Isolation of development-related genes in somatic embryo radicle of carrot (Daucus carota L.) using modified cDNA RDA

Using modified cDNA RDA capitalizing on the high affinity of streptavidin for biotin and magnetic-absorption-based separation, we have obtained four bands of specifically expressed cDNA in the carrot somatic embryo deregulated for 12 h, which were designated as NR-1, NR-2, NR-3 and NR-4, respectively. As revealed by homology analysis of their DNA sequences after cloning them into pBS, remarkable homology was demonstrated in NR-2, NR-3 and NR-4 with the genes coding for LEA (late embryogenesis abundant protein), Dna J and xyloglucan endo-transglycosylase in plants. On the other hand, NR-1 showing no homology with any known sequence may have come from unknown genes. Using³²P-labeled NR-1 as probe, hybridization with cDNA fragment population has shown that we have actually cloned a new gene fragment related to radicle development. As shown by further Southern hybridization, these genes may be present in carrot genome in the form of single or low copies.     

A gene expressed preferentially in the globular stage of somatic embryogenesis encodes elongation-factor 1 alpha in carrot.

We have isolated cDNA of genes that are preferentially expressed during somatic embryogenesis of carrot (Daucus carota L.) by differential screening of globular embryos and cells that are dividing in an unorganized manner. As a result of Northern-blot analysis, one of the genes identified in this way, which we refer to as CEM1, was found to be expressed at high levels in somatic embryos at the globular and heart-shaped stages. In-situ hybridization using globular embryos revealed that the mRNA transcribed from CEM1 was located preferentially in the spherical region of the globular embryo. A homology search using the amino acid sequence deduced from the nucleotide sequence of the CEM1 cDNA revealed that CEM1 encodes the eukaryotic translational elongation-factor 1 alpha.     

草菇冷诱导相关基因的克隆及序列分析

利用差异显示技术分离获得草菇低温特异DNA片段,经与正常草菇和低温诱导草菇cDNA分别southern杂交验证后,得到低温特异性片段。采用PCR标记技术对获得的低温特异性片段进行DIG标记,以此为探针,对低温处理的草菇cDNA文库进行筛选,获得4个阳性克隆,分别进行测序。序列同源性比较分析发现,Cor3基因与s-腺苷-L-高半胱氨酸水解酶有很高的同源性,Cor4基因与40S核糖体蛋白S9有很高的同源性,这两个基因可能与草菇的低温自溶现象有关。Cor1基因与脉孢菌的保守假设蛋白(conservedhypotheticalprotein)有同源性,Cor2基因与辅酶A连接酶有同源性。半定量RT-PCR验证发现Cor1和Cor2基因在正常情况下没有表达,低温处理后有表达,Cor3和Cor4基因在正常情况下有表达,低温处理后表达量增加。     

Cytogenetic mapping of 31 functional genes on chicken chromosomes by direct R-banding FISH

Using direct R-banding fluorescence in situ hybridization, we determined the location of 31 functional genes on chicken chromosomes. Replication R-banded chromosomes were obtained by synchronizing splenocyte cultures with excessive thymidine, followed by BrdU treatment. Thirty-one functional genes were directly localized to banded chicken chromosomes using genomic DNA and cDNA fragments as probes. The possibility of conserved linkage homology between chicken and human chromosomes was demonstrated for seven chicken chromosome regions (1p, 1q, 2q, 4p, 4q, and 5q).     

Isolation and characterization of cDNA clones for plant cyclins.

We have isolated and sequenced a carrot cDNA and two soybean cDNAs encoding mitotic cyclin homologs. The soybean clones were derived from nearly identical cognate genes. The carrot cyclin and soybean cyclins were slightly more similar to A-type and B-type cyclins thus far defined, respectively. However, they had divergent amino acid sequences in the portion that is most highly conserved in known cyclins and we could not easily include them in either of the phylogenetic types. Since the homology between carrot and soybean cyclins was low, each of them might define a novel and distinct type. The mRNA of carrot cyclin, 1.5 kb in length, was expressed concomitant with somatic embryogenesis of cultured cells. Expression of soybean cyclin mRNAs, 1.6 kb in length, was localized in proliferating parts of seedlings. As in the case of cyclin genes of marine invertebrates, microinjection of a synthetic mRNA for the soybean cyclin induced the maturation of Xenopus oocytes. Other cyclin genes may be present because, on Southern blot analysis of soybean genomic DNA, the isolated soybean cDNA probe hybridized with additional genes under low stringency.     

