Analysis of fat body transcriptome from the adult tsetse fly, Glossina morsitans morsitans.

Tsetse flies (Diptera: Glossinidia) are vectors of pathogenic African trypanosomes. To develop a foundation for tsetse physiology, a normalized expressed sequence tag (EST) library was constructed from fat body tissue of immune-stimulated Glossina morsitans morsitans. Analysis of 20,257 high-quality ESTs yielded 6372 unique genes comprised of 3059 tentative consensus (TC) sequences and 3313 singletons (available at http://aksoylab.yale.edu). We analysed the putative fat body transcriptome based on homology to other gene products with known functions available in the public domain. In particular, we describe the immune-related products, reproductive function related yolk proteins and milk-gland protein, iron metabolism regulating ferritins and transferrin, and tsetse's major energy source proline biosynthesis. Expression analysis of the three yolk proteins indicates that all are detected in females, while only the yolk protein with similarity to lipases, is expressed in males. Milk gland protein, apparently important for larval nutrition, however, is primarily synthesized by accessory milk gland tissue.

Adult midgut expressed sequence tags from the tsetse fly Glossina morsitans morsitans and expression analysis of putative immune response genes.

BACKGROUND: Tsetse flies transmit African trypanosomiasis leading to half a million cases annually. Trypanosomiasis in animals (nagana) remains a massive brake on African agricultural development. While trypanosome biology is widely studied, knowledge of tsetse flies is very limited, particularly at the molecular level. This is a serious impediment to investigations of tsetse-trypanosome interactions. We have undertaken an expressed sequence tag (EST) project on the adult tsetse midgut, the major organ system for establishment and early development of trypanosomes. RESULTS: A total of 21,427 ESTs were produced from the midgut of adult Glossina morsitans morsitans and grouped into 8,876 clusters or singletons potentially representing unique genes. Putative functions were ascribed to 4,035 of these by homology. Of these, a remarkable 3,884 had their most significant matches in the Drosophila protein database. We selected 68 genes with putative immune-related functions, macroarrayed them and determined their expression profiles following bacterial or trypanosome challenge. In both infections many genes are downregulated, suggesting a malaise response in the midgut. Trypanosome and bacterial challenge result in upregulation of different genes, suggesting that different recognition pathways are involved in the two responses. The most notable block of genes upregulated in response to trypanosome challenge are a series of Toll and Imd genes and a series of genes involved in oxidative stress responses. CONCLUSIONS: The project increases the number of known Glossina genes by two orders of magnitude. Identification of putative immunity genes and their preliminary characterization provides a resource for the experimental dissection of tsetse-trypanosome interactions.

Novel genes are enriched in normalized cDNA libraries from drought-stressed seedlings of rice (Oryza sativa L. subsp. indica cv. Nagina 22).

We have utilized an efficient method to enrich cDNA libraries for novel genes and genes responsive to drought stress in rice (Oryza sativa L. subsp. indica). We separately constructed standard and normalized cDNA libraries from leaf tissue of rice seedlings grown under controlled drought stress. Sequencing from the 3' end was performed on 1000 clones from the normalized leaf cDNA library and 200 clones from the standard leaf cDNA library. For the first 200 clones, the clone redundancy in the non-normalized library was about 10%, compared with 3.5% in the normalized cDNA library. Comparison of these cDNAs with the sequences in public databases revealed that 28.2% of the expressed sequence tags (ESTs) from the normalized library were novel. Clones from the standard and normalized leaf libraries and a root library uncovered numerous cDNAs that are highly homologous to known drought-responsive genes including those that encode metallothioneins, late embroyonic abundant (LEA) proteins, heat-shock proteins, cytochrome P450 enzymes, catalases, peroxidases, kinases, phosphatases, and transcription factors.

The NIEHS Xenopus maternal EST project: interim analysis of the first 13,879 ESTs from unfertilized eggs.

The sequencing of expressed sequence tags (ESTs) from Xenopus laevis has lagged behind efforts on many other common experimental organisms and man, partly because of the pseudotetraploid nature of the Xenopus genome. Nonetheless, large collections of Xenopus ESTs would be useful in gene discovery, oligonucleotide-based knockout studies, gene chip analyses of normal and perturbed development, mapping studies in the related diploid frog X. tropicalis, and for other reasons. We have created a normalized library of cDNAs from unfertilized Xenopus eggs. These cells contain all of the information necessary for the first several cell divisions in the early embryo, as well as much of the information needed for embryonic pattern formation and cell fate determination. To date, we have successfully sequenced 13,879 ESTs out of 16,607 attempts (83.6% success rate), with an average sequence read length of 508 bp. Using a fragment assembly program, these ESTs were assembled into 8,985 'contigs' comprised of up to 11 ESTs each. When these contigs were used to search publicly available databases, 46.2% bore no relationship to protein or DNA sequences in the database at the significance level of 1e-6. Examination of a sample of 100 of the assembled contigs revealed that most ( approximately 87%) were comprised of two apparent allelic variants. Expression profiles of 16 of the most prominent contigs showed that 12 exhibited some degree of zygotic expression. These findings have implications for sequence-specific applications for Xenopus ESTs, particularly the use of allele-specific oligonucleotides for knockout studies, differential hybridization techniques such as gene chip analysis, and the establishment of accurate nomenclature and databases for this species.

