Two tobacco DNA-binding proteins with homology to the nuclear factor CREB

The 35S promoter of the cauliflower mosaic virus (CaMV) contains a tandem repeat of the sequence TGACG in the region -83 to -63. This 21-base pair (bp) sequence, called as-1, is involved in root expression of the 35S promoter. When inserted in a promoter of a gene expressed specifically in photosynthetic tissues, as-1 confers high level expression in roots. We have described a factor, ASF-1, that binds specifically to as-1 in vitro. There is a good correlation between ASF-1 binding affinity to as-1 related sequences in vitro and the function of these sequences in vivo. These results strongly suggest that ASF-1 is responsible for the function of as-1. Here we report the isolation of tobacco complementary DNA clones encoding two TGACG-sequence-specific binding-proteins (TGA1a and TGA1b). Sequence analysis of the cDNA clones shows that both proteins contain a basic region that shows high homology to a stretch of basic amino acids in the nuclear factors CREB, GCN4, and c-Jun to a 'leucine-zipper' region. On the basis of binding specificity we propose TGA1a to be a good candidate for ASF-1.     

Molecular tagging of a genic male-sterile gene in rice

The sterility of Pingxiang male-sterile rice (Pms), possibly derided from a spontaneous mutation in Pingxiang fertile rice (Pmf), was previously reported to be controlled by a single dominant nuclear gene. It can be restored to fertility either by a dominant epistatic gene or by higher temperature treatment at the early stage of inflorescence development. In order to tag the genic male-sterile gene, Pms, Pmf and Ce 64, a cytoplasmic male-sterile restoring line without the epistatic gene for Pms, were used to construct mapping populations. Two segregation populations, “(Pms/Ce 64) F1s (sterile plant)//Pmf ” F1 and “Pms//(Pmf/Ce 64) F1” F1, were simultaneously developed. Subsequently, the genic male- sterile gene was mapped between a simple sequence length polymorphism marker, RM228, and a restriction fragment length polymorphism marker, G2155, with distances of 14.9 and 2.6 cM, respectively. The tagged dominant genic male-sterile gene is temporarily designated Ms-p.     

mRNA transcripts related to full-length endogenous retroviral DNA in human cells

Mammalian cells contain multiple copies of endogenous type C retroviral DNA sequences. Among these sequences are complete, potentially infectious proviruses, proviral DNA that is expressed only in the form of viral antigens, retroviral segments that may contribute portions of envelope (env) genes during the generation of recombinant polytropic viruses, and many subgenomic viral DNA segments that may not be expressed at all. We have previously reported the identification and molecular cloning of type C retroviral sequences from human DNA and have shown that the partial nucleotide and deduced amino acid sequences of one of the clones obtained (lambda 51) are homologous to Moloney MuLV (MoMuLV) in the gag and pol regions. The lambda 51 clone as well as several others isolated from a human DNA library contained approximately 4.3 kilobases (kb) of retroviral sequences, were deleted in the env region, and were flanked by tandem repeats unlike the long terminal repeats (LTRs) typically found in proviral DNAs (P.E.S., in preparation). We describe here the characterization of a full-length human retroviral clone (lambda 4-1) containing LTR elements as well as a putative env region. DNA-RNA hybridization experiments reveal that human cells contain species of poly(A)+ RNA that anneal to segments of the full-length retroviral DNA clone.     

HPV-58型E7蛋白抗原模拟表位的筛选及分析

利用该实验室成熟的噬菌体随机十二肽库筛选平台,对HPV-58型E7蛋白单克隆抗体株4C4a、6C7a进行3轮亲和筛选,得到2组、各8个阳性噬菌体克隆.阳性噬菌体氨基酸序列用以模拟单抗(靶蛋白)特异性识别的HPV-58型E7蛋白的抗原表位.将测得的阳性噬菌体多肽序列及HPV-58型E7蛋白氨基酸序列进行生物信息学分析后,进行线性序列比对及构象性表位预测及分析.构象性表位的预测借力于EpiSearch Server和Pepitope Server,对构象性表位匹配及簇的寻找提供有力支持,也为早期诊断及研制多肽疫苗提供参考.     