Curcumin-induced differentiation of mouse embryonal carcinoma PCC4 cells.

Curcumin, a natural component of turmeric extracted from the rhizomes of Curcuma longa, is known to exhibit a number of biological properties. In the present study, curcumin, at low concentration, was shown to induce differentiation in embryonal carcinoma cell line PCC4. In response to curcumin, PCC4 cells ceased to proliferate and showed cell cycle arrest at G1 phase after 4 hours of treatment, followed by their differentiation which is characterized by increase of nuclear/cytoplasmic ratio. The expression of hsp 70 was also seen upon 8 h of curcumin treatment, and it remained constant up to 48 h. Differentiated cells also expressed a series of differentiation markers such as lamin A, well-established actin, and keratin cytoskeleton. We used mRNA differential display analysis to identify the genes that are regulated during curcumin-induced differentiation of PCC4 cells. We cloned and sequenced three partial cDNAs that were differentially expressed in normal and differentiated cells. Sequence comparison of one downregulated cDNA (Al) has shown homology to a gene present on mouse chromosome five, while the two upregulated cDNA (C1 and C7) are homologous to several mouse ESTs clones from organs of mesodermal origin. We have identified the full-length coding sequence of the Cl fragment with a putative amino acid sequence. Tissue-specific Northern with RNA from adult mouse organs with the C1 fragment alone showed hybridization with mRNA from several tissues, whereas the same Northern with only the coding sequence showed expression of C1 gene mainly in the adult kidney. Homology search revealed that C1 sequence is part of the 3' UTR and may be common to several genes expressed in many tissues. Thus, curcumin appears to differentiate embryonal carcinoma cell PCC4, and one of the upregulated genes seems to be expressed mainly in the adult kidney.     

Identification and mapping of swine cyclin-dependent kinase inhibitor CDKN2A and CDKN2B exon 2 sequences

We have isolated the swine homologs of human CDKN2A and CDKN2B exon 2 sequences. As in the human and mouse genomes, the exon 2 sequences of these two genes present a high level of sequence homology and are tightly linked. Using fluorescence in situ hybridization, we have mapped swine CDKN2A and CDKN2B to chromosome 1q25. This confirms the comparative mapping data among man, mouse, and swine, showing a conserved synteny among chromosome segments 9p21, 4C3-C6, and 1q25, respectively.     

Analysis of multiple nuclear receptor binding sites for CAR/RXR in the phenobarbital responsive unit of CYP2B2

The phenobarbital (PB) responsive enhancers in CYP2B genes contain a core of two direct repeat-4 nuclear receptor binding sites, NR-1 and NR-2, which flank an NF-1 site and appear to be most important for PB responsiveness. Additional sequences outside the core are required for maximal PB responsiveness, including a third direct repeat-4 site, NR-3. The PB response is mediated by constitutive androstane receptor (CAR) which binds as a CAR/RXR heterodimer to the NR sites. To determine the relative importance of the third NR site, each of the NR sites was mutated individually and in all combinations in the rat PB responsive unit (PBRU). Mutation of NR-3 resulted in similar effects on transactivation of the PBRU by CAR in HepG2 cells as did mutations of NR-1 and NR-2. The recruitment of GRIP1/SRC-2 by CAR/RXR to the PBRU assessed by gel shift assays was cooperatively enhanced if more than one NR site in the PBRU was occupied by CAR/RXR. NR-3 in combination with NR-1 or NR-2 was equal to NR-1 and NR-2 in mediating this cooperative recruitment. Recruitment of SRC-1 and GRIP1/SRC-2 was similar for all NR sites, while some selectivity of NR-1 for SRC-3 was observed. SRC-3 also exhibited CAR-independent activation of the PBRU in HepG2 cells. Micrococcal nuclease mapping of nucleosomes revealed that the NR-1/NR-2 core of the PBRU is present in a nucleosome while NR-3 is present in the linker adjacent to the nucleosome. In the linear sequence NR-3 is further from NR-1 than NR-2 is, but in a nucleosomal structure, NR-3 is well positioned for cooperative recruitment of GRIP1/SRC-2 by CAR/RXR that is bound to NR-3 and either NR-1 or NR-2, while NR-1 and NR-2 are on opposite sides of the nucleosome separated by the histone core. These results demonstrate that NR-3 is functionally similar to NR-1 and NR-2 in CAR transactivation of the PBRU in vitro and suggest that NR-3 may have a greater role in a chromatin context in vivo than is apparent from transient transfection studies.     

