Genome size estimations on Ulmus minor Mill., Ulmus glabra Huds., and Celtis australis L. using flow cytometry

The Ulmaceae family is composed of nearly 2000 species widely distributed in the northern hemisphere. Despite their wide distribution area, there are only four native species in the Iberian Peninsula. In this work the genome size of three of those species (ULMUS MINOR, U. GLABRA, and CELTIS AUSTRALIS) was estimated using flow cytometry. The nuclear DNA content of C. AUSTRALIS was estimated as 2.46 +/- 0.061 pg/2C, of U. MINOR as 4.25 +/- 0.158 pg/2C, and of U. GLABRA as 4.37 +/- 0.103 pg/2C of DNA. No statistically significant differences were detected among individuals of the same species. These species revealed to be problematic for flow cytometric analyses, due to the release of mucilaginous compounds into the nuclear suspension. Despite that, the modified protocol here presented ensured high quality analyses (low coefficient of variation and background debris and nuclear fluorescence stability), opening good perspectives on its application to estimate the genome size of species with similar problems.     

Large genomes among caridean shrimp.

Recent genome size estimates for Arctic amphipods have revealed the largest genomes known in the Crustacea. Here we provide additional data for 7 species of caridean shrimp collected from the Canadian Arctic and the Gulf of St. Lawrence. Genome sizes were estimated by flow cytometry and haploid C-values ranged from 8.53 +/- 0.30 pg in Pandalus montagui (Pandalidae) to 40.89 +/- 1.23 pg in Sclerocrangon ferox (Crangonidae). The value for S. ferox represents the largest decapod genome yet recorded and indicates a 38-fold variation in genome size within this order. These data suggest that large genomes may be relatively common in Arctic crustaceans, and underline the need for further comparative studies.     

Amphipod genome sizes: first estimates for Arctic species reveal genomic giants.

The genome sizes of 8 species of amphipods collected from the Canadian Arctic were estimated by flow cytometry. Haploid genome sizes ranged from 2.94 +/- 0.04 pg DNA in Acanthostepheia malmgreni (Oedicerotidae) to 64.62 +/- 2.85 pg in Ampelisca macrocephala (Ampeliscidae). The value for Ampelisca macrocephala represents the largest crustacean genome size recorded to date (and also the largest within the Arthropoda) and indicates a 400-fold variation in genome size among crustaceans. The presence of such large genomes within a relatively small sample of Arctic amphipods is striking and highlights the need to further explore the relationships between genome size, development rates, and body size in both Arctic and temperate amphipods.     

Nuclear DNA content of the whitefly Bemisia tabaci (Aleyrodidae: Hemiptera) estimated by flow cytometry

The nuclear DNA content of the whitefly Bemisia tabaci (Gennnadius) was estimated using flow cytometry. Male and female nuclei were stained with propidium iodide and their DNA content was estimated using chicken red blood cells and Arabidopsis thaliana L. (Brassicaceae) as external standards. The estimated nuclear DNA content of male and female B. tabaci was 1.04 and 2.06 pg, respectively. These results corroborated previous reports based on chromosome counting, which showed that B. tabaci males are haploid and females are diploid. Conversion between DNA content and genome size (1 pg DNA=980 Mbp) indicate that the haploid genome size of B. tabaci is 1020 Mbp, which is approximately five times the size of the genome of the fruitfly Drosophila melanogaster Meigen. These results provide an important baseline that will facilitate genomics-based research for the B. tabaci complex.     

