Molecular cloning of DNA fragments produced by restriction endonucleases Sa1I and BamI.

The highly specific restriction endonucleases Sa1I and BamI produce DNA fragments with complementary, cohesive termini that can be covalently joined by DNA ligase. The Escherichia coli kanamycin resistance factor pML21 has one SalI site, at which DNA can be inserted without interfering with the expression of drug resistance or replication of the plasmid. A more convenient cloning vehicle can be made with the tetracycline resistance factor pSC101, since insertion of DNA either at its single site for Sa1I or at that for BamI inactivates plasmid-specified drug resistance but not replication. To take advantage of this insertional inactivation, pSC101 was joined to a Co1E1-ampicillin resistance plasmid having no Sa1I site, and to a Co1E1-kanamycin resistance plasmid having no BamI site. Chimeras formed with the resulting hybrid vehicles can be identified simply by replica plating. These three vehicles, which all replicate under relaxed control, have been used to clone and amplify Drosophila melanogaster DNA fragments.     

Ctk: a protein-tyrosine kinase related to Csk that defines an enzyme family.

We used the polymerase chain reaction with degenerate oligonucleotide primers to search for Csk-related kinases. A cDNA coding for a Csk-like protein-tyrosine kinase was cloned from mouse brain and was designated ctk, for csk-type protein-tyrosine kinase. The 1.9-kb ctk mRNA was found to be expressed predominantly in brain and capable of encoding a 52-kDa protein-tyrosine kinase. The amino acid sequence of Ctk was found to possess 53% identity with mouse Csk, shared all the predicted structural features of Csk, and was capable of phosphorylating the carboxyl-terminal conserved tyrosine of Src family members. Our results thereby indicate that ctk represents a gene that defines a family of structurally and functionally related Csk-like protein-tyrosine kinases.     

The adenylyltransferase domain of bacterial Pnkp defines a unique RNA ligase family

Pnkp is the end-healing and end-sealing component of an RNA repair system present in diverse bacteria from ten different phyla. To gain insight to the mechanism and evolution of this repair system, we determined the crystal structures of the ligase domain of Clostridium thermocellum Pnkp in three functional states along the reaction pathway: apoenzyme, ligase•ATP substrate complex, and covalent ligase-AMP intermediate. The tertiary structure is composed of a classical ligase nucleotidyltransferase module that is embellished by a unique α-helical insert module and a unique C-terminal α-helical module. Structure-guided mutational analysis identified active site residues essential for ligase adenylylation. Pnkp defines a new RNA ligase family with signature structural and functional properties.     

Subtypes of herpes simplex virus type 1 in Japan: classification by restriction endonucleases and analysis of distribution

An attempt was made to classify herpes simplex virus type 1 (HSV-1) isolates into subtypes on the basis of the combination of the gain or loss of specific cleavage sites of HSV-1 genomes with each of three restriction endonucleases (Bam HI, Kpn I, and Sal I). According to the criteria we used for the determination of HSV-1 subtypes, 93 strains of HSV-1 that were isolated in three areas of Japan (Sapporo, Tottori, and Kagawa) were tentatively classified into eight subtypes: subtypes A-H. The bulk of the strains (84 of 93) fell into three subtypes: A, C, and H. There were highly significant differences (P less than .01) in the proportion of subtypes A and H that were isolated in Sapporo as compared with those isolated in Tottori and in Kagawa, which are geographically far from Sapporo. No significant differences, however, were found in subtypes isolated in Tottori as compared with those isolated in Kagawa, which are geographically close to each other. These data suggest that there might be a correlation between the genome structure of HSV-1 and the areas of their isolation in Japan.     

Multicopy plasmid with a structure related to the polyoma virus genome.

In a subclone derived from mouse L(tk-) cells, we found a plasmid present in a high copy number (greater than 5000 copies per cell) that was stably maintained extrachromosomally without any cytopathic effect to the host cells. This plasmid, termed L factor, has two forms: 5.3 and 5.5 kilobase pairs. DNA sequencing and restriction enzyme mapping showed that, although the structure contains DNA sequences common to polyoma virus, plasmid sequences belonging to the regulatory region (the enhancer region) and other regions are quite different from those in polyoma. In cells bearing the plasmid, we detected a low level of material that cross-reacts with antibody to polyoma tumor antigens, suggesting that the plasmids replicate and are maintained in the cells by a mechanism different from that functioning during propagation following infection of papovaviruses.     

Muscle-specific trk-related receptor with a kringle domain defines a distinct class of receptor tyrosine kinases.

