Determination of the copy number of the nuclear rDNA and beta-tubulin genes of Pneumocystis carinii f. sp. hominis using PCR multicompetitors

Multiple copies of a gene may lead to difficulty in the interpretation of typing results because polymorphism of the copies may wrongly lead to the conclusion that different types are present in a specimen. To determine the copy number per genome of the nuclear rDNA and beta-tubulin genes analyzed for the typing of Pneumocystis carinii f. sp. hominis, we developed a strategy based on the use of the same multicompetitor molecule in two different quantitative-competitive PCRs, one for the gene under study and the other for a reference single copy gene, allowing direct comparison of the results of both PCRs. Control experiments showed that the strategy was sensitive enough to detect duplication of a gene. The copy number of the nuclear rDNA operon was determined by amplification of the intron of the 26S rDNA gene and that of the beta-tubulin by amplification of the region surrounding the intron no. 6. The method was first tested on P. c. carinii, the special form commonly infecting rats. Pneumocystis c. carinii was found to contain a single copy of the rDNA operon. The method was then applied to P. c. hominis. The results confirmed that P. c. hominis genome contains a single copy of the nuclear rDNA and beta-tubulin genes.     

Vitamin D receptor is not required for the rapid actions of 1,25-dihydroxyvitamin D3 to increase intracellular calcium and activate protein kinase C in mouse osteoblasts

The rapid, non-genomic actions of 1,25-dihydroxyvitamin D(3) [1,25(OH)(2)D(3)] have been well described, however, the role of the nuclear vitamin D receptor (VDR) in this pathway remains unclear. To address this question, we used VDR(+/+) and VDR(-/-) osteoblasts isolated from wild-type and VDR null mice to study the increase in intracellular calcium ([Ca(2+)](i)) and activation of protein kinase C (PKC) induced by 1,25(OH)(2)D(3). Within 1 min of 1,25(OH)(2)D(3) (100 nM) treatment, an increase of 58 and 53 nM in [Ca(2+)](i) (n = 3) was detected in VDR(+/+) and VDR(-/-) cells, respectively. By 5 min, 1,25(OH)(2)D(3) caused a 2.1- and 1.9-fold increase (n = 6) in the phosphorylation of PKC substrate peptide acetylated-MBP(4-14) in VDR(+/+) and VDR(-/-) osteoblasts. The 1,25(OH)(2)D(3)-induced phosphorylation was abolished by GF109203X, a general PKC inhibitor, in both cell types, confirming that the secosteroid induced PKC activity. Moreover, 1,25(OH)(2)D(3) treatment resulted in the same degree of translocation of PKC-alpha and PKC-delta, but not of PKC-zeta, from cytosol to plasma membrane in both VDR(+/+) and VDR(-/-) cells. These experiments demonstrate that the 1,25(OH)(2)D(3)-induced rapid increases in [Ca(2+)](i) and PKC activity are neither mediated by, nor dependent upon, a functional nuclear VDR in mouse osteoblasts. Thus, VDR is not essential for these rapid actions of 1,25(OH)(2)D(3) in osteoblasts.     

Dynamics of mitochondrial inheritance in the evolution of binary mating types and two sexes

The uniparental inheritance (UPI) of mitochondria is thought to explain the evolution of two mating types or even true sexes with anisogametes. However, the exact role of UPI is not clearly understood. Here, we develop a new model, which considers the spread of UPI mutants within a biparental inheritance (BPI) population. Our model explicitly considers mitochondrial mutation and selection in parallel with the spread of UPI mutants and self-incompatible mating types. In line with earlier work, we find that UPI improves fitness under mitochondrial mutation accumulation, selfish conflict and mitonuclear coadaptation. However, we find that as UPI increases in the population its relative fitness advantage diminishes in a frequency-dependent manner. The fitness benefits of UPI ‘leak’ into the biparentally reproducing part of the population through successive matings, limiting the spread of UPI. Critically, while this process favours some degree of UPI, it neither leads to the establishment of linked mating types nor the collapse of multiple mating types to two. Only when two mating types exist beforehand can associated UPI mutants spread to fixation under the pressure of high mitochondrial mutation rate, large mitochondrial population size and selfish mutants. Variation in these parameters could account for the range of UPI actually observed in nature, from strict UPI in some Chlamydomonas species to BPI in yeast. We conclude that UPI of mitochondria alone is unlikely to have driven the evolution of two mating types in unicellular eukaryotes.     