Cloning and functional expression of a first inducible avian cytochrome P450 of the CYP3A subfamily (CYP3A37)

CYP3As represent a family of cytochromes P450 involved in the metabolism of both endogenous and exogenous natural and synthetic compounds. Well described in mammals, none have yet been cloned and characterized in avian species. In this paper, we report the cloning and analysis of an avian CYP3A (CYP3A37). Using an RNA differential display approach, an 80-bp phenobarbital-inducible cDNA fragment was amplified from chicken embryo liver. Based on its homology with mammalian CYP3As, this fragment was used to clone a full-length cDNA consisting of 1638 bp encoding a putative protein of 509 amino acids. The sequence shares between 57.4 and 62% identity at the amino acid level with CYP3As of other species. This cDNA was designated CYP3A37 according to the current cytochrome P450 nomenclature. When expressed in COS1 cells, the CYP3A37 cDNA produced a protein of congruent with55 kDa, which was recognized by polyclonal anti-rat CYP3A1 antiserum. In a bacterial expression system, the CYP3A37 cDNA produced a protein capable of steroid 6beta-hydroxylation. At a substrate concentration of 100 microM, progesterone, testosterone, and androstenedione were found to be 6beta-hydroxylated at a rate of 15.4, 11.7, 12.2 nmol/min/nmol P450, respectively. Used as control, the human CYP3A4 gave similar hydroxylation rates. Finally, in both chicken embryo liver and chicken hepatoma cells (LMH), CYP3A37 mRNA was increased after treatment with typical CYP3A inducers, such as metyrapone, phenobarbital, dexamethasone, and pregnenolone 16alpha-carbonitrile, but not rifampicin. CYP2H1, a well-characterized inducible chicken cytochrome P450, also was induced by the same compounds, suggesting similar regulation of CYP3 and CYP2 genes in this species.     

我国非肠道传播非甲非乙型肝炎(丙型)病毒NS3蛋白的部份cDNA克隆及序列分析

用15ml非肠道传播非甲非乙型肝炎病人的混合血清提取病毒RNA,经逆转录和聚合酶链反应(PCR)扩增,获得583个核苷酸的非肠道传播非甲非乙型肝炎病毒(HCV)NS3蛋白的cDNA片段.该片段与美国报道的同片段HCV cDNA原型比较,核酸序列同源性为80.9％,氨基酸序列同源性为93.2％。与日本报道的同片段J1 HCV cDNA相比较,核酸序列和氨基酸序列的同源性分别为92.6％和95.2％。用α-~(32)P同位素标记该片段,与HCV病人血清出现杂交反应。     

The Arabidopsis functional homolog of the p34cdc2 protein kinase.

The p34cdc2 protein kinase is a key component of the eukaryotic cell cycle, which is required for G1 to S-phase transition and for entry into mitosis. Using a 380-base pair DNA fragment obtained by polymerase chain reaction amplification from an Arabidopsis thaliana flower cDNA library as a probe, we isolated and sequenced a cdc2-homologous cDNA from Arabidopsis. The encoded polypeptide has extensive homology with cdc2-like kinases. Furthermore, when expressed in a CDC28ts Saccharomyces strain, it partially restores the capacity to grow at 36 degrees C, indicating that the plant cDNA is a functional homolog of the p34cdc2 kinase. Genomic hybridization demonstrated that there is one copy of the cdc2 gene per Arabidopsis haploid genome. Using RNA gel blot analysis, we found that cdc2 mRNA is present in all plant organs.     

Analysis of highly expressed genes in the late zygotene to pachytene stages of meiotic prophase I in david lily

Plant meiotic prophase I is a complex process involving the late zygotene and pachytene stages, crucial for both completing synapsis and recombination. Using David lily (Lilium davidii var. Willmottiae) as research material, we performed suppressive subtractive hybridization to construct expessed sequence tag (EST) library of anthers at various stages of development by the pollen mother cells. From this library, we identified 34 genes with significantly enhanced expression during the late zygotene to pachytene stages. The cDNA fragment sequences were compared with data in GenBank by BLASTN and BLASTX, and 18 unique ESTs were shown to exhibit significant homology to the data in GenBank. They were classified into eight different groups: metabolism, protein modification, signal transduction, etc. Through the study of classification and functions of these highly expressed genes during the late zygotene to pachytene stages, we obtained much information about the complex biological progress of meiotic prophase I, especially during chromosome synapsis and recombination.     

