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N R Sturm

Publications and source records attributed to N R Sturm.

31 records · Page 2Linked to original sources
PubMed1994

petD mRNA maturation in Chlamydomonas reinhardtii chloroplasts: role of 5' endonucleolytic processing.

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.

Animals↗
PubMed1992

Generation of unexpected editing patterns in Leishmania tarentolae mitochondrial mRNAs: misediting produced by misguiding.

We have analyzed the generation of unexpected patterns of RNA editing, i.e., those not following a strict 3' to 5' progression, which occur in junction regions between fully edited and preedited sequences. Evidence is presented that these patterns are generated by misediting due to specific events of misguiding. Misediting can occur through the interaction of inappropriate gRNAs with mRNAs or appropriate gRNAs in an incorrect fashion. Four possible mechanisms for the generation of misedited sequences are presented. Chimeric molecules have been detected in steady-state mitochondrial RNAs that are composed of misguiding gRNAs covalently linked to mRNAs at misediting sites by the 3' oligo(U) tail. We propose that misediting within junction regions can be corrected by appropriately acting gRNAs.

Animals↗
PubMed1992

An intergenic G-rich region in Leishmania tarentolae kinetoplast maxicircle DNA is a pan-edited cryptogene encoding ribosomal protein S12.

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.

Amino Acid Sequence↗
PubMed1991

Leishmania tarentolae minicircles of different sequence classes encode single guide RNAs located in the variable region approximately 150 bp from the conserved region.

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.

Animals↗
PubMed1991

Chimeric gRNA-mRNA molecules with oligo(U) tails covalently linked at sites of RNA editing suggest that U addition occurs by transesterification.

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.

Animals↗
PubMed1990

Partially edited mRNAs for cytochrome b and subunit III of cytochrome oxidase from Leishmania tarentolae mitochondria: RNA editing intermediates.

Partially edited mRNAs were selected by the polymerase chain reaction and sequenced. In the case of cytochrome b, 102 out of 106 clones displayed patterns of editing that were consistent with a strictly progressive 3' to 5' editing process, as predicted by the guide RNA model of RNA editing. In the case of cytochrome oxidase subunit III (COIII), 177 out of 304 clones displayed strictly progressive 3' to 5' patterns of editing. However, the remaining 127 COIII clones displayed unexpected patterns in which upstream editing preceded downstream editing, uridines were inserted at sites not normally edited, and purine residues were deleted. We suggest that many of these RNAs are produced by normal 3' to 5' editing of the COIII mRNA with incorrect guide RNA molecules.

Animals↗
PubMed1990

Kinetoplast DNA minicircles encode guide RNAs for editing of cytochrome oxidase subunit III mRNA.

Guide RNAs (gRNAs) for the editing of sites 1-8 of COIII mRNA and an "unexpected" partially edited COIII mRNA are encoded in the variable regions of specific kinetoplast DNA minicircles. The gRNAs can form 37 and 44 nucleotide perfect hybrids (allowing for G-U base pairs) with edited mRNAs. The gRNAs were detected on Northern blots and shown to have unique 5' ends situated close to the beginning of the potential base pairing with the edited mRNAs. We suggest that kinetoplast DNA minicircle molecules in general may encode gRNAs for editing of cryptogene mRNAs by a mechanism similar to that previously proposed for editing by maxicircle-encoded gRNAs.

Animals↗
PubMed1989

Sensitive detection and schizodeme classification of Trypanosoma cruzi cells by amplification of kinetoplast minicircle DNA sequences: use in diagnosis of Chagas' disease.

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.

Animals↗
PubMedDate not supplied

Mini-exon gene sequences define six groups within the genus Crithidia.

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.

Animals↗
PubMedDate not supplied

5S ribosomal RNA gene repeat sequences define at least eight groups of plant trypanosomatids (Phytomonas spp.): phloem-restricted pathogens form a distinct section.

Trypanosomatids isolated from plants have been assigned typically into the genus Phytomonas. Such designations do not reflect the biology of the diverse isolates; confusion may arise due to the transient presence in plants of monogenetic (insect) trypanosomatids deposited by phytophagous bugs. To develop further molecular markers for the plant kinetoplastids, we have obtained the DNA sequence of the 5S ribosomal RNA gene from 24 isolates harvested from phloem, latex, and fruit. Small, distinct sequence differences were found at the 3'-ends of the transcribed regions; substantial sequence and size differences were found in the non-transcribed regions. Alignment of the gene sequences from all the isolates suggested the presence of eight groupings. While six groups contained isolates from single plant tissues, groups C and A contained isolates from both fruit and latex. The DNA sequences of the 10 phloem-restricted pathogenic isolates from South America and the Carribean were highly conserved and thus comprised a single group (H). The conserved nature of the 5S ribosomal RNA genes in these plant pathogens supports the proposal that they be considered as a distinct section, the phloemicola.

Animals↗
PubMedDate not supplied

Diplonema spp. possess spliced leader RNA genes similar to the Kinetoplastida.

The phylogenetic placement of the genus Diplonema in relation to fellow phylum members Euglena and Trypanosoma has been uncertain. The spliced leader RNA gene, present in the euglenids and kinetoplastids in distinct forms, was a potential target for resolving this question. The first indication supporting a closer relationship to the kinetoplastids was the recognition of potential spliced leader RNA exon sequences in the genomic DNA of two Diplonema isolates. Examination of total cell RNA revealed transcripts in the anticipated size range at approximately 120 and 130 nt. Specific PCR amplification of a spliced leader RNA gene repeat was performed. The hallmark features of the kinetoplastid-type spliced leader RNA, specifically the 39-nt exon, splice-donor site, Sm-binding site and poly-T tract and the potential to form the requisite stem-loop structures, were found. Diplonema spp. are different from the kinetoplastids by virtue of C residues at positions 4 and 18 in the exon. While the intergenic spacer regions varied in size, each contained the complete sequence or remnants of a 5S ribosomal RNA gene. Possession of a functional spliced leader RNA gene of the kinetoplastid variety in Diplonema supports a closer evolutionary relationship with this group than with the euglenids.

Animals↗