San Ming Wang

The birth and death of microRNA genes in Drosophila

MicroRNAs (miRNAs) are small, endogenously expressed RNAs that regulate mRNAs post-transcriptionally. The class of miRNA genes, like other gene classes, should experience birth, death and persistence of its members. We carried out deep sequencing of miRNAs from three species of Drosophila, and obtained 107,000 sequences that map to no fewer than 300 loci that were not previously known. We observe a large class of miRNA genes that are evolutionarily young, with a rate of birth of 12 new genes per million years (Myr). Most of these new miRNAs originated from non-miRNA sequences. Among the new genes, we estimate that 96% disappeared quickly in the course of evolution; only 4% of new miRNA genes were retained by natural selection. Furthermore, only 60% of these retained genes became integrated into the transcriptome in the long run (60 Myr). This small fraction (2.5%) of surviving miRNAs may later on become moderately or highly expressed. Our results suggest that there is a high birth rate of new miRNA genes, accompanied by a comparably high death rate. The estimated net gain of long-lived miRNA genes, which is not strongly affected by either the depth or the breadth (number of tissues) of sequencing, is 0.3 genes per Myr in Drosophila.

Identifying novel transcripts and novel genes in the human genome by using novel SAGE tags

The number of genes in the human genome is still a controversial issue. Whereas most of the genes in the human genome are said to have been physically or computationally identified, many short cDNA sequences identified as tags by use of serial analysis of gene expression (SAGE) do not match these genes. By performing experimental verification of more than 1,000 SAGE tags and analyzing 4,285,923 SAGE tags of human origin in the current SAGE database, we examined the nature of the unmatched SAGE tags. Our study shows that most of the unmatched SAGE tags are truly novel SAGE tags that originated from novel transcripts not yet identified in the human genome, including alternatively spliced transcripts from known genes and potential novel genes. Our study indicates that by using novel SAGE tags as probes, we should be able to identify efficiently many novel transcripts/novel genes in the human genome that are difficult to identify by conventional methods.

SAGE is far more sensitive than EST for detecting low-abundance transcripts

BackgroundIsolation of low-abundance transcripts expressed in a genome remains a serious challenge in transcriptome studies. The sensitivity of the methods used for analysis has a direct impact on the efficiency of the detection. We compared the EST method and the SAGE method to determine which one is more sensitive and to what extent the sensitivity is great for the detection of low-abundance transcripts.ResultsUsing the same low-abundance transcripts detected by both methods as the targeted sequences, we observed that the SAGE method is 26 times more sensitive than the EST method for the detection of low-abundance transcripts.ConclusionsThe SAGE method is more efficient than the EST method in detecting the low-abundance transcripts.

Oligo(dT) primer generates a high frequency of truncated cDNAs through internal poly(A) priming during reverse transcription

We have analyzed a systematic flaw in the current system of gene identification: the oligo(dT) primer widely used for cDNA synthesis generates a high frequency of truncated cDNAs through internal poly(A) priming. Such truncated cDNAs may contribute to 12% of the expressed sequence tags in the current dbEST database. By using a synthetic transcript and real mRNA templates as models, we characterized the patterns of internal poly(A) priming by oligo(dT) primer. We further demonstrated that the internal poly(A) priming can be effectively diminished by replacing the oligo(dT) primer with a set of anchored oligo(dT) primers for reverse transcription. Our study indicates that cDNAs designed for genomewide gene identification should be synthesized by use of the anchored oligo(dT) primers, rather than the oligo(dT) primers, to diminish the generation of truncated cDNAs caused by internal poly(A) priming.

Gene Transfer Efficiency and Genome-Wide Integration Profiling of Sleeping Beauty, Tol2, and PiggyBac Transposons in Human Primary T Cells

In this study, we compared the genomic integration efficiencies and transposition site preferences of Sleeping Beauty (SB or SB11), Tol2, and piggyBac (PB) transposon systems in primary T cells derived from peripheral blood lymphocytes (PBL) and umbilical cord blood (UCB). We found that PB demonstrated the highest efficiency of stable gene transfer in PBL-derived T cells, whereas SB11 and Tol2 mediated intermediate and lowest efficiencies, respectively. Southern hybridization analysis demonstrated that PB generated the highest number of integrants when compared to SB and Tol2 in both PBL and UCB T cells. Tol2 and PB appeared more likely to promote clonal expansion than SB, which may be in part due to the dysregulated expression of cancer-related genes near the insertion sites. Genome-wide integration analysis demonstrated that SB, Tol2, and PB integrations occurred in all the chromosomes without preference. Additionally, Tol2 and PB integration sites were mainly localized near transcriptional start sites (TSSs), CpG islands and DNaseI hypersensitive sites, whereas SB integrations were randomly distributed. These results suggest that SB may be a preferential choice of the delivery vector in T cells due to its random integration site preference and relatively high efficiency, and support continuing development of SB-mediated T-cell phase I trials.

The pattern of gene expression in human CD34+ stem/progenitor cells

We have analyzed the pattern of gene expression in human primary CD34+ stem/progenitor cells. We identified 42,399 unique serial analysis of gene expression (SAGE) tags among 106,021 SAGE tags collected from 2.5 × 106 CD34+ cells purified from bone marrow. Of these unique SAGE tags, 21,546 matched known expressed sequences, including 3,687 known genes, and 20,854 were novel without a match. The SAGE tags that matched known sequences tended to be at higher levels, whereas the novel SAGE tags tended to be at lower levels. By using the generation of longer sequences from SAGE tags for gene identification (GLGI) method, we identified the correct gene for 385 of 440 high-copy SAGE tags that matched multiple genes and we generated 198 novel 3′ expressed sequence tags from 138 high-copy novel SAGE tags. We observed that many different SAGE tags were derived from the same genes, reflecting the high heterogeneity of the 3′ untranslated region in the expressed genes. We compared the quantitative relationship for genes known to be important in hematopoiesis. The qualitative identification and quantitative measure for each known gene, expressed sequence tag, and novel SAGE tag provide a base for studying normal gene expression in hematopoietic stem/progenitor cells and for studying abnormal gene expression in hematopoietic diseases.

