Zea ribosomal repeat evolution and substitution patterns

Zea and Tripsacum nuclear ribosomal internal transcribed spacer (ITS) sequences were used to evaluate patterns of concerted evolution, rates of substitutions, patterns of methylation-induced deamination, and structural constraints of the ITS. ITS pseudogenes were identified by their phylogenetic position, differences in nucleotide composition, extensive deamination at ancestral methylation sites, and substitutions resulting in low-stability secondary RNA structures. Selection was important in shaping the kinds of polymorphisms and substitutions observed in the ITS. ITS substitution rates were significantly different among the Zea taxa. Deamination of cytosines at methylation sites was a potent mutation source, but selection appeared to maintain high methylation site density throughout the ribosomal repeat except for the gene promoter. Nucleotide divergence statistics identified selectively constrained regions at the 5' ends of the ITS1 and ITS2.      

Spinocerebellar ataxia type 1 and Machado-Joseph disease: incidence of CAG expansions among adult-onset ataxia patients from 311 families with dominant, recessive, or sporadic ataxia.

The ataxias are a complex group of diseases with both environmental and genetic causes. Among the autosomal dominant forms of ataxia the genes for two, spinocerebellar ataxia type 1 (SCA1) and Machado-Joseph disease (MJD), have been isolated. In both of these disorders the molecular basis of disease is the expansion of an unstable CAG trinucleotide repeat. To assess the frequency of the SCA1 and MJD trinucleotide repeat expansions among individuals diagnosed with ataxia we have collected DNA from individuals representing 311 families with adult-onset ataxia of unknown etiology and screened these samples for trinucleotide repeat expansions within the SCA1 and MJD genes. Within this group there are 149 families with dominantly inherited ataxia. Of these, 3% had SCA1 trinucleotide repeat expansions, whereas 21% were positive for the MJD trinucleotide expansion. Thus, together SCA1 and MJD represent 24% of the autosomal dominant ataxias in our group, and the frequency of MJD is substantially greater than that of SCA1. For the 57 patients with MJD trinucleotide repeat expansions, a strong inverse correlation between CAG repeat size and age at onset was observed (r = -.838). Among the MJD patients, the normal and affected ranges of CAG repeat size are 14-40 and 68-82 repeats, respectively. For SCA1 the normal and affected ranges are much closer, containing 19-38 and 40-81 CAG repeats, respectively.     

Quantitative fluorescence in situ hybridization of Bifidobacterium spp. with genus-specific 16S rRNA-targeted probes and its application in fecal samples.

Three 16S rRNA hybridization probes were developed and tested for genus-specific detection of Bifidobacterium species in the human fecal flora. Variable regions V2, V4, and V8 of the 16S rRNA contained sequences unique to this genus and proved applicable as target sites for oligodeoxynucleotide probes. Determination of the genus specificity of the oligonucleotides was performed by whole-cell hybridization with fluorescein isothiocyanate-labelled probes. To this end, cells were fixed on glass slides, hybridized with the probes, and monitored by videomicroscopy. In combination with image analysis, this allowed quantification of the fluorescence per cell and objective evaluation of hybridization experiments. One of the probes developed was used to determine the population of Bifidobacterium spp. in human fecal samples. A comparison was made with results obtained by cultural methods for enumeration. Since both methods gave similar population estimates, it was concluded that all bifidobacteria in feces were culturable. However, since the total culturable counts were only a fraction of the total microscopic counts, the contribution of bifidobacteria to the total intestinal microflora was overestimated by almost 10-fold when cultural methods were used as the sole method for enumeration.     

Generation and spectroscopic characterization of new 18+δ electron complexes. Relationship between the stability of 18+δ electron organometallic complexes and their ligand reduction potentials

The relationship between the electrochemical reduction potential of a ligand and the ability of that ligand to form a kinetically inert 18+δ complex in a reaction with a 17-electron radical was investigated. (18+δ complexes are 19-electron adducts in which the unpaired electron is primarily located on a ligand orbital.) To probe the relationship, a series of 18+δ complexes was generated by irradiating the Cp′₂Mo₂(CO)₆, Cp₂Fe₂(CO)₄ and Co₂(CO)₈ dimers in the presence of a series of bidentate phosphorus ligands. (Irradiation of the dimers formed 17-electron metal radicals by photolysis of the metal-metal bonds.) These experiments showed that bidentate phosphorus ligands with reduction potentials more positive than −1 volt (versus SCE) formed long-lived 18+δ complexes (in THF or CH₂Cl₂ solutions at 23 °C), while ligands with potentials more negative than −1 V formed reactive 18+δ complexes. The inability to detect 18+δ complexes in the latter case is attributed to kinetic factors: the 18+δ complexes are powerful reductants and they readily initiate a chain disproportionation of the dimers by electron transfer. Analogous experiments with bidentate nitrogen ligands did not produce any detectable 18+δ complexes. In this case, the undetectability of the 18+δ complexes is probably thermodynamic in origin: the hard nitrogen ligands and soft metal centers form adducts that are unstable with respect to metal-nitrogen bond cleavage. 18+δ complexes are the subject of increasing interest, especially as models for their more reactive 19-electron-complex counterparts. These results provide some guidelines for the design of 18+δ complexes that can be synthesized, isolated and characterized for such studies.     

