Whole Genome Amplification for Sequencing and Applications in Conservation Genetics

Abstract: We describe a method for rapidly amplifying whole genomes via a Phi29 DNA polymerase-mediated strand displacement reaction (SDR). Genomic amplification products derived from the SDR reaction resulted in high quantities of DNA suitable for polymerase chain reaction (PCR) amplification and sequencing of mitochondrial genomes. Control region sequences of DNA derived directly from PCR amplicons of extracted DNA were identical to those derived from PCR amplification of SDR genomic DNA. Effective SDR amplification and subsequent sequencing was successful across tissues sources ranging in age from 1 year to 19 years. Strand replacement reaction genomic amplification offers a means of obtaining large quantities of DNA from small amounts of tissue.     

Salivary alpha amylase and salivary cortisol response to fluid consumption in exercising athletes

The objective of the study was to examine salivary biomarker response to fluid consumption in exercising athletes. Exercise induces stress on the body and salivary alpha amylase (sAA) and salivary cortisol are useful biomarkers for activity in the sympathoadrenal medullary system and the hypothalamic pituitary adrenal axis which are involved in the stress response. Fifteen college students were given 150 ml and 500 ml of water on different days and blinded to fluid condition. The exercise protocol was identical for both fluid conditions using absolute exercise intensities ranging from moderate to high. Saliva was collected prior to exercise, post moderate and post high intensities and analyzed by Salimetrics assays. Exercise was significant for sAA with values different between pre-exercise (85 ± 10 U · ml⁻¹) and high intensity (284 ± 30 U · ml⁻¹) as well as between moderate intensity (204 ± 32 U · ml⁻¹) and high intensity. There was no difference in sAA values between fluid conditions at either intensity. Exercise intensity and fluid condition were each significant for cortisol. Cortisol values were different between pre-exercise (0.30 ± 0.03 ug · dL⁻¹) and high intensity (0.45 ± 0.05 ug · dL⁻¹) as well as between moderate intensity (0.33 ± 0.04 ug · dL⁻¹) and high intensity. Moderate exercise intensity cortisol was lower in the 500 ml condition (0.33 ± 0.03 ug · dL⁻¹) compared with the 150 ml condition (0.38 ± 0.03 ug · dL⁻¹). This altered physiological response due to fluid consumption could influence sport performance and should be considered. In addition, future sport and exercise studies should control for fluid consumption.     

Cryphonectria nitschkei virus 1 structure shows that the capsid protein of chrysoviruses is a duplicated helix-rich fold conserved in fungal double-stranded RNA viruses

Cryoelectron microscopy reconstruction of Cryphonectria nitschkei virus 1, a double-stranded RNA (dsRNA) virus, shows that the capsid protein (60 copies/particle) is formed by a repeated helical core, indicative of gene duplication. This unusual organization is common to chrysoviruses. The arrangement of many of these putative α-helices is conserved in the totivirus L-A capsid protein, suggesting a shared motif. Our results indicate that a 120-subunit T=1 capsid is a conserved architecture that optimizes dsRNA replication and organization.     

The restoration of Lake Apopka in relation to alternative stable states: an alternative view to that of Bachmann et al. (1999)

Bachmann et al. (1999) postulated that wind energy initiated, and has maintained, high turbidity in hypertrophic (mean chlorophyll a = 92 g l^–1) Lake Apopka, Florida (mean depth = 1.6 m; area = 12500 ha). They asserted that the turbid condition was initiated by a hurricane in late 1947 that destroyed submersed plant beds and that high turbidity has since been maintained by wind-driven resuspension of fluid sediments. In their view, there has been sufficient light for re-establishment of submersed plants over about 38% of the lake bottom, but plant growth has been precluded by the fluid character of the sediments. They concluded that the restoration program of the St. Johns River Water Management District, which includes reduction of the phosphorus (P) loading rate, will not restore water clarity or submersed vegetation. An alternative explanation for Lake Apopka's turbid state is that it was initiated, and has been maintained, by excessive P loading that led to algal blooms and elimination of submersed vegetation through light limitation. The transition to the turbid state was contemporaneous with drainage of 7300 ha of the floodplain wetland to create polders for farming, beginning in the early 1940s. Lake P budgets indicate that drainage of the farms caused a seven-fold increase in the P loading rate (0.08 g TP m^–2 yr^–1 to 0.55 g TP m^–2 yr^–1). Paleolimnological analysis of lake sediments also indicates an increase in the P loading rate in mid-century, concomitant with the decline in submersed vegetation and the increase in phytoplankton abundance. After the increase in P loading, wind disturbance may have accelerated the transition to the turbid state; but, before the increase in P loading, wind disturbance was insufficient to elicit the turbid state, as evidenced by the stability of the clear-water state in the face of 14 hurricanes and 41 tropical storms from 1881 to 1946. Measurements of photosynthetically active radiation (PAR) indicate that light limitation has inhibited submersed plant growth except on the shallowest 5% of the lake bottom. Further, the correlation between the diffuse attentuation coefficient (K _PAR) and chlorophyll a (CHLA) indicates that light limitation would be removed over about 82% of the lake bottom with a reduction in CHLA from 92 g l^–1 to 25 g l^–1. Recently, following a 40% reduction in the P loading rate, the mean total P (TP) concentration, mean CHLA, and total suspended solids fell by about 30% while mean Secchi depth increased by more than 20%. Submersed plant beds appeared in areas devoid of macrophytes for nearly 50 years. These improvements, during a period with no change in mean wind speeds measured at Lake Apopka, provide the strongest evidence that the turbid state has been maintained by excessive P loading and that the current restoration program, which combines P load reduction with planting and removal of planktivorous fish, will be effective.     