Development of simple sequence repeats (SSR) markers of ramie and comparison of SSR and inter-SSR marker systems

Ramie (Boehmeria nivea L. ) is an important bast fiber crop. To study genetic background of this species, we isolated and characterized microsatellite markers of ramie. A genomic library containing inserts of rapid amplification of polymorphic DNA (RAPD)fragments was constructed, and screened by PCR amplification using anchored simple sequence repeats as primers. A total of 26 clones were identified as positives, and 13 microsatellite loci were found after sequencing. The polymorphism of these 13 microsatellite loci was examined and the utility of simple sequence repeats (SSR) and inter-SSR (ISSR) marker systems for genetic characterization compared using 19 selected ramie cultivars. Both approaches successfully discriminated the 19 cultivars which differed in the amount of polymorphism detected. The level of polymorphism detected by SSR was 95.0 %, higher than that by ISSR (72.3 % ), but the average polymorphism information content (PIC) of ISSR (0. 651) was higher than that of SSR (0. 441). The higher PIC value of ISSR suggests that ISSR is more efficient for fingerprinting ramie cultivars than SSR markers. However, because the SSR loci are codominant, they are more suitable for determining the homozygosity levels of ramie, constructing linkage map, quantitative trait loci study of complex traits and marker-as-sisted selection.     

Isolation of cDNAs Enconding Two Distinct Transcription Factors Binding to DRE cis-acting Element Involved in Cold- and drought-induced Expression of Arabidopsis rd29A Gene

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Somatic variants of murine immunoglobulin lambda light chains

Studies of the murine lambda light chains produced by myeloma cells provided the first evidence for somatic point mutation of germ-line variable (V) region genes. An examination of the variable regions of 19 lambda 1 chains revealed seven which differed from a common sequence by one to three amino acid substitutions. Subsequently, one of these presumed somatic variants of the single lambda 1 V gene was characterized by DNA sequence analysis of the rearranged functional gene. The predicted DNA sequence alteration was observed and no silent mutation was evident. These studies of lambda chain variants suggested that the hypervariable, complementarity-determining regions (CDRs) ht be a preferred site of somatic mutation because all seven characterized variants contained substitutions only in these regions. By contrast, comparisons of closely related kappa chain variable region amino acid sequences, and more recently VK and VH genes, have suggested that somatic mutation probably occurs in codons for both framework and CDR residues. To examine this apparent discrepancy between the sites of somatic mutations in lambda and kappa genes, we have determined the nucleotide sequence of two lambda 1 gene from hybridomas and a lambda 2 gene from a myeloma. These sequences demonstrate that somatic mutation in lambda genes can occur in both the framework and CDR residues.     

野生一粒小麦着丝粒RCS1相关序列的克隆鉴定

用水稻着丝粒重复序列RCS1为探针 ,与 30 72个克隆进行菌落杂交 ,得到了 32个阳性克隆 ,用RCS1与拟斯卑尔脱山羊草着丝粒重复序列Tcs2 5 0为探针进一步筛选 ,在 32个RCS1相关的阳性克隆中任选 10个克隆进行点杂交 ,分别有 6个和 5个阳性克隆 .为了克隆RCS1相关片段 ,依据RCS1的序列设计了三对引物 ,将引物 3从上述阳性克隆中扩增的一个 5 4 3bp的片段克隆测序 ,发现与水稻RCS1部分片段达到约 83%的同源 ,与大麦的反转座子 (Ty3 gypsy)部分序列同源性达到了 92 % ,与节节麦中着丝粒的整合酶基因部分序列同源性达到了 96 % ,命名为TBRCS1.TBRCS1可能是野生一粒小麦着丝粒区的组成部分     