Little is known about the signaling pathways by which motoneurons induce synapses on muscle fibers, and no receptors for synapse-inducing signals have yet been identified. Because several other inductive events in development are mediated by receptor tyrosine kinases (RTKs), and because phosphotyrosine staining within muscle fibers is concentrated at synaptic sites, one possibility is that synapse-inducing signals are transduced by a RTK within the muscle fiber. We have used PCR to search for tyrosine kinases within the electric organ of the electric ray Torpedo californica, since this tissue is homologous to muscle but is much more densely innervated and is therefore a rich source of synaptic molecules. We have isolated a RTK that is specifically expressed in electric organ and skeletal muscle. The kinase domain of this receptor is related to the trk family of neurotrophin receptors, but unlike any previously described receptor, the extracellular region of this Torpedo RTK contains a kringle domain close to the transmembrane domain.     

Application of restriction endonucleases as a tool for studying the action of 4-S-ethanolsulfido-cyclophosphamide on DNA with known sequences

Summary Lambda-DNA and plasmid pBR 322-DNA, respectively, were treated in vitro with increasing amounts of 4-S-ethanolsulfido-cyclophosphamide (CPA-P1). Subsequent digestion with restriction endonuclease Pvu II or Mbo II revealed discrete alterations in the cleavage patterns as compared to the controls, indicating subtile changes in DNA structure due to CPA-P1 interaction. In the case of pBR 322-DNA the CPA-P1 treatment of supercoiled circular DNA was more inhibitory to subsequent digestion as compared to the treatment of linearized plasmid DNA molecules.     

Sir2 mediates longevity in the fly through a pathway related to calorie restriction

Calorie restriction can extend life span in a variety of species including mammals, flies, nematodes, and yeast. Despite the importance of this nearly universal effect, little is understood about the molecular mechanisms that mediate the life-span-extending effect of calorie restriction in metazoans. Sir2 is known to be involved in life span determination and calorie restriction in yeast mother cells. In nematodes increased Sir2 can extend life span, but a direct link to calorie restriction has not been demonstrated. We now report that Sir2 is directly involved in the calorie-restriction life-span-extending pathway in Drosophila. We demonstrate that an increase in Drosophila Sir2 (dSir2) extends life span, whereas a decrease in dSir2 blocks the life-span-extending effect of calorie reduction or rpd3 mutations. These data lead us to propose a genetic pathway by which calorie restriction extends life span and provides a framework for genetic and pharmacological studies of life span extension in metazoans.     

EPEC粘附相关蛋白融合表达载体的构建及表达

目的构建与EPEC粘附相关蛋白IntiminC300、EspA、BfpA融合表达载体,并进行表达。方法用PCR方法从EPEC染色体中调取intiminC300、espA、bfpA基因,用基因重组的方法把3个基因片段用2个(Gly4Ser)3连接肽串联克隆到载体pQE30的多克隆区,转化宿主菌M15,用IPTG诱导表达,用SDS-PAGE和Western blot检测表达的融合蛋白。结果酶切和测序表明成功构建了融合表达载体,SDS-PAGE检测表达蛋白的分子质量单位分别为36、43和76kd,与理论值相符,Western blot显示表达的融合蛋白均能被抗His标签抗体识别。结论本研究成功构建了EPEC粘附相关融合蛋白表达载体,并表达目的蛋白,为进一步研究融合蛋白的免疫原性奠定了基础。     

AGTR1 overexpression defines a subset of breast cancer and confers sensitivity to losartan, an AGTR1 antagonist

Breast cancer patients have benefited from the use of targeted therapies directed at specific molecular alterations. To identify additional opportunities for targeted therapy, we searched for genes with marked overexpression in subsets of tumors across a panel of breast cancer profiling studies comprising 3,200 microarray experiments. In addition to prioritizing ERBB2, we found AGTR1, the angiotensin II receptor type I, to be markedly overexpressed in 10–20% of breast cancer cases across multiple independent patient cohorts. Validation experiments confirmed that AGTR1 is highly overexpressed, in several cases more than 100-fold. AGTR1 overexpression was restricted to estrogen receptor-positive tumors and was mutually exclusive with ERBB2 overexpression across all samples. Ectopic overexpression of AGTR1 in primary mammary epithelial cells, combined with angiotensin II stimulation, led to a highly invasive phenotype that was attenuated by the AGTR1 antagonist losartan. Similarly, losartan reduced tumor growth by 30% in AGTR1-positive breast cancer xenografts. Taken together, these observations indicate that marked AGTR1 overexpression defines a subpopulation of ER-positive, ERBB2-negative breast cancer that may benefit from targeted therapy with AGTR1 antagonists, such as losartan.     

