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.     

Five major nuclear ribosomal repeats represent a large and variable fraction of the genomic DNA of Picea rubens and P. mariana.

The nuclear ribosomal repeats for the 18S, 5.8S, and 26S RNAs of two closely related Picea (spruce) species were characterized by restriction mapping and Southern blot hybridization. Restriction polymorphisms were identified in the IGS and ITS sequences; however, no polymorphism was species specific. As many as five different rDNA repeat units were observed in individual genomes. The repeat size for these gymnosperms ranged from a minimum of 32 kbp to greater than 40 kbp, two- to threefold larger than the typical angiosperm rDNA unit. Slot-blot hybridizations were used to determine the nuclear rDNA copy concentration. Among P. rubens individuals threefold variation was observed in the rDNA copy concentration, and among P. mariana individuals such variation was as much as sixfold. At a size greater than 32 kbp and at a concentration averaging 1.2-1.3 x 10(4) copies/pg, the rDNA constitutes approximately 4% of the total genome. Regression analysis revealed a significant relationship between copy concentration of the rDNA repeat unit in P. rubens and geographic origins. Differences in the rDNA content in Picea could contribute to the variation, in overall genome size, that has been observed within conifer species.     

Development of an integrated laboratory information management system for the maize mapping project

MOTIVATION: The development of an integrated genetic and physical map for the maize genome involves the generation of an enormous amount of data. Managing this data requires a system to aid in genotype scoring for different types of markers coming from both local and remote users. In addition, researchers need an efficient way to interact with genetic mapping software and with data files from automated DNA sequencing. They also need ways to manage primer data for mapping and sequencing and provide views of the integrated physical and genetic map and views of genetic map comparisons. RESULTS: The MMP-LIMS system has been used successfully in a high-throughput mapping environment. The genotypes from 957 SSR, 1023 RFLP, 189 SNP, and 177 InDel markers have been entered and verified via MMP-LIMS. The system is flexible, and can be easily modified to manage data for other species. The software is freely available. AVAILABILITY: To receive a copy of the iMap or cMap software, please fill out the form on our website. The other MMP-LIMS software is freely available at http://www.maizemap.org/bioinformatics.htm.     

Differential regulation of growth and checkpoint control mediated by a Cdc25 mitotic phosphatase from Pneumocystis carinii

Pneumocystis carinii is an opportunistic fungal pathogen phylogenetically related to the fission yeast Schizosaccharomyces pombe. P. carinii causes severe pneumonia in immunocompromised patients with AIDS and malignancies. Although the life cycle of P. carinii remains poorly characterized, morphologic studies of infected lung tissue indicate that P. carinii alternates between numerous small trophic forms and fewer large cystic forms. To understand further the molecular mechanisms that regulate progression of the cell cycle of P. carinii, we have sought to identify and characterize genes in P. carinii that are important regulators of eukaryotic cell cycle progression. In this study, we have isolated a cDNA from P. carinii that exhibits significant homology, but unique functional characteristics, to the mitotic phosphatase Cdc25 found in S. pombe. P. carinii Cdc25 was shown to rescue growth of the temperature-sensitive S. pombe cdc25-22 strain and thus provides an additional tool to investigate the unique P. carinii life cycle. Although P. carinii Cdc25 could also restore the DNA damage checkpoint in cdc25-22 cells, it was unable to restore fully the DNA replication checkpoint. The dissociation of checkpoint control at the level of Cdc25 indicates that Cdc25 may be under distinct regulatory control in mediating checkpoint signaling.     