利用流式细胞仪测定荚蒾属植物的基因组大小

荚蒾属 (Viburnum) 植物在园林中广泛用作观赏灌木,并且其具有优良园艺性状的杂交种在世界范围内越来越受欢迎。本研究利用流式细胞仪测定了14种荚蒾属植物的基因组大小。二倍体中基因组大小变化范围是255pg（陕西荚蒾）到426pg（琼花）。同时,琼花的核型也较不对称,这可能反应了它的育种历史。四倍体物种珊瑚树的基因组大小（762pg）是其他二倍体物种的两倍还多,这揭示了该属的多倍化在进化中可能并不遥远。该研究为荚蒾属细胞遗传学和分类学的深入研究奠定了基础,并为该属杂交育种提供有用的信息。     

Genome size variation in the common frog Rana temporaria

Genome size variation in the common frog (Rana temporaria) was investigated with flow cytometry in three latitudinally separated populations in Sweden to see whether it could provide a useful tool for sex-identification in this species. Depending on the sex and population, per cell DNA content (2C value) varied from 8.823 to 11.266 pg with a mean (+/- SE) 2C value of 9.961+/-0.083 pg. Analysis of variance revealed significant differences in genome size among populations and between sexes. Females had ca 3% larger genomes (x=10.133+/-0.068 pg) than males (x=9.832+/-0.068 pg) in all of the populations (sex x population interaction: P>0.10). Individuals from the southern-most population had significantly (x=9.330+/-0.081 pg) smaller genomes than those from the more northern populations (x=10.032+/-0.085 and x=10.584+/-0.085 pg, respectively). These results are in line with the interpretation that males in the common frog are the heterogametic sex, and that there exists large (up to 12%) geographic variation in genome size in this species. However, the sex differences in the genome size are too small to be useful in individual sex identification.     

Genome size variation in Corchorus olitorius (Malvaceae s.l.) and its correlation with elevation and phenotypic traits

In this study, we report genome size variations in Corchorus olitorius L. (Malvaceae s.l.), a crop species known for its morphological plasticity and broad geographical distribution, and Corchorus capsularis L., the second widely cultivated species in the genus. Flow cytometric analyses were conducted with several tissues and nuclei isolation buffers using 69 accessions of C. olitorius and 4 accessions of C. capsularis, representing different habitats and geographical origins. The mean 2C nuclear DNA content (± SD) of C. olitorius was estimated to be 0.918 ± 0.011 pg, with a minimum of 0.882 ± 0.004 pg, and a maximum of 0.942 ± 0.004 pg. All studied plant materials were found to be diploid with 2n = 14. The genome size is negatively correlated with days to flowering (r = -0.29, p < 0.05) and positively with seed surface area (r = 0.38, p < 0.05). Moreover, a statistically significant positive correlation was detected between genome size and growing elevation (r = 0.59, p < 0.001) in wild populations. The mean 2C nuclear DNA content of C. capsularis was estimated to be 0.802 ± 0.008 pg. In comparison to other economically important crop species, the genome sizes of C. olitorius and C. capsularis are much smaller, and therewith closer to that of rice. The relatively small genome sizes will be of general advantage for any efforts into genomics or sequencing approaches of these species.     

DNA probes for the rapid identification of medically important Candida species using a multianalyte profiling system

Recently, a new flow cytometric technology to detect multiple DNA target sequences in a single microtiter well plate was developed [multianalyte profiling (MAP) System, Luminex Corp., Austin, TX]. DNA probes, directed to the internal transcribed spacer 2 region of ribosomal DNA, were therefore designed to detect and differentiate PCR amplicons from six medically important Candida species using this system. Each probe was covalently linked to one of 100 available microsphere (bead) sets. Biotinylated PCR amplicons were then hybridized to the complementary probe on each bead set. Bound amplicons were detected fluorometrically using a streptavidin-linked reporter dye, R-phycoerythrin. Specific hybridization was noted for all six Candida species probes (mean sample-to-background ratio+/-standard error: Candida albicans, 58.7+/-1.2; Candida tropicalis, 53.2+/-3.8; Candida glabrata, 46.9+/-2.1; Candida parapsilosis, 59.9+/-1.6; Candida krusei, 54.7+/-3.7 vs. 0.9+/-0.03 for all heterologous Candida species DNA targets and vs. 1.0+/-0.1 for samples containing water instead of DNA; P < 0.001). The limit of test sensitivity was 0.5 pg of DNA. A sample could be processed and analyzed within 1 h post-PCR amplification. Therefore, the multianalyte profiling system was rapid, sensitive and specific for the detection and differentiation of the most medically important species of Candida.     

