Screening of a broad range of rice (<Emphasis Type="Italic">Oryza sativa</Emphasis> L.) germplasm for <Emphasis Type="Italic">in vitro</Emphasis> rapid plant regeneration and development of an early prediction system

Rice has emerged as a model monocot for studies in agriculture and biotechnology due to its relatively small genome and a ready accessibility to plant material. Tissue culture is one of the tools required for genetic transformation and some breeding programs, and the selection of high-frequency regenerator types is essential for success in these technologies. Thirty-three rice entries with agricultural and biotechnological characteristics of interest were screened with the aim to identify the best regenerators. Entries that exhibited between 50% and 90% regeneration frequencies include ‘Taipei-309,’ ‘Super Dwarf,’ ‘Norin’ (japonica types), PI 312777, ‘Ali Combo’ (indica types), ‘STG-S,’ and ‘LA3’ (red rice types). One third of the entries tested were at least two times better at regeneration than the often-cited regenerator ‘Nipponbare.’ Those entries showing at least 85% frequency of greening or somatic embryo formation at 15 or 30 d on regeneration medium ultimately produced whole plants after 45 d on regeneration medium at high frequency (at least 40%); those entries not reaching the 85% threshold of greening by Days 15 or 30 exhibited moderate (15–40%) to low (less than 10%) frequency of whole plant regeneration. This greening response suggests the means for an early prediction system for identification of useful rice regenerator lines, which would be beneficial for high-throughput screening of germplasm as well as for decreasing the time and cost of in vitro culture.     

Evaluating human autosomal loci for sexually antagonistic viability selection in two large biobanks

Sex and sexual differentiation are pervasive across the tree of life. Because females and males often have substantially different functional requirements, we expect selection to differ between the sexes. Recent studies in diverse species, including humans, suggest that sexually antagonistic viability selection creates allele frequency differences between the sexes at many different loci. However, theory and population-level simulations indicate that sex-specific differences in viability would need to be very large to produce and maintain reported levels of between-sex allelic differentiation. We address this contradiction between theoretical predictions and empirical observations by evaluating evidence for sexually antagonistic viability selection on autosomal loci in humans using the largest cohort to date (UK Biobank, n = 487,999) along with a second large, independent cohort (BioVU, n = 93,864). We performed association tests between genetically ascertained sex and autosomal loci. Although we found dozens of genome-wide significant associations, none replicated across cohorts. Moreover, closer inspection revealed that all associations are likely due to cross-hybridization with sex chromosome regions during genotyping. We report loci with potential for mis-hybridization found on commonly used genotyping platforms that should be carefully considered in future genetic studies of sex-specific differences. Despite being well powered to detect allele frequency differences of up to 0.8% between the sexes, we do not detect clear evidence for this signature of sexually antagonistic viability selection on autosomal variation. These findings suggest a lack of strong ongoing sexually antagonistic viability selection acting on single locus autosomal variation in humans.     

Comparative genomic hybridization in combination with flow cytometry improves results of cytogenetic analysis of spontaneous abortions

More than 50% of spontaneous abortions (SAs) have abnormal chromosomes; the most common abnormalities are trisomy, sex chromosome monosomy, and polyploidy. Conventional cytogenetic analysis of SAs depends on tissue culturing and is associated with a significant tissue culture failure rate and contamination by maternally derived cells. Comparative genomic hybridization (CGH), in combination with flow cytometry (FCM), can detect numerical and unbalanced structural chromosomal abnormalities associated with SAs while avoiding the technical problems associated with tissue culture. Routine cytogenetic and CGH analysis was performed independently on tissue from 301 SAs. Samples shown to be chromosomally balanced by CGH were analyzed by FCM to determine ploidy. Of 253 samples successfully analyzed by both approaches, there was an absolute correlation of results in 235 (92.8%). Of the 18 cases with discrepancies between cytogenetic and CGH/FCM results, an explanation could be found in 17. Twelve samples produced a 46,XX karyotype by cytogenetics, whereas CGH/FCM demonstrated aneuploidy/polyploidy or a male genome, indicating maternal contamination of the tissue cultures. In two cases, where tetraploidy was demonstrated by cytogenetics and diploidy by FCM, tissue culture artifact is implied. In three cases, CGH demonstrated an aneuploidy, and cytogenetics demonstrated hypertriploidy. In one unexplainable case, aneuploidy demonstrated by CGH could not be detected by repeat CGH analysis, conventional cytogenetic, or FISH analysis. These results demonstrate that CGH supplemented with FCM can readily identify chromosomal abnormalities associated with SAs and, by avoiding maternal contamination and tissue culture artifacts, can do so with a lower failure rate and more accuracy than conventional cytogenetic analysis.     