An integrated BAC and genome sequence physical map of Phytophthora sojae

Phytophthora spp. are serious pathogens that threaten numerous cultivated crops, trees, and natural vegetation worldwide. The soybean pathogen P. sojae has been developed as a model oomycete. Here, we report a bacterial artificial chromosome (BAC)-based, integrated physical map of the P. sojae genome. We constructed two BAC libraries, digested 8,681 BACs with seven restriction enzymes, end labeled the digested fragments with four dyes, and analyzed them with capillary electrophoresis. Fifteen data sets were constructed from the fingerprints, using individual dyes and all possible combinations, and were evaluated for contig assembly. In all, 257 contigs were assembled from the XhoI data set, collectively spanning approximately 132 Mb in physical length. The BAC contigs were integrated with the draft genome sequence of P. sojae by end sequencing a total of 1,440 BACs that formed a minimal tiling path. This enabled the 257 contigs of the BAC map to be merged with 207 sequence scaffolds to form an integrated map consisting of 79 superscaffolds. The map represents the first genome-wide physical map of a Phytophthora sp. and provides a valuable resource for genomics and molecular biology research in P. sojae and other Phytophthora spp. In one illustration of this value, we have placed the 350 members of a superfamily of putative pathogenicity effector genes onto the map, revealing extensive clustering of these genes.     

The gene encoding the cytosolic form of phosphoenolpyruvate carboxykinase (GTP) from the chicken

The gene for cytosolic phosphoenolpyruvate carboxykinase (GTP) (EC 4.1.1.32) from the chicken was isolated from a recombinant library containing the chicken genome in phage lambda Charon 4A. The isolated clone, lambda PCK1cc, contains the complete gene for the enzyme as well as both 5' and 3' flanking sequences. The gene is approximately 8 kilobases in length divided into 8 exons, as demonstrated by restriction endonuclease mapping and DNA-RNA heteroduplex analysis. Southern blotting of chicken chromosomal DNA digested with various restriction enzymes shows a pattern predicted from the restriction map of lambda PCK1cc. The phosphoenolpyruvate carboxykinase gene is present as a single copy in the haploid chicken genome. The 5' region of the gene was defined by S1 nuclease mapping and by sequencing. Two mRNA species with discrete 5' ends were observed using S1 nuclease mapping. The ratio between the amounts of these multiple forms of mRNA is the same in chicken kidney and liver and is not affected by induction of the enzyme mRNA by cAMP. Examination of sequence homologies with the gene for rat cytosolic phosphoenolpyruvate carboxykinase indicates a putative control region contained in flanking sequences at the 5' end of the gene.     

Essential eukaryotic core

The AIDs-related fungal pathogen Pneumocystis carinii is unusual in having a remarkably compact genome of 7.7 megabase pairs (mbp) whose small size presents the opportunity to identify the essential eukaryotic core of genes. The essential eukaryotic core is defined to be a collection of essential genes shared by all eukaryotes. Sequencing the 3' ends of more than 5500 cDNAs from P. carinii allowed us to identify about 200 genes shared with its nearest known but distant relative, Schizosaccharomyces pombe and also Saccharomyces cerevisiae, and with homologs known to be essential in S. pombe or S. cerevisae. As the cDNA library contains about one half of the P. carinii genes, the size of the essential eukaryotic core (approximately 400) is slightly larger than the prokaryotic core (265-350) being identified by studies of the bacterial pathogen Mycoplasma genitalium. The collection of genes in the essential eukaryotic core may prove useful in identifying new broad spectrum antifungal drug targets.     

An integrated physical and genetic map of the rice genome

Rice was chosen as a model organism for genome sequencing because of its economic importance, small genome size, and syntenic relationship with other cereal species. We have constructed a bacterial artificial chromosome fingerprint–based physical map of the rice genome to facilitate the whole-genome sequencing of rice. Most of the rice genome (~90.6%) was anchored genetically by overgo hybridization, DNA gel blot hybridization, and in silico anchoring. Genome sequencing data also were integrated into the rice physical map. Comparison of the genetic and physical maps reveals that recombination is suppressed severely in centromeric regions as well as on the short arms of chromosomes 4 and 10. This integrated high-resolution physical map of the rice genome will greatly facilitate whole-genome sequencing by helping to identify a minimum tiling path of clones to sequence. Furthermore, the physical map will aid map-based cloning of agronomically important genes and will provide an important tool for the comparative analysis of grass genomes.     