Linkage relationships of seven enzyme and two pigmentation loci in the snail Biomphalaria glabrata

Crossing experiments with inbred stocks of the snail (Biomphalaria glabrata) demonstrated that variants at two loci determining pigmentation and seven enzyme-determining loci exhibited normal Mendelian segregation ratios in F2 progeny. Among 39 pairwise comparisons for joint segregation, there was evidence of genetic linkage between a locus controlling mantle pigmentation (S) and 6-phosphogluconate dehydrogenase (Pgd) and confirmation of a previously described linkage between esterase-2 (Est-2) and catalase (Cat). Recombination fractions were estimated to be 17 +/- 4 for S-Pgd and 33 +/- 5 for Est-2-Cat. The remaining five loci--Acon-1, Pgm-1, Lap-1, Lap-2, and Pgd--assorted independently. This brings to 17 the number of loci examined for segregation and assortment in this medically important species. As Biomphalaria has a chromosome number n = 18, markers should soon be available for most or all of the linkage groups.     

The snail (Biomphalaria glabrata) genome project

In 2001, ideas for a snail genome project were discussed at the American Society of Parasitologists meeting (New Mexico) and a snail genome consortium was subsequently established (the first consortium meeting was held in 2005). A proposal for sequencing the snail genome was submitted to the National Human Genome Research Institute, and Biomphalaria glabrata was prioritized as a non-mammalian sequencing target in 2004. The sequencing of the genome of this medically important snail is now underway.     

Plasma somatostatin-like immunoreactivity responses to a mixed meal and the heterogeneity in healthy and non-insulin-dependent (NIDDM) diabetics

Peripheral plasma somatostatin-like immunoreactivity (SLI) was estimated in non-extracted plasma using a specific somatostatin-14 (SS-14) antiserum. The basal plasma SLI level in healthy subjects (n = 18) was 43 +/- 2.9 pg/ml (mean +/- SE) and rose significantly to 8.3 +/- 2.7, 7.3 +/- 1.1 and 5.8 +/- 2.1 pg/ml above the mean basal level 20, 30, and 40 min after a mixed meal, respectively (P less than 0.05). Basal plasma SLI levels in diet (n = 8), sulfonyl urea (n = 8), and insulin groups (n = 8) of non-insulin-dependent maturity onset diabetics (NIDDM) were 50 +/- 1.6, 59 +/- 4.5, and 74 +/- 5.8 pg/ml, respectively. The basal levels for patients with NIDDM were significantly higher than those for healthy subjects (P less than 0.05). No significant increases in plasma SLI were observed after a mixed meal in any group of NIDDM subjects. Elevated plasma SLI levels are considered to be closely related to the severity of the diabetes. The ratios of SS-14 and SS-28 to the total amount of basal plasma SLI were analyzed using high pressure liquid chromatography (HPLC). The ratio of SS-14 to the total SLI was 71-80% in healthy subjects. The ratio of SS-28 to the total SLI increased from 26-30% in the diet group to 50-55% in the group on insulin. These findings suggest a possible pathophysiological role for gastrointestinal somatostatin in NIDDM.     

The ACTH content of plasma in fetal and newborn swine]

ACTH concentration has been estimated radioimmunologically in fetal plasma (100th day of gestation) and in plasma of newborn piglets during the first 24 hours of life and in sows. In comparison to the values of ACTH in sows at the 100th day of gestation during anaesthesia (175 pg/ml) and sows at parturition (235 +/- 77 pg/ml) the concentration in fetal (558 +/- 163 pg/ml) and newborn piglets (448 +/- 158 pg/ml) was much higher. On an average ACTH concentration increased during the first 24 hours of life up to 998 +/- 628 pg/ml. The results are compared to those in other species.     