Isolation and Characterization of Rat and Human cDNAs Encoding a Novel Putative Peroxisomal Enoyl-CoA Hydratase

We have used a PCR-based subtractive hybridization method to identify upregulated cDNAs in the livers of rats treated with a peroxisome proliferator [clofibrate or di(2-ethylhexyl) phthalate]. After four rounds of subtractive hybridization 62 differentially hybridizing clones were partially sequenced and analyzed by sequence homology searching. Of 62, 49 were identical to 14 different upregulated rat sequences in the databank (mostly genes encoding microsomal or peroxisomal enzymes), 4 of 62 were fragments of three previously unknown genes, and 9 of 62 were false positives. Two of the unknown fragments hybridized to a single novel cDNA that was found to be more than 20-fold induced by both peroxisome proliferators. The 36-kDa predicted protein product of this cDNA shows a high degree of sequence homology to enoyl-CoA hydratases of several different species and has a C-terminal peroxisomal targeting sequence. An epitope-tagged protein product of a full-length cDNA was targeted to peroxisomes in a human cell line. We named this gene, which encodes an apparent peroxisomal enoyl-CoA hydratase, ECH1. We have also identified human ECH1 cDNA and mapped its structural gene to 19q13, 3′ to the ryanodine receptor, by hybridization to somatic cell hybrid DNA and chromosome 19-specific cosmid arrays. Possible roles for the ECH1 protein product in peroxisomal β-oxidation are discussed.     

Localization of the acetylcholine receptor gamma subunit gene to human chromosome 2q32----qter

The nicotinic acetylcholine receptor of skeletal muscle (CHRN in man, Acr in mouse) is a transmembrane protein composed of four different subunits (alpha, beta, gamma, and delta) assembled into the pentamer alpha 2 beta gamma delta. These subunits are encoded by separate genes which derive from a common ancestral gene by duplication. We have used a murine full-length 1,900-bp-long cDNA encoding the gamma subunit subcloned into M 13 (clone gamma 18) to prepare single-stranded probes for hybridization to EcoRI-digested DNA from a panel of human x rodent somatic cell hybrids. Using conditions of low stringency to favor cross-species hybridization, and prehybridization with rodent DNA to prevent rodent background, we detected a single major human band of 30-40 kb. The pattern of segregation of this 30-40 kb band correlated with the segregation of human chromosome 2 within the panel and the presence of a chromosomal translocation in the distal part of the long arm of this t(X;2)(p22;q32.1) chromosome allowing the localization of the gamma subunit gene (CHRNG) to 2q32----qter. The human genes encoding the gamma and delta subunits have been shown to be contained in an EcoRI restriction fragment of approximately 20 kb (Shibahara et al., 1985). Consequently, this study also maps the delta subunit gene (CHRND) to human chromosome 2q32.1----qter. In the mouse, the Acrd and Acrg genes have been shown to be linked to Idh-1, Mylf (IDH1 and MYL1 in humans, respectively) and to the gene encoding villin on chromosome 1. Interestingly, we have recently localized the human MYL1 gene to the same chromosomal fragment of human chromosome 2. These results clearly demonstrate a region of chromosomal homoeology between mouse chromosome 1 and human chromosome 2.     

用人染色体14q24.3区带探针池直接分离表达顺序

本文报道了从显微切割的人染色体区带直接分离区带专一性表达序列的方法和结果。     

Cloning of cDNA encoding a soybean allergen, Gly m Bd 28K

A cDNA clone encoding a soybean allergen, Gly m Bd 28K, has been isolated. The clone has a 1567-bp cDNA insert with a 1419-bp open reading frame and a 148-bp 3'-untranslated region, followed by a polyadenylation tail. The open reading frame was shown to encode a polypeptide composed of 473 amino acids. The chemically determined amino acid sequences of the peptides obtained from the allergen, including its N-terminal peptide, were shown to be contained in the N-terminal region of the amino acid sequence deduced from the cDNA, showing that the first half of the cDNA encodes the allergen with a preceding segment of 21 amino acids. The peptide fragment including the allergen was expressed as a fusion protein with glutathione S-transferase in Escherichia coli and immunoblotted with the sera of soybean-sensitive patients and the monoclonal antibody against the allergen. Furthermore, homology analyses demonstrate that the polypeptide for the cDNA exhibits high homology with the MP27/MP32 proteins in pumpkin seeds and the carrot globulin-like protein. This finding suggests that the polypeptide may consist of a 21-amino acid segment as a part of the signal peptide and the proprotein, which may be converted to two mature proteins, Gly m Bd 28K and a 23-kDa protein, during the development of soybean cotyledons.