Development of pachytene FISH maps for six maize chromosomes and their integration with other maize maps for insights into genome structure variation

Integrated cytogenetic pachytene fluorescence in situ hybridization (FISH) maps were developed for chromosomes 1, 3, 4, 5, 6, and 8 of maize using restriction fragment length polymorphism marker-selected Sorghum propinquum bacterial artificial chromosomes (BACs) for 19 core bin markers and 4 additional genetic framework loci. Using transgenomic BAC FISH mapping on maize chromosome addition lines of oats, we found that the relative locus position along the pachytene chromosome did not change as a function of total arm length, indicative of uniform axial contraction along the fibers during mid-prophase for tested loci on chromosomes 4 and 5. Additionally, we cytogenetically FISH mapped six loci from chromosome 9 onto their duplicated syntenic regions on chromosomes 1 and 6, which have varying amounts of sequence divergence, using sorghum BACs homologous to the chromosome 9 loci. We found that successful FISH mapping was possible even when the chromosome 9 selective marker had no counterpart in the syntenic block. In total, these 29 FISH-mapped loci were used to create the most extensive pachytene FISH maps to date for these six maize chromosomes. The FISH-mapped loci were then merged into one composite karyotype for direct comparative analysis with the recombination nodule-predicted cytogenetic, genetic linkage, and genomic physical maps using the relative marker positions of the loci on all the maps. Marker colinearity was observed between all pair-wise map comparisons, although marker distribution patterns varied widely in some cases. As expected, we found that the recombination nodule-based predictions most closely resembled the cytogenetic map positions overall. Cytogenetic and linkage map comparisons agreed with previous studies showing a decrease in marker spacing in the peri-centromeric heterochromatin region on the genetic linkage maps. In fact, there was a general trend with most loci mapping closer towards the telomere on the linkage maps than on the cytogenetic maps, regardless of chromosome number or maize inbred line source, with just some of the telomeric loci exempted. Finally and somewhat surprisingly, we observed considerable variation between the relative arm positions of loci when comparing our cytogenetic FISH map to the B73 genomic physical maps, even where comparisons were to a B73-derived cytogenetic map. This variation is more evident between different chromosome arms, but less so within a given arm, ruling out any type of inbred-line dependent global features of linear deoxyribonucleic acid compared with the meiotic fiber organization. This study provides a means for analyzing the maize genome structure by producing new connections for integrating the cytogenetic, linkage, and physical maps of maize.     

Preliminary profile of the Cryptosporidium parvum genome: an expressed sequence tag and genome survey sequence analysis

Cryptosporidium parvum is a protozoan enteropathogen that infects humans and animals and causes a pronounced diarrheal disease that can be life-threatening in immunocompromised hosts. No specific chemo- or immunotherapies exist to treat cryptosporidiosis and little molecular information is available to guide development of such therapies. To accelerate gene discovery and identify genes encoding potential drug and vaccine targets we constructed sporozoite cDNA and genomic DNA sequencing libraries from the Iowa isolate of C. parvum and determined approximately 2000 sequence tags by single-pass sequencing of random clones. Together, the 567 expressed sequence tags (ESTs) and 1507 genome survey sequences (GSSs) totaled one megabase (1 mb) of unique genomic sequence indicating that approximately 10% of the 10.4 mb C. parvum genome has been sequence tagged in this gene discovery expedition. The tags were used to search the public nucleic acid and protein databases via BLAST analyses, and 180 ESTs (32%) and 277 GSSs (18%) exhibited similarity with database sequences at smallest sum probabilities P(N)< or =10(-8). Some tags encoded proteins with clear therapeutic potential including S-adenosylhomocysteine hydrolase, histone deacetylase, polyketide/fatty-acid synthases, various cyclophilins, thrombospondin-related cysteine-rich protein and ATP-binding-cassette transporters. Several anonymous ESTs encoded proteins predicted to contain signal peptides or multiple transmembrane spanning segments suggesting they were destined for membrane-bound compartments, the cell surface or extracellular secretion. One-hundred four simple sequence repeats were identified within the nonredundant sequence tag collection with (TAA)(> or =6)/(TTA)(> or =6) and (TA)(> or = 10)/(AT)(> or =10 ) being the most prevalent, occurring 40 and 15 times, respectively. Various cellular RNAs and their genes were also identified including the small and large ribosomal RNAs, five tRNAs, the U2 small nuclear RNA, and the small and large virus-like, double-stranded RNAs. This investigation has demonstrated that survey sequencing is an efficient procedure for gene discovery and genome characterization and has identified and sequence tagged many C. parvum genes encoding potential therapeutic targets.     

