Biomedical subjects

D R Pring

Publications and source records attributed to D R Pring.

At least 19 recordsLinked to original sources

PubMed2001

Mitochondrial atp6 transcript editing during microgametogenesis in male-sterile sorghum.

A marked reduction of mitochondrial atp6 transcript-editing capability in sorghum anthers and pollen has been invoked as a factor in the loss of viability of male gametophytes in lines carrying the IS1112C male-sterile cytoplasm. We initiated a systematic examination of transcript editing of sorghum atp6 during microgametogenesis, from microspores through pollen, in two sets of male-fertile and near-isogenic, male-sterile lines. Transcript editing in cDNA populations from fertile lines indicated an average of about 90% editing among 15 sites examined. Parallel samples from sterile lines demonstrated a detectable reduction in editing efficiency, most evident in late pollen samples. However, editing efficiency in these cells averaged 80%, substantially higher than the 20% previously observed. Thus male-sterile lines retain substantial atp6-editing efficiency in an environment where the sterility and fertility restoration traits are stably expressed. Drastically reduced editing of atp6 transcripts in anthers and pollen of male-sterile sorghum is not demonstrable in a field environment; and thus it is not a factor in the loss of microgametophyte viability in this system.

Base Sequence↗

PubMed1999

Nuclear genes from Tx CMS maintainer lines are unable to maintain atp6 RNA editing in any anther cell-type in the sorghum bicolor A3 cytoplasm.

RNA editing and cytoplasmic male sterility are two important phenomena associated with higher plant mitochondria. We recently have shown a potential function of RNA editing in CMS development. The frequency of atp6 RNA editing was specifically reduced in anthers of male-sterile Sorghum bicolor, which increased in frequency in partially restored progeny. Here we present data that show that the loss of RNA editing capability also occurs in a second nuclear background that allows the expression of male sterility. Loss of RNA editing thus appears to be associated with unique combinations of male-sterile cytoplasm and non-restoring nuclear backgrounds. In addition, the reduction of RNA editing affects both gametophytic and sporophytic anther cell-types but not other floral tissues. An analysis of F(2) plants exhibiting different levels of fertility indicates a co-segregation of fertility restoration and atp6 RNA editing. The atp6 transcript abundance is similar in seedlings and anthers of male-sterile, partially restored, and male-fertile lines and thus is not associated with loss of atp6 RNA editing in anthers. A model for RNA editing and male sterility based on the data available is presented. Functional correlations with other CMS systems are also discussed.

Cell Nucleus↗

PubMed1998

Mutations at specific atp6 codons which cause human mitochondrial diseases also lead to male sterility in a plant.

Defects in the human mitochondrial genetic system result in some diseases. These disorders are the result of rearrangements or point mutations in mitochondrial genes. In higher plants mutations and rearrangements in the mitochondrial DNA are believed to cause cytoplasmic male sterility (CMS), a mitochondrially inherited inability to produce viable pollen. In sorghum, formation of CMS is strongly correlated with anther-specific loss of mitochondrial atp6 RNA editing. Here we show that this loss of atp6 RNA editing mimics point mutations at codons that cause severe disorders in humans. We conclude that (i) loss of RNA editing in sorghum anthers probably causes CMS, (ii) similarities exist in the onset of mitochondrial dysfunction in plant and human tissues, and (iii) the evolutionary appearance of RNA editing provided a mechanism to compensate for otherwise lethal point mutations.

Amino Acid Sequence↗

PubMed1998

Interaction of mitochondrial RNA editing and nucleolytic processing in the restoration of male fertility in sorghum.