Molecular characterization of Macrophomina phaseolina and Fusarium species by a single primer RAPD technique

Charcoal root rot and wilt, are two economically important diseases of many crop plants in North and South America, Asia and Africa and some parts of Europe. Genetic variation in 43 isolates of Macrophomina phaseolina and 22 isolates of Fusarium species, collected from geographically distinct regions over a range of hosts, was studied using random amplified polymorphic DNA (RAPD) markers. Initially, 210 arbitrary nucleotide (10-mer) primers were tested for amplification of genomic DNA of one M. phaseolina isolate, 70 primers amplified the genomic DNA of M. phaseolina. One primer OPA-13 (5'-CAGCACCCAC-3') produced fingerprint profiles, which clearly distinguished between the different isolates of M. phaseolina. UPGMA analysis classified these isolates into five major groups. By primer OPA-13, 22 isolates of pathogenic and non-pathogenic Fusarium species of different formae-speciales and races, were also distinguished from M. phaseolina. This marker is useful for distinguishing between these two important plant pathogens irrespective of hosts, virulence spectrum and races. This is the first report of reliable diagnosis of two soilborne pathogens (root/collar rot and wilt causing pathogens) at the level of isolates, formae-speciales and races by a single primer RAPD procedure with uniform PCR conditions.     

3q26.31–q29 duplication and 9q34.3 microdeletion associated with omphalocele,ventricular septal defect,abnormal first-trimester maternal serum screening and increased nuchal translucency: Prenatal diagnosis and aCGH characterization

We present prenatal diagnosis and array comparative genomic hybridization characterization of 3q26.31–q29 duplication and 9q34.3 microdeletion in a fetus with omphalocele, ventricular septal defect, increased nuchal translucency, abnormal first-trimester maternal screening and facial dysmorphism with distinct features of the 3q duplication syndrome and Kleefstra syndrome. The 26.61-Mb duplication of 3q26.31–q29 encompasses EPHB3, CLDN1 and CLDN16, and the 972-kb deletion of 9q34.3 encompasses EHMT1. We review the literature of partial trisomy 3q associated with omphalocele and discuss the genotype–phenotype correlation in this case.     

Within-population structure highlighted by differential introgression across semipermeable barriers to gene flow in Anguilla marmorata

In the marine environment, differential gene exchange between partially reproductively isolated taxa can result in introgression that extends over long distances due to high larval dispersal potential. However, the degree to which this process contributes to interlocus variance of genetic differentiation within introgressed populations remains unclear. Using a genome-scan approach in the Indo-Pacific eel Anguilla marmorata, we investigated the degree of interpopulation genetic differentiation, the rate of introgression, and within-population genetic patterns at 858 AFLP markers genotyped in 1117 individuals. Three divergent populations were identified based on clustering analysis. Genetic assignments of individuals revealed the existence of different types of hybrids that tended to co-occur with parental genotypes in three population contact zones. Highly variable levels of genetic differentiation were found between populations across the AFLP markers, and reduced rates of introgression were shown at some highly differentiated loci. Gene flow across semipermeable genetic barriers was shown to generate spatial introgression patterns at some loci which define within-population structure over long distances. These results suggest that differential introgression in subdivided populations may be relevant when interpreting spatial variation patterns displayed by outlying loci in other marine fish populations.     

A genome map of Salmonella enterica serovar Agona: numerous insertions and deletions reflecting the evolutionary history of a human pathogen

Salmonella enterica serovar Agona is an important zoonotic pathogen, causing serious human illness worldwide, but knowledge about its genetics and evolution, especially regarding the genomic events that might have contributed to the formation of S . Agona as an important pathogen, is lacking. As a first step toward understanding this pathogen and characterizing its genomic differences with other salmonellae, we constructed a physical map of S . Agona in strain SARB1 using I-CeuI, XbaI, AvrII and Tn 10 insertions with pulsed-field gel electrophoresis techniques. On the 4815-kb genomic map, we located 82 genes, revealed one inversion of about 1000 kb and resolved seven deletions and seven insertions ranging from 10 to 67 kb relative to the genome of Salmonella typhimurium LT2. These genomic features clearly distinguish S . Agona from other previously analyzed salmonellae and provide clues to the molecular basis for its genomic divergence. Additionally, these kinds of physical maps, combined with emerging high-speed sequencing technologies, such as the Solexa or SOLiD techniques, which require a pre-existing high-resolution physical map such as the S . Agona map reported here, will play important roles in genomic comparative studies of bacteria involving large numbers of strains.     

Genomic Diversity in the pfoA Region of Theta-Toxin-Deficient Strains of Clostridium perfringens

The genomic structure of the pfoA-colA region in six theta-toxin-deficient strains of Clostridium perfringens was examined by Southern hybridization using the pfoR, pfoA, pbg, arcABDC and colA genes, encoding regulator for pfoA, theta-toxin, beta-galactosidase, arginine metabolism enzymes and kappa-toxin, respectively, as gene probes. It is suggested that the productivity of theta-toxin in these strains is diverse because of the multiple genetic backgrounds including single deletion of pfoA, large deletion of the pfoA-colA region and the putative point mutations.