An expressed sequence tag analysis of the life-cycle of the parasitic nematode Strongyloides ratti

14,761 expressed sequence tags (ESTs) were generated, representing five stages during the parasitic and free-living phases of the life-cycle of the parasitic nematode Strongyloides ratti. These ESTs formed 4152 clusters, of which 97% contained 10 or fewer ESTs and 66% were singletons. These 4152 clusters are likely to represent approximately 20% of S. ratti's genes. The clusters' consensus sequences were used to assign each cluster to one of three databases: (i) Caenorhabditis elegans and C. briggsae sequences; (ii) other nematode sequences; (iii) non-nematode sequences. This approach has identified putative nematode-specific genes, that may be targets for developing approaches for parasitic nematode control. Approximately 25% of the clusters have no significant alignments and may therefore represent novel genes. The EST representation between the libraries was used to analyse stage-specific or -biased expression in silico. This showed that 81% of clusters are present in only one library and 12% are present in any two libraries, indicating substantial stage-specificity of gene expression. The 30-most abundantly expressed clusters were analysed in further detail. Many of these have significantly different parasitic- or free-living-specific or -biased expression. Many of the parasitic-specific genes are, as yet, uncharacterised: one of these represents 25% of all ESTs obtained from the parasitic stage.     

严重烧伤F344大鼠免疫细胞中一个EST全长序列的扩增

目的:探讨免疫细胞在大面积烧伤后全身免疫功能紊乱中的变化,对F344大鼠烧伤后血液淋巴细胞和单核细胞中的1个差异表达基因片段(AI764697.1)进一步研究。方法:根据该片段核苷酸序列设计双向引物和巢式引物,应用cDNA末端快速扩增技术(SMARTRACE)对该片段进行双向扩增,扩增后结果经测序,用Northernblot杂交验证。结果:设计的两对引物经扩增都得到了产物,克隆测序后得到1条长度为592bp的片段。Northernblot杂交在烧伤后得到1个长度约700bp的产物,在脾脏中表达水平较高,与RACE结果符合。该核苷酸序列已得到GenBank登录号AF244895。结论:在严重烧伤F344大鼠的血液淋巴细胞和单核细胞中分离并扩增出一个新的基因全长序列,其来源、定位和功能需要进一步研究。     

Resolution of fluorescence in-situ hybridization mapping on rice mitotic prometaphase chromosomes, meiotic pachytene chromosomes and extended DNA fibers

Fluorescence in-situ hybridization (FISH) is a quick and affordable approach to map DNA sequences to specific chromosomal regions. Although FISH is one of the most important physical mapping techniques, research on the resolution of FISH on different cytological targets is scarce in plants. In this study, we report the resolution of FISH mapping on mitotic prometaphase chromosomes, meiotic pachytene chromosomes and extended DNA fibers in rice. A majority of the FISH signals derived from bacterial artificial chromosome (BAC) clones separated by approximately 1 Mb of DNA cannot be resolved on mitotic prometaphase chromosomes. In contrast, the relative positions of closely linked or even partially overlapping BAC clones can be resolved on a euchromatic region of rice chromosome 10 at the early pachytene stage. The resolution of pachytene FISH is dependent on early or late pachytene stages and also on the location of the DNA probes in the euchromatic or heterochromatic regions. We calibrated the fiber-FISH technique in rice using seven sequenced BAC clones. The average DNA extension was 3.2 kb/μm among the seven BAC clones. Fiber-FISH results derived from a BAC contig that spanned 1 Mb DNA matched remarkably to the sequencing data, demonstrating the high resolution of this technique in cytological mapping. This revised version was published online in July 2006 with corrections to the Cover Date.     