Cell-to-cell contact is critical for the survival of hematopoietic progenitor cells on osteoblasts

Osteoblasts constitute part of the stromal cell support system in marrow for hematopoiesis, however little is known as to how they interact with hematopoietic stem cells (HSCs). In vitro studies have demonstrated that the survival of HSCs in co-culture with osteoblasts requires intimate cell-to-cell contact. This suggests that the osteoblast-derived factor(s) that supports stem cell activities are produced in very small quantities, are rapidly turned over, may be membrane-anchored and/or require the engagement of cell-cell adhesion molecules that are yet to be determined. In the present report we found that the survival of hematopoietic progenitor cells on osteoblasts is dependent upon the engagement of VLA-4 (alpha4beta1) and VLA-5 (alpha5beta1) receptors using function blocking antibodies. Cell-to-cell contact is required to support progenitor activity, but can be replaced if receptor-ligand engagement of the VLA-4 and LFA-1 complexes is provided through the use of recombinant ligands (fibronectin, ICAM-1, VCAM-1). Moreover, once these receptors were engaged, conditioned medium derived from HSCs grown on osteoblast ligands supported significantly greater hematopoietic progenitors in vitro than did osteoblast-conditioned or HSC-conditioned medium alone. While the molecules present in the co-cultured medium remain to be identified, the data suggest that hematopoietic cells cooperate with osteoblasts to assemble the various marrow microenvironments by directing the synthesis of osteoblast-derived cytokines to improve HSC survival.     

Ontogenetic approach reveals how cognitive capability and motivation shape prey‐searching behavior in Pholcus phalangioides cellar spiders

An important part of understanding the evolution of behavior is understanding how and why behavior develops and changes throughout ontogeny. Patterns of behavior are shaped by an animal's capabilities as well as its motivations, both of which are subject to selection. We ran an experiment to see how spiders' efforts to recover lost prey change with age and to determine the relative contributions of shifts in capability and motivation. We found that as spiders mature, they spend less time searching to recover lost prey, and they discriminate less between prey of different sizes. We also found that even the youngest, least experienced spiders are cognitively equipped to search for lost prey. Thus, predatory behavior in spiders fluctuated primarily with each age group's motivations to capture and consume prey, and did not seem to be hindered by behavioral or cognitive limitations at young ages.     

Behavioral stimulation of ovarian growth

The present study was conducted to test the following hypothesis: male courtship induced egg-laying behavior in the female ring dove is mediated by the female's nest-coo display which feeds back to stimulate her ovarian system through the mechanisms of audio- and proprioceptive feedback. In Experiment I, females were exposed to 24 hr male courtship and then received playback of different nest-coos in the absence of their mates. All playback was effective in inducing the females to perform nest-coo displays, but none as effective as presence of the courting males. A specified level of the female's nest-coo display determined whether there would be changes in follicular diameter. The females which received no playback after 24 hr pairing with the male (control) did not show any change in the follicular diameter. In Experiment II, females were deafened by removal of cochleae and upon recovery paired with males. Half of those deafened females performed nest-coo displays and subsequently laid clutches. The others failed to perform the display and did not lay. We conclude that both proprioceptive feedback and audio-feedback must be involved to yield maximal stimulation of follicular growth by the female's nest-coo display.     

Cloning and gene map assignment of the Xiphophorus DNA ligase 1 gene

Fishes represent the stem vertebrate condition and have maintained several gene arrangements common to mammalian genomes throughout the 450 Myr of divergence from a common ancestor. One such syntenic arrangement includes the GPI-PEPD enzyme association on Xiphophorus linkage group IV and human chromosome 19. Previously we assigned the Xiphophorus homologue of the human ERCC2 gene to linkage group U5 in tight association with the CKM locus. CKM is also tightly linked to the ERCC2 locus on human chromosome 19, leading to speculation that human chromosome 19 may have arisen by fusion of two ancestral linkage groups which have been maintained in fishes. To investigate this hypothesis further, we isolated and sequenced Xiphophorus fish genomic regions exhibiting considerable sequence similarity to the human DNA ligase 1 amino acid sequence. Comparison of the fish DNA ligase sequence with those of other species suggests several modes of amino acid conservation in this gene. A 2.2-kb restriction fragment containing part of an X. maculatus DNA ligase 1 exon was used in backcross hybrid mapping with 12 enzyme or RFLP loci. Significant linkage was observed between the nucleoside phosphorylase (NP2) and the DNA ligase (LIG1) loci on Xiphophorus linkage group VI. This assignment suggests that the association of four DNA repair-related genes on human chromosome 19 may be the result of chance chromosomal rearrangements.