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Identification and initial characterization of 6,000 expressed sequenced tags (ESTs) from rat normal-growing cartilage and swarm rat chondrosarcoma cDNA libraries.

ABSTRACT: Chondrosarcoma is the second most common type of skeletal malignancy with a survival rate at five years for histological grade III of only 29 percent. The development of a reliable chondrosarcoma animal model could enable the study of tumor growth and progression, the effect of the host on tumor behavior, and the effectiveness of various therapeutic modalities. The Swarm rat chondrosarcoma is a tumor tissue line that has been maintained through the years by serial subcutaneous injections, and the histochemical characteristics of the tumor have remained essentially similar in all transplants over the years. This study was designed to initiate the characterization of the Swarm rat chondrosarcoma model by gene expression profiling as compared to normal-growing rat cartilage. Analysis of the gene expression from both libraries revealed a complex pattern of gene expression, including many genes not yet reported to be expressed by chondrocytes. It suggests that the biochemical characterization of growing cartilage and chondrosarcoma reported to date has only begun to describe the complexity of these tissues.

SUBMITTER: Morcuende JA

PROVIDER: S-EPMC1888380 | biostudies-literature | 2002

REPOSITORIES: biostudies-literature

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Identification and initial characterization of 6,000 expressed sequenced tags (ESTs) from rat normal-growing cartilage and swarm rat chondrosarcoma cDNA libraries.

Morcuende Jose A JA Huang Xiao Dong XD Stevens Jeff J Kucaba Tammy A TA Brown Bart B Abdulkawy Hakeem H Scheetz Todd E TE Malchenko Sergey S Bonaldo Fatima F Casavant Thomas L TL Soares Bento B

The Iowa orthopaedic journal 20020101

Chondrosarcoma is the second most common type of skeletal malignancy with a survival rate at five years for histological grade III of only 29 percent. The development of a reliable chondrosarcoma animal model could enable the study of tumor growth and progression, the effect of the host on tumor behavior, and the effectiveness of various therapeutic modalities. The Swarm rat chondrosarcoma is a tumor tissue line that has been maintained through the years by serial subcutaneous injections, and th ...[more]

PMID: 12180607

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Project description:BackgroundChondrosarcomas are malignant cartilage tumors that do not respond to traditional chemotherapy or radiation. The 5-year survival rate of histologic grade III chondrosarcoma is less than 30%. An animal model of chondrosarcoma has been established--namely, the Swarm Rat Chondrosarcoma (SRC)--and shown to resemble the human disease. Previous studies with this model revealed that tumor microenvironment could significantly influence chondrosarcoma malignancy.MethodsTo examine the effect of the microenvironment, SRC tumors were initiated at different transplantation sites. Pyrosequencing assays were utilized to assess the DNA methylation of the tumors, and SAGE libraries were constructed and sequenced to determine the gene expression profiles of the tumors. Based on the gene expression analysis, subsequent functional assays were designed to determine the relevancy of the specific genes in the development and progression of the SRC.ResultsThe site of transplantation had a significant impact on the epigenetic and gene expression profiles of SRC tumors. Our analyses revealed that SRC tumors were hypomethylated compared to control tissue, and that tumors at each transplantation site had a unique expression profile. Subsequent functional analysis of differentially expressed genes, albeit preliminary, provided some insight into the role that thymosin-?4, c-fos, and CTGF may play in chondrosarcoma development and progression.ConclusionThis report describes the first global molecular characterization of the SRC model, and it demonstrates that the tumor microenvironment can induce epigenetic alterations and changes in gene expression in the SRC tumors. We documented changes in gene expression that accompany changes in tumor phenotype, and these gene expression changes provide insight into the pathways that may play a role in the development and progression of chondrosarcoma. Furthermore, specific functional analysis indicates that thymosin-?4 may have a role in chondrosarcoma metastasis.