Emergence of a new satellite RNA from cucumber mosaic virus isolate P1

The cucumber mosaic virus (CMV) isolate P1 caused very mild symptoms on many plant species.After serial passages by mechanical inoculation over five years, CMV P1 caused severe symptoms on several tobacco cultivars and tomato. A specific band of approximately 0.3 kb in length was amplified by RT-PCR with primers synthesized based on reported CMV satellite RNA (satRNA) sequences. Sequence analysis showed there were two satRNAs (Sat-Pl-1 and Sat-P1-2). Sat-Pl-1 contained 335 nucleotides, and Sat-P1-2 contained 394 nucleotides. These two satRNAs shared 64% overall nucleotide sequence homology, and differences between the two satRNAs included mutations as well as deletions. Sat-Pl-1 was identical to a satRNA (Z96099) reported in 1995 in CMV P1. Based on differences in the sequence and secondary structure between these two satRNAs, we conclude that Sat-P1-2 represents the emergence of a new satellite ( necrotic satellite) from attenuated satRNA populations. The possible effect of the emergence of this new satRNA is discussed.     

Sequence identification of 2,375 human brain genes.

We recently described a new approach for the rapid characterization of expressed genes by partial DNA sequencing to generate 'expressed sequence tags'. From a set of 600 human brain complementary DNA clones, 348 were informative nuclear-encoded messenger RNAs. We have now partially sequenced 2,672 new, independent cDNA clones isolated from four human brain cDNA libraries to generate 2,375 expressed sequence tags to nuclear-encoded genes. These sequences, together with 348 brain expressed sequence tags from our previous study, comprise more than 2,500 new human genes and 870,769 base pairs of DNA sequence. These data represent an approximate doubling of the number of human genes identified by DNA sequencing and may represent as many as 5% of the genes in the human genome.     

亲环素A抗原表位在三维结构中的初步定位

以抗人亲环素A单克隆抗体D4作捕获抗体，用噬菌体展示库鉴定出亲环素A模拟抗原表位的氨基酸序列为WSLQSFL，在亲环素A的一级结构中没有相同的序列，提示亲环素A抗原表位为构象型的，亲环素A的三维空间结构已测定，利用RasMol等蛋白质三维结构观察软件，可以初步确定亲环素A的抗原表位的时间位置，此抗原表位与环孢素的结合位点有重叠。     

Comparison of the community structure of planktonic bacteria in ballast water from entry ships and local sea water in Xiamen Port

In this study, the bacterial community structures in samples of ballast water collected from a ship from Singapore and of local sea water collected from Xiamen Port were compared using restriction fragment length polymorphism (RFLP) and 16S rDNA sequence analysis. Except for dominant α-Proteobacteria that are common to both systems, the bacterial community structures of the two systems were quite different. Most of the clones derived from the different systems were grouped into different phylogenetic clusters, and the sys-tems share only one common RFLP pattern. The ballast water, which is likely from clean offshore waters, contains sequences specific to α- and γ-Proteobacteria. Phylogenetic analysis revealed that the ballast water contained sequences belonging to attached bacteria and bacteria commonly found in the open sea, as well as many novel sequences. In addition, no known pathogenic bacteria were detected in the ballast water samples. Conversely, water samples from Xiamen Port were apparently affected by the near shore environments.Specifically, in addition to α- and γ-Proteobacteria, water from Xiamen Port contained β- and δ-Proteobacteria, Synechococcus, Bacter-oidetes and Actinobacteria, which are common in coastal environments. Additionally, four pathogenic bacterial sequences and one plas-mid sequence of a potential red tide forming alga were detected in the water from Xiamen Port, which suggests that the local sea water is polluted. The results of this study can be used as background information to assess the risk associated with the introduction of non-indig-enous species to local systems and to establish ballast water management systems.     

Cloning, sequence and expression of human interleukin-2 receptor

T lymphocytes, essential for the generation of a normal immune response, require the presence of the lymphokine interleukin-2 (IL-2) in order to proliferate. Cells that respond to IL-2 possess a surface receptor glycoprotein specific for this lymphokine. We have recently purified and chemically characterized the IL-2 receptor from both phytohaemagglutinin-activated human T cells and the human T-cell lymphoma HUT-102 (ref. 5). From the NH2-terminal protein sequence obtained in that study, we have now used synthetic oligonucleotides to probe a complementary DNA library, prepared from HUT-102 messenger RNA, for the presence of cDNA clones that might code for the IL-2 receptor. Two cDNA clones were isolated which had closely related DNA sequences. Interestingly, only one coded for an active receptor when transfected into COS-7 cells. This clone contained a 216-base pair (bp) insert that was not present in the other clone. The insert was flanked by an 8-bp direct repeat reminiscent of a transposable element, and appeared to code for a region of marked structural homology to the NH2-terminal region of the receptor molecule.     

