Preparation of chloroplast DNA from pea plastids isolated in a medium of high ionic strength

A simple, rapid, and inexpensive method for the preparation and purification of chloroplast DNA (cpDNA) from pea has been developed. The crucial step is the isolation of chloroplasts in a medium of high ionic strength (I congruent equal to 1.40 M). CpDNA from pea prepared according to this method has successfully been used for restriction enzyme mapping, Southern transfers, and cloning.     

An anatomical study of anther development in Acorus L.: Phylogenetic implications

 Critical morphological synapomorphies have not been found in support of the Acoranan hypothesis, the molecular phylogenetic discovery that Acoranae are the basal monocots. The previously undetermined pattern of anther wall development in Acorus has been suggested to be one such character. Two main types of anther wall development have been recognized: 1) the “monocotyledonous” type, which characterizes both monocots and dicots, and 2) the “dicotyledonous” type, which is almost exclusively found among dicots. An anatomical study of anther wall development in Acorus was here undertaken using the electron microscope. Development of the anther wall in Acorus was found to be somewhat irregular or perhaps even intermediate between the two types although largely consistent with the “monocotyledonous” type. The presumed significance of anther wall development and other critical morphological characters to the Acoranan hypothesis in the absence of knowledge about the sister group to the monocots is evaluated. Received August 28, 2000 Accepted February 19, 2001     

MicroID2: A Novel Biotin Ligase Enables Rapid Proximity-Dependent Proteomics

Identifying protein–protein and other proximal interactions is central to dissecting signaling and regulatory processes in cells. BioID is a proximity-dependent biotinylation method that uses an “abortive” biotin ligase to detect proximal interactions in cells in a highly reproducible manner. Recent advancements in proximity-dependent biotinylation tools have improved efficiency and timing of labeling, allowing for measurement of interactions on a cellular timescale. However, issues of size, stability, and background labeling of these constructs persist. Here we modified the structure of BioID2, derived from Aquifex aeolicus BirA, to create a smaller, highly active, biotin ligase that we named MicroID2. Truncation of the C terrminus of BioID2 and addition of mutations to alleviate blockage of biotin/ATP binding at the active site of BioID2 resulted in a smaller and highly active construct with lower background labeling. Several additional point mutations improved the function of our modified MicroID2 construct compared with BioID2 and other biotin ligases, including TurboID and miniTurbo. MicroID2 is the smallest biotin ligase reported so far (180 amino acids [AAs] for MicroID2 versus 257 AAs for miniTurbo and 338 AAs for TurboID), yet it demonstrates only slightly less labeling activity than TurboID and outperforms miniTurbo. MicroID2 also had lower background labeling than TurboID. For experiments where precise temporal control of labeling is essential, we in addition developed a MicroID2 mutant, termed lbMicroID2 (low background MicroID2), that has lower labeling efficiency but significantly reduced biotin scavenging compared with BioID2. Finally, we demonstrate utility of MicroID2 in mass spectrometry experiments by localizing MicroID2 constructs to subcellular organelles and measuring proximal interactions.     

Localization of99mTc in bone by means of autoradiography

The uptake of two different preparations of^99mTechnetium-methylene diphosphonate in fetal rat calvaria is compared. The localization of^99mTc after administration of^99mTc(Sn)-MDP and^99mTc-MDP showed equal distribution in autoradiography.     

Restriction mapping of the rRNA genes from Artemia larvae

A restriction endonuclease analysis of the genes coding for the ribosomal RNA from Artemia larvae has shown that these genes consist of a repeat unit of 16.2 kilobase pairs (10.7 Mdaltons) and that the repeat unit seems to be homogeneous in size.     

Genetic diversity and molecular fingerprinting of Prunus cerasus var. austera from central Italy

In central Italy, Prunus cerasus var. austera is cultivated as small stands or scattered trees in marginal areas for the production of jam and wine. Thanks to the healthy attributes of its products and its ability to grow in different environmental conditions, this variety has gained new interest in the development of marginal areas. We assessed the level of the genetic variability of P. cerasus var. austera germplasm from central Italy and identified a ‘core collection’ representative of the present genetic diversity. A total of 161 trees, morphologically identified as var. austera, and one tree, identified as var. caproniana were collected and genotyped by 14 SSRs. Two individuals provided by a commercial plant nursery, one of P. cerasus var. caproniana and one of P. cerasus var. austera, were used as control. Thirteen SSRs presented private alleles in austera. Seven individuals morphologically identified as austera revealed private alleles specific to caproniana. The PCoA and Bayesian clustering analysis showed a main genetic group including var. austera, while a second group included all the caproniana-like genotypes. A core collection of 31 trees (46% of austera genotypes) was selected. This study can be considered as a starting point for future investigations on this variety.     

