Nancy D. Moncrief

Evolution of EF-hand calcium-modulated proteins. I. Relationships based on amino acid sequences

SummaryThe relationships among 153 EF-hand (calcium-modulated) proteins of known amino acid sequence were determined using the method of maximum parsimony. These proteins can be ordered into 12 distinct subfamilies-calmodulin, troponin C, essential light chain of myosin, regulatory light chain, sarcoplasmic calcium binding protein, calpain, aequorin,Strongylocentrotus purpuratus ectodermal protein, calbindin 28 kd, parvalbumin, α-actinin, and S100/intestinal calcium-binding protein. Eight individual proteins-calcineurin B fromBos, troponin C fromAstacus, calcium vector protein fromBranchiostoma, caltractin fromChlamydomonas, cell-division-cycle 31 gene product fromSaccharomyces, 10-kd calcium-binding protein fromTetrahymena, LPS1 eight-domain protein fromLytechinus, and calcium-binding protein fromStreptomyces—are tentatively identified as unique; that is, each may be the sole representative of another subfamily. We present dendrograms showing the relationships among the subfamilies and uniques as well as dendrograms showing relationships within each subfamily.The EF-hand proteins have been characterized from a broad range of organismal sources, and they have an enormous range of function. This is reflected in the complexity of the dendrograms. At this time we urge caution in assigning a simple scheme of gene duplications to account for the evolution of the 600 EF-hand domains of known sequence.

The EF-hand family of calcium-modulated proteins

The EF-hand homolog proteins bind calcium (Ca2+) with dissociation constants in the micromolar range and are modulated by stimulus-induced increases in cytosolic free Ca2+. We have grouped over 160 different EF-hand homolog proteins into ten subfamilies and ten unique categories. Except for troponin-C, all subfamilies and unique EF-hand homologs represented in vertebrates can be found in the CNS. In this review, structural and functional characteristics of these proteins are discussed, with special emphasis on the multifunctional regulatory protein, calmodulin. The possible function of bending within the central helix of calmodulin is considered and is illustrated with a model calmodulin--target complex.

Evolution of EF-hand calcium-modulated proteins. II. Domains of several subfamilies have diverse evolutionary histories.

SummaryIn the first report in this series we described the relationships and evolution of 152 individual proteins of the EF-hand subfamilies. Here we add 66 additional proteins and define eight (CDC, TPNV, CLNB, LPS, DGK, 1 F8, VIS, TCBP) new subfamilies and seven (CAL, SQUD, CDPK, EFH5, TPP, LAV, CRGP) new unique proteins, which we assume represent new subfamilies.The main focus of this study is the classification of individual EF-hand domains. Five subfamilies—calmodulin, troponin C, essential light chain, regulatory light chain, CDC31/caltractin-and three uniques—call, squidulin, and calcium-dependent protein kinase-are congruent in that all evolved from a common four-domain precursor. In contrast calpain and sarcoplasmic calcium-binding protein (SARC) each evolved from its own one-domain precursor. The remaining 19 subfamilies and uniques appear to have evolved by translocation and splicing of genes encoding the EF-hand domains that were precursors to the congruent eight and to calpain and to SARC.The rates of evolution of the EF-hand domains are slower following formation of the subfamilies and establishment of their functions. Subfamilies are not readily classified by patterns of calcium coordination, interdomain linker stability, and glycine and proline distribution. There are many homoplasies indicating that similar variants of the EF-hand evolved by independent pathways.

Maximum parsimony approach to construction of evolutionary trees from aligned homologous sequences

Publisher Summary This chapter describes the significance of maximum parsimony approach to the construction of evolutionary trees from aligned homologous sequences. A maximum parsimony tree accounts for the evolutionary descent or related sequences by the fewest possible genie changes. Such a tree maximizes the genetic likenesses associated with common ancestry while minimizing the incidence of convergent mutations. Calculation of tree length is simplified by removing the root from the tree. Such an unrooted tree or network still retains the interior nodes and the exterior nodes (the OTUs).The maximum parsimony procedure can reconstruct ancestral sequences for each interior node of a tree but cannot determine which interior node or which pair of adjacent interior nodes is closest to the root. The problem of finding the maximum parsimony tree can be broken down into two parts. The first part proved to be easy and was solved by Fitch for homologous nucleotide sequences. The algorithm requires as input data both the OTUs, which are contemporary homologous nucleotide sequences already aligned against one another, and the instructions for a tree or dendrogram specifying any one of the possible dichotomous branching orders for the OTUs.

