Identification, purification, and molecular cloning of autonomously replicating sequence-binding protein 1 from fission yeast Schizosaccharomyces pombe.

Autonomously replicating sequence (ARS) elements of the fission yeast Schizosaccharomyces pombe contain multiple imperfect copies of the consensus sequence reported by Maundrell et al. [Maundrell K., Hutchison, A. & Shall, S. (1988) EMBO J. 7, 2203-2209]. When cell free extracts of S. pombe were incubated with a dimer or tetramer of an oligonucleotide containing the ARS consensus sequence, several complexes were detected using a gel mobility-shift assay. The proteins forming these complexes also bind ars3002, which is the most active origin in the ura4 region of chromosome III of S. pombe. One protein, partly responsible for the binding activity observed with crude extracts, was purified to near homogeneity. It is a 60-kDa protein and was named ARS-binding protein 1 (Abp1). Abp1 preferentially binds to multiple sites in ARS 3002 and to the DNA polymer poly[d(A.T)]. The cloning and sequence of the gene coding for Abp1 revealed that it encodes a protein of 59.8 kDa (522 amino acids). Abp1 has significant homology (25% identity, 50% similarity) to the N-terminal region (approximately 300 amino acids) of the human and mouse centromere DNA-binding protein CENP-B. Because centromeres of S. pombe contain a high density of ARS elements, Abp1 may play a role connecting DNA replication and chromosome segregation.     

An IgE-binding protein with a distinctive repetitive sequence and homology with an IgG receptor.

Proteins that bind IgE play important roles in both the synthesis and function of IgE are therefore intimately involved in IgE-mediated human allergic disorders. This report describes the structure of an IgE-binding protein, as predicted from sequencing a cDNA cloned from rat basophilic leukemia cells. This protein contains two domains: the amino-terminal domain (140 amino acids) consists of a highly conserved repetitive amino acid sequence, Tyr-Pro-Gly-Pro/Gln-Ala/Thr-Pro/Ala-Pro-Gly-Ala, whereas the carboxyl-terminal domain (122 amino acids) shares significant sequence homology with a domain of lymphocyte/macrophage receptor for the Fc portion of IgG. Other proteins with this type of structure but with affinity for other immunoglobulin isotypes may exist and may represent a heretofore unidentified component of the immune system.     

Identification and molecular cloning of a soluble human granulocyte-macrophage colony-stimulating factor receptor.

Granulocyte-macrophage colony-stimulating factor (GM-CSF) plays an important role in hematopoiesis and host defense via interaction with specific cell-surface receptors in target tissues. We identified a truncated, soluble form of the low-affinity GM-CSF receptor (GMR) in chorio-carcinoma cells. Low-affinity GMR cDNAs encoding both the membrane-bound and soluble receptors were obtained by PCR using primers corresponding to the published sequence. Clones encoding the soluble receptor were identical in sequence to the membrane-bound form but contained a 97-nucleotide internal deletion. The amino acid sequence of this deleted cDNA predicts a protein that lacks the 84 C-terminal amino acids of the membrane-bound receptor, including the transmembrane and cytoplasmic domains, and contains 16 different amino acids at its C terminus. Expression of the soluble GMR cDNA in murine psi-AM cells as well as GM-CSF-dependent myeloid 32Dc13 cells produced a secreted protein that retained its capacity to bind GM-CSF in solution. RNase protection analysis indicates that this variant cDNA is derived from a naturally occurring mRNA. Soluble receptors have been identified for several other hematopoietin receptors and may be a general feature of this class. The striking similarity between the soluble form of the GMR and other hematopoietin receptors suggests that soluble binding proteins may play an important role in regulating the broad spectrum of biological responses mediated by these cytokines.     

Identification of a protein kinase multigene family of Dictyostelium discoideum: molecular cloning and expression of a cDNA encoding a developmentally regulated protein kinase.

We have identified protein kinase genes of Dictyostelium by using highly conserved amino acid sequence motifs to design the synthesis and amplification of DNA fragments by polymerase chain reactions (PCRs). Cloning and sequencing the PCR products have revealed five different members of the protein kinase multigene family. These five putative kinases showed varying degrees of amino acid sequence similarity (40-70%) to protein kinases in data bases and contained invariant amino acid residues characteristic of protein kinases. DNA from PCR was labeled and used to isolate several lambda gt11 cDNA clones, including one full-length one (Dd kinase-2). The nucleotide sequence of Dd kinase-2 contained a region identical to one of the cloned kinase fragments amplified by PCR, and based on the deduced amino acid sequence Dd kinase-2 encodes a protein of 479 amino acids. A 350-amino acid kinase domain at the C-terminal end shows high homology to the catalytic domains of protein kinase A, protein kinase C, S-6 kinase of Xenopus, and the suppressor of cdc25 of yeast. The N-terminal domain is highly basic and also contains alternating threonine/proline residues. The cDNA hybridized to a single copy gene but to two differentially regulated mRNAs--a 2.0-kilobase mRNA that is expressed in vegetative cells and a 2.2-kilobase mRNA that is expressed during development. The larger mRNA is induced by cAMP by using a cell-surface receptor-mediated signal transduction pathway.     

