Comparison of endogenous phosphorylation of hen and rat spinal cord proteins and partial characterization of optimal phosphorylation conditions for hen spinal cord

The optimal conditions for the endogenous phosphorylation of hen spinal cord cytosolic and membrane proteins with 5 μM [γ-³²P]ATP, 10 mM MgCl₂, were determined by 10% SDS-polyacrylamide gel electrophoresis, autoradiography, and microdensitometry. Phosphate incorporation increased linearly with concentrations ranging from 35–75 μg/100 μl for cytosolic proteins and 21–125 μg/200 μl for membrane proteins. Optimal incubation times, temperatures, and pH values were 60 s, 30°C, and 6.0, respectively, for spinal cord cytosolic proteins and 15 s, 45°C, and 8.0, respectively, for spinal cord membranes. Prominent species differences in protein phosphorylation between these fractions in hens and similarly prepared fractions in rats, co-electrophoresed, include 80K and 30K protein phosphate acceptors unique to rat spinal cord cytosol, 60K and 16K protein phosphate acceptors characteristic of rat spinal cord membranes, a 50K protein phosphate acceptor present only in hen spinal cord membranes, and greater phosphorylation of a more abundant 20K protein in both hen spinal cord fractions. The functional significance of these differences is presently unclear. However, their characterization provides a basis from which to launch future investigations of the biochemistry, pharmacology, and toxicology of spinal cord protein phosphorylation and indicates that caution should be exercised in the choice of an animal model with characteristics appropriate to those of the system it is representing.     

Involvement of zinc in the regulation of pHi, motility, and acrosome reactions in sea urchin sperm

When sperm of Strongylocentrotus purpuratus or Lytechinus pictus are diluted into seawater, motility is initiated; and when exposed to egg jelly, an acrosome reaction is induced. In the presence of a variety of structurally different metal chelators (0.1-1 mM EDTA, EGTA, phenanthroline, dipyridyl, cysteine, or dithiothreitol), motility initiation is delayed and the acrosome reaction is inhibited. Of the metals detected in the sperm of these two species, very low levels of Zn+2 (0.1 microM free Zn+2) uniquely prevent this chelator inhibition. L. pictus sperm concentrate 65Zn+2 from seawater, and EDTA removes 50% of the accumulated 65Zn+2 by 5 min. Since both sperm motility and acrosome reactions are in part regulated by intracellular pH (pHi), the effect of chelators on the sperm pHi was examined by using the fluorescent pH sensitive probe, 9-aminoacridine, EDTA depresses sperm pHi in both species, and 0.1 microM free Zn+2 reverses this pHi depression. When sperm are diluted into media that contain chelators, both NH4Cl and monensin (a Na+/H+ ionophore) increase the sperm pHi and reverse the chelator inhibition of sperm motility and acrosome reactions. The results of this study are consistent with the involvement of a trace metal (probably zinc) in the pHi regulation of sea urchin sperm and indicate a likely mechanism for the previously observed effects of chelators on sperm motility and acrosome reactions.     

Homogenization of geographical variants at the nontranscribed spacer of rDNA in Drosophila mercatorum

rDNA nontranscribed spacer (NTS) lengths of Drosophila mercatorum have been measured in individuals from several geographic regions. Individuals from the different geographic subpopulations share some length fragments but are in general distinct. The length differences, both within and between individuals, arise from different copy numbers of a 250-bp repeating unit that is localized to one part of the NTS. In addition to the length differences caused by the 250-bp repeat, there is a Y chromosome (male)-specific length variant elsewhere in the NTS that is approximately 70 bp shorter than the NTS fragment from the X chromosome. Sexual dimorphism seems to be present in all Drosophila. Also, D. mercatorum has fewer NTS length variants per individual than does D. melanogaster while possessing comparable levels of restriction- site polymorphism. The mechanisms that may cause this pattern of variation are selection, gene conversion, and unequal recombination.      

