urf13-T of T cytoplasm maize mitochondria encodes a 13 kD polypeptide

Polyspecific antibody to a 17 amino acid synthetic peptide from the maize T-cytoplasm urf13-T mitochondrial open reading frame immunoprecipitated a 13 kD polypeptide from ³⁵S-methionine incorporations of T cytoplasm maize. Male-fertile, toxin-insensitive mutants in which the urf13-T sequence is deleted do not synthesize the 13 kD polypeptide. A mutant designated T-4, which carries a 5 bp insertion and a premature stop codon, synthesizes a truncated polypeptide, corresponding to an open reading frame of 8.3 kD. Thus the 13 kD polypeptide is trunctated or absent in mutants expressing male fertility and toxin insensitivity in T-cytoplasm maize.USDA-ARS     

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.     

Cloning and expression of the soybean DapA gene encoding dihydrodipicolinate synthase

The rate-limiting step in the pathway for lysine synthesis in plants is catalyzed by the enzyme dihydrodipicolinate synthase (DS). We have cloned the portion of the soybean (Glycine max cv. Century) DapA cDNA that encodes the mature DS protein. Expression of the cloned soybean cDNA as a lacZ fusion protein was selected in a dapA ^- Escherichia coli auxotroph. The DS activity of the fusion protein was characterized in E. coli extracts. The DS activity of the fusion protein was inhibited by lysine concentrations that also inhibited native soybean DS, while E. coli DS activity was much less sensitive to inhibition by lysine.     

Direct genetic selection of a maize cDNA for dihydrodipicolinate synthase in an Escherichia coli dapA- auxotroph

Summary Dihydrodipicolinate synthase (DHPS; EC 4.2.1.52) is the first committed enzyme in the lysine branch of the aspartate-derived amino acid biosynthesis pathway and is common to bacteria and plants. Due to feedback inhibition by lysine, DHPS serves in a regulatory role for this pathway in plant metabolism. To elucidate the molecular genetic characteristics of DHPS, we isolated a putative full-length cDNA clone for maize DHPS by direct genetic selection in an Escherichia coli dapA ^–auxotroph. The maize DHPS activity expressed in the complemented E. coli auxotroph showed the lysine inhibition characteristics of purified maize DHPS, indicating that the cDNA encoded sequences for both the catalytic function and regulatory properties of the enzyme. The N-terminal amino acid sequence of purified maize DHPS was determined by direct sequencing and showed homology to a sequence within the cDNA, indicating that the clone contained the entire coding region for a mature polypeptide of 326 amino acids plus a 54 amino acid transit peptide sequence. The molecular weight of 35854, predicted from the deduced amino acid sequence, was similar to the 38 000 M_r determined by SDS-polyacrylamide gel electrophoresis (SDS-PAGE) for the purified enzyme from maize. DHPS mRNAs complementary to the cDNA were detected in RNA isolated from developing maize endosperm and embryo tissues. Southern blots indicated the presence of more than one genomic sequence homologous to DHPS per haploid maize genome.     

Genetic and molecular analysis of tissue-culture-derived Ac elements

Summary Our previous experiments on maize (Zea mays L.) plants regenerated from tissue culture revealed genetic activity characteristic of the transposable element Activator (Ac) in the progeny of 2–3% of the plants tested, despite the lack of Ac activity in the progenitor plants. The objective of the present study was to determine whether the presence of Ac activity in tissue-culture-derived plants was associated with changes in the number or structure of Ac-homologous DNA sequences. Families segregating for Ac activity were obtained by crossing plants heterozygous for Ac activity onto Ac-responsive tester plants. A DNA probe derived from a previously isolated Ac sequence was used to examine the Ac-homologous sequences within individual progeny seedlings of segregating families and noncultured control materials. All plants tested had six or more Ac-homologous DNA sequences, regardless of whether Ac activity was present. In the segregating progeny of one tissue-culturederived plant, a 30-kb Ac-homologous SstI restriction fragment and a 10-kb Ac-homologous BglII restriction fragment were found to cosegregate with Ac activity. We propose that these fragments contained a previously silent Ac sequence that had been activated during tissue culture. Although one or more Ac sequences were often hypomethylated at internal PvuII and HpaII sites in plants with Ac activity, hypomethylation was not a prerequisite for activity. Reduced methylation at these sites may have been a result rather than a cause of Ac activity.     

Mitochondrial sensitivity to Drechslera maydis T-toxin and the synthesis of a variant mitochondrial polypeptide in plants derived from maize tissue cultures with texas male-sterile cytoplasm

Summary Tissue cultures of maize carrying cms-T cytoplasm have been found to regenerate fertile, T-toxin resistant plants, with and without a selective treatment with T-toxin. Progeny of these plants were tested for mitochondrial sensitivity to T-toxin and the translation products synthesised by isolated mitochondria were analysed. The results confirm previous indications of a close correlation between susceptibility to T-toxin and the synthesis of a variant 13,000 M_r mitochondrial polypeptide. Interestingly, there appeared to be a critical level at about 33% maximum synthesis of the 13,000 M_r polypeptide above which male sterility and sensitivity to T-toxin are jointly expressed. The possibility that there is a causal link between synthesis of this additional mitochondrial polypeptide, pollen abortion and sensitivity to T-toxin is discussed.     