Running and shipping elevate plasma levels of beta-endorphin-like substance (B-END-LI) in thoroughbred horses

A specific RIA for beta-endorphin (B-END) was developed to measure horse plasma levels of B-END-like material (B-END-LI) during exercises and shipping. Three exercise speeds and durations were: trot at 260-300 m/min for 10 min; slow gallop at 390-420 m/min for 5 min and fast gallop at 700-800 m/min for 2 min. Blood samples were taken from 4 horses before, immediately after, 30 and 60 min after exercise. Trotting increased plasma B-END-LI from a basal level of 109 +/- 7 pg/ml to 172 +/- 22 at the end of exercise and returned to 127 +/- 17 and 107 +/- 10 pg/ml at 30 and 60 min after exercise. Similar results were obtained in slow gallop (121 +/- 6 to 210 +/- 17 then 155 +/- 8 and 131 +/- 11 pg/ml). However, fast gallop caused the greatest increase (352%) in B-END-LI to concentrations of 544 +/- 93 pg/ml and 276 +/- 74 pg/ml at 5 and 30 min after exercise. Plasma B-END-LI returned to 199 +/- 46 pg/ml in 1 hr. Sequential exercises of trot, slow and fast gallop were conducted in 6 horses. Plasma B-END-LI were 116 +/- 19 pg/ml (pre-exercise), 198 +/- 21 (trot), 361 +/- 51 (slow gallop), 500 +/- 57 (fast gallop) and 248 +/- 29, 171 +/- 24, 143 +/- 20 and 139 +/- 21 pg/ml at 0.5, 1, 2 and 3 hr, respectively, following exercises. Transportation in horse trailer also significantly increased plasma levels of B-END-LI from a basal level of 138 +/- 12 to 196 +/- 24 pg/ml within 30 min and this levels were maintained at 45 min (177 +/- 3 pg/ml). Plasma levels of B-END-LI began to decline at 60 min of shipping. These results showed that plasma B-END-LI was increased in all speeds of exercise and by shipping and returned to pre-exercise and pre-shipping level in 30 min except fast gallop which returned to pre-exercise level in 1 hr.     

The effect of angiotensin II and ADH on the secretion of atrial natriuretic factor

Studies in intact animals have suggested that angiotensin II (AII) and antidiuretic hormone (ADH) increase the plasma concentration of atrial natriuretic factor (ANF). The purpose of these studies was to examine the effects of AII and ADH on ANF secretion in a rat heart-lung preparation under conditions where aortic pressure could be regulated and other indirect effects of these hormones eliminated. ANF secretion was estimated as the total amount of ANF present in a perfusion reservoir at the end of each 30-min period. A pump was used to deliver a fluorocarbon perfusate to the right atrium at rates of either 2 or 5 ml/min. In a time control series where venous return was maintained at 2 ml/min for three 30-min periods ANF secretion was 672 +/- 114, 794 +/- 91, and 793 +/- 125 pg/min (n = 6, P greater than 0.05). When venous return was increased from 2 to 5 ml/min ANF secretion increased from 669 +/- 81 to 1089 +/- 127 pg/min (P less than 0.01). The addition of AII to the perfusate in concentrations of 50, 100, or 200 pg/ml (n = 6 in each group) had no significant effect on basal ANF secretion or the ANF response to increasing venous return. Similarly, the addition of ADH to the perfusate in concentrations of 5, 25, or 100 pg/ml had no significant effect on ANF release from the heart. These results suggest that the ability of AII and ADH to increase plasma ANF concentration in vivo may be due to the effects of these hormones on right or left atrial pressure.     