A SNP-based high-density linkage map of zoysiagrass (<Emphasis Type="Italic">Zoysia japonica</Emphasis> Steud.) and its use for the identification of QTL associated with winter hardiness

Zoysia japonica Steud. (2n?=?4×?=?40) is a C₄ turfgrass well-adapted for the warm-humid and transitional climatic zones of the USA. Its use is limited to warmer climates because of a relative lack of winter hardiness compared to C₃ grasses. Molecular markers associated with this trait would be useful for effective selection of winter hardy germplasm before field testing. A pseudo-F₂ mapping population of 175 individuals was developed from crosses between Z. japonica cultivars “Meyer” (freeze-tolerant) and “Victoria” (freeze-susceptible) and used to generate a high-density genetic map of 104 SSR markers and 2359 sequencing-derived SNP markers. The map covers 324 Mbp and 2520 cM as well as the 20 chromosomes for the zoysiagrass haploid genome. Phenotypic data on winter injury, establishment, and turf quality collected in North Carolina and Indiana in 2014–2016 were used in conjunction with this map to identify quantitative trait loci (QTL) associated with winter hardiness. Fifty-six QTL associated with winter injury, establishment, and turf quality were identified over six environments. Twelve of those were identified in two or more environments. Furthermore, seven regions of interest were identified on chromosomes 8, 11, and 13 where co-location of QTL for three or more traits occurred. Within these regions, analysis with NCBI basic local alignment search tool (BLAST) identified proteins related to cold and other abiotic stresses tolerance. These QTL and associated markers could be valuable in implementing marker-assisted selection for winter hardiness in zoysiagrass breeding programs.     

Developmental Analysis of the Ovarian Tumor Gene during Drosophila Oogenesis

Severe alleles of the ovarian tumor (otu) and ovo genes result in female sterility in Drosophila melanogaster, producing adult ovaries that completely lack egg chambers. We examined the developmental stage in which the agametic phenotype first becomes apparent. Germ cell development in embryos was studied using a strategy that allowed simultaneous labeling of pole cells with the determination of embryonic genotype. We found that ovo(-) or otu(-) XX embryonic germ cells were indistinguishable in number and morphology from those present in wild-type siblings. The effects of the mutations were not consistently manifested in the female germline until pupariation, and there was no evidence that either gene was required for germ cell viability at earlier stages of development. The requirement for otu function in the pupal and adult ovary is supported by temperature-shift experiments using a heat-inducible otu gene construct. We demonstrate that otu activity limited to prepupal stages was not sufficient to support oogenesis, while induction during the pupal and adult periods caused suppression of the otu mutant phenotype.     

The influence of optimization target selection on the structure of arterial tree models generated by constrained constructive optimization

The computational method of constrained constructive optimization was used to generate complex arterial model trees by optimization with respect to a target function. Changing the target function also changes the tree structure obtained. For a parameterized family of target functions a series of trees was created, showing visually striking differences in structure that can also be quantified by appropriately chosen numerical indexes. Blood transport path length, pressure profile, and an index for relative segment orientation show clear dependencies on the optimization target, and the nature of changes can be explained on theoretical grounds. The main goal was to display, quantify, and explain the structural changes induced by different optimization target functions.     

Diverse reactions catalyzed by naphthalene dioxygenase fromPseudomonas sp strain NCIB 9816

Naphthalene dioxygenase (NDO) fromPseudomonas sp strain NCIB 9816 is a multicomponent enzyme system which initiates naphthalene catabolism by catalyzing the addition of both atoms of molecular oxygen and two hydrogen atoms to the substrate to yield enantiomerically pure (+)-cis-(1R,2S)-dihydroxy-1,2-dihydronaphthalene. NDO has a relaxed substrate specificity and catalyzes the dioxygenation of many related 2- and 3-ring aromatic and hydroaromatic (benzocyclic) compounds to their respectivecis-diols. Biotransformations with a diol-accumulating mutant, recombinant strains and purified enzyme components have established that in addition tocis-dihydroxylation, NDO also catalyzes a variety of other oxidations which include monohydroxylation, desaturation (dehydrogenation),O-andN-dealkylation and sulfoxidation reactions. In several cases, the absolute stereochemistry of the oxidation products formed by NDO are opposite to those formed by toluene dioxygenase (TDO). The reactions catalyzed by NDO and other microbial dioxygenases can yield specific hydroxylated compounds which can serve as chiral synthons in the preparation of a variety of compounds of interest to pharmaceutical and specialty chemical industries. We present here recent work documenting the diverse array of oxidation reactions catalyzed by NDO. The trends observed in the oxidation of a series of benzocyclic aromatic compounds are compared to those observed with TDO and provide the basis for prediction of regio- and stereospecificity in the oxidation of related substrates. Based on the types of reactions catalyzed and the biochemical characteristics of NDO, a mechanism for oxygen activation by NDO is proposed.     

