Levels of genetic polymorphism: marker loci versus quantitative traits.

Species are the units used to measure ecological diversity and alleles are the units of genetic diversity. Genetic variation within and among species has been documented most extensively using allozyme electrophoresis. This reveals wide differences in genetic variability within, and genetic distances among, species, demonstrating that species are not equivalent units of diversity. The extent to which the pattern observed for allozymes can be used to infer patterns of genetic variation in quantitative traits depends on the forces generating and maintaining variability. Allozyme variation is probably not strictly neutral but, nevertheless, heterozygosity is expected to be influenced by population size and genetic distance will be affected by time since divergence. The same is true for quantitative traits influenced by many genes and under weak stabilizing selection. However, the limited data available suggest that allozyme variability is a poor predictor of genetic variation in quantitative traits within populations. It is a better predictor of general phenotypic divergence and of postzygotic isolation between populations or species, but is only weakly correlated with prezygotic isolation. Studies of grasshopper and planthopper mating signal variation and assortative mating illustrate how these characters evolve independently of general genetic and morphological variation. The role of such traits in prezygotic isolation, and hence speciation, means that they will contribute significantly to the diversity of levels of genetic variation within and among species.     

Cell wall polysaccharides are informative in Porphyra species taxonomy

As part of systematic studies of the genus Porphyrain New Zealand, constituent sugar analyses of cell wall polysaccharidesin situ in dry thalli were found to yield data that weretaxonomically informative. Variation in constituent sugar levels betweenspecieswas sufficient in some cases to be useful in species differentiation. Thereproductive state of thallus regions had a significant impact on the levels ofconstituent sugars, whereas storage of dried thalli for eight months had noeffect. Three epiphytic taxa currently classified as species ofPorphyra appear to be incorrectly placed within the genus,as their constituent sugars and the levels of these sugars differed markedlyfrom those of all other species examined.     

A nonradioisotope biomedical assay for intact oligonucleotide and its chain-shortened metabolites used for determination of exposure and elimination half-life of antisense drugs in tissue.

Rigorous extraction methods coupled with capillary gel electrophoresis (CGE) provide a basis for a nonradiolabel assay for quantitation of intact antisense drug and its numerous chain-shortened metabolites. As part of the validation of the CGE method, we compared the quantitation of unlabeled ISIS 3521 (ISI 641A) and its chain-shortened metabolites with total radioactivity of [(35)S]-ISIS 3521. ISIS 3521 was labeled on the fifth nucleotide linkage from the 5'-end with (35)S by well-established methods. Multiple tissues collected from rats after administration of [(35)S]-ISIS 3521 were assayed by both radiolabel (liquid scintillation spectroscopy) and CGE methods. The CGE method provided accurate quantitation of the drug and its metabolites in kidney cortex and liver tissues. The correlation between methods for multiple tissues over time was excellent with 88.5% of the measurements being statistically equivalent. These data suggest that CGE is an accurate means of quantitating oligonucleotide in tissue and that it compares favorably with traditional radiochemical techniques. Clearance half-lives for total measurable oligonucleotides were equivalent to clearance of total radioactivity in both liver and kidney with the longest clearance half-life associated with the kidney.     

The generic placement of a morphologically enigmatic species in Asteraceae: evidence from ITS sequences

 Bidens cordylocarpa is a high polyploid species restricted in distribution to stream sides in the mountains of Jalisco, Mexico. The morphologically enigmatic species was originally described as a member of the genus Coreopsis, but later transferred to Bidens, largely because the involucral bracts appear most similar to Bidens. Characters of the cypselae, often useful in generic placement, are of no value for this species because the fruits have features not detected in either Bidens or Coreopsis. Sequences from the internal transcribed spacer region of nuclear ribosomal DNA (ITS) were used to assess the relationships of Bidens cordylocarpa. The molecular phylogeny places B. cordylocarpa in a strongly supported clade of Mexican and South American Bidens, and provides more definitive evidence of relationships than morphology, chromosome number, or secondary chemistry. Molecular, morphological, and chromosomal data suggest that B. cordylocarpa is an ancient polyploid, perhaps the remnant of a polyploid complex. Received August 28, 2000 Accepted February 11, 2001     

Rapid, simple and sensitive high-performance liquid chromatographic method for detection and determination of acyclovir in human plasma and its use in bioavailability studies

A rapid, simple and sensitive reversed-phase high-performance liquid chromatographic (HPLC) method has been developed for the measurement of acyclovir concentrations in human plasma and its use in bioavailability studies is evaluated. Unchanged acyclovir has been quantified without the introduction of an internal standard using the present method. Human plasma proteins were selectively precipitated by the addition of 7% perchloric acid to spiked plasma samples or to the plasma samples obtained after acyclovir administration to human volunteers and the mixture was spun at 1000 g for 10 min. The supernatant was directly injected into a Novaflex C₁₈ column and detected at 254 nm. The mobile phase consisted of octane sulfonic acid buffer (pH 2.5) and methanol (92:08). The limit of quantitation for acyclovir in plasma was 20 ng/ml, which enabled the determination of the area under the curve (AUC) more precisely, that is, it is much closer to its extrapolated value. The present method has been successfully applied to samples from bioavailability studies.     

