Isolation and characterization of male flower cDNAs from maize

Differential screening of two libraries made from whole, immature maize tassels was used to isolate six cDNAs which show enhanced levels of expression in male flowers. MFS1, MFS2, MFS4, MFS10 and MFS18, which were isolated from a 5 cm tassel library, are expressed throughout tassel growth up until mature pollen is produced in the anthers. MFS14, which was isolated from a 10–12 cm tassel library, has a narrower window of expression associated with microsporogenesis and declines as mature pollen is produced. MFS18 mRNA accumulates in the glumes and in anther walls, paleas and lemmas of mature florets. MFS18 mRNA is particularly associated with the vascular bundle in the glumes and encodes a polypeptide of 12 kDa, rich in glycine, proline and serine that has similarities with other plant structural proteins. In contrast, MFS14 mRNA accumulates in the tapetum and encodes a polypeptide of 13 kDa that is rich in alanine. The MFS14 and MFS18 proteins are basic (isolectric points of 11.56 and 9.54, respectively) and both have hydrophobic N-termini which display all the characteristics of signal peptides, indicating that these proteins may be secreted.     

Isolation of cell death-associated cDNAs from involuting mouse mammary epithelium

Although apoptosis is important in determining cell fate and maintaining tissue homeostasis, the initiation and control of apoptotic cell death in epithelium is not well understood. Post-lactationai involution of the mammary gland provides both an important developmental process and a normal physiological setting for studying apoptosis of epithelium. We used a differential screening strategy, based on previous studies correlating morphology with gene expression and nucleic acid integrity during mammary gland involution, to isolate genes involved in the regulation and execution of apoptotic cell death in regressing mammary epithelium. This screening strategy yielded a large number of genes the expression of which is significantly altered during mammary gland involution. These include genes associated with cell death processes, tissue remodelling and mesenchymal differentiation. In addition, a number of novel genes have been isolated. We have used Northern analysis and in situ hybridisation to study the expression of a selection of these putative death-associated genes during post-lactational mouse mammary gland involution.     

Isolation and analysis of two cellulase cDNAs from Orpinomyces joyonii

Two cellulase cDNAs, celB29 and celB2, were isolated from a cDNA library derived from mRNA extracted from the anaerobic fungus, Orpinomyces joyonii strain SG4. The nucleotide sequences of celB2 and celB29 and the primary structures of the proteins encoded by these cDNAs were determined. The larger celB29 cDNA was 1966bp long and encoded a 477 amino acid polypeptide with a molecular weight of 54kDa. Analysis of the 1451bp celB2 cDNA revealed an 1164bp open reading frame coding for a 44kDa protein consisting of 388 amino acids. Both deduced proteins had a high sequence similarity in central regions containing putative catalytic domains. Primary structure analysis revealed that CelB29 contained a Thr/Pro-rich sequence that separated the N-terminal catalytic domain from a C-terminal reiterated region of unknown function. Homology analysis showed that both enzymes belong to glycosyl hydrolase family 5 and were most closely related to endoglucanases from the anaerobic fungi Neocallimastic patriciarum, Neocallimastix frontalis and Orpinomyces sp. The classification of CelB29 and CelB2 as endoglucanases was supported by enzyme assays. The cloned enzymes had high activities towards barley beta-glucan, lichenan and carboxymethylcellulose (CMC), but not Avicel, laminarin, pachyman, xylan and pullulan. In addition, CelB29 and CelB2 showed activity against p-nitrophenyl-beta-D-cellobioside (pNP-G(2)) to p-nitrophenyl-beta-D-cellopentaoside (pNP-G(5)) but not p-nitrophenyl-beta-D-glucopyranoside (pNP-G(1)) with preferential activity against p-nitrophenyl-beta-D-cellotrioside (pNP-G(3)). Based on these results, we proposed that CelB29 and CelB2 are endoglucanases with broad substrate specificities for short- and long-chain beta-1,4-glucans.     

番茄叶绿体DNA的分离纯化及某些性质的研究

分离纯化了番茄的叶绿体DNA(ct DNA)。热变性分析测得Tm=82.1℃,由此计算得(G+C)％=31.2％。热变性微分曲线表明,在番茄ct NDA分子上存在着碱基组成比例分布上的异质性。分析超离心测得沉降系数S_(20w)=62.4。据此计算出分子量为80×10~6d。用电子显微镜观索了制备的ctDNA.     

Isolation of Mhc class I cDNAs from the axolotl Ambystoma mexicanum

 Class I major histocompatibility complex (Mhc) cDNA clones were isolated from axolotl mRNA by polymerase chain reaction (PCR) and by screening a cDNA phage library. The nucleotide and predicted amino acid sequences show definite similarities to the Mhc class Iα molecules of higher vertebrates. Most of the amino acids in the peptide binding region that dock peptides at their N and C termini in mammals are conserved. Several amino acids considered to be important for the interaction of β₂-microglobulin with the Mhc α chain are also conserved in the axolotl sequence. The fact that axolotl class I A cDNAs are ubiquitously expressed and highly polymorphic in the α1 and α2 domains suggests the classical nature of axolotl class I A genes. Received: 3 June 1996 / Revised: 14 October 1996     

