Electrophysiological and pharmacological properties of GluR1, a subunit of a glutamate receptor-channel expressed in Xenopus oocytes.

A cDNA clone encoding an excitatory amino acid receptor was isolated from a rat brain cDNA library by Hollmann et al. (Nature, 342 (1989) 643-648). In Xenopus oocytes, this clone, GluR1, expressed a functional receptor-channel activated by kainate (KA), domoate (D), glutamate and quisqualate (QA). The apparent affinity (EC50) for QA (0.1 microM) was higher than that for KA (50 microM). The maximal response to QA was about 1/10 of that to KA. QA inhibited the KA induced current. The N-methyl-D-aspartate (non-NMDA) receptor antagonist 6,7-dinitroquinoxaline-2,3 dione (DNQX) competitively blocked the effects of both agonists. Currents induced by KA, QA and D in oocytes expressing GluR1 showed identical voltage sensitivities. GluR1 and KA receptor-channels expressed from rat striatum poly(A)+ RNA showed the same ionic selectivity, being permeable mostly to Na+ and K+. The current-voltage relationships of GluR1 showed a strong inward rectification, whereas those of KA receptor-channels expressed from poly(A)+ RNA from various rat brain regions were more linear.     

Localization of three novel zinc finger genes to the centromeric region of human chromosome 10 by fluorescencein situ hybridization

An oligonucleotide probe for the consensus sequence of the linker region of zinc finger proteins was used to isolate cDNA clones from a human fetal heart cDNA library. Following DNA sequencing analysis and comparison, genes for the novel clones were mapped by fluorescencein situ hybridization. We report the chromosomal localization of three zinc finger-coding, genes to the region of centromere on human chromosome 10p11.1q-11.2, indicating involvement in gene duplication and chromosome rearrangement during primate evolution.     

人胎儿骨骼和关节RACE cDNA文库的构建

目的 建立人胎儿骨骼和关节快速扩增cDNA末端（rapid amplification of cDNA ends,RACE cDNA）文库,为分离骨骼和关节发育相关基因奠定基础。方法 采用改进的早硫氰酸胍－酚－氯仿－异戊醇一步法提取骨骼和关节总RNA,用TaKaRa公司生产的cDNA合成试剂盒合成平末端的双链cDNA,然后与衔接子连接,再用位于双链cDNA末端的通用引物扩增全部cDNA。结果 建立了从骨骼和关节构建RACE cDNA文库的方法,并用该方法成功地构建了人胎儿骨骼和关节RACE cDNA文库。结论 所构建的利用少量总RNA构建RACE cDNA文库方法切实可行,所构建的文库适用于用RACE方法从中分离骨骼和关节发育相关基因。     

Identification of Babesia bovis L-lactate dehydrogenase as a potential chemotherapeutical target against bovine babesiosis

In this study, we characterized a novel Babesia bovis cDNA clone obtained by immunoscreening the cDNA expression phage library with B. bovis-infected bovine serum. The genetic analyses showed that it contained an open reading frame of 993 bp, which was considered to encode B. bovis L-lactate dehydrogenase (BbLDH: E.C. 1.1.1.27) because of the strikingly high amino acid identities of its gene product to the LDHs of Plasmodium falciparum and Toxoplasma gondii. Immunological analyses with the anti-recombinant BbLDH mouse serum showed that 36 kDa of the native BbLDH was expressed not only in the cytoplasm of intra- and extraerythrocytic parasites but also along the membrane of infected erythrocytes. The kinetic properties of recombinant BbLDH proved a certain enzymatic activity of LDH, and the activity was significantly inhibited by the addition of gossypol, a competitive inhibitor of protozoan LDHs. Moreover, 100 microM of the gossypol irretrievably arrested the in vitro growth of B. bovis. The results demonstrated that BbLDH provides a suitable drug target for the design of novel babesial chemotherapeutics.     

Ranunculus latent virus: a strain of artichoke latent virus or a new macluravirus infecting artichoke?