Molecular genetics of peripheral populations of Nova Scotian Unionidae (Mollusca: Bivalvia)

Peripheral populations of eight species of freshwater bivalves (Unionidae.) extending their geographic ranges into Nova Scotia, Canada, were examined electrophoretically to determine both the extent of genetic variability within such populations, and whether the hypothesized pathway of colonization across the Isthmus of Chignecto is reflected in patterns of genetic resemblance among these populations. The Nova Scotian species examined could be separated into two groups based on levels of observed heterozygosity and levels of variability in allele frequencies. The first group is characterized by low levels of heterozygosity and polymorphism compared with north-eastern American populations, and in the case of one species, Elliptio complanala, considerable variability in allele frequencies among populations occurring in similar habitats in different drainages. Populations of E. complanata from Nova Scotia can be differentiated from conspecific populations on the southern Atlantic Slope by possession of fast alleles at two loci. Multivariate analyses define subgroups within populations of E. complanata consistent with hypothesis that the species invaded Nova Scotia by way of the Isthmus of Chignecto, and then split into two groups, one of which colonized Cape Breton to the north and the other of which colonized southern areas of the Province. The second group of Nova Scotian species is characterized by little reduction in heterozygosity and polymorphism compared with values observed among north-eastern American conspecifics or congeners, little variability in allele frequencies from population to population, and little evidence to suggest that these species were dependent on the land bridge to invade the Province. The type of dispersal is hypothesized to be responsible, in part, for these differences: larvae of species in the first group rely on a parasitic attachment to fish with territorial habits limited to fresh water, and are thus likely to invade new drainages separated by salt water by chance, in small numbers, and in stepping-stone fashion. Species in the second group parasitize anadromous or saltwater tolerant hosts, are likely to be introduced into new habitats in greater numbers and/or receive greater amounts of gene flow subsequent to colonization, and seem less dependent on land-bridges to colonize new habitats.     

Cloning of aroG, the gene coding for phospho-2-keto-3-deoxy-heptonate aldolase(phe), in Escherichia coli K-12, and subcloning of the aroG promoter and operator in a promoter-detecting plasmid

Defective transducing phages carrying aroG, the structural gene for phenylalanine (phe)-inhibitable phospho-2-keto-heptonate aldolase (EC 4.1.2.15; previously known as 3-deoxy-D-arabinoheptulosonate-7-phosphate synthetase[phe]), have been isolated, and DNA from two of these phages has been used to construct a restriction map of the region from att lambda to aroG. A 7.6-kb PstI-HindIII fragment from one of these phages was cloned into pBR322 and shown to contain aroG. The location of aroG within the 7.6 kb was established by subcloning and Tn3 transpositional mutagenesis. A fragment carrying the aroG promoter and operator has been cloned into a high copy number promoter-cloning vector (pMC489), and the resulting aroGpo-LacZ' (alpha) fusion subcloned in a low copy number vector. Strains with this fusion on the low copy number vector exhibit negative regulation of beta-galactosidase expression by both phenylalanine and tryptophan and positive regulation by tyrosine in a tyrR+ background.     

A rapid single-stranded cloning strategy for producing a sequential series of overlapping clones for use in DNA sequencing: application to sequencing the corn mitochondrial 18 S rDNA

A simple new procedure was described for producing a sequential series of overlapping clones for use in DNA sequencing. The technique used single-stranded M13 DNA and complementary DNA oligomers to form specific cleavage and ligation substrates. It was, therefore, independent of the sequence of the DNA cloned into the vector. Deletions of varying sizes were generated from one end of the insert through the 3' to 5' exonuclease activity of T4 DNA polymerase. The approximate size of the deletion and therefore the starting point for DNA sequencing could be estimated by electrophoresis of the subcloned phage DNA on a agarose gel. This greatly reduced the number of templates that must be sequenced to obtain a complete sequence. The entire procedure could be carried out in one tube in less than a day. The procedure was used to subclone and sequence the maize mitochondrial 18 S rDNA and 5' flanking region (2622 bases) in less than a week. Other applications of oligomers and single-stranded DNA in the construction of insertions, deletions, and cDNAs are discussed.