Identification, cloning, and characterization of an immune activation gene.

We have identified an immune activation gene, denoted Act-2, by differential hybridization screening of an activated T-cell library. The gene is induced rapidly after T-cell activation with phytohemagglutinin, B-cell activation with Staphylococcus aureus Cowan I, and monocyte activation with lipopolysaccharide. We have isolated a cDNA containing the full-length coding region. The deduced amino acid sequence predicts an open reading frame of 92 amino acids, including a very hydrophobic N terminus, which by weight matrix score is predicted to be a signal peptide. Using a baculovirus expression system, we have shown that this gene encodes a secreted product. It is therefore possible that Act-2 represents a newly discovered cytokine.     

Identification and molecular cloning of Moloney mouse sarcoma virus-specific sequences from uninfected mouse cells.

When uninfected mouse cell DNA is cleaved with restriction endonuclease EcoRI, a DNA fragment of 14.0 kilobases can be identified by hybridization to cloned DNA containing sarcoma specific sequences of Moloney mouse sarcoma virus (M-MSVsrc). The cellular DNA fragment contains the entire M-MSVsrc specific sequences. The 14.0-kilobase EcoRI DNA fragment was cloned in bacteriophage lambda. The sequence organization of a recombinant clone, lambda . MTX-1, was analyzed by restriction endonuclease mapping, nuclease S1 mapping, and electron microscopy. The results indicate that lambda . MTX-1 contains an uninterrupted stretch of 1.0 kilobase similar to that found in the M-MSV genome.     

Construction of chimeric vaccinia viruses by molecular cloning and packaging.

Foreign DNA was inserted into unique restriction endonuclease cleavage sites (Sma I or Not I) of the 200,000-base-pair vaccinia virus genome by direct molecular cloning. The modified vaccinia virus DNA was packaged in fowlpox virus-infected avian cells, and chimeric vaccinia virus was isolated from mammalian cells not supporting the growth of the fowlpox helper virus. In contrast to the classical "in vivo" recombination technique, chimeric viruses with inserts in both possible orientations and families of chimeras with multiple inserts were obtained. The different genomic configurations of chimeric viruses provide a broader basis for screening of optimal viruses. In addition to packaging in avian cells, a second packaging procedure for vaccinia DNA, based on the abortive infection of mammalian cells with the fowlpox helper virus, was developed. This procedure permits simultaneous packaging and host-range selection for the packaged virus. The cloning/packaging procedure allows the direct insertion of foreign DNA without the need for plasmids having flanking regions homologous to viral nonessential regions and is independent of inefficient in vivo recombination events. By direct cloning and packaging, about 5-10% of the total vaccinia virus yield consisted of chimeras. The procedure is, therefore, a useful tool in molecular virology.     

Formation of IgE-binding factors by human T-cell hybridomas.

Normal human T cells that proliferated in the presence of interleukin 2 (IL-2) formed IgE-binding factors when incubated with human IgE. These cells were then fused with a mutant of the human T-cell line CEM. Incubation of five hybridomas with human IgE or culture of the cells in IgE-coated wells resulted in the formation of IgE-binding factors. One hour of incubation with 10 micrograms of human IgE per ml was sufficient to induce the hybridomas to form IgE-binding factors. Polymerized IgE was much more efficient than monomeric IgE for the induction of the factor formation. As little as 10 ng of IgE dimer per ml was sufficient to induce factor formation. The IgE-binding factors produced by the hybridomas bound to human IgE-coated Sepharose and were recovered from the beads by elution at acid pH. The factors had low affinity for rat IgE but failed to bind to human IgG. The IgE-binding factors formed by four hybridomas had a Mr between 25,000 and 30,000, whereas one hybridoma formed IgE-binding factors of Mr 30,000 and Mr 15,000. The IgE-binding formed by all of the hybridomas had affinity for concanavalin A, indicating that the factors are glycoproteins.     

Identification of disease genes by positional cloning

Positional cloning or reverse genetics is a combination of techniques that has been extraordinarily successful in finding the genes that cause many inherited disorders, including some that affect the cardiovascular system. This approach consists of finding a DNA marker that cosegregates with the disorder and then using the tools of molecular biology to examine systematically the DNA in the vicinity of such a marker until the gene is identified. In addition to the availability of preclinical diagnostic tests for individuals at risk, the identification of such genes might also provide the basis for targeted drug design. In the longer term, with the emerging technologies for the delivery of genes into cells, finding the genes that cause inherited disorders raises the possibility of eventual therapeutic intervention.     

Identification of genes expressed in premalignant breast disease by microscopy-directed cloning.

