Depth profiling in membranes by fluorescence quenching

Membrane penetration depth is an important parameter in relation to membrane structure and organization. A methodology has been developed to analyze the membrane penetration depths of fluorescent molecules or groups utilizing differential fluorescence quenching caused by membrane embedded spin-label probes located at different depths. The method involves determination of the parallax in the apparent location of fluorophores, detected when quenching by phospholipids spin-labelled at two different depths is compared. By use of relatively simple algebraic expressions, the method allows calculation of depth in å. This method has been used to determine the location of fluorophores in NBD-labelled lipids and anthroyloxy-labelled fatty acids in model membranes and of the membrane embedded tryptophan residues in the reconstituted nicotinic acetylcholine receptor.     

LSU rDNA-BASED RFLP ASSAYS FOR DISCRIMINATING SPECIES AND STRAINS OF ALEXANDRIUM (DINOPHYCEAE)1

In a previous study large-subunit ribosomal RNA gene (LSU rDNA) sequences from the marine dinoflagellates Alexandrium tamarense (Lebour) Balech, A. catenella (Whedon et Kofoid) Balech, A. fundyense Balech, A. affine (Fukuyo et Inoue) Balech, A. minutum Halim, A. lusitanicum Balech, and A. andersoni Balech were compared to assess inter- and intraspecific relationships. Many cultures compared in that study contained more than one class of LSU rDNA. Sequencing pooled clones of rDNA from single cultures revealed length heterogeneities and sequence ambiguities. This complicated sequence comparisons because multiple rDNA clones from a single culture had to be sequenced individually to document the different classes of molecules present in that culture. A further complication remained as to whether or not the observed intraculture sequence variations were reliable genetic markers or were instead artifacts of the polymerase chain reaction (PCR) amplification, cloning, and/or sequencing methods employed. The goals of the present study were to test the accuracy of Alexandrium LSU rDNA sequences using restriction fragment-length polymorphism (RFLP) analysis and to devise RFLP-based assays for discriminating among representatives of that group. Computer-assisted examination of the sequences allowed us to identify a set of restriction enzymes that were predicted to reveal species, strain, and intraculture LSU rDNA heterogeneities. All groups identified by sequencing were revealed independently and repeatedly by RFLP analysis of PCR-amplified material. Five ambiguities and one length heterogeneity, each of which ascribes a unique group of Alexandrium species or strains, were confirmed by restriction digests. Observed intraculture LSU rDNA heterogeneities were not artifacts of cloning and sequencing but were instead a good representation of the spectrum of molecules amplified during PCR reactions. Intraculture LSU rDNA heterogeneities thus serve as unique genetic markers for particular strains of Alexandrium, particularly those of A. tamarense, A. catenella, and A. fundyense. However, some of these “signature heterogeneities” represented a smaller portion of PCR product than was expected given acquired sequences. Other deviations from predicted RFLP patterns included incomplete digestions and appearance of spurious products. These observations indicate that the diversity of sequences in PCR product pools were greater than that observed by cloning and sequencing. The RFLP tests described here are useful tools for characterizing Alexandrium LSU rDNA to define the evolutionary lineage of cultures and are applicable at a fraction of the time, cost, and labor required for sequencing.     

EPISTASIS AS A SOURCE OF INCREASED ADDITIVE GENETIC VARIANCE AT POPULATION BOTTLENECKS

The role of epistasis in evolution and speciation has remained controversial. We use a new parameterization of physiological epistasis to examine the effects of epistasis on levels of additive genetic variance during a population bottleneck. We found that all forms of epistasis increase average additive genetic variance in finite populations derived from initial populations with intermediate allele frequencies. Average additive variance continues to increase over many generations, especially at larger population sizes (N = 32 to 64). Additive-by-additive epistasis is the most potent source of additive genetic variance in this situation, whereas dominance-by-dominance epistasis contributes smaller amounts of additive genetic variance. With additive-by-dominance epistasis, additive genetic variance decreases at a relatively high rate immediately after a population bottleneck, rebounding to higher levels after several generations. Empirical examples of epistasis for murine adult body weight based on measured genotypes are provided illustrating the varying effects of epistasis on additive genetic variance during population bottlenecks.     

