Haplotype characterization of the COI mitochondrial gene in Chrysoperla externa (Neuroptera: Chrysopidae) from different environments in Jaboticabal, state of São Paulo, southeastern Brazil

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Molecular phylogeny of the genus Saguinus (Platyrrhini, Primates) based on the ND1 mitochondrial gene and implications for conservation

The systematics of the subfamily Callitrichinae (Platyrrhini, Primates), a group of small monkeys from South America and Panama, remains an area of considerable discussion despite many investigations, there being continuing controversy over subgeneric taxonomic classifications based on morphological characters. The purpose of our research was to help elucidate the phylogenetic relationships within the monkey genus Saguinus (Callitrichinae) using a molecular approach to discover whether or not the two different sections containing hairy-faced and bare-faced species are monophyletic, whether Saguinus midas midas and Saguinus bicolor are more closely related than are S. midas midas and Saguinus midas niger, and if Saguinus fuscicollis melanoleucus and Saguinus fuscicollis weddelli really are different species. We sequenced the 957 bp ND1 mitochondrial gene of 21 Saguinus monkeys (belonging to six species and nine morphotypes) and one Cebus monkey (the outgroup) and constructed phylogenetic trees using maximum parsimony, neighbor joining, and maximum likelihood methods. The phylogenetic trees obtained divided the genus Saguinus into two groups, one containing the small-bodied species S. fuscicollis and the other, the large-bodied species S. mystax, S. leucopus, S. oedipus, S. midas, S. bicolor. The most derived taxa, S. midas and S. bicolor, grouped together, while S. fuscicollis melanoleucus and S. f. weddelli showed divergence values that did not support the division of these morphotypes into subspecies. On the other hand, S. midas individuals showed divergence compatible with the existence of three subspecies, two of them with the same morphotype as the subspecies S. midas niger. The results of our study suggest that there is at least one Saguinus subspecies that has not yet been described and that the conservation status of Saguinus species and subspecies should be carefully revised using modern molecular approaches.

First assessment on genetic structure and phylogeography of Mediterranean blue shark (prionace glauca, L. 1758) population using mitochondrial gene variation: a comparison with the Atlantic.

The blue shark, Prionace glauca, is one of the most vagile shark species worldwide distributed. The particular body shape allows blue sharks make transoceanic movements, leading to a circumglobal distribution. Due to its reproductive cycle, an extraordinarily high number of specimens is globally registered but, even if it is still a major bycatch of longline fishery rather than a commercial target, it is characterized by a high vulnerability. In this perspective it is important to increase the amount of informations regarding its population extent in the different worldwide areas, evaluating the possible phylogeographic patterns between different locations. This study, included in the "MedBlueSGen" European project, aims exactly at filling a gap in knowledges regarding the genetic population structure of the Mediterranean blue sharks, which has never been investigated before, with a comparison with the North-Eastern Atlantic blue shark population. To reach this objective, we used a dataset of samples from different Mediterranean areas implementing it with some samples from North-Eastern Atlantic. Analyzing the variability of the two mitochondrial markers control region and cytochrome b, with the design of new species-specific primer pairs, we assessed the mitochondrial genetic structure of Mediterranean and North-Eastern Atlantic samples, focusing on the analysis of their possible connectivity, and we tried to reconstruct their demographic history and population size. Data analyses highlighted the absence of a genetic structuring within the Mediterranean and among it and North-Eastern Atlantic, suggesting that the Strait of Gibraltar doesn't represent a phylogeographic barrier. These results are coherent to what has been found in similar investigations on other worldwide blue shark populations. Analysis of the historical demographic trend revealed a general stable pattern for the cytochrome-b and a slightly population expansion for the control region marker.