Isolation and characterization of a cDNA encoding a novel member of the human regenerating protein family: Reg IV.

Human Reg and Reg-related genes constitute a multi-gene family belonging to the calcium (C-type) dependent lectin superfamily. Regenerating gene family members are expressed in the proximal gastrointestinal (GI) tract and ectopically at other sites in the setting of tissue injury. By high-throughput sequence analysis of a large inflammatory bowel disease library, two cDNAs have been isolated which encode a novel member of this multigene family. Based on primary sequence homology, tissue expression profiles, and shared exon-intron junction genomic organization, we assign this gene to the regenerating gene family. Specific protein structural differences suggest that the current three regenerating gene subtypes should be expanded to four. We demonstrate that Reg IV has a highly restricted tissue expression pattern, with prominent expression in the gastrointestinal tract. Reg IV mRNA expression is significantly up-regulated by mucosal injury from active Crohn's disease or ulcerative colitis.

The neuronal ceroid lipofuscinoses in human EPMR and mnd mutant mice are associated with mutations in CLN8.

The neuronal ceroid lipofuscinoses (NCLs) are a genetically heterogeneous group of progressive neurodegenerative disorders characterized by the accumulation of autofluorescent lipopigment in various tissues. Progressive epilepsy with mental retardation (EPMR, MIM 600143) was recently recognized as a new NCL subtype (CLN8). It is an autosomal recessive disorder characterized by onset of generalized seizures between 5 and 10 years, and subsequent progressive mental retardation. Here we report the positional cloning of a novel gene, CLN8, which is mutated in EPMR. It encodes a putative transmembrane protein. EPMR patients were homozygous for a missense mutation (70C-->G, R24G) that was not found in homozygosity in 433 controls. We also cloned the mouse Cln8 sequence. It displays 82% nucleotide identity with CLN8, conservation of the codon harbouring the human mutation and is localized to the same region as the motor neuron degeneration mouse, mnd, a naturally occurring mouse NCL (ref. 4). In mnd/mnd mice, we identified a homozygous 1-bp insertion (267-268insC, codon 90) predicting a frameshift and a truncated protein. Our data demonstrate that mutations in these orthologous genes underlie NCL phenotypes in human and mouse, and represent the first description of the molecular basis of a naturally occurring animal model for NCL.

Construction of a normalized cDNA library for the Trypanosoma cruzi genome project.

Sequencing of the Trypanosoma cruzi genome is underway. Expressed sequence tags, obtained from cDNA libraries, facilitate mapping and gene discovery. The efficiency of large-scale generation of such tags is increased when using normalized cDNA libraries, where the frequency of individual clones is brought within a narrow range. Repetitive sequencing of abundant clones is therefore minimized. We constructed a normalized cDNA library from epimastigotes of clone CL Brener, and the efficiency of normalization of representative clones was assessed and shown to be adequate. The normalized cDNA library has been distributed to several groups and large-scale sequencing is currently in progress.

High-resolution YAC-cosmid-STS map of human chromosome 13.

We have assembled a high-resolution physical map of human chromosome 13 DNA (approximately 114 Mb) from hybridization, PCR, and FISH mapping data using a specifically designed set of computer programs. Although the mapping of 13p is limited, 13q (approximately 98 Mb) is covered by an almost continuous contig of 736 YACs aligned to 597 contigs of cosmids. Of a total of 10,789 cosmids initially selected from a chromosome 13-specific cosmid library (16,896 colonies) using inter-Alu PCR probes from the YACs and probes for markers mapped to chromosome 13, 511 were assembled in contigs that were established from cross-hybridization relationships between the cosmids. The 13q YAC-cosmid map was annotated with 655 sequence tagged sites (STSs) with an average spacing of 1 STS per 150 kb. This set of STSs, each identified by a D number and cytogenetic location, includes database markers (198), expressed sequence tags (93), and STSs generated by sequencing of the ends of cosmid inserts (364). Additional annotation has been provided by positioning 197 cosmids mapped by FISH on 13q. The final (comprehensive) map, a list of STS primers, and raw data used in map assembly are available at our Web site (genome1.ccc.columbia.edu/ approximately genome/) and can serve as a resource to facilitate accurate localization of additional markers, provide substrates for sequencing, and assist in the discovery of chromosome 13 genes associated with hereditary diseases.

