Sequence of the supernumerary B chromosome of maize provides insight into its drive mechanism and evolution.

B chromosomes are enigmatic elements in thousands of plant and animal genomes that persist in populations despite being nonessential. They circumvent the laws of Mendelian inheritance but the molecular mechanisms underlying this behavior remain unknown. Here we present the sequence, annotation, and analysis of the maize B chromosome providing insight into its drive mechanism. The sequence assembly reveals detailed locations of the elements involved with the cis and trans functions of its drive mechanism, consisting of nondisjunction at the second pollen mitosis and preferential fertilization of the egg by the B-containing sperm. We identified 758 protein-coding genes in 125.9 Mb of B chromosome sequence, of which at least 88 are expressed. Our results demonstrate that transposable elements in the B chromosome are shared with the standard A chromosome set but multiple lines of evidence fail to detect a syntenic genic region in the A chromosomes, suggesting a distant origin. The current gene content is a result of continuous transfer from the A chromosomal complement over an extended evolutionary time with subsequent degradation but with selection for maintenance of this nonvital chromosome.

Quantitative Proteomics of Zea mays Hybrids Exhibiting Different Levels of Heterosis.

Maize hybrids exhibiting heterosis (hybrid vigor) were generated from inbred parents with increasing genetic distance. B73 was used as the common female parent in crosses with N192 (low heterosis), MO17 (high-heterosis 1), and NC350 (high-heterosis 2). Total and mitochondria-enriched proteomes were analyzed from ear shoots of field-grown hybrids and their inbred parents. GeLCMS (1D SDS-PAGE fractionation, trypsin digestion, LTQ Orbitrap nano-RP-LC MS/MS) was used to analyze proteins, and spectral counting was used for quantitation. In total, 3,568 proteins were identified and quantified in hybrids including 2,489 in the mitochondria-enriched fraction and 2,162 in the total protein fraction. Sixty-one proteins were differentially abundant (p < 0.05) in one or both of the high-heterosis hybrids compared with the low-heterosis hybrid. For the total proteome, eight of these showed similar trends in abundance in both of the higher-heterosis hybrids. Nine proteins showed this heterosis-correlated pattern in the mitochondrial proteome, including a mitochondria-associated target of rapamycin (TOR) protein. Although differentially abundant proteins belong to various pathways, protein, and RNA metabolism, and stress responsive proteins were the major classes changed in response to increasing heterosis.

Specific changes in total and mitochondrial proteomes are associated with higher levels of heterosis in maize hybrids.

The phenomenon of hybrid vigor (heterosis) has long been harnessed by plant breeders to improve world food production. However, the changes that are essential for heterotic responses and the mechanisms responsible for heterosis remain undefined. Large increases in biomass and yield in high-heterosis hybrids suggest that alterations in bioenergetic processes may contribute to heterosis. Progeny from crosses between various inbred lines vary in the extent of vigor observed. Field-grown maize F1 hybrids that consistently exhibited either low or high heterosis across a variety of environments were examined for changes in proteins that may be correlated with increased plant vigor and yield. Unpollinated ears at the time of flowering (ear shoots) were selected for the studies because they are metabolically active, rich in mitochondria, and the sizes of the ears are diagnostic of yield heterosis. Total protein and mitochondrial proteomes were compared among low- and higher-heterosis hybrids. Two-dimensional difference gel electrophoresis was used to identify allelic and/or isoform differences linked to heterosis. Identification of differentially regulated spots by mass spectrometry revealed proteins involved in stress responses as well as primary carbon and protein metabolism. Many of these proteins were identified in multiple spots, but analysis of their abundances by label-free mass spectrometry suggested that most of the expression differences were due to isoform variation rather than overall protein amount. Thus, our proteomics studies suggest that expression of specific alleles and/or post-translational modification of specific proteins correlate with higher levels of heterosis.

Community-based Agency Delivery of Parent-Child Interaction Therapy: Comparing Outcomes for Children with and Without Autism Spectrum Disorder and/or Developmental Delays.

While externalizing behaviors are common among children with autism spectrum disorder (ASD), there is a shortage of specialist community-based clinicians to provide treatment. Parent-Child Interaction Therapy (PCIT), an intervention designed to reduce child disruptive behaviors, may be effective for families of children with ASD but has rarely been studied outside of university-based research settings. We examined the effectiveness of PCIT delivered for children with (N = 109) and without (N = 2,324) ASD/developmental delays (DD) across community-based agencies in Oregon. Findings revealed significant reductions in disruptive behavior and positive changes in the parent-child relationship in both groups. These findings support PCIT as an efficacious intervention for children with ASD/DD and demonstrate PCIT's promise in community-based agencies with non-specialized clinicians.

