The Drosophila PROS-28.1 gene is a member of the proteasome gene family

In the present communication, we report the identification of a new gene family which encodes the protein subunits of the proteasome. The proteasome is a high-M_r complex possessing proteolytic activity. Screening a Drosophila λgt11 cDNA expression library with the proteasome-specific antibody N19-28 we isolated a clone encoding the 28-kDa No. 1 proteasome protein subunit. In accordance with the nomenclature of proteasome subunits in Drosophila, the corresponding gene is designated PROS-28.1, and it encodes an mRNA of 1.1 kb with an open reading frame of 249 amino acids (aa). Genomic Southern-blot hybridization shows PROS-28.1 to be a member of a family of related genes. Analysis of the predicted aa sequence reveals a potential nuclear targeting signal, a potential site for tyrosine kinase and a potential cAMP/cGMP-dependent phosphorylation site. The aa sequence comparison of the products of PROS-28.1 and PROS-35 with the C2 proteasome subunit of rat shows a strong sequence similarity between the different proteasome subunits. The data suggest that at least a subset of the proteasome-encoding genes belongs to a family of related genes (PROS gene family) which may have evolved from a common ancestral PROS gene.     

The Drosophila PROS-28.1 gene is a member of the proteasome gene family

In the present communication, we report the identification of a new gene family which encodes the protein subunits of the proteasome. The proteasome is a high-M_r complex possessing proteolytic activity. Screening a Drosophila λgt11 cDNA expression library with the proteasome-specific antibody N19-28 we isolated a clone encoding the 28-kDa No. 1 proteasome protein subunit. In accordance with the nomenclature of proteasome subunits in Drosophila, the corresponding gene is designated PROS-28.1, and it encodes an mRNA of 1.1 kb with an open reading frame of 249 amino acids (aa). Genomic Southern-blot hybridization shows PROS-28.1 to be a member of a family of related genes. Analysis of the predicted aa sequence reveals a potential nuclear targeting signal, a potential site for tyrosine kinase and a potential cAMP/cGMP-dependent phosphorylation site. The aa sequence comparison of the products of PROS-28.1 and PROS-35 with the C2 proteasome subunit of rat shows a strong sequence similarity between the different proteasome subunits. The data suggest that at least a subset of the proteasome-encoding genes belongs to a family of related genes (PROS gene family) which may have evolved from a common ancestral PROS gene.     

Nongranular proteolytic enzymes of rat IL-2–activated natural killer cells. II. Purification and identification of rat A-NKP 1 and A-NKP 2 as constituents of the multicatalytic proteinase (proteasome) complex

We have recently described nongranular, cytosolic, high-molecular-weight trypsin-like (A-NKP 1) and chymotrypsin-like (A-NKP 2) proteases of interleukin-2–activated rat natural killer (A-NK) cells. A functional correlation between the inactivation of A-NKP 2 and the inhibition of rat A-NK cell-mediated cytotoxicity was found. Herein we describe the 6,000-fold purification of A-NKP 2 to apparent homogeneity following: isopycnic sucrose gradient fractionation of postnuclear supernatants, molecular sieve chromatography, and heparin-Sepharose® chromatography. We also report the novel finding that A-NKP 2 as well as A-NKP 1, derived from either rat A-NK cells or the rat NK leukemic cell line CRNK-16, are constituents of the multicatalytic proteinase (MCP/proteasome) complexes of these cells. Characteristic biochemical, biophysical, and electron microscopic/ultrastructural similarity to the rat liver proteasome was observed. However, Western blot analysis using polyclonal antibodies to the rat liver proteasome clearly indicated differences in the rat hepatic proteasome and the CRNK-16–derived proteasomal subunits. The identification, characterization, and purification of A-NKP 1 and A-NKP 2, described herein, now allow for further investigation of the potential role of these proteasome components in NK cell function. Moreover, the proteasome of NK and A-NK cells can now be compared and contrasted to the granzymes of lytic granules with respect to their role in cell-mediated cytotoxicity. © 1994 Wiley-Liss, Inc.     

Construction of a human full-length cDNA bank

We aimed to construct a full-length cDNA bank from an entire set of human genes and to analyze the function of a protein encoded by each cDNA. To achieve this purpose, a multifunctional phagemid shuttle vector, pKAl, was constructed for preparing a high-quality cDNA library composed of full-length cDNA clones which can be sequenced and expressed in vitro and in mammalian cells without subcloning the cDNA fragment into other vectors. Using this as a vector primer, we have prepared a prototype of the bank composed of full-length cDNAs encoding 236 human proteins whose amino acid sequences are identical or similar to known proteins. Most cDNAs contain a putative cap site sequence, some of which show a pyrimidine-rich conserved sequence exhibiting polymorphism. It was confirmed that the vector permits efficient in vitro translation, expression in mammalian cells and the preparation of nested deletion mutants.     