Nucleolytic processing of transcripts within mitochondrial orf107, associated with male sterility in sorghum, is regulated by the fertility restoration gene Rf3, conferring 75% cleavage of whole-length transcripts. Two transcript editing sites are 81% and 61% edited in rf3rf3 lines, while these sites are 41% and 10% edited in the remaining whole-length transcripts in an Rf3Rf3 line. RNA editing and processing efficiency in F1 progeny were similar to the Rf3Rf3 parent, and analyses of backcross progeny indicated that all rf3rf3 lines were characterized by high editing efficiency. We postulate that highly edited transcripts within the population are quickly processed in lines carrying Rf3, generating a residual population of poorly edited transcripts. Thus, action of Rf3 may have no direct affect on RNA editing, and may be dependent on a substrate of highly edited transcripts. These data indicate a potentially novel role of RNA editing in gene expression through an influence on the efficiency of transcript processing.

Amino Acid Sequence↗

PubMed1998

Cosegregation of single genes associated with fertility restoration and transcript processing of sorghum mitochondrial orf107 and urf209.

Defective nuclear-cytoplasmic interactions leading to aberrant microgametogenesis in sorghum carrying the IS1112C male-sterile cytoplasm occur very late in pollen maturation. Amelioration of this condition, the restoration of pollen viability, involves a novel two-gene gametophytic system, wherein genes designated Rf3 and Rf4 are required for viability of individual gametes. Rf3 is tightly linked to, or represents, a single gene that regulates a transcript processing activity that cleaves transcripts of orf107, a chimeric mitochondrial open reading frame specific to IS1112C. The mitochondrial gene urf209 is also subject to nucleus-specific enhanced transcript processing, 5' to the gene, conferred by a single dominant gene designated Mmt1. Examinations of transcript patterns in F2 and two backcross populations indicated cosegregation of the augmented orf107 and urf209 processing activities in IS1112C. Several sorghum lines that do not restore fertility or confer orf107 transcript processing do exhibit urf209 transcript processing, indicating that the activities are distinguishable. We conclude that the nuclear gene(s) conferring enhanced orf107 and urf209 processing activities are tightly linked in IS1112C. Alternatively, the similarity in apparent regulatory action of the genes may indicate allelic differences wherein the IS1112C Rf3 allele may differ from alleles of maintainer lines by the capability to regulate both orf107 and urf209 processing activities.

Alleles↗

PubMed1997

Transcriptional initiation sites in sorghum mitochondrial DNA indicate conserved and variable features.

Transcriptional initiation and processing was examined for three sorghum mitochondrial DNA genes (atp6-1, atp6-2, urf209) and two open reading frames (orf265/130, orf107) to characterize sequences associated with initiation and other transcriptional strategies for this species. The 5' termini of ten transcripts were determined by primer extension, and mtRNA was capped with guanylyl transferase and annealed to anti-sense riboprobes to identify transcriptional initiation regions. Eight transcript termini were suitable substrates for guanylyl transferase, indicating the presence of one (atp6-1, atp6-2, urf209), two (orf265/130), or three (orf107) promoters for the five examples. The majority of the putative promoters were associated with single primer-extension termini, while two examples exhibited two transcript-initiation sites within the promoter. Four examples were characterized by initiated transcripts without subsequent processing, indicating that processing is not obligatory. Each of the putative promoter regions included significant A/T-rich 5' regions, consistent with previous examples, but four exceptions to a consensus core YRTA sequence were identified. The anomalies (AATA, CTTA) suggest plasticity in the primary structure of the core region of higher-plant mitochondrial DNA promoters.

Base Sequence↗

PubMed1997

Transcription initiation sites for sorghum mitochondrial atp9 are positioned immediately 3' to trnfM.

Sorghum mitochondrial atp9 is polymorphic among male-sterile cytoplasms, but each cytoplasm is characterized by a major 650 nt transcript, regardless of fertility status. The gene is positioned 323 bp 3' to trnfM. Primer extension revealed multiple atp9 5' transcript termini, distributed from +1 to +28 3' to trnfM; the termini could be labeled with polynucleotide kinase, suggesting that they result from the maturation of trnfM. Guanylyltransferase experiments, however, showed that four of the termini were capable. The juxtaposition of a putative promoter 3' to trnfM results in a unique atp9 transcript population consisting of primary and processed transcripts.