SGK and 14-3-3 protein are involved in the serine/ threonine phosphorylation mechanism for TPO/MPL signal transduction

Thrombopioetin (TPO), the critical regulator of platelet production, acts by binding to its cell surface receptor, c-Mpl. Yeast two-hybrid screening was performed to isolate the proteins interacting with the cytoplasmic domain of c-Mpl. 48 positive clones were isolated from 5 × 10⁶ independent transformants. The results of sequence analysis demonstrate that they represent 13 different protein encoding sequences. Among them there are a partial coding sequence of serine/threonine protein kinase SGK (serum and glucocorticoid-inducible kinase) and 14-3-3 theta protein partial coding sequence. GST-pull-down assay and co-immunoprecipitation in mammal cells have confirmed the interaction between these two proteins and c-Mpl. By constructing a series of deleted c-Mpl cytoplasmic domain, the interaction region in c-Mpl cytoplasmic tail was localized in amino acids 523–554. At the same time, the directed interaction between SGK and 14-3-3 proteins also has been verified by yeast two-hybrid assay. The present note is the first time to report that two proteins act with c-Mpl at the same time and put forward that SGK and 14-3-3 protein may be involved in the serine/threonine phosphorylation mechanism for signal transduction.     

Cloning defined regions of the human genome by microdissection of banded chromosomes and enzymatic amplification

The molecular analysis of many genetic diseases requires the isolation of probes for defined human chromosome regions. Existing techniques such as the screening of chromosome-specific libraries, subtractive DNA cloning and chromosome jumping are either tedious or not generally applicable. Microdissection and microcloning has successfully been applied to various chromosome regions in Drosophila and mouse, but conventional microtechniques are too coarse and inefficient for analysis of the human genome. Because microdissection has previously been used on unbanded chromosomes only, cell lines in which the chromosome of interest could be identified without banding had to be used. At least one hundred chromosomes were needed for dissection and lambda vectors used to achieve maximum cloning efficiency. Recombinant phage clones are, however, more difficult to characterize than plasmid clones. Here we describe the dissection of the Langer-Giedion syndrome region on chromosome 8 from GTG-banded metaphase chromosomes (G-banding with trypsin-Giemsa) and the universal enzymatic amplification of the dissected DNA. Eighty per cent of clones from this library (total yield 20,000) identify single-copy DNA sequences. Fifty per cent of clones detect deletions in two patients with Langer-Giedion syndrome. Although the other clones have not yet been mapped, this result demonstrates that thousands of region-specific probes can be isolated within ten days.     

Use of suppression subtractive hybridization strategy for cloning and identifying specifically expressed genes of renal cell carcinoma

Using suppression subtractive hybridization, a renal cell carcinoma (RCC) cDNA subtractive library which only contains differently expressed cDNAs between human RCC and normal kidney has been constructed. 200 clones were picked out randomly to perform enzyme digest analysis, a part of them underwent sequence analysis and Northern blot to identify RCC specially expressed genes. Results showed that 190 clones contain 50—400 bp inserts respectively. Sequence analysis was performed in 10 clones. All the 10 sequences were unknown before and derived from 6 unique novel genes among which the cDNA insert RCC18 has five copies. Northern blot analysis showed that RCC18 cDNA expressed highly in RCC, but there was no signal detected in normal kidney, and the full length of RCC18 was about 2.5 kb. The constructed cDNA subtractive library of human RCC is a highly efficient one and lays the solid foundation for large-scale screening and cloning new and specific oncogenes or tumor suppressor genes of RCC. The novel specially expressed genes provided an important clue for researching the mechanism of the occurrence and development of RCC.