Dispersal Abilities and Genetic Population Structure of Insular and Mainland Oryzomys palustris and Peromyscus leucopus

A comparative hierarchical approach was used to examine allozymic variability within and among nine populations of Oryzomys palustris (marsh rice rat) and seven populations of Peromyscus leucopus (white-footed mouse) from the Virginia barrier islands and southern Delmarva Peninsula. O. palustris is an effective disperser over water and is present on 21 of 24 islands. In contrast, P. leucopus is a less effective disperser over water and occurs on only four of 24 islands. Of 31 loci, four were variable in O. palustris ; six were variable in P. leucopus . The nine populations of O. palustris had an average heterozygosity of 2.4% with 6.7% polymorphic loci. For seven populations of P. leucopus , the average heterozygosity was 3.6% with 12.3% polymorphic loci. Both species had lower levels of variation among mainland populations than among island populations. Populations of P. leucopus exhibited considerable genetic differentiation ( F ST = 0.180) and lower levels of gene flow ( N m = 1.14) among populations, whereas O. palustris had moderate levels of differentiation ( F ST = 0.135) and higher levels of gene flow ( N m = 1.60) among populations. Mantel tests indicated a significant relationship between genetic distance and geographic distance in Oryzomys but not in Peromyscus .

Homology of Calcium-Modulated Proteins: Their Evolutionary and Functional Relationships

In 1972 Kretsinger published a “Gene Triplication Deduced from the Tertiary Structure” of parvalbumin. He then proposed (1975) that “ Calcium Modulated Proteins Contain EF-Hands”. Calcium-modulated proteins, as a distinct superfamily of all the proteins that bind calcium, are defined by two characteristics. They are found within the cytosol or on a membrane facing the cytosol. They bind calcium with a dissociation constant about 10-6 M under cytosolic conditions, i. e., about 10-3 M free Mg2+ ion. They are inferred to be involved in transmitting the information inherent in calcium’s functioning as a cytosolic messenger.

Geographic Variation in Fox Squirrels (Sciurus niger) and Gray Squirrels (S. carolinensis) of the Lower Mississippi River Valley

Geographic variation was studied in fox squirrels ( Sciurus niger ) and gray squirrels (5. carolinensis ) by comparing patterns of differentiation within and between these two sympatric species in the lower Mississippi River valley. Geographic patterns of variation were apparent in the morphology and allozymes of both species. Patterns of differentiation in morphologic and allozymic characters are similar between species; however, morphologic variation is not congruent with allozymic variation within either species. Fox squirrels and gray squirrels vary morphologically in that, within each species, individuals inhabiting the Mississippi River floodplain and delta region are smaller than animals from adjacent areas. Allozyme analyses revealed that within each species, there are differences among eastern and western populations, as defined by their geographic location relative to the present channel of the Mississippi River. This study provides considerable evidence that the lower Mississippi River has influenced morphologic differentiation in fox squirrels and gray squirrels and that the river has impeded (and may still impede) gene flow in these species.

Polymorphic microsatellites in the meadow vole Microtus pennsylvanicus: conservation of loci across species of rodents