An elongated virus was isolated from artichoke crops in Liguria, and a 700-bp fragment was amplified by RT-PCR using oligonucleotides to detect members of the family Potyviridae. Comparison of fragment sequences showed 98% identity at the nucleotide level with the ranunculus isolate of the macluravirus Ranunculus latent virus (RaLV). RaLV was then detected by DAS-ELISA in symptomatic and asymptomatic artichoke plants from Liguria, Sardinia and Latium. The sequence of a 5.5-kb region was assembled from a cDNA library, and a 500-bp NIa fragment showed 80% identity to Artichoke latent virus.     

Cloning and expression of MP13 gene from rat hippocampus, a new factor related to guanosine triphosphate regulation

C-Fos and the Fos-related antigens (FRA) are induced by various stimuli. A novel 35-37 kDa FRA was induced much longer after the treatment using kainic acid (KA) and may be very important for neuronal survival after brain damage. To identify this long-term FRA, we have constructed a cDNA library derived from hippocampus after KA treatment and screened it with an antibody highly conserved M-peptide region of FRAs. One gene, MP13, was cloned with a 1662 bp open reading frame and coded for a 554-amino acid protein. MP13 has a leucine zipper region, a glutamine repeat region, and has high similarity to the activator of the small guanosine triphosphate (GTP)ase Rab5. Gel retardation analysis revealed that MP13 functions as a GTP regulation related factor.     

Intranasal BCG vaccination protects BALB/c mice against virulent Mycobacterium bovis and accelerates production of IFN-γ in their lungs

Local immune reactivity in the lungs of BALB/c mice was studied following (i) intranasal (i.n.) vaccination with Mycobacterium bovis BCG, (ii) intravenous (i.v.) challenge with a virulent M. bovis field isolate and (iii) i.n. vaccination with M. bovis BCG followed by i.v. challenge with an M. bovis field isolate. The results demonstrated that i.n. vaccination with BCG induced a high degree of protection against systemic M. bovis challenge, and that this protection correlated with a rapid production of IFN-gamma after M. bovis challenge by lung T cells from vaccinated mice.     

Analysis of the shotgun expression library of the Mycobacterium tuberculosis genome for immunodominant polypeptides: potential use in serodiagnosis

A recombinant DNA strategy was applied to analyze and screen the shotgun expression library from a clinically confirmed local virulent isolate of Mycobacterium tuberculosis with sera from tuberculosis patients, which led to expression and purification of highly immunoreactive and specific mycobacterial antigens expressed during the course of active disease which could be of diagnostic significance. An enzyme-linked immunoassay for diagnosis of tuberculosis was devised by using a shotgun immunoexpression library in the lambdagt11 vector. DNA from a virulent M. tuberculosis patient isolate (TBW-33) confirmed with the BACTEC 460 system was sheared and expressed to generate shotgun polypeptides. beta-Galactosidase fusion proteins capable of demarcating active tuberculosis infections from Mycobacterium bovis BCG-vaccinated healthy subjects or people harboring environmental mycobacteria were selected by comparative immunoreactivity studies. Promising mycobacterial DNA cassettes were subcloned and expressed into the glutathione S-transferase (GST) fusion vector pGEX-5X-1 with a strong tac promoter and were expressed in Escherichia coli BL21. These fusion proteins were severed at a built-in factor Xa recognition site to separate the GST tags and were utilized in an indirect enzyme-linked immunoassay for serodiagnosis of patients with active tuberculosis. The system offered a clear demarcation between BCG-vaccinated healthy subjects and patients with active tuberculosis and proved to be effective in detecting pulmonary as well as extrapulmonary tuberculosis, with an overall sensitivity of 84.33% and an overall specificity of 93.62%.     