Genome evolution in the genus Sorghum (Poaceae)

BACKGROUND AND AIMS: The roles of variation in DNA content in plant evolution and adaptation remain a major biological enigma. Chromosome number and 2C DNA content were determined for 21 of the 25 species of the genus Sorghum and analysed from a phylogenetic perspective. METHODS: DNA content was determined by flow cytometry. A Sorghum phylogeny was constructed based on combined nuclear ITS and chloroplast ndhF DNA sequences. KEY RESULTS: Chromosome counts (2n = 10, 20, 30, 40) were, with few exceptions, concordant with published numbers. New chromosome numbers were obtained for S. amplum (2n = 30) and S. leiocladum (2n = 10). 2C DNA content varies 8.1-fold (1.27-10.30 pg) among the 21 Sorghum species. 2C DNA content varies 3.6-fold from 1.27 pg to 4.60 pg among the 2n = 10 species and 5.8-fold (1.52-8.79 pg) among the 2n = 20 species. The x = 5 genome size varies over an 8.8-fold range from 0.26 pg to 2.30 pg. The mean 2C DNA content of perennial species (6.20 pg) is significantly greater than the mean (2.92 pg) of the annuals. Among the 21 species studied, the mean x = 5 genome size of annuals (1.15 pg) and of perennials (1.29 pg) is not significantly different. Statistical analysis of Australian species showed: (a) mean 2C DNA content of annual (2.89 pg) and perennial (7.73 pg) species is significantly different; (b) mean x = 5 genome size of perennials (1.66 pg) is significantly greater than that of the annuals (1.09 pg); (c) the mean maximum latitude at which perennial species grow (-25.4 degrees) is significantly greater than the mean maximum latitude (-17.6) at which annual species grow. CONCLUSIONS: The DNA sequence phylogeny splits Sorghum into two lineages, one comprising the 2n = 10 species with large genomes and their polyploid relatives, and the other with the 2n = 20, 40 species with relatively small genomes. An apparent phylogenetic reduction in genome size has occurred in the 2n = 10 lineage. Genome size evolution in the genus Sorghum apparently did not involve a 'one way ticket to genomic obesity' as has been proposed for the grasses.     

Concentrations of oestradiol-17 beta in plasma and corpora lutea throughout pregnancy in the tammar, Macropus eugenii

Oestradiol-17 beta concentrations were measured by radioimmunoassay in peripheral blood samples from 10 tammar wallabies after their pouch young were removed to terminate embryonic diapause. Oestradiol concentrations rose from 8.3 +/- 1.2 pg/ml on Days 3 and 4 to peak of 15.8 +/- 2.9 pg/ml on Day 5, coincident with an increase in 'progesterone' concentrations, and then fell to 10.5 +/- 2.7 pg/ml on Day 7. No changes in oestradiol concentrations were associated with parturition. Five females came into oestrus and mated 9.8 +/- 6.1 h post partum; peak concentrations of plasma oestradiol (20.9 +/- 2.1 pg/ml) occurred around the time of mating. None of the females that did not mate up to the end of the experiment at Day 30 had a rise in plasma oestradiol concentrations. Corpora lutea contained 20-100 pg oestradiol during pregnancy. The highest ovarian oestradiol content (greater than 1200 pg) was measured in whole ovaries containing Graafian follicles from full-term pregnant females. The rise in oestradiol concentrations at Day 5 may be important in the termination of diapause. The post-partum increase in plasma oestradiol concentrations coincides with oestrus. The source of this oestrogen appears to be the preovulatory follicle.     

Variation in genome size of argasid and ixodid ticks

The suborder Ixodida includes many tick species of medical and veterinary importance, but little is known about the genomic characteristics of these ticks. We report the first study to determine genome size in two species of Argasidae (soft ticks) and seven species of Ixodidae (hard ticks) using flow cytometry analysis of fluorescent stained nuclei. Our results indicate a large haploid genome size (1C>1000 Mbp) for all Ixodida with a mean of 1281 Mbp (1.31+/-0.07 pg) for the Argasidae and 2671 Mbp (2.73+/-0.04 pg) for the Ixodidae. The haploid genome size of Ixodes scapularis was determined to be 2262 Mbp. We observed inter- and intra-familial variation in genome size as well as variation between strains of the same species. We explore the implications of these results for tick genome evolution and tick genomics research.     