Determinants of Acidobacteria activity inferred from the relative abundances of 16S rRNA transcripts in German grassland and forest soils

16S rRNA genes and transcripts of Acidobacteria were investigated in 57 grassland and forest soils of three different geographic regions. Acidobacteria contributed 9-31% of bacterial 16S rRNA genes whereas the relative abundances of the respective transcripts were 4-16%. The specific cellular 16S rRNA content (determined as molar ratio of rRNA:rRNA genes) ranged between 3 and 80, indicating a low in situ growth rate. Correlations with flagellate numbers, vascular plant diversity and soil respiration suggest that biotic interactions are important determinants of Acidobacteria 16S rRNA transcript abundances in soils. While the phylogenetic composition of Acidobacteria differed significantly between grassland and forest soils, high throughput denaturing gradient gel electrophoresis and terminal restriction fragment length polymorphism fingerprinting detected 16S rRNA transcripts of most phylotypes in situ. Partial least squares regression suggested that chemical soil conditions such as pH, total nitrogen, C:N ratio, ammonia concentrations and total phosphorus affect the composition of this active fraction of Acidobacteria. Transcript abundance for individual Acidobacteria phylotypes was found to correlate with particular physicochemical (pH, temperature, nitrogen or phosphorus) and, most notably, biological parameters (respiration rates, abundances of ciliates or amoebae, vascular plant diversity), providing culture-independent evidence for a distinct niche specialization of different Acidobacteria even from the same subdivision.

Characterization of fecal contamination targeting 16S rRNA bacteroides molecular markers in the Santa Cruz River, Arizona

Understanding the origins, transport and fate of contamination is essential to effective management of water resources and public health. Individuals and organizations with management responsibilities need to understand the risks to ecosystems and to humans from contact with contamination. Managers also need to understand how key contaminants vary over time and space in order to design and prioritize mitigation strategies. Tumacacori National Historic Park (NHP) is responsible for management of its water resources for the benefit of the park and for the health of its visitors. The existence of microbial contaminants in the park poses risks that must be considered in park planning and operations. The water quality laboratory at the Maricopa Agricultural Center (in collaboration with stakeholder groups and individuals located in the ADEQ-targeted watersheds) identified biological changes in surface water quality in impaired reaches rivers to determine the sources of Escherichia coli (E. coli); bacteria utilizing innovative water quality microbial/bacterial source tracking methods. The end goal was to support targeted watershed groups and ADEQ towards E. coli reductions. In the field monitoring was conducted by the selected targeted watershed groups in conjunction with The University of Arizona Maricopa Agricultural Center Water Quality Laboratory. This consisted of collecting samples for Bacteroides testing from multiple locations on select impaired reaches, to determine contamination resulting from cattle, human recreation, and other contributions. Such testing was performed in conjunction with high flow and base flow conditions in order to accurately portray water quality conditions and variations. Microbial monitoring was conducted by The University of Arizona Water Quality Laboratory at the Maricopa Agricultural Center using genetic typing to differentiate among two categories of Bacteroides: human and all (total). Testing used microbial detection methodologies and molecular source tracking techniques.^

Mechanism of molecular regulation of nuclear -encoded mitochondrial gene expression by electrical stimulation in the cultured neonatal rat cardiac myocytes