Identification of genes with a biocontrol function in Trichoderma harzianum mycelium using the expressed sequence tag approach

Species of Trichoderma are commercially applied as biological control agents against plant fungal pathogens based on different mechanisms, such as the production of antifungal metabolites, competition for space and nutrients and mycoparasitism. But the integrated biocontrol mechanism of Trichoderma harzianum is not well explored at the genetic level. This study aimed to initiate the preliminary development of an expressed sequence tag (EST) database for T. harzianum and thereby gain potentially useful information on Trichoderma gene sequences in order to elucidate the integrated biocontrol mechanism. Partial sequencing of anonymous cDNA clones is a widely used technique for gene identification. A directional cDNA library has been constructed from mycelium of T. harzianum and 3298 clones have been randomly selected, subjected to single-pass sequencing from the 5' end of the vector, and identified by sequence similarity searches against gene sequences in international databases. Of the 3298 mycelium clones, 2174 exhibit similarity to known genes and 451 to known ESTs, while 673 represent novel gene sequences. Analysis of the identified clones indicated sequence similarity to a broad diversity of genes encoding proteins such as enzymes, structural proteins, and regulatory factors. A significant proportion of genes identified in the mycelium were involved in processes related to mycoparasitism and fungicidal metabolites, as would be expected in biocontrol fungus. These results present the successful application of EST analysis in T. harzianum and provide a preliminary indication of gene expression in mycelium.     

Assignment of genetic linkage maps to diploid Solanum tuberosum pachytene chromosomes by BAC-FISH technology

A cytogenetic map has been developed for diploid potato (Solanum tuberosum), in which the arms of the 12 potato bivalents can be identified in pachytene complements using multicolor fluorescence in situ hybridization (FISH) with a set of 60 genetically anchored bacterial artificial chromosome (BAC) clones from the RHPOTKEY BAC library. This diagnostic set of selected BACs (five per chromosome) hybridizes to euchromatic regions and corresponds to well-defined loci in the ultradense genetic map, and with these probes a new detailed and reliable pachytene karyotype could be established. Chromosome size has been estimated both from microscopic length measurements and from 4′,6-diamidino-2-phenylindole fluorescence-based DNA content measurements. In both approaches, chromosome 1 is the largest (100–115 Mb) and chromosome 11 the smallest (49–53 Mb). Detailed measurements of mega-base-pair to micrometer ratios have been obtained for chromosome 5, with average values of 1.07 Mb/μm for euchromatin and 3.67 Mb/μm for heterochromatin. In addition, our FISH results helped to solve two discrepancies in the potato genetic map related to chromosomes 8 and 12. Finally, we discuss the significance of the potato cytogenetic map for extended FISH studies in potato and related Solanaceae, which will be especially beneficial for the potato genome-sequencing project.     

Uneven size distribution of mammalian genes in the number of tissues expressed and in the number of co-expressed genes

Tissue specificity, the traditional predictor of gene function, has recently been used to interpret the selective pressure associated with gene architecture. In this work, we examine gene structures and their relation to the number of tissues expressed and to the number of co-expressed genes, using a recent atlas of microarray-based mouse gene expression in 55 normal tissues. We define tissue specificity and expression-pattern specificity according to the number of tissues expressed and the number of co-expressed genes, respectively. We find that, consistent with previous findings, tissue non-specific (housekeeping) genes are short in all gene regions (coding regions, intron, 5' and 3' untranslated regions). However, in contrast to previous suggestion that tissue-specific genes are long, the genes that are the most tissue-specific (expressed only in one tissue) are also short. We further show that both expression-pattern-specific and non-specific genes are long in coding and non-coding regions. The origins for short tissue-specific genes and long expression-pattern-specific genes are not clear. Genes with highly non-specific expression patterns (i.e. genes with a large number of co-expressed genes) are composed of genes that spread all tissues but are overwhelmingly enriched in the central nervous system (e.g. brain). Thus, the large sizes of these genes are possibly related to the functional complexity and/or accelerated evolutions of the central nervous system.     