Isolation and Properties of Intact Chromoplasts from Tomato Fruits

Intact chromoplasts were isolated from tomato fruits at differentripening stages by Percoll density gradient centrifugation.The isolated chromoplast fractions were contaminated very littleby other organelles, although the fraction from fully ripenedfruits contained some mitochondria and microbodies. As the transformationof chloroplasts to chromoplasts proceeded, the density of theplastids decreased from 1.096 to 1.075 g-cm^-3 and the decreasewas related to a decrease in chlorophyll and an increase inlycopene in the plastids. (Received December 21, 1983; Accepted April 20, 1984)     

Isolation and characterization of cDNAs encoding imidazoleglycerolphosphate dehydratase from Arabidopsis thaliana.

cDNA clones encoding imidazoleglycerolphosphate dehydratase (IGPD; EC 4.2.1.19) from Arabidopsis thaliana were isolated by complementation of a bacterial auxotroph. The predicted primary translation product shared significant identity with the corresponding sequences from bacteria and fungi. As in yeast, the plant enzyme is monofunctional, lacking the histidinol phosphatase activity present in the Escherichia coli protein. IGPD mRNA was present in major organs at all developmental stages assayed. The Arabidopsis genome appears to contain two genes encoding this enzyme, based on DNA gel blot and polymerase chain reaction analysis.     

侵染番茄的番茄花叶病毒的研究

从种传番茄苗中获得一病毒分离物Ｔｏ－Ｓｌ,人工摩擦接种７科２４种植物,Ｔｏ～Ｓｌ能侵染４科１５种植物,在番茄上产生花叶,在白肋烟上为局部枯斑。Ｔｏ－Ｓｌ的钝化温度为８５～９０℃,稀释限点为１０￣（－６）～１０￣（－７）．体外保毒期在一个月以上。病毒粒体杆状,长度主要分布于２８１～３００ｎｍ之间,平均长度２８８ｎｍ。病毒衣壳蛋白亚基只有一条多肽链,分子量为２１ｋＤａ。ｄｓＲＮＡ分析测得其基因组长度为６．４ｋｂ。琼胎糖双扩散和胶内交叉吸附试验证明,Ｔｏ－Ｓｌ与ＴＭＶ有血清关系,但有一定的差异,病毒粒体电泳分析也表明Ｔｏ－Ｓｌ与ＴＭＶ粒体有差异。在交叉保护试验中,ＴＭＶ和Ｔｏ－Ｓｌ之间均无保护作用。根据以上试验结果Ｔｏ－Ｓｌ被鉴定为番茄花叶病毒。这是我国首次系统报道番茄上番茄花叶病毒的侵染。     

Isolation of cDNAs encoding GTP cyclohydrolase II from arabidopsis thaliana

A GTP cyclohydrolase II-encoding gene from Arabidopsis thaliana was isolated through functional complementation of a mutant of Escherichia coli, BSV18, deficient in this protein. The derived amino-acid sequence constitutes a polypeptide of 27 kDa and shows 37–58% identity with previously published sequences of Escherichia coli, Bacillus subtilis, Photobacterium leiognathi and P. phosphoreum.     

Isolation of pathogen/stress-inducible cDNAs from alfalfa by mRNA differential display

Differential display of mRNA was used to isolate a full-length (SRG1) and a partial (SRG2) alfalfa cDNA induced during infection with the fungal pathogen Colletotrichum trifolii. The deduced amino acid sequences are similar to each other and resemble plant defense-related proteins and tree pollen allergens. SRG1 is a member of a gene family in alfalfa, which may also include the putative defense-related gene PR10. Unlike many defense-related genes described in similar systems, expression of SRG1-like genes does not correlate with resistance to C. trifolii. We speculate SRG1 is induced in response to plant stress.     

Isolation and Mapping of a Family of Putative Zinc-finger Protein cDNAs from Rice

To understand the functions of rice homologues of the Arabidopsisflowering-time gene CONSTANS (CO) and salt-tolerance gene STO,we performed a similarity search of the single-run sequencedata of cDNA clones accumulated by the Rice Genome ResearchProgram, and isolated seven rice cDNA clones (S3574, C60910,S12569, R2931, R1479, R1577, and E10707) coding for proteinscontaining one or two zinc-finger-like motifs. Comparison ofthe deduced amino acid sequences between these cDNAs and theCO gene revealed significant similarities (46%-;61%) in theregion of zinc-finger motifs. A domain having a high contentof basic amino acids at the C-terminus of the CO protein wasfound in the corresponding region of proteins predicted fromcDNAs S3574, C60910, and S12569. Two amino acid sequences, "CCADEAAL"and "FCV(L)EDRA," which were present inside each zinc-fingerin the Arabidopsis regulatory protein STO, were also found ineach of the two zinc-finger regions of proteins predicted fromcDNAs R2931, R1479, R1577, and E10707. Using restriction fragmentlength polymorphism (RFLP) linkage analysis, we determined thechromosomal location of the seven cDNA clones. The positionof R2931 on the RFLP linkage map was closely linked to Hd-3,one of the putative quantitative trait loci (QTL) controllingheading date in rice.