Cloning and gene mapping of the chromosome 13q14 region deleted in chronic lymphocytic leukemia.

Frequent deletions and loss of heterozygosity in a segment of chromosome 13 (13q14) in cases of B-cell chronic lymphocytic leukemia (CLL) have suggested that this malignancy is caused by inactivation of an unknown tumor suppressor gene located in this region. Toward the identification of the putative CLL tumor suppressor, we have constructed a high-resolution physical map of YAC, PAC, and cosmid contigs covering 600 kb of the 13q14 genomic region. In addition to densely positioned genetic markers and STSs, this map was further annotated by localization of 32 transcribed sequences (ESTs) using a combination of exon trapping, direct cDNA selection, sample sequencing of cosmids and PACs, and homology searches. On the basis of these mapping data, allelic loss analyses at 13q14 using CLL tumor samples allowed narrowing of the genomic segment encompassing the putative CLL gene to <300 kb. Twenty-three ESTs located within this minimally deleted region are candidate exons for the CLL-associated tumor suppressor gene.

Identification of a novel member of the TGF-beta superfamily highly expressed in human placenta.

While conducting a gene discovery effort targeted to transcripts of the prevalent and intermediate frequency classes in placenta throughout gestation, we identified a novel member of the TGF-beta superfamily that is expressed at high levels in human placenta. Hence, we named this factor 'Placental Transforming Growth Factor Beta' (PTGFB). The full-length sequence of the 1.2-kb PTGFB mRNA has the potential of encoding a putative pre-pro-PTGFB protein of 295 amino acids and a putative mature PTGFB protein of 112 amino acids. Multiple sequence alignments of PTGFB and representative members of all TGF-beta subfamilies evidenced a number of conserved residues, including the seven cysteines that are almost invariant in all members of the TGF-beta superfamily. The single-copy PTGFB gene was shown to be composed of only two exons of 309 bp and 891 bp, separated by a 2.9-kb intron. The gene was localized to chromosome 19p12-13.1 by fluorescence in-situ hybridization. Northern analyses revealed a complex tissue-specific pattern of expression and a second transcript of 1.9 kb that is predominant in adult skeletal muscle. Most importantly, the 1.2-kb PTGFB transcript was shown to be expressed in placenta at much higher levels than in any other human fetal or adult tissue surveyed.

Normalization and subtraction: two approaches to facilitate gene discovery.

Large-scale sequencing of cDNAs randomly picked from libraries has proven to be a very powerful approach to discover (putatively) expressed sequences that, in turn, once mapped, may greatly expedite the process involved in the identification and cloning of human disease genes. However, the integrity of the data and the pace at which novel sequences can be identified depends to a great extent on the cDNA libraries that are used. Because altogether, in a typical cell, the mRNAs of the prevalent and intermediate frequency classes comprise as much as 50-65% of the total mRNA mass, but represent no more than 1000-2000 different mRNAs, redundant identification of mRNAs of these two frequency classes is destined to become overwhelming relatively early in any such random gene discovery programs, thus seriously compromising their cost-effectiveness. With the goal of facilitating such efforts, previously we developed a method to construct directionally cloned normalized cDNA libraries and applied it to generate infant brain (INIB) and fetal liver/spleen (INFLS) libraries, from which a total of 45,192 and 86,088 expressed sequence tags, respectively, have been derived. While improving the representation of the longest cDNAs in our libraries, we developed three additional methods to normalize cDNA libraries and generated over 35 libraries, most of which have been contributed to our integrated Molecular Analysis of Genomes and Their Expression (IMAGE) Consortium and thus distributed widely and used for sequencing and mapping. In an attempt to facilitate the process of gene discovery further, we have also developed a subtractive hybridization approach designed specifically to eliminate (or reduce significantly the representation of) large pools of arrayed and (mostly) sequenced clones from normalized libraries yet to be (or just partly) surveyed. Here we present a detailed description and a comparative analysis of four methods that we developed and used to generate normalize cDNA libraries from human (15), mouse (3), rat (2), as well as the parasite Schistosoma mansoni (1). In addition, we describe the construction and preliminary characterization of a subtracted liver/spleen library (INFLS-SI) that resulted from the elimination (or reduction of representation) of -5000 INFLS-IMAGE clones from the INFLS library.

Generation and analysis of 280,000 human expressed sequence tags.

