转化的大鼠胚胎成纤维细胞系差异表达基因的筛选研究

来源于转化的大鼠胚胎成纤维细胞系的两株细胞,A1－5细胞与B4细胞相比表现出非常强的抗辐射性并伴随不同寻常强的G2延迟效应;用PCR选择性抑制消减杂交方法对这两株细胞进行差减,希望找到对A1－5细胞表现出的不同寻常的表型起关键作用的某一个或某一些基因。结果得到了160个差减转化子,逐个进行序列测定,并进行Dot blot杂交,共得到35个差异表达基因片段（EST）。通过对美国国家生物技术信息中心（NCBI）的非冗余序列库（NT）、鼠EST库及人EST库的BLAST进行同源检索,发现其中21个代表了尚未登录的新基因,另外14个分别与已知基因高度同源。     

Human trash ESTs--sequences from cDNA collection that are not aligned to genome assembly

Expressed sequence tags (ESTs) represent 500-1000-bp-long sequences corresponding to mRNAs derived from different sources (cell lines, tissues, etc.). The human EST database contains over 8,000,000 sequences, with over 4,000,000,000 total nucleotides. RNA molecules are transcribed from a genomic DNA template; therefore, all ESTs should match corresponding genomes. Nevertheless, we have found in the human EST database approximately 11,000 ESTs not matching sequences in the human genome database. The presence of "trash" ESTs (TESTs) in the EST database could result from DNA or RNA contamination of the laboratory equipment, tissues, or cell lines. TESTs could also represent sequences from unidentified human genes or from species inhabiting the human body. Here, we attempt to identify the sources of human EST database contaminations. In particular, we discuss systematic contamination of the mammalian EST databases with sequences of plants.     

Census of orthologous genes and self-organizing maps of biologically relevant transcriptional patterns in chickens (Gallus gallus)

The launch of large-scale chicken expressed sequence tags (EST) projects has placed the chicken in the lead for the number of EST sequences in agriculturally important animals. More than 451,000 chicken ESTs derived from over 158 libraries have been deposited in the NCBI dbEST database as of December 2003. But how many genes these ESTs represent and how they are expressed in different chicken tissues/organs remain undetermined. In the present research, we developed a human gene-based strategy for census of chicken orthologous genes and identification of their expression patterns. Among 34,157 human coding genes used in the study, BLAST analysis revealed that 11,066 genes provisionally matched 248,628 chicken ESTs. Based on the average EST abundance of the orthologous genes, the current public repository of chicken ESTs could represent 20,000 provisional genes. Analysis of gene expression in 14 single tissues/organs showed that approximately 15% of genes were expressed exclusively in single tissue/organ whereas the remaining 85% of genes were co-expressed in two or more tissues/organs. A majority (91.15%) of genes expressed in chicken embryos were also expressed at post-hatch stages, indicating that most genes activated in chicken embryos could serve housekeeping functions. Self-organizing maps (SOM) analysis organized 8807 provisional genes in selected chicken tissues into 98 clusters with each cluster being indicative of common regulatory factors and pathways. A total of 969 provisional orthologous genes were identified as preferentially expressed genes (PEGs) in various chicken tissues/organs (LOD>3.0). No doubt, the present study on gene expression patterns will provide insight into dynamics of metabolic pathways and tissue/organ programming and reprogramming in chickens.     

一个鼻咽癌相关EST的鉴定及其全长cDNA序列分析

鼻咽癌是我国南方及东南亚地区常见的恶性肿瘤之一.通过对鼻咽癌染色体高频率杂合性丢失区域3p21的表达序列标签(expressedsequencetag,EST)进行同源性比较分析,运用逆转录聚合酶链式反应的方法,筛选到一个在41.18%(14/34)的鼻咽癌活检组织及20.0%(1/5)的鼻咽癌细胞系中表达下调的ESTBG772301;并用Northern杂交方法,检测了该EST在多种正常成人组织中的表达状况及其所代表基因的转录本大小.在此基础上,对该EST来源的cDNA克隆(IMAGE:4839190)进行直接测序,获得了一个全长为2377bp的新cDNA序列;经生物信息学分析,发现它与已知基因序列无明显同源性,属于一个新基因,定位于染色体3p21.3,被命名为鼻咽癌表达下调基因(NPCEDRG,GenBank登录号:AF538150).其编码的蛋白质含169个氨基酸,与一个已报道的在进化上相对保守、功能未知的人类蛋白Nicolin1(简称NICN1)N端170个氨基酸残基的序列同源性为97%,但缺少NICN1蛋白C端43个氨基酸残基,可能是nicolin1基因不同剪接本的编码产物.     

Gene expression profiling of hereditary exencephaly in chickens

In this preliminary study, differentially expressed genes were investigated in cranial tissues from chickens with hereditary exencephaly using cDNA microarrays containing 1,152 genes and expressed sequence tags (ESTs). Genes showing twofold or greater differences at P < 0.05 between affected and normal cranial cells were considered to be candidates for hereditary exencephaly in chicken. Eighteen ESTs (11 known genes/homologues) were upregulated and 108 ESTs (51 known genes/homologues) were downregulated. The EST AL584231 (ROS006C9), orthologous to human MTHFD1, a known candidate gene for human neural tube defects (NTDs), was expressed at the same level both in normal and affected chicken cranial tissues. ESTs AL584253 (ROS006F7, thioredoxin reductase 1) and AL585511 (ROS024H9, thioredoxin), both involved in NTD pathogenic pathways in mice, were downregulated and had mean ratios of 0.41 and 0.04 for expression in affected vs. normal cells respectively. Expression differences of these two ESTs were confirmed by quantitative real-time polymerase chain reaction. These data indicate that ESTs AL584253 and AL585511 are candidates for hereditary exencephaly in chickens.     

Gene expression profile of human bone marrow stromal cells: high-throughput expressed sequence tag sequencing analysis

Human bone marrow stromal cells (HBMSC) are pluripotent cells with the potential to differentiate into osteoblasts, chondrocytes, myelosupportive stroma, and marrow adipocytes. We used high-throughput DNA sequencing analysis to generate 4258 single-pass sequencing reactions (known as expressed sequence tags, or ESTs) obtained from the 5' (97) and 3' (4161) ends of human cDNA clones from a HBMSC cDNA library. Our goal was to obtain tag sequences from the maximum number of possible genes and to deposit them in the publicly accessible database for ESTs (dbEST of the National Center for Biotechnology Information). Comparisons of our EST sequencing data with nonredundant human mRNA and protein databases showed that the ESTs represent 1860 gene clusters. The EST sequencing data analysis showed 60 novel genes found only in this cDNA library after BLAST analysis against 3.0 million ESTs in NCBI's dbEST database. The BLAST search also showed the identified ESTs that have close homology to known genes, which suggests that these may be newly recognized members of known gene families. The gene expression profile of this cell type is revealed by analyzing both the frequency with which a message is encountered and the functional categorization of expressed sequences. Comparing an EST sequence with the human genomic sequence database enables assignment of an EST to a specific chromosomal region (a process called digital gene localization) and often enables immediate partial determination of intron/exon boundaries within the genomic structure. It is expected that high-throughput EST sequencing and data mining analysis will greatly promote our understanding of gene expression in these cells and of growth and development of the skeleton.