (VII), 6.20 (VIII), 5.85 (IX), 6.20 (X), and 6.00 (XI). Putative topographical requirements of the glucagon receptor for the aromatic side chain conformation in position 10 of glucagon antagonists are discussed.

The tandem mini-exon gene repeat is an ideal diagnostic target for trypanosomatids because it includes sequences that are conserved absolutely coupled with regions of extreme variability. We have exploited these features and the polymerase chain reaction to differentiate Phytomonas strains isolated from phloem, fruit or latex of various host plants. While the transcribed regions are nearly identical, the intergenic sequences are variable in size and content (130-332 base pairs). The mini-exon genes of these phytomonads can therefore be distinguished from each other and from the corresponding genes in insect trypanosomes, with which they are oft confused.

FUD6, a nonphotosynthetic mutant of Chlamydomonas reinhardtii, was previously found to be deficient in the synthesis of subunit IV of the cytochrome b6/f complex, the chloroplast petD gene product (C. Lemaire, J. Girard-Bascou, F.-A. Wollman, and P. Bennoun, Biochim. Biophys. Acta 851:229-238, 1986). The lesion in FUD6 is a 236-bp deletion between two 11-bp direct repeats in the chloroplast genome. It extends from 82 to 72 bp upstream of the 5' end of wild-type petD mRNA to 156 to 166 bp downstream of the 5' end. Thus, the deletion extends into the putative promoter and 5' untranslated region of petD. No petD mRNA of the normal size can be detected in FUD6 cells, but a low level of a dicistronic message accumulates, which contains the coding regions for subunit IV and cytochrome f, the product of the upstream petA gene. petD transcriptional activity in FUD6 is not significantly altered from the wild-type level. This transcriptional activity was eliminated by petA promoter disruptions, suggesting that it originates at the petA promoter. We conclude that the petD-coding portion of most cotranscripts is rapidly degraded in FUD6, possibly following processing events that generate the 3' end of petA mRNA. A chloroplast transformant was constructed in which only the sequence from -81 to -2 relative to the major 5' end of the petD transcript was deleted. Although this deletion eliminates all detectable petD promoter activity, the transformant grows phototrophically and accumulates high levels of monocistronic petD mRNA. We conclude that the petD gene can be transcribed by functionally redundant promoters. In the absence of a functional petD promoter, a lack of transcription termination allows the downstream petD gene to be cotranscribed with the petA coding region and thereby expressed efficiently.

Complex processing of primary transcripts occurs during the expression of higher-plant chloroplast genes. In Chlamydomonas reinhardtii, most chloroplast genes appear to possess their own promoters, rather than being transcribed as part of multicistronic operons. By generating specific deletion mutants, we show that petD, which encodes subunit IV of the cytochrome b6/f complex, has an RNA processing site that is required for accumulation of monocistronic petD mRNA in petD promoter deletion mutants; in such mutants, transcription of petD originates from the upstream petA promoter. The 5' ends of transcripts initiated at the petD promoter are probably also generated by processing, since the 5' end of monocistronic petD mRNA is the same in wild-type strains as it is in the petD promoter mutants. The location and function of the processing site were further examined by inserting petD-uidA fusion genes into the chloroplast genome (uidA is an Escherichia coli gene that encodes beta-glucuronidase). When a promoterless petD-uidA fusion gene was inserted downstream of petA, a monocistronic uidA transcript accumulated, which was apparently initiated at the petA promoter and was processed at a site corresponding precisely to the petD mRNA 5' end. When a construct including only sequences downstream of +25 relative to the mature mRNA 5' end was inserted into the same site, a dicistronic petA-uidA transcript accumulated but no monocistronic uidA transcript could be detected, suggesting that a processing site lies at least partially within the region from -1 to +25. Beta-glucuronidase activity was not detected in transformants that accumulated only the dicistronic petA-uidA transcript, suggesting that the first 25 bp of the 5' untranslated region are required for translation initiation. One explanation for this translational defect is that Chlamydomonas chloroplasts cannot translate the second coding region of some dicistronic messages.