Isolation of the Pneumocystis carinii dihydrofolate synthase gene and functional complementation in Saccharomyces cerevisiae

The Pneumocystis carinii gene encoding the enzyme dihydrofolate synthase (DHFS), which is involved in the essential biosynthesis of folates, was isolated from clones of the Pneumocystis genome project, and sequenced. The deduced P. carinii DHFS protein shares 38% and 35% identity with DHFS of Schizosaccharomyces pombe and Saccharomyces cerevisiae, respectively. P. carinii DHFS expressed from a plasmid functionally complemented a S. cerevisiae mutant with no DHFS. Comparison of available DHFSs with highly similar folylpolyglutamate synthases allowed the identification of potential signatures responsible for the specificities of these two classes of enzymes. The results open the way to experimentally analyse the structure and function of P. carinii mono-functional enzyme DHFS, to investigate a possible role of DHFS in the resistance to antifolates of P. jirovecii, the species infecting specifically humans, and to develop a new class of antifolates.     

Fourteen chromosomal localizations and an update of the cytogenetic map of the rabbit

In order to improve the informativeness of the cytogenetic map of the rabbit genome, fourteen markers were regionally mapped to individual chromosomes. The localizations comprise eleven gene loci (PRLR, GHR, HK1, ACE, TF, 18S+28S rDNA, CYP2C4, PMP2, TCRB, ALOX15 and MT1) and three microsatellite loci (Sat13, Sol33 and D1Utr6). Five of the genes contain known microsatellite sequences. To achieve these localizations, homologous and heterologous small insert clones, and clones from a rabbit Bacterial Artificial Chromosome (BAC) library were used as probes for fluorescence in situ hybridization experiments. Results indicate that especially BAC clones are a valuable tool for cytogenetic mapping. Some of the genes were selected for mapping on the basis of human- rabbit comparative painting data, to achieve localizations on gene-poor rabbit chromosomes. Our data are, in general, in agreement with the human-rabbit comparative painting data. By mapping microsatellite sequences that have also been used in linkage studies, links are provided between the genetic and physical maps of the rabbit genome. Linkage groups I, VI and XI could be assigned to chromosomes 1, 5 and 3 respectively. Moreover, in this paper we give an overview of the current status of the rabbit cytogenetic map. This map now comprises 62 physically mapped genes, which are scattered over all autosomes, except chromosome 2, and the X chromosome.     

The RNA components of Schizosaccharomyces pombe RNase P are essential for cell viability

The fission yeast Schizosaccharomyces pombe contains in the haploid genome one copy of the gene (designated rrkl) for the RNA components of RNase P. Gene disruption in diploid cells of one copy of rrkl resulted in a moderate reduction of the level of cellular RNase P activity. Haploidization by meiosis demonstrated that rrkl is required for cell growth. Thus, the RNA components of S. pombe RNase P are essential in vivo. This is similar to the situation in Escherichia coli.     

Microsatellite markers and genetic mapping in Plasmodium falciparum

Whole-genome methods are changing the scope of biological questions that can be addressed in malaria research. In the rich context provided by Plasmodium falciparum genome sequencing, genetic mapping is a powerful tool for identifying genes involved in parasite development, invasion, transmission and drug resistance. The recent development of a high-resolution P. falciparum linkage map consisting of hundreds of microsatellite markers will facilitate an integrated genomic approach to understanding the relationship between genetic variations and biological phenotypes. Here, Michael Ferdig and Xin-zhuan Su provide an overview for applying microsatellite markers and genetic maps to gene mapping, parasite typing and studies of parasite population changes.     

Alignment of Sfi I sites with the Not I restriction map of Schizosaccharomyces pombe genome.

A Sfi I restriction map of the fission yeast Schizosaccharomyces pombe genome was aligned with the Not I restriction map. There are 16 Sfi I sites in the S. pombe genome. Three Sfi I sites are on chromosome III which is devoid of Not I sites. The sizes of the entire genome and individual chromosomes, calculated from the Sfi I fragment sizes, are consistent with that calculated from the Not I fragment sizes. The Sfi I map provides greater physical characterization of the S. pombe genome and further validates the use of S. pombe chromosomal DNA as size standard. These maps have allowed detection of polymorphism on all three chromosomes.