Characterization of a novel thermosensitive restorer of fertility for cytoplasmic male sterility in maize.

S-type cytoplasmic male sterility (CMS-S) in maize is associated with high levels of a 1.6-kb RNA in mitochondria. This RNA contains two chimeric open reading frames (ORFs), orf355 and orf77. The previously described nuclear restorer-of-fertility allele Rf3 causes the processing of all transcripts that contain these chimeric ORFs. The Lancaster Surecrop-derived inbred line A619 carries a restorer that is distinct from Rf3 in that it selectively reduces only the CMS-S-specific 1.6-kb RNA. We have found that 10 additional Lancaster lines carry a single restoring allele traceable to either of two inbred lines, C103 and Oh40B. The C103 and Oh40B restorers are allelic to each other, but not to Rf3. Thus, this restoring allele, designated Rf9, represents a second naturally occurring CMS-S restorer in maize. Rf9 is a less effective restorer of fertility than is Rf3; its expression is influenced by both inbred nuclear background and temperature. Rf9 acts to reduce the amounts of orf355/orf77-containing linear mitochondrial subgenomes, which are generated by recombination of circular subgenomes with CMS-S-specific linear plasmids. The 1.6-kb RNA, which is transcribed only from linear ends, is correspondingly reduced.

Mitochondrial DNA transfer to the nucleus generates extensive insertion site variation in maize.

Mitochondrial DNA (mtDNA) insertions into nuclear chromosomes have been documented in a number of eukaryotes. We used fluorescence in situ hybridization (FISH) to examine the variation of mtDNA insertions in maize. Twenty overlapping cosmids, representing the 570-kb maize mitochondrial genome, were individually labeled and hybridized to root tip metaphase chromosomes from the B73 inbred line. A minimum of 15 mtDNA insertion sites on nine chromosomes were detectable using this method. One site near the centromere on chromosome arm 9L was identified by a majority of the cosmids. To examine variation in nuclear mitochondrial DNA sequences (NUMTs), a mixture of labeled cosmids was applied to chromosome spreads of ten diverse inbred lines: A188, A632, B37, B73, BMS, KYS, Mo17, Oh43, W22, and W23. The number of detectable NUMTs varied dramatically among the lines. None of the tested inbred lines other than B73 showed the strong hybridization signal on 9L, suggesting that there is a recent mtDNA insertion at this site in B73. Different sources of B73 and W23 were examined for NUMT variation within inbred lines. Differences were detectable, suggesting either that mtDNA is being incorporated or lost from the maize nuclear genome continuously. The results indicate that mtDNA insertions represent a major source of nuclear chromosomal variation.

Reconceptualizing attrition in Parent-Child Interaction Therapy: "dropouts" demonstrate impressive improvements.

Purpose: Behavior disorders in early childhood are linked to a variety of negative outcomes for both children and families. Parent-Child Interaction Therapy (PCIT), an evidence-based parent-training program, demonstrates large effect sizes in reducing child problem behavior for dyads who complete treatment; however, a high number of families seeking treatment in community-based settings terminate from PCIT prior to meeting the protocol's strict graduation criteria. The purpose of this study was to examine the impact of PCIT on child behavior problems for families who received at least a small dose of PCIT but not enough to meet the strict mastery criteria required for graduation. Patients and methods: This study employed one of the largest community research samples conducted with PCIT (2,787 children and their families across the state of Oregon, 1,318 with usable data) to determine how PCIT impacts both graduates and early terminators. Results: While families who graduated from PCIT (17.7% of the sample) demonstrated a very large effect size in problem behavior intensity improvements (d=1.65), families who terminated treatment early, but after attending at least four treatment sessions (51.7% of the sample), still showed significant improvements in behavior problems with a medium-to-large effect size (d=0.70). In contrast, very early terminators (those attending fewer than four treatment sessions, 0.3% of the sample), demonstrated little improvement at the time of dropout from services (d=0.12). Conclusion: Though early terminators in PCIT have previously been identified as treatment failures, the present study discusses the reconceptualization of "dropouts" in relation to some positive evidence of treatment outcomes, the implications for community-based service delivery, and possible future directions.

Comparisons among two fertile and three male-sterile mitochondrial genomes of maize.