Evidences for genetic differentiation within the highly endemic and endangered annual fish Austrolebias nigrofasciatus (Cyprinodontiformes: Rivulidae)

Samples of Austrolebias nigrofasciatus (n = 103), an endangered species of annual fish endemic to a small area of the Patos-Mirim lagoon system encompassing the São Gonçalo Channel lowlands, were collected from eight isolated temporary ponds, four located at the known distribution range of the species and four located along the Piratini River lowlands, where morphologically different individuals were found. In the laboratory, fragments of the mitochondrial cytochrome c oxidase I (coI), cytochrome b (cytb) and nuclear rhodopsin (rho) genes were amplified, purified and sequenced for 100, 99 and 58 of these individuals, respectively. Samples were further analysed using phylogenetic and phylogeographic methods to evaluate the patterns of genetic diversity and differentiation presented within and between populations, while assessing their evolutionary history, in order to guide the application of further conservation strategies. We found that the four new populations from the Piratini River lowlands encompass a different lineage of A. nigrofasciatus that diverged from that encountered in the São Gonçalo Channel at approximately 0.165 M years before present, during a population expansion and did not yet attain reciprocal monophyly. This divergence was associated with a glacial event that was preceded by an interglacial period putatively associated with the dispersal. Moreover, significant levels of genetic differentiation and a high number of exclusive haplotypes could be encountered even in micro-geographical scales, as in the comparisons between populations located within the same major lineage, indicating each of them may encompass independent management units. Conservation actions are certainly urgent, especially in the face of signs of a recent bottleneck.     

Detection of haplotypic variation and natural hybridization in halepensis-complex pine species using chloroplast simple sequence repeat (SSR) markers

In this investigation, nine chloroplast, paternally inherited simple-sequence repeat (cpSSR) markers were used to describe genetic variation of three closely related species belonging to the halepensis complex ( Pinus halepensis Ait., P. brutia Mill. and P. eldarica Medwed.). Both the infinite allele model (IAM) and stepwise-mutation model (SMM) have been applied to the analysis of the genetic structure of natural populations and the geographical distribution of haplotypic variation. SMM-based estimators performed better than IAM-based estimators for large values of within-population diversity and divergence between population pairs. Overall, large haplotypic variation and high genetic divergence were detected for both P. halepensis and P. brutia . The genetic structures of the three species are discussed with consideration to the evolutionary and ecological characteristics of these species. Three highly informative markers showing size variants distinguishing P. halepensis from the other two species were used to provide more information on the occurrence of natural hybridization in a Turkish sympatric population of P. halepensis and P. brutia . Strong evidence of introgression of ' halepensis ' chloroplast haplotypes into P. brutia seeds (but not vice versa) was detected. According to previous evidence from controlled crossings, matings between the above species seem to be successful only when P. halepensis is the pollen donor and P. brutia is the female parent (but not reciprocally). The existence of unidirectional gene flow in sympatric populations confirms previous evidence about partial reproductive barriers between P. halepensis and P. brutia . Implications of the above evidence for the evolutionary history of these species are discussed.     

Resource competition and fire‐regulated nutrient demand in carnivorous plants of wet pine savannas

Question: What are the mechanisms by which fire reduces competition for both a short‐lived and a long‐lived species in old‐growth ground‐cover plant communities of wet pine savannas (originally Pinus palustris, replaced by P. elliottii)? Location: Outer coastal plain of southeastern Mississippi, USA. Methods: I reviewed previous competition experiments and proposed a new hypothesis to explain the relationship between fire, competition, and species co‐existence in wet longleaf pine savannas. The first study is about growth and seedling emergence responses of a short‐lived carnivorous plant, Drosera capillaris, to reduction in below‐ground competition and above‐ plus below‐ground competition. The second study deals with growth and survival responses of a long‐lived perennial carnivorous plant, Sarracenia alata, to neighbour removal and prey‐exclusion to determine if a reduction in nutrient supply increased the intensity of competition in this nutrient‐poor system. Results: Fire increased seedling emergence of the short‐lived species by reducing above‐ground competition through the destruction of above‐ground parts of plants and the combustion of associated litter. Prey exclusion did not increase competitive effects of neighbours on the long‐lived species. However, because the experiment was conducted in a year without fire, shade reduced nutrient demand, which may have obviated competition for soil nutrients between Sarracenia alata and its neighbours. Conclusion: Repeated fires likely interact with interspecific differences in nutrient uptake to simultaneously reduce both above‐ground competition and competition for nutrients in old‐growth ground cover communities in pine savannas. Restoration practitioners should consider the possibility that the composition of the plant community is just as important as fire in ensuring that frequent fires maintain species diversity.