小鼠巨噬细胞cDNA酵母表达文库的构建与鉴定

目的 采用Gateway技术构建适合酵母表达的小鼠巨噬细胞cDNA文库并进行鉴定.方法 将小鼠巨噬细胞的mRNA分离纯化后,以生物素标记的寡聚胸苷酸Oligo(dT)为引物反转录后连接attB衔接子(attB Adapter),层析柱纯化,通过BP重组反应将500 bp以上的片段克隆到含attP衔接子的pDONRTM222载体,电转化入ElectroMAXTM DH10BTMT1 Phage Resistant Cells,构建Gateway入门cDNA文库,并完全随机挑取单菌落,提取质粒酶切鉴定重组子插入片段大小.构建Gateway目的 载体,通过LR重组反应将入门文库转入此目的 载体成为酵母表达文库,挑取单克隆鉴定重组子插入片段大小.结果 经鉴定,入门文库平均滴度为(6.80±0.10)×105 cfu/ml,文库总容量为7.48×106 cfu,平均插入片段为(2.20±0.20)kb,重组率为100%.酵母表达文库平均滴度为(3.24±0.10)×106 cfu/ml,文库总容量为3.89×107 cfu,平均插入的片段为(2.27±0.15)kb,重组率为95.83%(23/24).结论 构建的小鼠巨噬细胞cDNA入门文库和酵母表达文库都符合高质量文库的要求,可用于进一步的研究.     

Cloning and expression of a chitinase gene from the hyperparasitic fungus Aphanocladium album

Summary Recombinant clones from a cDNA library of an Aphanocladium album chitinase-overproducing mutant strain were isolated by screening with antiserum against a 39 kDa chitinase purified from this hyperparasitic fungus. Analysis of the isolated positive clones indicated that most of them carried the same cDNA. A cDNA from this group was used as a hybridization probe to isolate an 8 kb DNA fragment from a genomic library of the wild-type strain. The chitinase 1 gene was mapped to this fragment by two independent approaches. Its partial DNA sequence was in perfect agreement with an amino-terminal peptide sequence obtained by sequencing 23 amino acids of the 39 kDa chitinase. Its transfer in Fusarium oxysporum resulted in a transformant producting both a protein of about 39 kDa that cross-reacted with the chitinase antiserum and a chitinase activity that was inhibited by the same antiserum. Northern blot analysis indicates that the cloned chitinase gene was subject to catabolite repression and appeared inducible by chitin.     

Genomic cloning and partial characterization of human chymotrypsinogen gene

Summary Chymotrypsinogen is a principal precursor of pancreatic proteolytic enzymes. We previously isolated a cDNA clone for human prechymotrypsinogen from a human pancreatic cDNA library. In the present study, we used this cDNA sequences to isolate genomic DNA clones. Three overlapping cosmid clones spanning approximately 65-kb genomic sequences were isolated from a human cosmid library. The genomic DNA clones were characterized by restriction enzyme mapping and by hybridizing them to subfragments of the cDNA. The sequence tagged sites for human chymotrypsinogen gene were created by designing two oligonucleotides. Furthermore, the isolated genomic clones were confirmed to be localized on chromosome 16q23 by fluorescencein situ hybridization and G-banding analysis.     

Reevaluation of Streptococcus bovis endocarditis cases from 1975 to 1985 by 16S ribosomal DNA sequence analysis

Studies that detected an association between Streptococcus bovis endocarditis and colon carcinoma have not taken into account the recently identified genetic diversity among organisms historically classified as S. bovis. With near full-length 16S ribosomal DNA sequence analysis, organisms cultured from the blood of endocarditis patients at the Mayo Clinic from 1975 to 1985 and previously identified as S. bovis or streptococcus group D nonenterococci were shown to represent S. bovis biotypes I (11 isolates) and II/2 (1 isolate), S. salivarius (1 isolate), and S. macedonicus (1 isolate). Two of the S. bovis biotype I cases were associated with colon cancer. Whether S. bovis biotype II or other organisms closely related to and historically identified as S. bovis (e.g., S. macedonicus) are associated with malignant (or premalignant) colon lesions in humans remains to be definitively determined.     