We have sequenced five distinct mitochondrial genomes in maize: two fertile cytotypes (NA and the previously reported NB) and three cytoplasmic-male-sterile cytotypes (CMS-C, CMS-S, and CMS-T). Their genome sizes range from 535,825 bp in CMS-T to 739,719 bp in CMS-C. Large duplications (0.5-120 kb) account for most of the size increases. Plastid DNA accounts for 2.3-4.6% of each mitochondrial genome. The genomes share a minimum set of 51 genes for 33 conserved proteins, three ribosomal RNAs, and 15 transfer RNAs. Numbers of duplicate genes and plastid-derived tRNAs vary among cytotypes. A high level of sequence conservation exists both within and outside of genes (1.65-7.04 substitutions/10 kb in pairwise comparisons). However, sequence losses and gains are common: integrated plastid and plasmid sequences, as well as noncoding "native" mitochondrial sequences, can be lost with no phenotypic consequence. The organization of the different maize mitochondrial genomes varies dramatically; even between the two fertile cytotypes, there are 16 rearrangements. Comparing the finished shotgun sequences of multiple mitochondrial genomes from the same species suggests which genes and open reading frames are potentially functional, including which chimeric ORFs are candidate genes for cytoplasmic male sterility. This method identified the known CMS-associated ORFs in CMS-S and CMS-T, but not in CMS-C.

Angiosperm mitochondrial genomes and mutations.

Flowering plants harbor the largest mitochondrial genomes reported so far. At present, the nucleotide sequences of 15 mitochondrial genomes from seven angiosperm species are available, making detailed comparative analysis feasible. The gene content is variable among the species, but the most striking feature is the fluidity of intergenic regions, where species-specific sequences predominate. Additionally, angiosperm mitochondrial genomes, even within a species, show a remarkable amount of rearrangement. We also review mitochondrial mutants in angiosperms from a genomic viewpoint, and discuss how they have arisen. The involvement of nuclear genes in mitochondrial genome stability and organization is currently being revealed through the analysis of mutants.

Plant Mitochondrial Genomes: Dynamics and Mechanisms of Mutation.

The large mitochondrial genomes of angiosperms are unusually dynamic because of recombination activities involving repeated sequences. These activities generate subgenomic forms and extensive genomic variation even within the same species. Such changes in genome structure are responsible for the rapid evolution of plant mitochondrial DNA and for the variants associated with cytoplasmic male sterility and abnormal growth phenotypes. Nuclear genes modulate these processes, and over the past decade, several of these genes have been identified. They are involved mainly in pathways of DNA repair by homologous recombination and mismatch repair, which appear to be essential for the faithful replication of the mitogenome. Mutations leading to the loss of any of these activities release error-prone repair pathways, resulting in increased ectopic recombination, genome instability, and heteroplasmy. We review the present state of knowledge of the genes and pathways underlying mitochondrial genome stability.

Cytogenetic and Sequence Analyses of Mitochondrial DNA Insertions in Nuclear Chromosomes of Maize.

The transfer of mitochondrial DNA (mtDNA) into nuclear genomes is a regularly occurring process that has been observed in many species. Few studies, however, have focused on the variation of nuclear-mtDNA sequences (NUMTs) within a species. This study examined mtDNA insertions within chromosomes of a diverse set of Zea mays ssp. mays (maize) inbred lines by the use of fluorescence in situ hybridization. A relatively large NUMT on the long arm of chromosome 9 (9L) was identified at approximately the same position in four inbred lines (B73, M825, HP301, and Oh7B). Further examination of the similarly positioned 9L NUMT in two lines, B73 and M825, indicated that the large size of these sites is due to the presence of a majority of the mitochondrial genome; however, only portions of this NUMT (~252 kb total) were found in the publically available B73 nuclear sequence for chromosome 9. Fiber-fluorescence in situ hybridization analysis estimated the size of the B73 9L NUMT to be ~1.8 Mb and revealed that the NUMT is methylated. Two regions of mtDNA (2.4 kb and 3.3 kb) within the 9L NUMT are not present in the B73 mitochondrial NB genome; however, these 2.4-kb and 3.3-kb segments are present in other Zea mitochondrial genomes, including that of Zea mays ssp. parviglumis, a progenitor of domesticated maize.

Unique changes in mitochondrial genomes associated with reversions of S-type cytoplasmic male sterility in maizemar.