A MARKER-BASED METHOD FOR INFERENCES ABOUT QUANTITATIVE INHERITANCE IN NATURAL POPULATIONS

A marker-based method for studying quantitative genetic characters in natural populations is presented and evaluated. The method involves regressing quantitative trait similarity on marker-estimated relatedness between individuals. A procedure is first given for estimating the narrow sense heritability and additive genetic correlations among traits, incorporating shared environments. Estimation of the actual variance of relatedness is required for heritability, but not for genetic correlations. The approach is then extended to include isolation by distance of environments, dominance, and shared levels of inbreeding. Investigations of statistical properties show that good estimates do not require great marker polymorphism, but rather require significant variation of actual relatedness; optimal allocation generally favors sampling many individuals at the expense of assaying fewer marker loci; when relatedness declines with physical distance, it is optimal to restrict comparisons to within a certain distance; the power to estimate shared environments and inbreeding effects is reasonable, but estimates of dominance variance may be difficult under certain patterns of relationship; and any linkage of markers to quantitative trait loci does not cause significant problems. This marker-based method makes possible studies with long-lived organisms or with organisms difficult to culture, and opens the possibility that quantitative trait expression in natural environments can be analyzed in an unmanipulative way.     

INFERENCES ABOUT QUANTITATIVE INHERITANCE BASED ON NATURAL POPULATION STRUCTURE IN THE YELLOW MONKEYFLOWER,MIMULUS GUTTATUS

We used a nonmanipulative, marker-based method to study quantitative genetic inheritance in two habitats of a common monkeyflower population. The method involved regressing quantitative trait similarity on marker-estimated relatedness between individuals sampled in the field. We sampled 300 adult plants from each of two transects, one along a stream habitat and another through a meadow habitat. For each plant we measured 10 quantitative characters and assayed 10 polymorphic isozyme loci. In the meadow habitat, relatedness of plants within 1 m was moderate (r = 0.125, corresponding to half-sibs) as was actual variance of relatedness (V_r = 0.044). Significant heritabilities of 50–70% were found for corolla width and the fitness characters of flower number and plant weight. Genetic correlations were strongly positive, but sharing of environmental effects within 1 m was weak. In the stream habitat, levels of relatedness were lower and similar heritabilities were indicated. To detect dominance variance and the correlation of phenotypes due to shared inbreeding, we also estimated higher-order coefficients of relationship and inbreeding, but these did not significantly differ from zero. Laboratory-based estimates of heritability in the field were lower than the marker-based estimates, indicating that natural heritabilities and genetic correlations may be stronger than indicated by controlled studies.     

Characterization and application of soybean YACs to molecular cytogenetics

Yeast artificial chromosomes (YACs) are widely used in the physical analysis of complex genomes. In addition to their value in chromosome walking for map-based cloning, YACs represent excellent probes for chromosome mapping using fluorescence in situ hybridization (FISH). We have screened such a library for low-copy-number clones by hybridization to total genomic DNA. Four clones were chosen for chromosome tagging based upon their low or moderate signal. By using degenerate oligonucleotide-primed PCR (DOP-PCR), we were able to use relatively small amounts of soybean YAC DNA, isolated directly by preparative pulsed-field gel electrophoresis, as FISH probes for both metaphase chromosome spreads and interphase nuclei. FISH chromosomal analysis using the three of the clones as probes resulted in relatively simple hybridization patterns consistent with a single homologous locus or two homoeologous loci. The fourth YAC probe resulted in a diffuse hybridization pattern with signal on all metaphase chromosomes. We conclude that YACs represent a valuable source of probes for chromosomal analysis in soybean.     