To answer the question whether increased energy demand resulting from myocyte hypertrophy and enhanced $\beta$-myosin heavy chain mRNA, contractile protein synthesis and assembly leads to mitochondrial proliferation and differentiation, we set up an electrical stimulation model of cultured neonatal rat cardiac myocytes. We describe, as a result of increased contractile activity, increased mitochondrial profiles, cytochrome oxidase mRNA, and activity, as well as a switch in mitochondrial carnitine palmitoyltransferase-I (CPT-I) from the liver to muscle isoform. We investigate physiological pathways that lead to accumulation of gene transcripts for nuclear encoded mitochondrial proteins in the heart. Cardiomyocytes were stimulated for varying times up to 72 hr in serum-free culture. The mRNA contents for genes associated with transcriptional activation (c-fos, c-jun, junB, nuclear respiratory factor 1 (Nrf-1)), mitochondrial proliferation (cytochrome c (Cyt c), cytochrome oxidase), and mitochondrial differentiation (carnitine palmitonyltransferase I (CPT-I) isoforms) were measured. The results establish a temporal pattern of mRNA induction beginning with c-fos (0.25-3 hr) and followed by c-jun (0.5-3 hr), junB (0.5-6 hr), NRF-1 (1-12 hr), Cyt c (12-72 hr), cytochrome c oxidase (12-72 hr). Induction of the latter was accompanied by a marked decrease in the liver-specific CPT-I mRNA. Electrical stimulation increased c-fos, $\beta$-myosin heavy chain, and Cyt c promoter activities. These increases coincided with a rise in their respective endogenous gene transcripts. NRF-1, cAMP response element (CRE), and Sp-1 site mutations within the Cyt c promoter reduced luciferase expression in both stimulated and nonstimulated myocytes. Mutations in the Nrf-1 and CRE sites inhibited the induction by electrical stimulation or by transfection of c-jun into non-paced cardiac myocytes whereas mutation of the Sp-1 site maintained or increased the fold induction. This is consistent with the appearance of NRF-1 and fos/jun mRNAs prior to that of Cyt c. Overexpression of c-jun by transfection also activates the Nrf-1 and Cyt c mRNA sequentially. Electrical stimulation of cardiac myocytes activates the c-Jun-N-terminal kinase so that the fold-activation of the cyt c promoter is increased by pacing when either c-jun or c-fos/c-jun are cotransfected. We have identified physical association of Nrf-1 protein with the Nrf-1 enhancer element and of c-Jun with the CRE binding sites on the Cyt c promoter. This is the first demonstration that induction of Nrf-1 and c-Jun by pacing of cardiac myocytes directly mediates Cyt c gene expression and mitochondrial proliferation in response to hypertrophic stimuli in the heart.^ Subsequent to gene activation pathways that lead to mitochondrial proliferation, we observed an isoform switch in CPT-I from the liver to muscle mRNA. We have found that the half-life for the muscle CPT-I is not affected by electrical stimulation, but electrical decrease the T1/2 in the liver CPT-I by greater than 50%. This suggests that the liver CPT-I switch to muscle isoform is due to (1) a decrease in T1/2 of liver CPT-I and (2) activation of muscle CPT-Itranscripts by electrical stimulation. (Abstract shortened by UMI.) ^

Genetic integration of a nuclear -encoded mitochondrial gene with cardiac function

Cardiovascular disease (CVD) is the leading cause of death in the United States. One manifestation of CVD known to increase mortality is an enlarged, or hypertrophic heart. Hypertrophic cardiomyocytes adapt to increased contractile demand at the genetic level with a re-emergence of the fetal gene program and a downregulation of fatty acid oxidation genes with concomitant increased reliance on glucose-based metabolism. To understand the transcriptional regulatory pathways that implement hypertrophic directives we analyzed the upstream promoter region of the muscle specific isoform of the nuclear-encoded mitochondrial gene, carnitine palmitoyltransferase-1β (CPT-1β) in cultured rat neonatal cardiac myocytes. This enzyme catalyzes the rate-limiting step of fatty acid entry into β-oxidation and is downregulated in cardiac hypertrophy and failure, making it an attractive model for the study of hypertrophic gene regulation and metabolic adaptations. We demonstrate that the muscle-enriched transcription factors GATA-4 and SRF synergistically activate CPT-1β; moreover, DNA binding to cognate sites and intact protein structure are required. This mechanism coordinates upregulation of energy generating processes with activation of the energy consuming contractile promoter for cardiac α-actin. We hypothesized that fatty acid or glucose responsive transcription factors may also regulate CPT-1β. Oleate weakly stimulates CPT-1β activity; in contrast, the glucose responsive Upstream Stimulatory Factors (USF) dramatically depresses the CPT-1β reporter. USF regulates CPT-1β through a novel physical interaction with the cofactor PGC-1 and abrogation of MEF2A/PGC-1 synergistic stimulation. In this way, USF can inversely regulate metabolic gene programs and may play a role in the shift of metabolic substrate preference seen in hypertrophy. Failing hearts have elevated expression of the nuclear hormone receptor COUP-TF. We report that COUP-TF significantly suppresses reporter transcription independent of DNA binding and specific interactions with GATA-4, Nkx2.5 or USF. In summary, CPT-1β transcriptional regulation integrates mitochondrial gene expression with two essential cardiac functions: contraction and metabolic substrate oxidation. ^

A possible mitochondrial gene in the early-branching amitochondriate protist Trichomonas vaginalis

Trichomonads are anaerobic flagellated protists that, based on analyses of ribosomal RNA sequences, represent one of the earliest branching lineages among the eukaryotes. The absence of mitochondria in these organisms coupled with their deep phylogenetic position has prompted several authors to suggest that trichomonads, along with other deeply-branching amitochondriate protist groups, diverged from the main eukaryotic lineage prior to the endosymbiotic origin of mitochondria. In this report we describe the presence of a gene in Trichomonas vaginalis specifically related to mitochondrial chaperonin 60 (cpn60). A recent study indicates that a protein immunologically related to cpn60 is located in trichomonad hydrogenosomes. Together, these data provide evidence that ancestors of trichomonads perhaps harbored the endosymbiotic progenitors of mitochondria, but that these evolved into hydrogenosomes early in trichomonad evolution.