A single-molecule Hershey-Chase experiment

Ever since Hershey and Chase used phages to establish DNA as the carrier of genetic information in 1952, the precise mechanisms of phage DNA translocation have been a mystery. Although bulk measurements have set a timescale for in vivo DNA translocation during bacteriophage infection, measurements of DNA ejection by single bacteriophages have only been made in vitro. Here, we present direct visualization of single bacteriophages infecting individual Escherichia coli cells. For bacteriophage λ, we establish a mean ejection time of roughly 5 min with significant cell-to-cell variability, including pausing events. In contrast, corresponding in vitro single-molecule ejections are more uniform and finish within 10 s. Our data reveal that when plotted against the amount of DNA ejected, the velocity of ejection for two different genome lengths collapses onto a single curve. This suggests that in vivo ejections are controlled by the amount of DNA ejected. In contrast, in vitro DNA ejections are governed by the amount of DNA left inside the capsid. This analysis provides evidence against a purely intrastrand repulsion-based mechanism and suggests that cell-internal processes dominate. This provides a picture of the early stages of phage infection and sheds light on the problem of polymer translocation.     

Reversal of ketamine/xylazine anesthesia in the rabbit with yohimbine

Ketamine and xylazine used in combination have been shown to be effective, easily administered, cost efficient agents for surgical anesthesia in the rabbit. The effect of xylazine on the central nervous system has been shown to be mediated through alpha-2 adrenergic receptors. Yohimbine, an alpha-2 adrenergic antagonist has been shown to reverse xylazine induced depression and partially antagonize ketamine in other species. We evaluated the antagonistic effect of yohimbine on ketamine/xylazine anesthesia in the rabbit. Six New Zealand White rabbits were anesthetized with intramuscular ketamine (50 mg/kg) and xylazine (10 mg/kg) to establish baseline parameters including respiratory rate, heart rate, and palpebral, pedal and postural reflex activity. Fourteen days later each rabbit was subjected to the same anesthetic regimen followed 30 minutes later by the intravenous administration of yohimbine (0.2 mg/kg). The duration of anesthesia estimated by the time elapsed between the loss and return of the palpebral reflex was reduced in the yohimbine treated trial (means = 29.7 +/- 1.9 minutes) compared to the control trial (means = 67.0 +/- 13.5 minutes). The palpebral reflex returned within 5 minutes following yohimbine treatment. Our results indicated that yohimbine is an effective antagonist of ketamine/xylazine anesthesia in the rabbit. Yohimbine decreases anesthetic duration after intravenous administration and also may aid in the control of undesirable anesthetic effects and overdosage.     

Exogenous GnRH overrides the endogenous annual reproductive rhythm in green iguanas, Iguana iguana

Female green iguanas, Iguana iguana, were caught in Belize, Central America (17 degrees N), in December, at the onset of seasonal gonadal activity. The animals were immediately transferred to San Diego (32 degrees N). Ovarian follicular development continued, with peak plasma hormone levels measured in January and February; 200 pg/ml for progesterone (P) and 800 pg/ml for total estrogens (Et = estradiol [E2] + estrone [E1]). E2 was the predominant estrogen throughout the cycle. Follicular atrophy was indicated in April with circulating progesterone and estrogen levels decreasing to baseline (refractory phase) levels (P = 20 pg/ml; Et = 50 pg/ml). Approximately midway through the refractory phase of their annual reproductive cycle (late May), either the D-Arg6 analog of Chicken II or mammalian GnRH was administered via intraperitoneal osmotic pumps for 14 days to nine females. The analog of chicken II induced a fivefold increase in total circulating estrogens within 3-4 days after implantation. Both continuous and pulsatile delivery of the chicken II analog produced a similar pattern of steroidogenic response. A radical sham control animal showed no increase in steroidogenesis. Mammalian GnRH produced a pattern of similar duration, although the magnitude of the steroidogenic response was only half that produced by the chicken II analog. Estrogen titers approached baseline levels in all treatment groups two days after treatment ceased. Progesterone levels increased in all treatment groups during the delivery of exogenous GnRH, although the increases were not consistent. Untreated male cagemates housed with treated females exhibited increased territoriality, courtship behavior, and mating, which began on day 4 or 5 of the treatment period. The control female was not courted by its male cagemate.     

Polymerization of G-actin by myosin subfragment 1

The polymerization of actin from rabbit skeletal muscle by myosin subfragment 1 (S-1) from the same source was studied in the depolymerizing G-actin buffer. The polymerization reactions were monitored in light-scattering experiments over a wide range of actin/S-1 molar rations. In contrast to the well resolved nucleation-elongation steps of actin assembly by KC1 and Mg2+, the association of actin in the presence of S-1 did not reveal any lag in the polymerization reaction. Light scattering titrations of actin with S-1 and vice versa showed saturation of the polymerization reaction at stoichiometric 1:1 ratios of actin to S-1. Ultracentrifugation experiments confirmed that only stoichiometric amounts of actin were incorporated into a 1:1 acto-S-1 polymer even at high actin/S-1 ratios. These polymers were indistinguishable from standard complexes of S-1 with F-actin as judged by electron microscopy, light scattering measurements, and fluorescence changes observed while using actin covalently labeled with N-(1-pyrenyl)iodoacetamide. F-actin obtained by polymerization of G-actin by S-1 could initiate rapid assembly of G-actin in the presence of 10 mM KC1 and 0.5 mM MgCl2 and showed normal activation of MgATPase hydrolysis by myosin.