毛竹基因组大小测定

毛竹(Phyllostachys edulis)属禾本科(Poaceae)竹亚科(Bambusoideae)刚竹属(Phyllostachys), 是我国分布和栽培面积最大的经济竹种, 有着广泛的开发前景。本实验以水稻(Oryza sativa)为内标, 用流式细胞仪对水稻和竹子样品的PI发射荧光强度进行测定, 通过比较水稻与毛竹样品峰值的倍数关系, 计算出毛竹的基因组大小。对24组样品进行重复测试, 测得毛竹基因组大小为 2 075.025±13.08 Mb, 即2 C DNA含量为4.24 pg(以1 pg DNA = 0.978×109 bp计算)。毛竹基因组大小测定为毛竹基因组文库的建立及其基因组学研究奠定了重要基础。     

Phylogeography of Biomphalaria glabrata and B. pfeifferi, important intermediate hosts of Schistosoma mansoni in the New and Old World tropics

The historical phylogeography of the two most important intermediate host species of the human blood fluke Schistosoma mansoni, B. glabrata in the New World, and B. pfeifferi in the Old World, was investigated using partial 16S and ND1 sequences from the mitochondrial genome. Nuclear sequences of an actin intron and internal transcribed spacer (ITS)-1 were also obtained, but they were uninformative for the relationships among populations. Phylogenetic analyses based on mtDNA revealed six well-differentiated clades within B. glabrata: the Greater Antilles, Venezuela and the Lesser Antilles, and four geographically overlapping Brazilian clades. Application of a Biomphalaria-specific mutation rate gives an estimate of the early Pleistocene for their divergence. The Brazilian clades were inferred to be the result of fragmentation, due possibly to climate oscillations, with subsequent range expansion producing the overlapping ranges. Within the Venezuela and Lesser Antilles clade, lineages from each of these areas were estimated to have separated approximately 740 000 years ago. Compared to B. glabrata, mitochondrial sequences of B. pfeifferi are about 4x lower in diversity, reflecting a much younger age for the species, with the most recent common ancestor of all haplotypes estimated to have existed 880 000 years ago. The oldest B. pfeifferi haplotypes occurred in southern Africa, suggesting it may have been a refugium during dry periods. A recent range expansion was inferred for eastern Africa less than 100 000 years ago. Several putative species and subspecies, B. arabica, B. gaudi, B. rhodesiensis and B. stanleyi, are shown to be undifferentiated from other B. pfeifferi populations.     

Uptake of pyrimidines and their derivatives into Candida glabrata and Candida albicans

The uptake of pyrimidines and their derivatives into Candida glabrata and Candida albicans was measured using a novel technique in which the cells were rapidly separated from their suspending medium by centrifugation through a layer of an inert oil. The uptake of [14C]cytosine was linear for 30 s for all concentrations of pyrimidine tested. In C. glabrata but not C. albicans cytosine transport was mediated by both a high affinity (Km 0.8 +/- 0.1 microM), low capacity [V 40 +/- 4 pmol (microliters cell water)-1 s-1] and a low affinity [Km 240 +/- 35 microM], high capacity system [V 770 +/- 170 pmol (microliters cell water)-1 s-1]. The cytosine permease in C. glabrata was specific for cytosine and 5-fluorocytosine. In C. albicans there was only one cytosine transport system [Km 2.4 +/- 0.3 microM; V 50 +/- 4 pmol (microliters cell water)-1 s-1]; this system also transported adenine, guanine and hypoxanthine. Differences in nucleoside transport were also observed for C. glabrata and C. albicans, with the uridine permease in C. glabrata transporting only uridine and 5-fluorouridine whereas cytidine and adenosine were also transported by the uridine permease in C. albicans. Studies on the effect of nucleoside analogues on uridine transport in C. glabrata demonstrated the importance of the sugar moiety in determining the specificity of transport, with a hydroxyl residue on C-2 being apparently essential for transport.