Anti-Lu4: a new antibody related to the Lutheran blood group system

Abstract. A new antibody, named anti-Lu4, has been discovered, that reacts with a high frequency red cell antigen. The antibody does not react with dominant or recessive Lu(a-b-) red cells or with the red cells of the propositus and two of her five siblings. Because the propositus and her sibs are all Lu(a-b+), anti-Lu4 cannot be the same as anti-Lu^aLu^b. Attempts to identify a low frequency allele to Lu4 were unsuccessful. The results suggest that Lu^a, Lu^b and Lu4 have a common precursor.     

Positive selection of primate TRIM5alpha identifies a critical species-specific retroviral restriction domain

Primate genomes encode a variety of innate immune strategies to defend themselves against retroviruses. One of these, TRIM5alpha, can restrict diverse retroviruses in a species-specific manner. Thus, whereas rhesus TRIM5alpha can strongly restrict HIV-1, human TRIM5alpha only has weak HIV-1 restriction. The biology of TRIM5alpha restriction suggests that it is locked in an antagonistic conflict with the proteins encoding the viral capsid. Such antagonistic interactions frequently result in rapid amino acid replacements at the protein-protein interface, as each genetic entity vies for evolutionary dominance. By analyzing its evolutionary history, we find strong evidence for ancient positive selection in the primate TRIM5alpha gene. This selection is strikingly variable with some of the strongest selection occurring in the human lineage. This history suggests that TRIM5alpha evolution has been driven by antagonistic interactions with a wide variety of viruses and endogenous retroviruses that predate the origin of primate lentiviruses. A 13-aa "patch" in the SPRY protein domain bears a dense concentration of positively selected residues, potentially implicating it as an antiviral interface. By using functional studies of chimeric TRIM5alpha genes, we show that this patch is generally essential for retroviral restriction and is responsible for most of the species-specific antiretroviral restriction activity. Our study highlights the power of evolutionary analyses, in which positive selection identifies not only the age of genetic conflict but also the interaction interface where this conflict plays out.     

The WD40 and FYVE domain containing protein 2 defines a class of early endosomes necessary for endocytosis

The FYVE domain binds with high specificity and avidity to phosphatidylinositol 3-phosphate. It is present in approximately 30 proteins in humans, some of which have been implicated in functions ranging from early endosome fusion to signal transduction through the TGF-beta receptor. To develop a further understanding of the biological roles of this protein family, we turned to the nematode Caenorhabditis elegans, which contains only 12 genes predicted to encode for phosphatidylinositol 3-phosphate binding, FYVE domain-containing proteins, all of which have homologs in the human genome. Each of these proteins was targeted individually by RNA interference. One protein, WDFY2, produced a strong inhibition of endocytosis when silenced. WDFY2 contains WD40 motifs and a FYVE domain, is highly conserved between species, and localizes to a set of small endosomes that reside within 100 nm from the plasma membrane. These endosomes are involved in transferrin uptake but lack the classical endosomal markers Rab5 and EEA1. Silencing of WDFY2 by siRNA in mammalian cells impaired transferrin endocytosis. These studies reveal the important, conserved role of WDFY2 in endocytosis, and the existence of a subset of early endosomes, closely associated with the plasma membrane, that may constitute the first stage of endocytic processing of internalized cargo.     

Murine Th1 clone defines a 60 kD tachyzoite antigen of Toxoplasma gondii

Toxoplasma gondii -specific murine CD4⁺ T cell clone 3Tx9 belongs to the Th1 subtype by virtue of secreting high levels of interleukin(IL)-2, interferon-γ and tumor necrosis factor without producing IL4 and IL10. To characterize the clonal antigen, Toxoplasma lysate was separated by SDS-PAGE and probed in T cell blot analysis with 3Tx9 T cells, revealing a fraction of about 60 kD molecular weight. This fraction proved highly stimulatory also for CD4⁺ splenocytes isolated from infected mice. The expression pattern of the relevant 60 kD antigen was determined by challenge of clone 3Tx9 with T. gondii strains from all three intraspecies subgroups and tachyzoites versus bradyzoites isolated from two strains as a source of antigen. While the T cell clone reacted with tachyzoites of all strains tested, bradyzoites lacked antigenic activity. Parallel T cell blot and ELISA confirmed co-migration of the T cell-stimulatory antigen p60 and rhoptry proteins ROP1, ROP2,3,4, and ROP5 among which ROP1 is a molecule of similar size and has only been shown on tachyzoites. However, a ROP1 knock-out Toxoplasma mutant still had antigen activity for 3Tx9 T cells. Since the two known tachyzoite-specific proteins, surface antigens SAG1/p30 and SAG2/p22, have a much lower molecular weight, we suggest that p60 represents a new T. gondii tachyzoite marker which is defined by clone 3Tx9.