Multiple independent origins of mitochondrial gene order in birds

Mitochondrial genomes of all vertebrate animals analyzed to date have the same 37 genes, whose arrangement in the circular DNA molecule varies only in the relative position of a few genes. This relative conservation suggests that mitochondrial gene order characters have potential utility as phylogenetic markers for higher-level vertebrate taxa. We report discovery of a mitochondrial gene order that has had multiple independent originations within birds, based on sampling of 137 species representing 13 traditionally recognized orders. This provides evidence of parallel evolution in mitochondrial gene order for animals. Our results indicate operation of physical constraints on mitochondrial gene order changes and support models for gene order change based on replication error. Bird mitochondria have a displaced OL (origin of light-strand replication site) as do various other Reptilia taxa prone to gene order changes. Our findings point to the need for broad taxonomic sampling in using mitochondrial gene order for phylogenetic analyses. We found, however, that the alternative mitochondrial gene orders distinguish the two primary groups of songbirds (order Passeriformes), oscines and suboscines, in agreement with other molecular as well as morphological data sets. Thus, although mitochondrial gene order characters appear susceptible to some parallel evolution because of mechanistic constraints, they do hold promise for phylogenetic studies.

Phylogenetic relationships of the hoatzin, an enigmatic South American bird.

The hoatzin (Opisthocomus hoazin) lives in the humid lowlands of northern and central South America, often in riparian habitats. It is a slender bird approximately 65 cm in length, brownish with lighter streaks and buffy tips to the long tail feathers. The small head has a ragged, bristly crest of reddish-brown feathers, and the bare skin of the face is bright blue. It resembles a chachalaca (Ortalis, Cracidae) in size and shape, but its plumage and markings are similar to those of the smaller guira cuckoo (Guira guira). The hoatzin (pronounced Watson) has been a taxonomic puzzle since it was described in 1776. It usually has been viewed as related to the gallinaceous birds, but alliances to other groups have been suggested, including the cuckoos. We present DNA sequence evidence from the 12S and 16S rRNA mitochondrial genes, and from the nuclear gene that codes for the eye lens protein, alpha A-crystallin. The results indicate that the hoatzin is most closely related to the typical cuckoos and that the divergence occurred at or near the base of the cuculiform phylogenetic tree.

Genetic and comparative analyses reveal an alternative secondary structure in the region of nt 912 of Escherichia coli 16S rRNA.

Mutations at position 912 of Escherichia coli 16S rRNA result in two notable phenotypes. The C-->U transition confers resistance to streptomycin, a translational-error-inducing antibiotic, while a C-->G transversion causes marked retardation of cell growth rate. Starting with the slow-growing G912 mutant, random mutagenesis was used to isolate a second site mutation that restored growth nearly to the wild-type rate. The second site mutation was identified as a G-->C transversion at position 885 in 16S rRNA. Cells containing the G912 mutation had an increased doubling time, abnormal sucrose gradient ribosome/subunit profile, increased sensitivity to spectinomycin, dependence upon streptomycin for growth in the presence of spectinomycin, and slower translation rate, whereas cells with the G912/C885 double mutation were similar to wild type in these assays. Comparative analysis showed there was significant covariation between positions 912 and 885. Thus the second-site suppressor analysis, the functional assays, and the comparative data suggest that the interaction between nt 912 and nt 885 is conserved and necessary for normal ribosome function. Furthermore, the comparative data suggest that the interaction extends to include G885-G886-G887 pairing with C912-U911-C910. An alternative secondary structure element for the central domain of 16S rRNA is proposed.