RNA editing in the mitochondria of kinetoplastid protoza involves the insertion and/or deletion of precise numbers of uridine residues at precise locations in the numbers of uridine residues at precise locations in the transcribed RNA of certain genes. These genes are known as cryptogenes. In this paper we study computational algorithms to search for unknown cryptogenes and for the associated templates for insertion of uridines, gRNA sequences. The pairwise similarity search algorithm of Smith and Waterman (1) is modified to study this problem. The algorithm searches for unknown gRNAs given the cryptogene sequence. The method is tested on 4 known cryptogenes from L.tarentolae which are known to have 7 associated gRNAs. The statistical distribution of the longest gRNA when comparing random sequences is derived. Finally we develop an algorithm to search for cryptogenes using amino acid sequences from related proteins.

Six short G-rich intergenic regions in the maxicircle of Leishmania tarentolae are conserved in location and polarity in two other kinetoplastid species. We show here that G-rich region 6 (G6) represents a pan-edited cryptogene which contains at least two domains edited independently in a 3'-to-5' manner connected by short unedited regions. In the completely edited RNA, 117 uridines are added at 49 sites and 32 uridines are deleted at 13 sites, creating a translated 85-amino-acid polypeptide. Similar polypeptides are probably encoded by pan-edited G6 transcripts in two other species. The G6 polypeptide has significant sequence similarity to the family of S12 ribosomal proteins. A minicircle-encoded gRNA overlaps 12 editing sites in G6 mRNA, and chimeric gRNA/mRNA molecules were shown to exist, in agreement with the transesterification model for editing.

The complete sequences of three kinetoplast DNA minicircles (B4, D3 and D12) from Leishmania tarentolae are reported. All L. tarentolae minicircles encode single gRNAs localized within the variable region approximately 150 bp from the conserved region. The 5' termini and tentative 3' termini of the new gRNAs were determined and the gene sequences and flanking sequences of all minicircle gRNA genes compared for conserved motifs of possible transcriptional regulatory significance. All minicircle gRNAs possess 3' oligo-[U] tails of variable length similar to maxicircle gRNAs. A role for the D3 minicircle gRNA in the editing of the 5' pan-edited MURF4 mRNA was suggested by sequence analysis, and a role for the D12 minicircle gRNA in the editing of the COIII mRNA and another minicircle gRNA (Lt154) in the editing of the pan-edited G6 mRNA have been previously reported. The cryptogene mRNAs edited by the B4 and Lt19 minicircle gRNAs are yet undetermined.

Chimeric RNA molecules were detected by polymerase chain reaction amplification of kinetoplast RNA using a 3' primer specific to mRNA and a 5' primer specific to guide RNA (gRNA), and directly by Northern analysis. Covalent linkage of the 3' oligo(U) tail of the gRNA to the mRNA occurs at editing sites. Chimeric molecules were isolated for NADH dehydrogenase subunit 7 and cytochrome oxidase subunits II and III. We propose that these molecules are intermediates in the editing process and that successive transesterifications result in the transfer of uridine residues from the gRNA 3' oligo(U) tail to an editing site, with the number of uridine residues determined by base pairing with adenine and guanine "guide" nucleotides in the gRNA.

Amplification of DNA sequences from the kinetoplast minicircle DNA was employed as a method for the detection and classification of small numbers of Trypanosoma cruzi cells. Two overlapping fragments from the conserved 120 bp minirepeat regions of the minicircle DNA and one fragment covering the adjacent variable regions were amplified. The minimal amount of minicircle DNA required to detect a product by hybridization with an oligonucleotide probe was 0.015 fg, which represents approximately 10 molecules or 0.1% of the minicircle DNA component of a single cell. The amplification worked equally well with kDNA from several strains of T. cruzi and did not occur with kDNA from several other kinetoplastids. kDNA recovered from less than 10 trypanosomes in whole blood could be used as a template for amplification; the presence of a several billion fold excess of human DNA had no effect on the amplification process. Schizodeme analysis by hybridization with specific oligonucleotides or by direct restriction enzyme digestion could be performed on the amplified fragments representing the minicircle conserved region or variable regions. This method should prove useful as a rapid, specific and sensitive assay for Chagas' disease in chronic patients as well as for epidemiological studies of infected animals and insects.