Isolation and characterization of a human gene that encodes a new subclass of protein tyrosine kinases

We describe the isolation and cDNA sequence of a novel human gene, which is distinct from all known members of the human src family of proto-oncogenes. In contrast to these, an autophosphorylation site corresponding to Tyr416, as well as the equivalent of Tyr527 in p60c-src, are missing in the amino acid (aa) sequence deduced from this gene. Furthermore, no N-terminal myristylation site is found. Our human clone is 98% identical at the aa level to a gene which was isolated independently from neonatal rat brain and was termed csk for c-src kinase. We, therefore, propose to designate the present human gene CSK. In Northern blot experiments, CSK was found to be expressed in human lung and macrophages. Due to its extreme conservation across species barriers, the CSK product is likely to exert important regulatory functions. On the basis of its expression in tissues, not typically expressing high c-src levels, it can be assumed that its regulatory role is more general and may also involve other tyrosine kinases.     

Cloning of a gene encoding thermostable cellobiohydrolase from the thermophilic fungus Chaetomium thermophilum and its expression in Pichia pastoris

Aims:  A new cellobiohydrolase (CBH) gene ( cbh3 ) from Chaetomium thermophilum was cloned, sequenced and expressed in Pichia pastoris .
Methods and Results:  Using RACE-PCR, a new thermostable CBH gene ( cbh3 ) was cloned from C. thermophilum . The cDNA of the CBH was 1607 bp and contained a 1356 bp open reading frame encoding a protein CBH precursor of 451 amino acid residues. The mature protein structure of C. thermophilum CBH3 only comprises a catalytic domain and lacks cellulose-binding domain and a hinge region. The gene was expressed in P. pastoris . The recombinant CBH purified was a glycoprotein with a size of about 48·0 kDa, and exhibited optimum catalytic activity at pH 5·0 and 60 °C. The enzyme was more resistant to high temperature. The CBH could hydrolyse microcrystalline cellulose and filter paper.
Conclusions:  A new thermostable CBH gene of C. thermophilum was cloned, sequenced and overexpressed in P. pastoris .
Significance and Impact of the Study:  This CBH offers an interesting potential in saccharification steps in both cellulose enzymatic conversion and alcohol production.     

Improved method for cloning DNA complementary to minor mRNAs: preparation of a hybridization probe from purified mRNAs encoding intermediate filament proteins

A procedure for the rapid fractionation of mRNA has been used to enrich mRNAs encoding a set of intermediate filament proteins in trophoblastoma cells. The procedure involves sucrose-gradient fractionation followed by high-resolution preparative gel electrophoresis. Part of the enriched mRNA preparation has been used to prepare a hybridization probe to screen a trophoblastoma cDNA library in Escherichia coli. A small proportion of the clones hybridized to the probe, and among these a specific clone was identified.     

Cloning and sequencing of cDNA encoding for a human sperm antigen involved in fertilization

A cDNA encoding for a sperm antigen, designated NZ-2, was cloned and sequenced from human testis cDNA-λgt11 expression library by using antibodies to human sperm surface antigens belonging to 14–18 kD molecular regions. These sperm antigens are involved in binding to zona pellucida of the human oocyte. Computer generated translation analysis of 963-bp cDNA yielded an open reading frame (ORF) of 163 amino acids (aa) with first ATG, Met start codon at nucleotide (nt) 335 and the stop codon TAA at nt 824. The NZ-2 cDNA has 335-bp 5′ and 139-bp 3′ noncoding regions. The translated protein has a calculated molecular weight of ∼19 kD, and has two casein kinase II (CK-2) sites at aa 94–97 and 149–152, respectively. Extensive computer search in the GenBank, National Biomedical Research Foundation (NBRF), and Swiss database indicates it to be a novel protein, having 99.5% nt sequence similarity, except for the first 40-bp, only with the human bacterial artificial chromosome (BAC) containing cloned human sperm DNA, at position 76935–76009. The in vitro translated product of T3 RNA polymerase by using NZ-2 cDNA digested with XhoI yielded a protein band of ∼20 kD, indicating it to be sense strand. The in vitro translated product of T7 RNA polymerase by using NZ-2 cDNA digested with NotI did not yield any protein band, indicating it to be antisense strand. The ∼20 kD protein was recognized specifically by the antisperm IgG, not by the control IgG in the Western blot procedure. Neither antisperm IgG nor control IgG recognized any protein band in the in vitro translation products of the antisense strand. The human genomic DNAs from three different cells/tissues namely, sperm, kidney, and testis when cut by HindIII, and then hybridized with the NZ-2 cDNA probe in the Southern blot procedure, showed restriction fragment length polymorphism (RFLP). The recombinant human sperm NZ-2 antigen may find applications in the development of a contraceptive vaccine, and diagnosis and treatment of infertility in humans. Mol. Reprod. Dev. 51:176–183, 1998. © 1998 Wiley-Liss, Inc.     