Project description:BACKGROUND:Melon (Cucumis melo), an economically important vegetable crop, belongs to the Cucurbitaceae family which includes several other important crops such as watermelon, cucumber, and pumpkin. It has served as a model system for sex determination and vascular biology studies. However, genomic resources currently available for melon are limited. RESULT:We constructed eleven full-length enriched and four standard cDNA libraries from fruits, flowers, leaves, roots, cotyledons, and calluses of four different melon genotypes, and generated 71,577 and 22,179 ESTs from full-length enriched and standard cDNA libraries, respectively. These ESTs, together with ~35,000 ESTs available in public domains, were assembled into 24,444 unigenes, which were extensively annotated by comparing their sequences to different protein and functional domain databases, assigning them Gene Ontology (GO) terms, and mapping them onto metabolic pathways. Comparative analysis of melon unigenes and other plant genomes revealed that 75% to 85% of melon unigenes had homologs in other dicot plants, while approximately 70% had homologs in monocot plants. The analysis also identified 6,972 gene families that were conserved across dicot and monocot plants, and 181, 1,192, and 220 gene families specific to fleshy fruit-bearing plants, the Cucurbitaceae family, and melon, respectively. Digital expression analysis identified a total of 175 tissue-specific genes, which provides a valuable gene sequence resource for future genomics and functional studies. Furthermore, we identified 4,068 simple sequence repeats (SSRs) and 3,073 single nucleotide polymorphisms (SNPs) in the melon EST collection. Finally, we obtained a total of 1,382 melon full-length transcripts through the analysis of full-length enriched cDNA clones that were sequenced from both ends. Analysis of these full-length transcripts indicated that sizes of melon 5' and 3' UTRs were similar to those of tomato, but longer than many other dicot plants. Codon usages of melon full-length transcripts were largely similar to those of Arabidopsis coding sequences. CONCLUSION:The collection of melon ESTs generated from full-length enriched and standard cDNA libraries is expected to play significant roles in annotating the melon genome. The ESTs and associated analysis results will be useful resources for gene discovery, functional analysis, marker-assisted breeding of melon and closely related species, comparative genomic studies and for gaining insights into gene expression patterns.

Project description:BackgroundSubtractive hybridization methods are valuable tools for identifying differentially regulated genes in a given tissue avoiding redundant sequencing of clones representing the same expressed genes, maximizing detection of low abundant transcripts and thus, affecting the efficiency and cost effectiveness of small scale cDNA sequencing projects aimed to the specific identification of useful genes for breeding purposes. The objective of this work is to evaluate alternative strategies to high-throughput sequencing projects for the identification of novel genes differentially expressed in sunflower as a source of organ-specific genetic markers that can be functionally associated to important traits.ResultsDifferential organ-specific ESTs were generated from leaf, stem, root and flower bud at two developmental stages (R1 and R4). The use of different sources of RNA as tester and driver cDNA for the construction of differential libraries was evaluated as a tool for detection of rare or low abundant transcripts. Organ-specificity ranged from 75 to 100% of non-redundant sequences in the different cDNA libraries. Sequence redundancy varied according to the target and driver cDNA used in each case. The R4 flower cDNA library was the less redundant library with 62% of unique sequences. Out of a total of 919 sequences that were edited and annotated, 318 were non-redundant sequences. Comparison against sequences in public databases showed that 60% of non-redundant sequences showed significant similarity to known sequences. The number of predicted novel genes varied among the different cDNA libraries, ranging from 56% in the R4 flower to 16 % in the R1 flower bud library. Comparison with sunflower ESTs on public databases showed that 197 of non-redundant sequences (60%) did not exhibit significant similarity to previously reported sunflower ESTs. This approach helped to successfully isolate a significant number of new reported sequences putatively related to responses to important agronomic traits and key regulatory and physiological genes.ConclusionsThe application of suppressed subtracted hybridization technology not only enabled the cost effective isolation of differentially expressed sequences but it also allowed the identification of novel sequences in sunflower from a relative small number of analyzed sequences when compared to major sequencing projects.

Dataset Information

Identification and initial characterization of 6,000 expressed sequenced tags (ESTs) from rat normal-growing cartilage and swarm rat chondrosarcoma cDNA libraries.

Publications

Identification and initial characterization of 6,000 expressed sequenced tags (ESTs) from rat normal-growing cartilage and swarm rat chondrosarcoma cDNA libraries.

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