Detection and Identification of Yersinia pestis by Polymerase Chain Reaction (PCR) Using Multiplex Primers

A PCR method for detection of Yersinia pestis-virulence determinants by the use of multiplex primers was developed. Four pairs of oligonucleotide primers were designed from each gene of three kinds of virulent plasmids and a chromosomal DNA; 60-Md plasmid-located gene (caf1) encoding Y. pestis-specific capsular antigen fraction 1, a Y. pestis-specific region of a yopM gene encoded on 42-Md virulent plasmid, a plasminogen activator gene (pla) encoded on Y. pestis-specific 7-Md plasmid and an invasin protein gene (inv) encoded on chromosomal DNA. This multiplex-primer system was specific for the detection of Y. pestis among pathogenic Yersinia species and other enterobacteriaceae having antigens common to Y. pestis. Since this method is simple and safe, it will be useful to identify and confirm Y. pestis in cases of emergency and for the surveillance of epidemics.     

Typing of Verotoxins by DNA Colony Hybridization with Poly- and Oligonucleotide Probes,a Bead-Enzyme-Linked Immunosorbent Assay,and Polymerase Chain Reaction

To identify the type of Verotoxins (VT) produced by Verocytotoxin-producing Escherichia coli (VTEC), a sensitive bead-enzyme-linked immunosorbent assay and polymerase chain reaction with common and specific primers to various VTs (VT1, VT2, VT2vha, VT2vhb, and VT2vp1) were developed. Together with colony hybridization tests with oligo- and polynucleotide probes, these methods were applied to VTEC isolates to type the VT produced. The toxin types of 26 of 37 strains were identified, but the reaction profiles in assays of the remaining 11 strains suggested the existence of new VT2 variants. The application of these identification procedures may be useful as a tool for clinical and epidemiological studies of VTEC infection.     

新疆亚鳞木（比较种）角质层特征

报道了产自江苏宜兴晚泥盆世五通组新疆亚鳞木（比较种）Ｓｕｂｌｅｐｉｄｏｄｅｎｄｒｏｎ ｃｆ． ｘｉｎｊｉａｎｇｅｎｓｅＳｕｎ）的表皮角质层用荧光分析显示的特征．该种茎干表皮角质层覆于叶座及叶座间隔带,其中间隔带角质层厚于叶座．表皮细胞在间隔带与叶座表现特征不同．间隔带中部,表皮细胞呈纵长的多边形,其纵长方向与茎干延伸方向相同,细胞壁略有弯曲．间隔带靠近叶座之表皮细胞,细胞壁直,形状类似于前者;但大小仅为前者的１／２左右,且其纵长方向逐渐向叶座边缘偏转．叶座表皮细胞呈近等多边形,有胞间隙．该种茎表皮无气孔     

CCD imaging of the electrical activity in the leech nervous system

A single ganglion of the nervous system of the leechHirudo medicinalis was isolated. One or both roots emerging from each side of the ganglion were sucked into suction pipettes used either for extracellular stimulation or for recording the gross electrical activity. The ganglion was stained with the fluorescence voltage sensitive dye Di-4-Anepps. The fluorescence was measured with a nitrogen cooled CCD camera. Our recording system allowed us to measure in real time slow optical signals corresponding to changes in light intensity of at least 5. These signals were caused by the direct polarization of neuronal structures, the afterhyperpolarization or the afterdischarge induced by a prolonged stimulation. When images were acquired at fixed times, several of them could be averaged and optical signals of at least 2 could be reliably measured. These optical signals originated from well identified neurons, such as T, P and N sensory neurons. By taking images at different times and at different focal planes, electrical events could be followed at a temporal resolution of 50 Hz. The three dimensional dynamics of electrical events, initiated by a specific stimulation, was imaged and the spread of excitation among leech neurons was followed. When two roots were selectively stimulated, their neuronal interactions could be imaged and the linear and non-linear terms of the interaction could be characterized.