Mutation of PTB binding sites causes misregulation of alternative 3' splice site selection in vivo.

Alternative splicing of pre-mRNA is a commonly used mechanism to regulate gene expression in higher eukaryotes. However, with the exception of regulated cascades in Drosophila, the cis-acting elements and the trans-acting factors that control tissue- and/or developmentally regulated splicing remain largely unidentified. Cis-acting elements that control smooth muscle-specific repression of exon 3 of alpha-tropomyosin (alpha-TM) have been identified recently and consist of two regions that flank this exon. Deletion of either element causes misregulated splicing of alpha-TM in transfected smooth muscle cells. In experiments designed to characterize essential sequences within each element and the factors that interact with these sequences, we have identified two overlapping sequences within the downstream regulatory element (DRE) that are identical to binding sites for polypyrimidine tract binding protein (PTB) that were identified using iterative selection techniques. Mutation of these sites caused aberrant splicing regulation in transfected smooth muscle cells. In addition, sequences identical to high-affinity PTB binding sites were also detected upstream of exon 3 and mutation of these sites also resulted in misregulation of splicing in vivo, suggesting that PTB binding to specific sequences flanking exon 3 is responsible, in part, for the repression of exon 3. Consistent with this hypothesis, UV crosslinking and equilibrium binding assays confirm that the same mutations that cause misregulated splicing also disrupt PTB binding to RNA.     

Phylogenetic evidence of mitochondrial DNA introgression among pocket gophers in New Mexico (family Geomyidae)

Mitochondrial DNA (mtDNA) variation in the cytochrome b gene was determined for two divergent taxa of pocket gophers, Thomomys bottae actuosus and T. b. ruidosae . These two taxa hybridize in a narrow contact zone, but introgression of nuclear markers such as allozymes or chromosomes does not extend much beyond the hybrid zone (Patton et al. 1979). We found that despite their distinctness, the two subspecies shared very similar mtDNA haplotypes. By a comparison of phylogenetic histories derived from nuclear markers (allozymes) and from mtDNA haplotypes sampled in different populations of T. bottae from New Mexico, we show that apparent similarity is due to an introgression of T. b. ruidosae mtDNA into T. b. actuosus nuclear background. Evidence of introgression is not limited to the present-day contact zone between these two taxa, but extends at least 75 km away from it. The actuosus haplotype coexists along with the ruidosae mtDNA in the Gallinas Mts., which are inhabited by otherwise pure T. b. actuosus , while further north only typical actuosus haplotypes were detected. Of several potential mechanisms which could lead to such a geographical pattern of variation, we argue that a combination of range shifts due to climatic fluctuations, and genetic drift are most likely. Horizontal gene transfers due to hybridization are historical events which seem rather common among pocket gophers. Although they can be identified with careful phylogenetic study using independent data sets, the potential for misinterpreting a gene tree as an organismal tree is great in this and other groups of animals.     

Morphological and dietary responses of chipmunks to a century of climate change

Predicting how individual taxa will respond to climatic change is challenging, in part because the impacts of environmental conditions can vary markedly, even among closely related species. Studies of chipmunks (Tamias spp.) in Yosemite National Park provide an important opportunity to explore the reasons for this variation in response. While the alpine chipmunk (T. alpinus) has undergone a significant elevational range contraction over the past century, the congeneric and partially sympatric lodgepole chipmunk (T. speciosus) has not experienced an elevational range shift during this period. As a first step toward identifying the factors underlying this difference in response, we examined evidence for dietary changes and changes in cranial morphology in these species over the past century. Stable isotope analyses of fur samples from modern and historical museum specimens of these species collected at the same localities indicated that signatures of dietary change were more pronounced in T. alpinus, although diet breadth did not differ consistently between the study species. Morphometric analyses of crania from these specimens revealed significant changes in cranial shape for T. alpinus, with less pronounced changes in shape for T. speciosus; evidence of selection on skull morphology was detected for T. alpinus, but not for T. speciosus. These results are consistent with growing evidence that T. alpinus is generally more responsive to environmental change than T. speciosus, but emphasize the complex and often geographically variable nature of such responses. Accordingly, future studies that make use of the taxonomically and spatially integrative approach employed here may prove particularly informative regarding relationships between environmental conditions, range changes, and patterns of phenotypic variation.