用定量PCR从杂交阳性融合噬菌斑中分离候选插入片段

目的为了在ｃＤＮＡ文库杂交筛选目的基因过程中，当复筛的杂交信号对应于１个多克隆融合噬菌斑时，能够快速、准确地分离出特异克隆的插入片段。方法依据定量ＰＣＲ扩增的原理，利用初始模板量的不同，通过两次ＰＣＲ反应，鉴定和分离出所需ｃＤＮＡ片段。结果利用这一方法，在β－１，４－半乳糖苷转移酶ｃＤＮＡ的“步移”复筛中，从一个双克隆融合斑中，得到了特异性克隆的插入片段，经测序证实为β－１，４－半乳糖苷转移酶的５′ｃＤＮＡ序列，长１．９ｋｂ，从而完成了全长ｃＤＮＡ的克隆，节省了进行再次复筛的时间和材料。结论该方法简便、快捷，可以显著地节省时间和实验材料，在新基因克隆中有一定的实用价值。     

Cloning of a species-specific antigen of Mycobacterium bovis.

A DNA library from a virulent strain of Mycobacterium bovis was constructed in the expression vector lambda gt11, and the library was probed with antisera to M. bovis. Clones expressing M. bovis antigens were isolated and characterized by using M. bovis-specific monoclonal antibodies that recognize a 22,000-molecular-weight protein (MPB70). MPB70 is a major protein antigen of the vaccine strain of M. bovis BCG and of virulent M. bovis, the causative agent of bovine tuberculosis. Of 32 clones selected by using polyclonal affinity-purified anti-M. bovis sera, 5 were recognized by the anti-MPB-70 monoclonal antibodies, and one monoclonal antibody, SB10, recognized all 5 clones. Characterization of these clones showed that one clone containing a 253-base-pair insert expressed a polypeptide bound by all of the MPB70-specific monoclonal antibodies. Western blots (immunoblots) showed that this cloned protein was recognized by sera from M. bovis-infected cattle, although not all cattle with bovine tuberculosis produced antibodies reactive to this clone. DNA sequencing of the clone showed that it coded for 84 amino acids from positions 17 to 114 of the 161-amino-acid protein, with a 16-peptide deletion between positions 79 and 94. Apart from this deletion, there were seven other variations between the cloned sequence and that deduced from M. bovis BCG MPB70.     

Repetitive DNA sequences as probes for Mycobacterium tuberculosis.

Three cloned segments of Mycobacterium tuberculosis DNA which are promising as clinical probes were identified. An MboI digest of DNA from a clinical isolate of M. tuberculosis was cloned into bacteriophage M13. To identify recombinants specific for the M. tuberculosis complex, plaque lifts were hybridized with M. bovis and M. kansasii DNA. Recombinants which selectively hybridized with M. bovis DNA were characterized by probing slot blots and restriction digests of DNA from various mycobacteria. Three recombinants that did not hybridize to a significant extent with DNA from nontuberculous mycobacteria were identified. These three probes are of special interest because they are each repeated multiple (10 to 16) times in the M. tuberculosis chromosome. These probes were also shown to be useful for fingerprinting strains for epidemiological studies.     

Molecular characterization of a putative heat shock protein cognate gene in Rhynchosciara americana

An hsc70 homologue gene (Rahsc70) of the diptera Rhynchosciara americana was isolated and characterized. We were able to determine the mRNA sequence from an EST of salivary gland cDNA library, and a Rahsc70 cDNA cassette was used as a probe to isolate the genomic region from a genomic library. The mRNA expression of this gene parallels the 2B puff expansion, suggesting its involvement in protein processing, since this larval period corresponds to a high synthetic activity period. During heat shock stress conditions, hsc70 expression decreased. In situ hybridization of polytene chromosomes showed that the Rahsc70 gene is located near the C3 DNA puff. The cellular localization of Hsc70 protein showed this protein in the cytoplasm and in the nucleus.     

Isolation of a cDNA clone and localization of the human glutathione S-transferase 3 genes to chromosome bands 11q13 and 12q13-14

A partial ^cDNA clone of the glutathione S-transferase 3 gene (GST3) was obtained by screening a Agtll human lung cDNA library with antiserum to human lung GST3. The sequence of this cDNA showed two base differences from the coding sequence of a GST3 cDNA isolated from a human placental cDNA library.
Hybridization of the cloned GST3 cDNA to human chromosomes resulted in a primary peak of grains over band 11q13, a localization predicted by prior experiments. An unexpected strong secondary peak of grains was obtained over bands 12q13 and 12q14, indicating that there is a GST3-like gene on the long arm of chromosome 12 in man.