Concerted transpositions of mobile genetic elements coupled with fitness changes in Drosophila melanogaster

In an inbred low-activity (LA) strain of Drosophila melanogaster with a low level of fitness and a complex of inadaptive characters, in situ hybridization reveals an invariant pattern of distribution of three copia-like elements (mdg-1, mdg-3, and copia). Rare, spontaneous, multiple transpositions of mobile elements in the LA strain were shown to be coupled with a drastic increase of fitness. A changed pattern of various types of mobile elements was also observed on selecting the LA strain for higher fitness. High-fitness strains show transpositions of mobile elements to definite chromosomal sites ("hot spots"). Concerted changes in the location of three different mobile elements were found to be coupled with an increase of fitness. The mdg-1 distribution patterns were also examined in two low-fitness strains independently selected from the high-fitness ones. Fitness decrease was accompanied by mdg-1 excision from the hot spots of their location usually detected in the high-fitness strains. The results suggest the existence of a system of adaptive transpositions of mobile elements that takes part in fitness control.      

Munc18-1 redistributes in nerve terminals in an activity- and PKC-dependent manner

Munc18-1 is a soluble protein essential for synaptic transmission. To investigate the dynamics of endogenous Munc18-1 in neurons, we created a mouse model expressing fluorescently tagged Munc18-1 from the endogenous munc18-1 locus. We show using fluorescence recovery after photobleaching in hippocampal neurons that the majority of Munc18-1 trafficked through axons and targeted to synapses via lateral diffusion together with syntaxin-1. Munc18-1 was strongly expressed at presynaptic terminals, with individual synapses showing a large variation in expression. Axon–synapse exchange rates of Munc18-1 were high: during stimulation, Munc18-1 rapidly dispersed from synapses and reclustered within minutes. Munc18-1 reclustering was independent of syntaxin-1, but required calcium influx and protein kinase C (PKC) activity. Importantly, a PKC-insensitive Munc18-1 mutant did not recluster. We show that synaptic Munc18-1 levels correlate with synaptic strength, and that synapses that recruit more Munc18-1 after stimulation have a larger releasable vesicle pool. Hence, PKC-dependent dynamic control of Munc18-1 levels enables individual synapses to tune their output during periods of activity.     

Reverse breeding: a novel breeding approach based on engineered meiosis

Reverse breeding (RB) is a novel plant breeding technique designed to directly produce parental lines for any heterozygous plant, one of the most sought after goals in plant breeding. RB generates perfectly complementing homozygous parental lines through engineered meiosis. The method is based on reducing genetic recombination in the selected heterozygote by eliminating meiotic crossing over. Male or female spores obtained from such plants contain combinations of non-recombinant parental chromosomes which can be cultured in vitro to generate homozygous doubled haploid plants (DHs). From these DHs, complementary parents can be selected and used to reconstitute the heterozygote in perpetuity . Since the fixation of unknown heterozygous genotypes is impossible in traditional plant breeding, RB could fundamentally change future plant breeding. In this review, we discuss various other applications of RB, including breeding per chromosome.     

Use of Camera-Trapping to Estimate Puma Density and Influencing Factors in Central Brazil

Abstract: We used remotely triggered cameras to collect data on Puma (Puma concolor) abundance and occupancy in an area of tropical forest in Brazil where the species' status is poorly known. To evaluate factors influencing puma occupancy we used data from 5 sampling campaigns in 3 consecutive years (2005 to 2007) and 2 seasons (wet and dry), at a state park and a private forest reserve. We estimated puma numbers and density for the 2007 sampling data by developing a standardized individual identification method. We based individual identification on 1) time-stable parameters (SP; physical features that do not change over time), and 2) time-variable parameters (VP; marks that could change over time such as scars and botfly marks). Following individual identification we established a capture-recapture history and analyzed it using closed population capture-mark-recapture models. Puma capture probability was influenced by camera placement (roads vs. trails), sampling year, and prey richness. Puma occupancy was positively associated with species richness and there was a correlation between relative puma and jaguar (Panthera onca) abundance. Identifications enabled us to generate 8 VP histories for each photographed flank, corresponding to 8 individuals. We estimated the sampled population at 9 pumas (SE = 1.03, 95% CI = 8–10 individuals) translating to a density of 3.40 pumas/100 km². Information collected using camera-traps can effectively be used to assess puma population size in tropical forests. As habitat progressively disappears and South American felines become more vulnerable, our results support the critical importance of private forest reserves for conservation.