Serial Analysis of Gene Expression Study of a Hybrid Rice Strain (LYP9) and Its Parental Cultivars

Using the serial analysis of gene expression technique, we surveyed transcriptomes of three major tissues (panicles, leaves, and roots) of a super-hybrid rice (Oryza sativa) strain, LYP9, in comparison to its parental cultivars, 93-11 (indica) and PA64s (japonica). We acquired 465,679 tags from the serial analysis of gene expression libraries, which were consolidated into 68,483 unique tags. Focusing our initial functional analyses on a subset of the data that are supported by full-length cDNAs and the tags (genes) differentially expressed in the hybrid at a significant level (P < 0.01), we identified 595 up-regulated (22 tags in panicles, 228 in leaves, and 345 in roots) and 25 down-regulated (seven tags in panicles, 15 in leaves, and three in roots) in LYP9. Most of the tag-identified and up-regulated genes were found related to enhancing carbon- and nitrogen-assimilation, including photosynthesis in leaves, nitrogen uptake in roots, and rapid growth in both roots and panicles. Among the down-regulated genes in LYP9, there is an essential enzyme in photorespiration, alanine:glyoxylate aminotransferase 1. Our study adds a new set of data crucial for the understanding of molecular mechanisms of heterosis and gene regulation networks of the cultivated rice.

2.45 GHz radiofrequency fields alter gene expression in cultured human cells

The biological effect of radiofrequency (RF) fields remains controversial. We address this issue by examining whether RF fields can cause changes in gene expression. We used the pulsed RF fields at a frequency of 2.45 GHz that is commonly used in telecommunication to expose cultured human HL‐60 cells. We used the serial analysis of gene expression (SAGE) method to measure the RF effect on gene expression at the genome level. We observed that 221 genes altered their expression after a 2‐h exposure. The number of affected genes increased to 759 after a 6‐h exposure. Functional classification of the affected genes reveals that apoptosis‐related genes were among the upregulated ones and the cell cycle genes among the downregulated ones. We observed no significant increase in the expression of heat shock genes. These results indicate that the RF fields at 2.45 GHz can alter gene expression in cultured human cells through non‐thermal mechanism.

Consistent deregulation of gene expression between human and murine MLL rearrangement leukemias.

Important biological and pathologic properties are often conserved across species. Although several mouse leukemia models have been well established, the genes deregulated in both human and murine leukemia cells have not been studied systematically. We performed a serial analysis of gene expression in both human and murine MLL-ELL or MLL-ENL leukemia cells and identified 88 genes that seemed to be significantly deregulated in both types of leukemia cells, including 57 genes not reported previously as being deregulated in MLL-associated leukemias. These changes were validated by quantitative PCR. The most up-regulated genes include several HOX genes (e.g., HOX A5, HOXA9, and HOXA10) and MEIS1, which are the typical hallmark of MLL rearrangement leukemia. The most down-regulated genes include LTF, LCN2, MMP9, S100A8, S100A9, PADI4, TGFBI, and CYBB. Notably, the up-regulated genes are enriched in gene ontology terms, such as gene expression and transcription, whereas the down-regulated genes are enriched in signal transduction and apoptosis. We showed that the CpG islands of the down-regulated genes are hypermethylated. We also showed that seven individual microRNAs (miRNA) from the mir-17-92 cluster, which are overexpressed in human MLL rearrangement leukemias, are also consistently overexpressed in mouse MLL rearrangement leukemia cells. Nineteen possible targets of these miRNAs were identified, and two of them (i.e., APP and RASSF2) were confirmed further by luciferase reporter and mutagenesis assays. The identification and validation of consistent changes of gene expression in human and murine MLL rearrangement leukemias provide important insights into the genetic base for MLL-associated leukemogenesis.

Poly A- Transcripts Expressed in HeLa Cells

Background Transcripts expressed in eukaryotes are classified as poly A+ transcripts or poly A- transcripts based on the presence or absence of the 3′ poly A tail. Most transcripts identified so far are poly A+ transcripts, whereas the poly A- transcripts remain largely unknown. Methodology/Principal Findings We developed the TRD (Total RNA Detection) system for transcript identification. The system detects the transcripts through the following steps: 1) depleting the abundant ribosomal and small-size transcripts; 2) synthesizing cDNA without regard to the status of the 3′ poly A tail; 3) applying the 454 sequencing technology for massive 3′ EST collection from the cDNA; and 4) determining the genome origins of the detected transcripts by mapping the sequences to the human genome reference sequences. Using this system, we characterized the cytoplasmic transcripts from HeLa cells. Of the 13,467 distinct 3′ ESTs analyzed, 24% are poly A-, 36% are poly A+, and 40% are bimorphic with poly A+ features but without the 3′ poly A tail. Most of the poly A- 3′ ESTs do not match known transcript sequences; they have a similar distribution pattern in the genome as the poly A+ and bimorphic 3′ ESTs, and their mapped intergenic regions are evolutionarily conserved. Experiments confirmed the authenticity of the detected poly A- transcripts. Conclusion/Significance Our study provides the first large-scale sequence evidence for the presence of poly A- transcripts in eukaryotes. The abundance of the poly A- transcripts highlights the need for comprehensive identification of these transcripts for decoding the transcriptome, annotating the genome and studying biological relevance of the poly A- transcripts.