Histopathologic study of human breast biopsy samples has identified specific lesions which are associated with a high risk of development of invasive breast cancer. Presumably, these lesions (collectively termed premalignant breast disease) represent the earliest recognizable morphologic expression of fundamental molecular events that lead to the development of invasive breast cancer. To study molecular events underlying premalignant breast disease, we have developed a method for isolating RNA from histologically identified lesions from frozen human breast tissue. This method specifically obtains mRNA from breast epithelial cells and has identified three genes which are differentially expressed in premalignant breast epithelial lesions. One gene identified by this method is overexpressed in four of five noncomedo ductal carcinoma in situ lesions and appears to be the human homologue of the gene encoding the M2 subunit of ribonucleotide reductase, an enzyme involved in DNA synthesis.     

Ascaris suum: molecular cloning of an intermediate filament*

It has been proposed that intermediate filament proteins are involved in force transduction from the muscle cells through the hypodermis to the cuticle of nematodes. An additional role of intermediate filaments as excretory/secretory components of parasitic nematodes is under discussion. We report on the molecular characterization of the cDNA clone AsIF of the intestinal nematode parasite Ascaris suum, encoding a member of the intermediate filament protein family by sequence comparison with intermediate filaments of other nematodes. We also show the precise location of the product encoded by AsIF within the organism by immunoelectron microscopy.     

Identification and sequence analysis of a second form of prolactin receptor by molecular cloning of complementary DNA from rabbit mammary gland.

Two lambda gt11 clones containing fragments of cDNA encoding the prolactin receptor from rabbit mammary gland were isolated using a rat liver prolactin receptor cDNA probe. An 1848-base-pair open reading frame encodes a mature prolactin-binding protein of 592 amino acids that contains three domains: (i) the extracellular, amino-terminal, prolactin-binding region of 210 residues; (ii) the transmembrane region of 24 residues; and (iii) the intracellular, carboxyl-terminal domain of 358 residues. This latter domain is much longer than the cytoplasmic domain (57 residues) previously described for the rat liver prolactin receptor. In addition, the sequence identity of this form of prolactin receptor with the growth hormone receptor is extended in the cytoplasmic domain.     

乙型肝炎病毒X蛋白反式激活基因XTP3的克隆化研究

目的 应用抑制性消减杂交(SSH)技术筛选乙型肝炎病毒(HBV)x蛋白(HBxAg)反式激活基因，克隆HBxAg反式激活相关靶基因。方法以HBxAg表达质粒PcDNA3．1(-)-x转染HepG2细胞，以空载体PcDNA3．1(-)为对照；制备转染后的细胞裂解液，提取mRNA并逆转录为cDNA，经RsaI酶切后，将实验组cDNA分成两组，分别与两种不同的接头衔接，再与对照组cDNA进行两次消减杂交及两次抑制性PCR，将产物与PGEM-Teasy载体连接，构建cDNA消减文库，并转染大肠杆菌进行文库扩增，随机挑选克隆PCR扩增后进行测序及同源性分析，发现其中之一为未知基因片段，与GenBank中注册的已知功能基因序列没有同源性。通过序列同源性搜索比对，电子拼接成功，根据基因起始密码子的Kozak规则和终止密码子下游保守的多聚腺苷信号序列，初步确定新型基因序列。从转染了pcDNA3．1(-)-x的HePG2细胞提取总RNA，以RT-PCR技术扩增获得该新基因的全长序列，并测序加以证实。结果 发现的新基因命名为XTP3，在GenBank中注册，注册号为AF490252。xTP3基因的编码序列全长为1020个核苷酸(nt)，编码产物由340个氨基酸残基(aa)组成。结论 应用抑制性消减杂交技术成功筛选与克隆HBxAg反式激活新型靶基因XTP3，为进一步阐明HBxAg反式调节作用及其在HBV感染中的分子生物学机制提供理论依据和研究方法。     

A putative Ca2+-binding protein: structure of the light subunit of porcine calpain elucidated by molecular cloning and protein sequence analysis.

cDNA clones specific for the light subunit of porcine calpain I have been isolated from a porcine kidney cDNA library. The complete primary structure of the light subunit has been revealed by nucleotide sequence analysis of the cDNA clones isolated and amino acid sequence analysis of peptides isolated from the purified mature protein. We found that the light subunit contains two distinct domains. Domain I, the amino-terminal half, has two unusually long, paired polyglycyl sequences and may serve as a binding site to the heavy subunit. Domain II, the carboxyl-terminal half, is a region highly homologous to the putative Ca2+-binding domain of the heavy subunit of chicken calpain elucidated recently. This region has four potential Ca2+-binding sites, each having the "E-F hand" structure. Our results suggest that the Ca2+-mediated proteolytic activity of calpain is controlled through the cooperative and/or sequential actions of multiple Ca2+-binding sites present in both two-subunit molecules, heavy and light subunits of calpain.