Analysis of the genes expressed in<Emphasis Type="Italic"> Clonorchis sinensis</Emphasis> adults using the expressed sequence tag approach

Clonorchis sinensis is a biliary tract parasite, which infects over 30 million people in China, Korea and Southeast Asia through the ingestion of undercooked freshwater fish that harbour the infective metacercariae. The genes expressed in C. sinensis adults were identified in order to develop novel drugs, better diagnostics and vaccines for the parasite. The C. sinensis cDNA library was constructed and DNA sequencing was performed with 450 randomly selected clones. Four hundred and fifteen clones contained the amino-acid-encoding sequences. The functions of these genes could be assigned by DNA sequence homology. Basic Local Alignment Search Tool X analysis showed that 277 of the 415 clones were strongly matched (P<10^-9) to previously identified proteins, while the remaining 138 fell into the "no database match" category. Among the clones matching previously identified proteins, 220 putatively identified genes were sorted into seven functional categories. These included the genes associated with energy metabolism (38), gene expression/RNA metabolism (21), regulatory/signalling components (14), protein metabolism/sorting (98), the structure/cytoskeleton (29), membrane transporters (ten) and antigenic proteins (ten). The remaining 57 clones were not included in these categories. The dataset included the genes encoding the proteases, a lipid binding protein, the antigen proteins and the other genes of interest from a diagnostics, drug or vaccine development viewpoint. The present expressed sequence tag analysis proved to be an effective tool for examining gene expression and identified several important genes which increase and complement our knowledge of the biology of C. sinensis.     

The distribution of topoisomerase II on mammalian chromosomes

The distribution of topoisomerase II (Topo II) has been studied using immunofluorescence on cytocentrifuged preparations of mammalian chromosomes. Immunolabelling of Topo II is affected by choice of fixative, by barriers to accessibility and by the lability of the enzyme. Chromosomes still embedded in cytoplasm remain unlabelled, while in contrast Topo II can easily be lost from some sites in chromosomes free of cytoplasm. The definitive distribution of Topo II consists of a line along the centre of each chromatid, corresponding to the chromosome core or scaffold, and quantities of Topo II elsewhere in the chromosomes which vary during the course of mitosis. A strong reaction for Topo II can be seen throughout prophase chromosomes, consistent with a role in condensation and/or segregation of the chromosome arms at this stage. At metaphase, Topo II is restricted to the centromeric regions, the only parts of the chromosomes that still have to be separated at this stage, while in anaphase, after segregation has occurred, this centromeric concentration of Topo II is lost. The distribution and quantity of Topo II in mammalian chromosomes is thus wholly consistent with the known functions of this enzyme in chromosome condensation and segregation.accepted for publication by H. C. Macgregor     

Uneven distribution of sialic acids on the luminal surface of aortic endothelium

Lectins which bind sialic acid (aprotinin, wheat germ) were used to measure sialic acid density on the luminal surface of aortic endothelium of rabbit. The bound fluorescein-labelled lectin was released from the sialic acids by the competing ligand N-acetyl-D-glucosamine and measured fluorimetrically. The specificity of the technique was shown by an 80% decrease of bound fluorescence after neuraminidase treatment of the endothelium. Endothelial sialic acid density was the same in the thoracic and abdominal parts of the aorta but significantly less in the arch. Around the orifices of the branches of the intercostal arteries, less surface fluorescence was observed. Ultrastructural studies have shown much less staining of the surface membranes with peroxidase-conjugated wheat germ lectin at these places than elsewhere. These areas, where less sialic acid density was found, coincide with the sites where the atherosclerotic process is known to develop.