Ribosome Shut-Down by 16S rRNA Fragmentation in Stationary-Phase Escherichia coli

Stationary-phase bacterial cells are characterized by vastly reduced metabolic activities yielding a dormant-like phenotype. Several hibernation programs ensure the establishment and maintenance of this resting growth state. Some of the stationary phase-specific modulations affect the ribosome and its translational activity directly. In stationary-phase Escherichia coli, we observed the appearance of a 16S rRNA fragmentation event at the tip of helix 6 within the small ribosomal subunit (30S). Stationary-phase 30S subunits showed markedly reduced activities in protein biosynthesis. On the other hand, the functional performance of stationary-phase large ribosomal subunits (50S) was indistinguishable from particles isolated from exponentially growing cells. Introduction of the 16S rRNA cut in vitro at helix 6 of exponential phase 30S subunits renders them less efficient in protein biosynthesis. This indicates that the helix 6 fragmentation is necessary and sufficient to attenuate translational activities of 30S ribosomal subunits. These results suggest that stationary phase-specific cleavage of 16S rRNA within the 30S subunit is an efficient means to reduce global translation activities under non-proliferating growth conditions.

A novel screen for nuclear mitochondrial gene associations with Parkinson's disease

Genetic factors play an important role in the aetiology of Parkinson's disease (PD). We have screened nuclear genes encoding subunits of mitochondrial complex I for associations between single nucleotide polymorphisms (SNPs) and PD. Abnormal functioning of complex I is well documented in human PD. Moreover, toxicological inhibition of complex I can lead to parkinsonism in animals. Thus, commonly occurring variants in these genes could potentially influence complex I function and the risk of developing PD. A sub-set of 70 potential SNPs in 31 nuclear complex I genes were selected and association analysis was performed on 306 PD patients plus 321 unaffected control subjects. Genotyping was performed using the DASH method. There was no evidence that the examined SNPs were significant genetic risk factors for PD, although this initial screen could not exclude the possibility that other disease-influencing variations exist within these genes.

Bacterial community structure associated with white band disease in the elkhorn coral Acropora palmata determined using culture-independent 16S rRNA techniques

Culture-independent molecular (16S ribosomal RNA) techniques showed distinct differences in bacterial communities associated with white band disease (WBD) Type I and healthy elkhorn coral Acropora palmata. Differences were apparent at all levels, with a greater diversity present in tissues of diseased colonies. The bacterial community associated with remote, non-diseased coral was distinct from the apparently healthy tissues of infected corals several cm from the disease lesion. This demonstrates a whole-organism effect from what appears to be a localised disease lesion, an effect that has also been recently demonstrated in white plague-like disease in star coral Montastraea annularis. The pattern of bacterial community structure changes was similar to that recently demonstrated for white plague-like disease and black band disease. Some of the changes are likely to be explained by the colonisation of dead and degrading tissues by a micro-heterotroph community adapted to the decomposition of coral tissues. However, specific ribosomal types that are absent from healthy tissues appear consistently in all samples of each of the diseases. These ribotypes are closely related members of a group of alpha-proteobacteria that cause disease, notably juvenile oyster disease, in other marine organisms. It is clearly important that members of this group are isolated for challenge experiments to determine their role in the diseases.

Association of extended-spectrum β-lactamase VEB-5 and 16S rRNA methyltransferase armA in Salmonella enterica from the United Kingdom

Aminoglycosides and beta-lactams are used for the treatment of a wide range of infections due to both Gram-negative and Gram-positive. An emerging aminoglycoside resistance mechanism, methylation of the aminoacyl site of the 16S rRNA, confers high-level resistance to clinically important aminoglycosides such as amikacin, tobramycin and gentamicin. Eight 16S rRNA methyltransferase genes, armA, rmtA, rmtB, rmtC, rmtD, rmtE, rmtF and npmA, have been identified in several species of enterobacteria worldwide (2, 6, 7, 9, 11, 13, 14). Resistance to extended spectrum β-lactams remains additionally an important clinical problem. Apart from the large TEM, SHV, and CTX-M families, several other extended-spectrum β-lactamases (ESBLs) have been identified, including VEB enzymes, which confer high-level resistance to cephalosporins and monobactams. Although 16S rRNA methyltransferases have been frequently identified associated with different ESBLs, there has been no report of association of a 16S rRNA methyltransferase with a VEB enzyme, except for the identification of rmtC with blaVEB-6 (14)