Cytoplasmic male sterility (CMS) in plants is usually associated with the expression of specific chimeric regions within rearranged mitochondrial genomes. Maize CMS-S plants express high amounts of a 1.6-kb mitochondrial RNA during microspore maturation, which is associated with the observed pollen abortion. This transcript carries two chimeric open reading frames, orf355 and orf77, both unique to CMS-S. CMS-S mitochondria also contain free linear DNA plasmids bearing terminal inverted repeats (TIRs). These TIRs recombine with TIR-homologous sequences that precede orf355/orf77 within the main mitochondrial genome to produce linear ends. Transcription of the 1.6-kb RNA is initiated from a promoter within the TIRs only when they are at linear ends. Reversions of CMS-S to fertility occur in certain nuclear backgrounds and are usually associated with loss of the S plasmids and/or the sterility-associated region. We describe an unusual set of independently recovered revertants from a single maternal lineage that retain both the S plasmids and an intact orf355/orf77 region but which do not produce the 1.6-kb RNA. A 7.3-kb inversion resulting from illegitimate recombination between 14-bp microrepeats has separated the genomic TIR sequences from the CMS-associated region. Although RNAs containing orf355/orf77 can still be detected in the revertants, they are not highly expressed during pollen development and they are no longer initiated from the TIR promoter at a protein-stabilized linear end. They appear instead to be co-transcribed with cytochrome oxidase subunit 2. The 7.3-kb inversion was not detected in CMS-S or in other fertile revertants. Therefore, this inversion appears to be a de novo mutation that has continued to sort out within a single maternal lineage, giving rise to fertile progeny in successive generations.

Impact of combination medical therapy on mortality in vascular surgery patients.

BACKGROUND: Th use of beta-blockers or statins has been associated with decreased mortality after noncardiac surgery. There are no prior perioperative studies of concurrent use of other cardioprotective drugs. OBJECTIVE: To ascertain whether combinations of aspirin, beta-blockers, statins, and/or angiotensin-converting enzyme (ACE) inhibitors were associated with decreased mortality 6 months after vascular surgery. PATIENTS AND DESIGN: We performed a retrospective cohort study on the 3020 patients who underwent vascular surgery between January 1998 and March 2005 at 5 regional Veterans Affairs (VA) medical centers. The Cochran-Mantel-Haenszel test was used to assess associations with 6-month all-cause mortality for the combination drug exposures compared to no exposure while adjusting for propensity score. RESULTS: Exposure to all 4 of the study drugs compared to none had a propensity-adjusted relative risk (aRR) of 0.52 (95% confidence interval [CI], 0.26-1.01; P = 0.052), number needed to treat (NNT) 19; 3 drugs vs. none, aRR 0.60 (95% CI, 0.38-0.95; P = 0.030), NNT 38; 2 drugs vs. none, aRR 0.68 (95% CI, 0.46-0.99; P = 0.043), NNT 170; and 1 drug vs. none, aRR 0.88 (95% CI, 0.63-1.22; P = 0.445). ACE inhibitor exposure was common in all combinations. CONCLUSIONS: Combination use of 2 to 3 study drugs, some of which included ACE inhibitors, was associated with decreased mortality after vascular surgery. Combination use of all 4 study drugs was not statistically significant due to the small number of events in this group. Further prospective studies of combination perioperative aspirin, beta-blockers, statins, and ACE inhibitors are warranted.

A mitochondrial mutator system in maize.

The P2 line of maize (Zea mays) is characterized by mitochondrial genome destabilization, initiated by recessive nuclear mutations. These alleles alter copy number control of mitochondrial subgenomes and disrupt normal transfer of mitochondrial genomic components to progeny, resulting in differences in mitochondrial DNA profiles among sibling plants and between parents and progeny. The mitochondrial DNA changes are often associated with variably defective phenotypes, reflecting depletion of essential mitochondrial genes. The P2 nuclear genotype can be considered a natural mutagenesis system for maize mitochondria. It dramatically accelerates mitochondrial genomic divergence by increasing low copy-number subgenomes, by rapidly amplifying aberrant recombination products, and by causing the random loss of normal components of the mitochondrial genomes.

Biochemical and molecular characterization of photosystem I deficiency in the NCS6 mitochondrial mutant of maize.