dynamics, immigration, colonization and extinction (Stenseth & Lidicker 1992; Lidicker 1994; Chitty 1996; Krebs 1996). Our studies of rodent populations that inhabit the Virginia barrier islands have prompted us to seek genetic techniques capable of detecting rare events such as interisland dispersal without disrupting the study system. Queller et al. (1993) and others (Bruford & Wayne 1993; Engel et al. 1996) have identified microsatellite DNA loci as highly polymorphic, single-locus, codominant genetic markers that can be unambiguously and consistently scored. After primer sequences are identified, these loci can be amplified by polymerase chain reaction (PCR), which allows the use of nondestructive sampling of tissues from living organisms. We have initiated population studies of the meadow vole Microtus pennsylvanicus, a species that is frequently the subject of investigations combining ecology and genetics (Gaines 1985). Primers unique to M. pennsylvanicus have not been reported in the literature. To expedite our efforts, we therefore used commercially available microsatellite primers for Mus sp. (Murine Screening Set, 410 Mouse MapPairsTM, Research Genetics, Inc., Huntsville, AL, USA), as well as five primers recently reported for an arvicoline rodent, the grey red-backed vole Clethrionomys rufocanus bedfordiae (Ishibashi et al. 1995). This approach allowed us to identify potentially useful primers for subsequent population studies of M. pennsylvanicus and simultaneously to test the efficacy of heterologous primers in rodents. A total of 24 meadow voles were collected from four sites (six animals per site) in Cache County, Utah during August 1995. Maximum distance between sites was 3 km. We killed the animals and harvested at least 2.5 g liver tissue to ensure availability of large quantities of genomic DNA; we also removed a 3 mm2 sample of skin (ear clip) to test its feasibility for subsequent nondestructive sampling. Tissues were collected from 23 animals immediately after death and stored at – 70 °C until analysis. One animal was frozen intact at – 20 °C for 4 months prior to tissue extraction. Fifty-two Mus primers were selected from 410 Mouse MapPairsTM in the Murine Screening Set (Research Genetics, Inc.). These primers were selected from across the Mus genome, with at least one primer from every chromosome; each was reported to be highly polymorphic in Mus, with 5–10 alleles per locus. Additionally, the five Clethrionomys primers reported by Ishibashi et al. (1995) were synthesized by the staff of the Utah State University Biotechnology Centre. Genomic DNA was extracted from 1 g of frozen liver for each animal (Ausubel et al. 1990); parallel analyses of ear tissue for several individuals produced identical genotype results. The DNA yield for liver samples and ear clips was about 1 mg and 15 μg, respectively. PCR was carried out in a 10 μL reaction containing 30 ng of genomic DNA, 10 μM of each primer, 20 μM of dATP, dGTP, dCTP, and dTTP, 0.1% gelatin, 1 μCi [α32P]dCTP (3000 Ci/mM, DuPont NEN Research Products, Boston, MA, USA), 50 mM KCl, 10 mM Tris (pH 8.3), 1.5 mM MgCl2, and 1 unit of AmpliTaq polymerase (Perkin Elmer Cetus, Norwalk, CT, USA). Samples were overlaid with mineral oil and processed through 1 cycle consisting of 5 min at 95 °C, 2 min at 55 °C, and 2 min at 72 °C, followed by 25 cycles consisting of 1 min at 94 °C, 1 min at 55 °C, and 1 min at 72 °C. Amplification was performed in a Techne PHC-3 (Princeton, NJ, USA). Aliquots of amplified DNA were mixed with 1 vol. of formamide sample buffer (80% formamide, 10 mM EDTA pH 8.0, 1 mg/mL xylene cyanol FF, 1 mg/mL bromophenol blue) and electrophoresed on a standard denaturing polyacrylamide DNA sequencing gel (7% acrylamide, 5.6 M urea, 32% formamide, 1 × TBE, 0.05% ammonium persulphate). Gels were exposed to Kodak XAR-5 diagnostic film for 48 h. PRIMER NOTE

Diagnostic Genetic Marker That Differentiates Eastern Fox Squirrels From Eastern Gray Squirrels

Abstract The Delmarva fox squirrel (Sciurus niger cinereus) has been listed as endangered by the United States Department of Interior since 1967. A high-priority task for the recovery of this taxon is to determine its current geographic distribution. Toward this end, we have identified a microsatellite locus that unambiguously differentiates Delmarva fox squirrels from eastern gray squirrels (S. carolinensis), which frequently co-occur with Delmarva fox squirrels. Analysis of this marker in noninvasively collected hair samples will allow unequivocal identification of localities occupied by Delmarva fox squirrels with a minimum investment of funds, time, and effort because handling individuals will be unnecessary. This protocol will expedite site review in connection with the Endangered Species Act consultation process.

Overwater Movement of Raccoons (Procyon lotor) in a Naturally Fragmented Coastal Landscape

Abstract Procyon lotor (Raccoon) is a major predator of beach-nesting and colonial waterbirds on the Virginia barrier islands. An understanding of water as a barrier to inter-island movement by Raccoons will be essential to effective management of these predators in this naturally fragmented coastal environment. We examined 4 independent lines of direct evidence for Raccoon movement between 1999 and 2007: 1) locations of recaptured, ear-tagged Raccoons on both the islands and the adjacent mainland, 2) overland movements of radio-collared Raccoons, 3) inter-island movements of radio-collared resident Raccoons, and 4) movements of translocated Raccoons. We recaptured 78 of 177 ear-tagged island Raccoons, all on the same island as the initial capture. We also tagged and released 65 mainland Raccoons, none of which was ever recaptured on an island. We often observed overland movements >1 km per day by radio-collared animals on both the islands and the mainland. Nevertheless, only 3 of 51 (6%) collared animals (2 males and 1 female) moved overwater from the location where they were captured. None of the 4 Raccoons radio-collared on the mainland moved to an island. Although Raccoons in this system are highly mobile, overwater movements seem to be infrequent events; only 3 of 234 tagged/ collared island individuals moved between islands, and none of the 69 tagged/collared mainland individuals moved to an island. Finally, we observed return movements by 22 of the 32 (69%) animals (11 males and 11 females) that were translocated either from the mainland to a nearby island or between adjacent islands. Translocated animals exhibited a much greater tendency than resident animals to make overwater crossings. In all cases of overwater movement, the water channels were relatively shallow and relatively slow moving. None of the 335 marked animals in this study crossed a tidal inlet. The mobility observed here is consistent with the idea that the distribution of Raccoons on the islands has expanded in recent decades. Predation management on these islands will require a strategic approach that takes into account both island isolation and Raccoon mobility.