Amino Acid Sequence↗

PubMed1996

Sorghum mitochondrial orf25 and a related chimeric configuration of a male-sterile cytoplasm.

We describe fundamental characteristics of sorghum mitochondrial orf25, urf209, and a related chimeric configuration, orf265/130, which is restricted to the IS1112C source of cytoplasmic male sterility in sorghum. Transcripts of urf209 are edited at ten nucleotides, resulting in nine amino-acid changes predicted from genomic sequences. The cDNA-predicted polypeptide product is 23.6 kDa, while Western blot analyses identify a product of 20k Da. Transcription of urf209 is characterized by one or two transcripts, dependent on nuclear background, but this difference is not related to male fertility status. The orf265/130 chimeric region includes 288 bp 95% identical to sequences 5' to maize T-cytoplasm T-urf13 and atp6, which includes a common transcription initiation site, and terminates with a recombinational event involving urf209. The urf209 similarity extends 189 bp, followed by sequences duplicated 5' to sorghum atp6-2. Sequences immediately 3' to the atp6-2 similarity include a second in-frame start codon, defining orf130. Structural features 5' to orf130 are shared with motifs found 5' to several translated mitochondrial open reading frames. The orf265/orf130 configuration is uniquely transcribed, and transcripts of orf130 exhibit one silent RNA editing event. Transcription in somatic cells is not altered by male fertility status.

Amino Acid Sequence↗

PubMed1996

Transcript processing internal to a mitochondrial open reading frame is correlated with fertility restoration in male-sterile sorghum.

A chimeric mitochondrial DNA (mtDNA) configuration of the cytoplasmic male-sterile (cms) sorghum line IS1112C includes a 321 bp open reading frame designated orf107, encoding a predicted polypeptide product of 11.85 kDa. The open reading frame, similar to several other genes associated with cms, consists of amino-terminal sequences derived from an obligate gene. Unlike other examples to date, however, the carboxy-terminal sequences are highly similar to the carboxy terminus of an open reading frame implicated in cms of rice, orf79. The amino-terminal 31 residues of orf107 are 84% similar to atp9, and the carboxy-terminal 49 residues are 57% identical and 80% similar to the carboxy terminus of orf79. Transcripts of orf107 are edited, with four C-to-U changes that alter amino acids. Sorghum lines partially or fully restored to fertility exhibit a high-efficiency internal-orf107 transcript processing activity, precluding abundant whole-length transcripts, while male-sterile lines exhibit only a trace of the activity. Previous data on the abundance of a 12kDa in organello-synthesized polypeptide in male-sterile versus male-fertile lines are correlated with differential orf107 transcript processing activity of these lines. Examinations of backcross and F2 lines suggest a gametophytic mode of restoration, and indicate that enhanced transcript processing activity is necessary, but not sufficient, to restore full fertility. These novel observations indicate that mitochondrial open reading frames associated with cms in different species can include highly similar motifs, and that fertility restoration could involve a mechanism by which synthesis of a cms-associated gene product may be precluded through internal transcript cleavage.

Amino Acid Sequence↗

PubMed1995

Analysis of silent RNA editing sites in atp6 transcripts of Sorghum bicolor.

We have observed numerous examples of silent or rare non-silent editing sites in the amino-extension and part of the conserved core of mitochondrial atp6 transcripts of Sorghum. In this region of the 1.4-kb atp6-2 mRNA (position 300 to 550) two editing sites, which alter the amino-acid sequence and occur in all cDNAs analysed, were already known, while nine others were found which are silent or occur in a few mRNAs only. Many aspects of RNA editing in the mitochondria of higher plants are still unknown. This includes the influence of genomic background or silent RNA editing. We were interested in the influence of nuclear and mitochondrial backgrounds on RNA editing. Previous preliminary results indicated the possibility of line-specific editing at silent sites. However, a more comprehensive approach gave no consistent evidence for such editing. These results are discussed with respect to their potential impact on the evolution of mitochondrial genes.