We report the generation of 319,311 single-pass sequencing reactions (known as expressed sequence tags, or ESTs) obtained from the 5' and 3' ends of 194,031 human cDNA clones. Our goal has been to obtain tag sequences from many different genes and to deposit these in the publicly accessible Data Base for Expressed Sequence Tags. Highly efficient automatic screening of the data allows deposition of the annotated sequences without delay. Sequences have been generated from 26 oligo(dT) primed directionally cloned libraries, of which 18 were normalized. The libraries were constructed using mRNA isolated from 17 different tissues representing three developmental states. Comparisons of a subset of our data with nonredundant human mRNA and protein data bases show that the ESTs represent many known sequences and contain many that are novel. Analysis of protein families using Hidden Markov Models confirms this observation and supports the contention that although normalization reduces significantly the relative abundance of redundant cDNA clones, it does not result in the complete removal of members of gene families.

Construction and characterization of a normalized cDNA library.

We have developed a simple procedure based on reassociation kinetics that can reduce effectively the high variation in abundance among the clones of a cDNA library that represent individual mRNA species. For this normalization, we used as a model system a library of human infant brain cDNAs that were cloned directionally into a phagemid vector and, thus, could be easily converted into single-stranded circles. After controlled primer extension to synthesize a short complementary strand on each circular template, melting and reannealing of the partial duplexes at relatively low C0t, and hydroxyapatite column chromatography, unreassociated circles were recovered from the flow through fraction and electroporated into bacteria, to propagate a normalized library without a requirement for subcloning steps. An evaluation of the extent of normalization has indicated that, from an extreme range of abundance of 4 orders of magnitude in the original library, the frequency of occurrence of any clone examined in the normalized library was brought within the narrow range of only 1 order of magnitude.

Selection of cDNAs using chromosome-specific genomic clones: application to human chromosome 13.

We have developed a general method for en masse isolation of cDNAs present in a normalized library by hybridization to arrayed chromosome-specific phage lambda clones; we have used this approach to initiate exon-mapping of human chromosome 13. An advantage of the simultaneous isolation of cDNA/lambda pairs is that it allows cytogenetic assignment of a bona fide genomic clone by in situ hybridization, which also verifies that the corresponding cDNA or a homologous expressed sequence resides on chromosome 13. This information is enriched by partial sequencing of a selected cDNA from both ends. The sequence of the 3' noncoding region provides an 'identifier' that is used to develop STSs, while the sequence from the 5' end, often corresponding to a coding region, is used for homology searches in databases that occasionally reveal gene functions.

A microsatellite genetic linkage map of human chromosome 13.

We have characterized 21 polymorphic (CA)n microsatellites for the development of a genetic map of chromosome 13. Fifteen markers were isolated from a flow-sorted chromosome 13 library, four CA repeats were derived from NotI-containing cosmid clones, and two polymorphic markers were described previously (J. L. Weber, A. E. Kwitek, and P. E. May, 1990, Nucleic Acids Res. 18: 4638; L. Warnich, I. Groenwald, L. Laubscher, and A. E. Retief, 1991, Am. J. Hum. Genet. 49(Suppl.): 372 (Abstract)). Regional localization for all of the markers was performed by amplification of DNA from five somatic cell hybrids containing different deletions of chromosome 13. Genetic markers were shown to be distributed throughout 6 of the 11 resolvable chromosomal subregions. Using data from nine families provided by the Centre d'Etude du Polymorphisme Humain (CEPH), a framework map of 12 of these 21 markers was developed. Six of the 12 markers form three pairs, with each two members of a pair being tightly linked, such that nine systems of markers can be distinguished. The average heterozygosity of these 12 markers is 0.75. The total length of the sex-averaged map is 65.4 cM (Kosambi), with an average distance of 8.2 cM between systems of markers (eight intervals). Seven remaining markers were placed provisionally into the framework map.

The transcript from a DNA puff of Rhynchosciara and its migration to the cytoplasm.

Electrophoretic analysis of 3H-RNA obtained from the proximal sections of Rhynchosciara salivary glands at two distinct developmental periods, one characterized by the presence and the other by the absence of the giant B-2 DNA puff, revealed that the appearance of a 14S poly(A)+ RNA is correlated with the opening of this puff. That this RNA species is transcribed from this puff is indicated by the fact that it is found in RNA extracted from B-2 puffs obtained by microdissection. This confirmed by the specific hybridization of the 14S poly(A)+ RNA to the B-2 locus. Our data indicate that the polyadenylation process takes place at the chromosome level, and that the nuclear sap is not an important compartment in the transport of polyadenylated RNA from the chromosome to the cytoplasm. The kinetics of migration of the 14S species to the cytoplasm were studied; the data indicate that this process is very rapid and, in addition, that the 14S RNA is unstable.

Distribution of repetitive DNA sequences in the polytene chromosomes of Rhynchosciara americana.

The distribution of fast, intermediate and slow renaturing fractions of Rhynchosciara americana DNA was examined in the polytene salivary gland chromosomes by in situ hybridization. Heterochromatic areas readily hybridized but hybrid formation in the euchromatin depended more on the repetitiveness of the RNA probe.