Interorganellar signaling interactions are poorly understood. The maize non-chromosomal stripe (NCS) mutants provide models to study the requirement of mitochondrial function for chloroplast biogenesis and photosynthesis. Previous work suggested that the NCS6 mitochondrial mutation, a cytochrome oxidase subunit 2 (cox2) deletion, is associated with a malfunction of Photosystem I (PSI) in defective chloroplasts of mutant leaf sectors (Gu et al., 1993). We have now quantified the reductions of photosynthetic rates and PSI activity in the NCS6 defective stripes. Major reductions of the plastid-coded PsaC and nucleus-coded PsaD and PsaE PSI subunits and of their corresponding mRNAs are seen in mutant sectors; however, although the psaA/B mRNA is greatly reduced, levels of PsaA and PsaB (the core proteins of PSI) are only slightly decreased. Levels of the PsaL subunit and its mRNA appear to be unchanged. Tested subunits of other thylakoid membrane complexes--PSII, Cyt b6/f, and ATP synthase, have minor (or no) reductions within mutant sectors. The results suggest that specific signaling pathways sense the dysfunction of the mitochondrial electron transport chain and respond to down-regulate particular PSI mRNAs, leading to decreased PSI accumulation in the chloroplast. The reductions of both nucleus and plastid encoded components indicate that complex interorganellar signaling pathways may be involved.

Mitochondrial respiratory deficiencies signal up-regulation of genes for heat shock proteins.

The consequences of mitochondrial dysfunction are not limited to the development of oxidative stress or initiation of apoptosis but can result in the establishment of stress tolerance. Using maize mitochondrial mutants, we show that permanent mitochondrial deficiencies trigger novel Ca(2+)-independent signaling pathways, leading to constitutive expression of genes for molecular chaperones, heat shock proteins (HSPs) of different classes. The signaling to activate hsp genes appears to originate from a reduced mitochondrial transmembrane potential. Upon depolarization of mitochondrial membranes in transient assays, gene induction for mitochondrial HSPs occurred more rapidly than that for cytosolic HSPs. We also demonstrate that in the nematode Caenorhabditis elegans transcription of hsp genes can be induced by RNA interference of nuclear respiratory genes. In both organisms, activation of hsp genes in response to mitochondrial impairment is distinct from their responses to heat shock and is not associated with oxidative stress. Thus, mitochondria-to-nucleus signaling to express a hsp gene network is apparently a widespread retrograde mechanism to facilitate cell defense and survival.

Differential expression of alternative oxidase genes in maize mitochondrial mutants.

We have examined the expression of three alternative oxidase (aox) genes in two types of maize mitochondrial mutants. Nonchromosomal stripe (NCS) mutants carry mitochondrial DNA deletions that affect subunits of respiratory complexes and show constitutively defective growth. Cytoplasmic male-sterile (CMS) mutants have mitochondrial DNA rearrangements, but they are impaired for mitochondrial function only during anther development. In contrast to normal plants, which have very low levels of AOX, NCS mutants exhibit high expression of aox genes in all nonphotosynthetic tissues tested. The expression pattern is specific for each type of mitochondrial lesion: the NADH dehydrogenase-defective NCS2 mutant has high expression of aox2, whereas the cytochrome oxidase-defective NCS6 mutant predominantly expresses aox3. Similarly, aox2 and aox3 can be induced differentially in normal maize seedlings by specific inhibitors of these two respiratory complexes. Translation-defective NCS4 plants show induction of both aox2 and aox3. AOX2 and AOX3 proteins differ in their ability to be regulated by reversible dimerization. CMS mutants show relatively high levels of aox2 mRNAs in young tassels but none in ear shoots. Significant expression of aox1 is detected only in NCS and CMS tassels. The induction pattern of maize aox genes could serve as a selective marker for diverse mitochondrial defects.

Sequence and comparative analysis of the maize NB mitochondrial genome.

The NB mitochondrial genome found in most fertile varieties of commercial maize (Zea mays subsp. mays) was sequenced. The 569,630-bp genome maps as a circle containing 58 identified genes encoding 33 known proteins, 3 ribosomal RNAs, and 21 tRNAs that recognize 14 amino acids. Among the 22 group II introns identified, 7 are trans-spliced. There are 121 open reading frames (ORFs) of at least 300 bp, only 3 of which exist in the mitochondrial genome of rice (Oryza sativa). In total, the identified mitochondrial genes, pseudogenes, ORFs, and cis-spliced introns extend over 127,555 bp (22.39%) of the genome. Integrated plastid DNA accounts for an additional 25,281 bp (4.44%) of the mitochondrial DNA, and phylogenetic analyses raise the possibility that copy correction with DNA from the plastid is an ongoing process. Although the genome contains six pairs of large repeats that cover 17.35% of the genome, small repeats (20-500 bp) account for only 5.59%, and transposable element sequences are extremely rare. MultiPip alignments show that maize mitochondrial DNA has little sequence similarity with other plant mitochondrial genomes, including that of rice, outside of the known functional genes. After eliminating genes, introns, ORFs, and plastid-derived DNA, nearly three-fourths of the maize NB mitochondrial genome is still of unknown origin and function.