Effect of iron on activity of soybean multi-subunit acetyl-coenzyme A carboxylase

Multi‐subunit acetyl‐coenzyme A carboxylase (MS‐ACCase; EC 6.4.1.2) isolated from soybean chloroplasts is a labile enzyme that loses activity during purification. We found that incubating the chloroplast stromal fraction under anaerobic conditions or in the presence of 5 mM FeSO₄ stimulated ACCase (acetyl‐CoA→malonyl‐CoA) and carboxyltransferase (malonyl‐CoA→acetyl‐CoA) activity. Fe‐stimulation of activity was associated with ⁵⁹Fe binding to a stromal protein fraction. ACCase and carboxyltransferase activities measured in the stromal protein fraction containing bound ⁵⁹Fe were 2‐fold and 6‐fold greater, respectively, than the control (stromal fraction not pretreated with FeSO₄). Superose 6 gel filtration chromatography indicated ⁵⁹Fe comigrated with stromal protein of approximately 180 kDa that exhibited carboxyltransferase activity, but lacked ACCase activity. Anion exchange (Mono‐Q) chromatography of the Superose 6 fraction yielded a protein peak that was enriched in carboxyltransferase activity and contained protein‐bound ⁵⁹Fe. Denaturing gels of the Mono‐Q fraction indicated that the 180‐kDa protein was composed of a 56‐kDa subunit that was bound by an antibody raised against a synthetic β‐carboxyltransferase (β‐CTase) peptide. Incubation of the Mono‐Q carboxyltransferase fraction with increasing concentrations of iron at a fixed substrate concentration resulted in increased initial velocities that fit well to a single rectangular three parameter hyperbola (v=v_o+V_max[FeSO₄]/K_m+[FeSO₄]) consistent with iron functioning as a bound activator of catalysis. UV/Vis spectroscopy of the partially purified fraction before and after iron incubation yielded spectra consistent with a protein‐bound metal cluster. These results suggest that the β‐CTase subunit of MS‐ACCase in soybean chloroplasts is an iron‐containing enzyme, which may in part explain its labile nature.     

Phylogenetic relationships of bolitoglossine salamanders: a demonstration of the effects of combining morphological and molecular data sets

We analyzed sequence data for 555 bp of the mitochondrial gene cytochrome b in plethodontid salamanders, taken from 18 ingroup (tribe Bolitoglossini) and 4 outgroup (tribe Plethodontini) taxa. There were 257 variable sites, of which 219 were phylogenetically informative. Sequence differences among taxa exceeded 20%, and there were up to 15% amino acid differences among the sequences. We also analyzed 37 morphological (including karyological) characters, taken from the literature. Data were analyzed separately and then combined using parsimony and likelihood approaches. There is little conflict between the morphological and DNA data, and that which occurs is at nodes that are weakly supported by one or both of the data sets. Treated separately, the morphological and DNA data provide strong support for some nodes but not for others. The combined data act synergistically so that good support is obtained for nearly all of the nodes in the tree. Recent divergences are supported by silent transitions, and older divergences are supported by a combination of morphological, karyological, DNA transversion, and amino acid changes. Eliminating silent changes from the DNA data improves the consistency index and improves some bootstrap and decay index values for several deeper branches in the tree. However, the combined data set with all characters included provides a better supported tree overall. Maximum likelihood and parsimony with all of the data give not only the same topology but also remarkably similar branch lengths. Results of this analysis support the monopoly of the supergenera Hydromantes and Batrachoseps, and of a sister group relationship of Batrachoseps and the supergenus Bolitoglossa (represented in this study one species of the genus Bolitoglossa).      

Salivary and serum procalcitonin and C-reactive protein as biomarkers of periodontitis in United States veterans with osteoarthritis or rheumatoid arthritis

Serum procalcitonin (ProCT) is elevated in response to bacterial infections, whereas high sensitivity C-reactive protein (hsCRP) is a nonspecific inflammatory marker that is increased by excess adipose tissue. We examined the efficacy of ProCT and hsCRP as biomarkers of periodontitis in the saliva and serum of patients with arthritis, which is characterized by variable levels of systemic inflammation that potentially can confound the interpretation of inflammatory biomarkers. Blood and unstimulated whole saliva were collected from 33 patients with rheumatoid arthritis (RA) and 50 with osteoarthritis (OA). Periodontal status was assessed by full mouth examination and patients were categorized as having no/mild, moderate or severe periodontitis by standard parameters. Salivary and serum ProCT and hsCRP concentrations were compared. BMI, diabetes, anti-inflammatory medications and smoking status were ascertained from the patient records. Differences between OA and RA in proportionate numbers of patients were compared for race, gender, diabetes, adiposity and smoking status. Serum ProCT was significantly higher in arthritis patients with moderate to severe and severe periodontitis compared with no/mild periodontitis patients. There were no significant differences in salivary ProCT or salivary or serum hsCRP in RA patients related to periodontitis category. Most of the OA and RA patients were middle aged or older, 28.9% were diabetic, 78.3% were overweight or obese, and slightly more than half were either current or past smokers. The OA and RA groups differed by race, but not gender; blacks and males were predominant in both groups. The OA and RA groups did not differ in terms of controlled or uncontrolled diabetes, smoking status or BMI. The RA patients had been prescribed more anti-inflammatory medication than the OA patients. Our results demonstrate that circulating ProCT is a more discriminative biomarker for periodontitis than serum hsCRP in patients with underlying arthritis. Any elevation in salivary and serum hsCRP due to periodontitis apparently was overshadowed by differences among these patients in factors that influence CRP, such as the extent of inflammation between RA and OA, the extent of adipose tissue, the use of anti- inflammatory medications and smoking status. Although our study showed no differences in salivary ProCT related to severity of periodontitis, this biomarker also may be useful with further refinement.