Vectors for ligation-independent construction of lacZ gene fusions and cloning of PCR products using a nicking endonuclease

Several ligation-independent cloning methods have been developed that offer advantages for construction of recombinant plasmids at high efficiency while minimizing cloning artifacts. Here we report new plasmid vectors that use the nicking endonuclease Nt.BspQI to generate extended single stranded tails for direct cloning of PCR products. The vectors include pLacCOs1, a ColE1-derivative plasmid imparting resistance to ampicillin, which allows facile construction of lacZ translational fusions and pKanCOs1, a pSC101-derivative cloning vector that imparts resistance to kanamycin, for cloning of PCR amplicons from genomic DNA as well as from ampicillin-based plasmids. We have successfully used these plasmids to directionally clone and characterize bacterial promoters that exhibit temperature regulated expression, as well as for cloning a variety of PCR products. In all cases, constructs with the correct configurations were generated at high efficiency and with a minimal number of manipulations. The cloning vectors can also be easily modified to incorporate additional reporter genes or to express epitope-tagged gene products.     

Molecular cloning and expression of a Tetrahymena pyriformis ubiquitin fusion gene coding for a 53-amino-acid extension protein.

Summary The genome of Tetrahymena pyriformis has been shown to contain a ubiquitin multigene family consisting of several polyubiquitin genes and at least one ubiquitin fusion gene. We report here the isolation and characterization of one genomic clone (pTUl1), that encodes a ubiquitin extension protein. A comparison of the predicted amino acid sequence of the ubiquitin extension protein gene of T. pyriformis with those from other organisms indicated a high degree of homology. However, the Tetrahymena ubiquitin extension protein contains 53 and not 52 amino acids. This feature is different from all ubiquitin 52-amino-acid extension protein genes thus far sequenced. Furthermore, we found an array of four cysteine residues similar to those found in nucleic acid binding proteins. Also, the C-terminal sequence possesses a conserved motif which may represent a nuclear translocation signal. The ubiquitin 53-amino-acid extension protein gene encodes the smallest class of ubiquitin mRNAs in T. pyriformis.     

Molecular analysis of the gene encoding a protein component of the Eikenella corrodens adhesin complex that is close to the carbohydrate recognition domain

A monoclonal antibody against a lectin-like substance (LS) of Eikenella corrodens (Ec) was used for screening the Ec DNA library. Three positive clones that carried an identical 12-kb segment were obtained. A 25-kDa protein, which specifically binds to the antibody, was overproduced in all of the Escherichia coli clones. Deletion analysis showed that the gene encoding the 25-kDa protein was located within a 1.2-kb segment. The nucleotide (nt) sequence of this segment contained an open reading frame encoding a protein of 24 600 Da. We purified the 25-kDa protein from the cloned E. coli strain. The sequence of the first 10 amino acids (aa) from the N-terminus of the purified 25-kDa protein agreed with that deduced from the nt sequence. Since the monoclonal antibody used in this study inhibits the physiological activity of EcLS, we concluded that the 25-kDa protein is a component of the adhesin complex, which is located near the carbohydrate recognition domain of lectin in EcLS.     

Genome-wide profiling of gene expression in 29 normal human tissues with a cDNA microarray.

We have performed a comprehensive analysis of the expression profiles in 25 adult and 4 fetal human tissues by means of a cDNA microarray consisting of 23,040 human genes. This study revealed a number of genes that were expressed specifically in each of those tissues. Among the 29 tissues examined, 4,080 genes were highly expressed (at least a five-fold expression ratio) in one or only a few tissues and 1,163 of those were expressed exclusively (more than a ten-fold higher expression ratio) in a particular tissue. Expression of some of the genes in the latter category was confirmed by northern analysis. A hierarchical clustering analysis of gene-expression profiles in nerve tissues (adult brain, fetal brain, and spinal cord), lymphoid tissues (bone marrow, thymus, spleen, and lymph node), muscle tissues (heart and skeletal muscle), or adipose tissues (mesenteric adipose and mammary gland) identified a set of genes that were commonly expressed among related tissues. These data should provide useful information for medical research, especially for efforts to identify tissue-specific molecules as potential targets of novel drugs to treat human diseases.     

烟实夜蛾性信息素合成激活肽基因的分子克隆

根据家蚕Bombyx mori和玉米夜蛾Helicoverpa zea的性信息素合成激活肽基因序列,设计若干套引物, 以烟实夜蛾Heliothis assulta基因组DNA为模板进行PCR扩增, 得到0.63 kb的特异性DNA片段。该片段克隆进适当载体,序列测定和同源比较, 查明烟实夜蛾的基因组中存在性信息素合成激活肽基因。烟实夜蛾的性信息素合成激活肽由33个氨基酸组成, C末端是FXPRL结构,是目前发现的第4种昆虫性信息素合成激活肽。在该神经肽第14和第15个氨基酸之间, 插入一个0.42 kb的内含子。 进一步的分析证明了烟实夜蛾的性信息素合成激活肽基因在潜成虫期的食道下神经节中表达。