Base Sequence↗

PubMed1992

Conserved sequence blocks 5' to start codons of plant mitochondrial genes.

Three sequence blocks of 10-12 bp are conserved in sequence and order 5' to putative start codons of several higher-plant mitochondrial genes. At least 25 examples were found, primarily associated with coxII, atp6, and orf25, in monocotyledons and dicotyledons. The proximal block can be 9 bp from start codons, and the three blocks generally occur within 100 bp 5' of start codons. In three examples 5' termini of the blocks represent recombination breakpoints, resulting in conservation of the blocks in resultant configurations. The two proximal blocks can form a secondary structure motif. The occurrence of the blocks near start codons, and conserved sequence and order, is consistent with a possible role in translation initiation or regulation.

Base Sequence↗

PubMed1992

Sorghum mitochondrial atp6: divergent amino extensions to a conserved core polypeptide.

Sorghum mitochondrial atp6 occurs as one copy in the line Tx398 and as two copies in IS1112C. In IS1112C a repeated sequence diverged within the atp6 open reading frames. The two open reading frames (1137 bp, atp6-1; 1002 bp, atp6-2) share an identical conserved region of 756 bp but are flanked 5' by divergent extensions of 246 (atp6-1) or 381 bp (atp6-2). Tx398 carried only atp6-2. The breakpoint of the repeated sequence of the conserved core region corresponds to the amino acid sequence Ser-Pro-Leu-Asp, which is the amino terminus of the proteolytically processed yeast ATP6. The 5' extensions of atp6-1 and atp6-2 were similar to those of rice and maize, respectively. Each open reading is transcribed, however nuclear background influenced transcriptional patterns of atp6-2 in IS1112C.

Amino Acid Sequence↗

PubMed1991

RNA editing of sorghum mitochondrial atp6 transcripts changes 15 amino acids and generates a carboxy-terminus identical to yeast.

Sequencing of sorghum mitochondrial atp6 cDNA clones revealed 19 C-to-U transcript editing events within a 756 bp-conserved core gene; three were silent and 16 resulted in 15 amino acid changes. Only one edit, which was silent, was found in the 381 bp amino-extension to the core gene. Eleven of the 15 changed amino acids were identical with or else represented conservative changes compared to yeast atp6. Editing of a CAA codon to TAA truncates the carboxy-terminus to a position identical to that of yeast. The frequency of editing at sites which change amino acids was very high in contrast to partial editing at silent, third base, sites.

Base Sequence↗

PubMed1991

Editing of mitochondrial atp9 transcripts from two sorghum lines.

Genomic and cDNA sequences of the ATP synthase complex subunit 9 (atp9) genes from two sorghum lines were determined. Sequences of cDNAs revealed eight C to U transcript editing events resulting in six amino acid changes and a new stop codon which eliminated 12 carboxy-terminal residues, compared to the genomic sequence. Sorghum atp9 has a unique five-residue amino-extension relative to other higher plants. The resulting predicted 79-residue gene product has a molecular weight of 8.179 kDa. The predicted phe-val-phe carboxy-terminus is identical to that from cDNA sequences of wheat, Oenothera, and petunia. Partial editing of transcripts was detected in each sorghum line.

Amino Acid Sequence↗

PubMed1990

Expression in Saccharomyces cerevisiae of a gene associated with cytoplasmic male sterility from maize: respiratory dysfunction and uncoupling of yeast mitochondria.

