Biomedical subjects

K D Pruitt

Publications and source records attributed to K D Pruitt.

25 records · Page 2Linked to original sources

PubMed1995

5-Fluoroindole resistance identifies tryptophan synthase beta subunit mutants in Arabidopsis thaliana.

A study of the biochemical genetics of the Arabidopsis thaliana tryptophan synthase beta subunit was initiated by characterization of mutants resistant to the inhibitor 5-fluoroindole. Thirteen recessive mutations were recovered that are allelic to trp2-1, a mutation in the more highly expressed of duplicate tryptophan synthase beta subunit genes (TSB1). Ten of these mutations (trp2-2 through trp2-11) cause a tryptophan requirement (auxotrophs), whereas three (trp2-100 through trp2-102) remain tryptophan prototrophs. The mutations cause a variety of changes in tryptophan synthase beta expression. For example, two mutations (trp2-5 and trp2-8) cause dramatically reduced accumulation of TSB mRNA and immunologically detectable protein, whereas trp2-10 is associated with increased mRNA and protein. A correlation exists between the quantity of mutant beta and wild-type alpha subunit levels in the trp2 mutant plants, suggesting that the synthesis of these proteins is coordinated or that the quantity or structure of the beta subunit influences the stability of the alpha protein. The level of immunologically detectable anthranilate synthase alpha subunit protein is increased in the trp2 mutants, suggesting the possibility of regulation of anthranilate synthase levels in response to tryptophan limitation.

Alleles↗

PubMed1993

Expression patterns of duplicate tryptophan synthase beta genes in Arabidopsis thaliana.

Expression of the two Arabidopsis thaliana genes encoding tryptophan synthase beta (TSB1 and TSB2) was investigated by gene-specific RNA blot hybridization and reporter gene analysis. TSB1 mRNA abundance varies in an organ-specific manner, whereas TSB2 mRNA does not. Quantitative analysis of transgenic plants expressing TSB1 and TSB2 translational fusions to the beta-glucuronidase (GUS) gene (gusA) indicates that TSB1-GUS activity is 15-fold higher than TSB2-GUS. Histochemical analysis of these transgenic A. thaliana plants indicates that GUS expression occurs in a developmentally regulated manner. GUS activity driven from the TSB1 promoter is predominantly associated with the stem, root tips, foliar vasculature, mesophyll cells, base of developing seed pods, and tips of anther filaments in plants 15 d and older. Sections through the vegetative stem reveal GUS staining in all cell types including the shoot apical meristem. Although TSB2-GUS expression is consistently detected in root tips and at the base of developing seed pods, it is observed later in plant development than is TSB1-GUS expression.

Arabidopsis↗

PubMed1991

Transcription of the Petunia mitochondrial CMS-associated Pcf locus in male sterile and fertility-restored lines.

Transcripts of the Petunia mitochondrial cytoplasmic male sterility (CMS)-associated S-Pcf locus, which consists of three co-transcribed genes (pcf, NADH dehydrogenase subunit 3, and ribosomal protein S12), have been characterized in reproductive tissues of CMS and fertility restored (Rf) Petunia lines by nuclease protection experiments and by RNA blot hybridization. Three 5' transcript termini have been previously described. Two 3' transcript termini and an additional S-Pcf locus transcript have now been identified. The relative abundance of the three 5' transcript termini is influenced by the presence of the nuclear Rf gene. A decrease in the abundance of the -121 5' transcript terminus relative to the -266 and -522 termini is consistently seen in Petunia lines which are restored to fertility by the Rf gene, compared to CMS Petunia lines. An additional transcript with a 5' terminus within the urf-s region of pcf is much more abundant in immature bud and anther tissue than in leaf or suspension cells. The total abundance of pcf transcripts varies greatly between plants of different nuclear backgrounds which lack the nuclear Rf allele, indicating that other nuclear genes also influence expression of the S-Pcf locus.

Autoradiography↗

PubMed1991

Splicing of the Petunia cytochrome oxidase subunit II intron.

A comparative analysis of the plant intron-containing mitochondrial cytochrome oxidase subunit II (coxII) genes provides an indication that four conserved sequence motifs, present in exon 1 (intron-binding sequences; IBS), and complementary motifs (exon-binding sequences; EBS), present in domain I of the group II intron, may be involved in splicing of the intron. Two of these potential IBS motifs (IBS1 and IBS2) have been previously discussed. Two further potential IBS motifs (IBSa and IBSb), which occur twice within exon 1, could be involved in specification of the 5' splice site and of a 5' cryptic splice site. Nuclease-protection experiments and DNA sequence analysis of a spliced coxII cDNA have confirmed the predicted positions of the petunia coxII 5' and 3' splice sites. Evidence for the occurrence of splicing in vivo at the putative 5' cryptic splice site in petunia is provided by the detection of a nuclease-protected fragment corresponding to the size which is predicted if splicing at the proposed cryptic splice site occurs. The existence and location of a cryptic splice site, upstream of the normal coxII 5' splice site, is consistent with the proposed derivation of the cytoplasmic male sterility (CMS)-associated pcf gene from an abnormally spliced coxII transcript (Pruitt and Hanson 1989).

Base Sequence↗

PubMed1991

Editing of pre-mRNAs can occur before cis- and trans-splicing in Petunia mitochondria.

Plant mitochondrial mRNAs have recently been shown to undergo editing, involving cytidine-to-uridine changes relative to the DNA sequence. We have examined the temporal relationship of editing and intron removal in coxII mRNAs in Petunia mitochondria. By using differential hybridization to probes specific for edited and unedited RNA and by sequencing of individual unspliced coxII pre-mRNA cDNAs, we found that RNA editing at any editing site can precede the splicing event. Similar results were obtained from examinations of pre-mRNA cDNAs of nad1, a gene composed of multiple exons that are both cis and trans spliced. Thus, intron removal is not required before editing can occur. The existence of editing intermediates indicates that the editing process is not strictly coincident with transcription.

Antisense Elements (Genetics)↗

PubMed1989

Cytochrome oxidase subunit II sequences in Petunia mitochondria: two intron-containing genes and an intron-less pseudogene associated with cytoplasmic male sterility.

The mitochondrial genome of Petunia hybrida contains two transcribed cytochrome oxidase subunit II (coxII) genes. The coding region of both genes is split by a 1.3 kb group II intron. Unlike coxII-1, which is similar to other sequenced plant coxII genes, the coxII-2 coding region is extended by 48 codons. The cytoplasmic male sterile (CMS) Petunia contains one coxII gene similar in structure and transcript pattern to the coxII-1 gene found in the fertile genome. Comparison of the sequenced coxII genes from the fertile mitochondrial genome with the coxII sequences present in the CMS-associated pcf gene from the CMS genome (Young and Hanson 1987) suggests that pcf is a processed pseudogene. A model for the generation of pcf is presented.

Amino Acid Sequence↗

PubMedDate not supplied

Editing of rps3/rpl16 transcripts creates a premature truncation of the rpl16 open reading frame.

Overlapping open reading frames corresponding to maize mitochondrial genes rps3 and rpl16 have been found in Petunia mitochondrial DNA. The DNA region associated with these two genes is part of the Petunia mitochondrial recombination repeat and is iterated three times. Analysis of transcripts from these genes shows that there is RNA editing of the coding regions and that one of the editing sites detected in the open reading frame overlap creates a premature stop codon in the rpl16 sequence. No transcripts were detected that were unedited at this site. Thus, in Petunia editing of rpl16 appears to render this gene nonfunctional.

Amino Acid Sequence↗