We asked whether the mitochondrial T-urf13 gene, associated with the male sterility phenotype of T cytoplasm in maize, can be expressed in Saccharomyces cerevisiae and whether this expression can mimic the effects observed in maize. We introduced the universal code equivalent of the T-urf13 gene into the S. cerevisiae nucleus by transformation and directed its translation product into mitochondria by means of a fusion with the targeting presequence from Neurospora crassa AT-Pase subunit 9. We show that expression of the universal code equivalent of the T-urf13 gene in the yeast nucleus does indeed mimic its effects in maize: respiratory growth of yeast is inhibited, respiration-deficient cytoplasmic mutants accumulate and NADH oxidation of isolated mitochondria is uncoupled. All these effects are observed only if the mitochondrial targeting peptide and methomyl or HmT toxin are present.

Chimera↗

PubMed1988

Recombination is associated with polymorphism of the mitochondrial genomes of maize and sorghum.

Extensive recombination events characterize higher-plant mitochondrial DNAs. Numerous recombination events resulted in the appearance of an unusual mitochondrial open reading frame, urf13-T, which encodes a 13 kDa polypeptide in the male-sterile T cytoplasm of maize. Maize lines with T cytoplasm are unusually susceptible to two fungal pathogens which produce host-selective toxins. Mutants derived from tissue culture expressing male fertility and toxin-insensitivity are characterized by truncation or deletion of urf13-T. These events result from a frameshift associated with a tandem 5 base pair repeat, placing a premature stop codon in frame, or from a recombination event, apparently limited to tissue culture, resulting in the deletion of urf13-T. Neither class of mutants produces the 13 kDa gene product. Repeated sequences that participate in recombination in sorghum appear to be randomly distributed among male-fertile or male-sterile cytoplasms. Processes involved in the evolution of mitochondrial DNAs in higher plants therefore include the generation and deletion of configurations through recombination.

Base Sequence↗

PubMed1988

Mitochondrial DNA rearrangement associated with fertility restoration and cytoplasmic reversion to fertility in cytoplasmic male sterile Phaseolus vulgaris L.

Restoration of pollen fertility to cytoplasmic male sterile (CMS) Phaseolus vulgaris by a nuclear restorer gene provides a system for studying nuclear-cytoplasmic interactions. Introduction of a nuclear restorer gene to this CMS line of P. vulgaris (CMS-Sprite) results in a mitochondrial genome rearrangement similar to that observed upon spontaneous cytoplasmic reversion to fertility. Three spontaneous heritable cytoplasmic revertants were derived from CMS-Sprite. Five fully fertile restored lines were also produced by using restorer line R-351 (BC(3)F(3) populations). Comparison of the mitochondrial DNA restriction patterns of CMS-Sprite, the three fertile revertants, and the five restored lines revealed loss of a 6.0-kilobase (kb) Pst I fragment in all restored and revertant lines. Southern hybridizations with a 1.3-kb BamHI clone, internal to the 6.0-kb Pst I fragment, as a probe revealed two configurations of 6.0-kb homologous sequences in the sterile cytoplasm; one of the configurations was lost upon reversion or restoration. Mitochondrial DNA rearrangement has thus been observed upon restoration by a nuclear restorer gene in this CMS system.

Journal Article↗

PubMed1987

Nucleotide sequence and molecular characterization of a maize mitochondrial plasmid-like DNA.

The mitochondrial genome of Black Mexican Sweet maize consists of the principal genome, a 2.3 kb minilinear DNA, a 1,913 bp (1.9 kb) and a 1,445 bp (1.4 kb) minicircular DNA. The three extrachromosomal DNAs exhibit characteristics of autonomous replication in cell suspension culture. The complete sequence of the 1.4 kb minicircle was determined. It has 61 bp of near perfect sequence homology to the 1.9 kb minicircle. Both minicircular DNAs are transcriptionally active; the longest open reading frame of the 1.4 kb minicircle was 231 bp. A putative origin of replication was identified as a high A + T sequence. These minicircles were present in some but not all of 20 maize lines surveyed. None of the lines examined carried the 1.4 kb minicircle without the 1.9 kb minicircle. Nuclear DNA of one line of the seven examined carried homology to both DNAs.

Cells, Cultured↗