Molecular cloning of the dog homologue of the lymphocyte antigen CD28

The nucleotide data reported in this paper have been submitted to the GenBank nucleotide sequence database and have been assigned the accession number L22178.     

Expression of the Blast-1 activation/adhesion molecule and its identification as CD48.

We have analyzed the induction and expression of Blast-1 at the mRNA and protein levels and demonstrated its identity with CD48. Blast-1/CD48 is expressed on a wider range of cell types, notably T cells and monocytes, than previously thought, but appears to be restricted to lymphoid and myeloid cells. Resting B and T cells express Blast-1/CD48 molecules at the cell surface; however, they lack the epitope recognized by the 17D6 mAb. Resting B cells express no detectable Blast-1/CD48 mRNA. Induction by EBV infection or stimulation with PMA, IL-4, or PHA results in increased levels of Blast-1/CD48 protein (both 6.28 and 17D6 epitopes) at the cell surface. Detailed analysis of EBV-induced expression revealed that it is due to increased steady-state levels of Blast-1/CD48 mRNA induced by transforming but not nontransforming strains of the virus. Induction by IL-1 beta, ionomycin, or suboptimal levels of PMA plus ionomycin results in increased expression of the 17D6 epitope only. In transfected Cos-7 cells Blast-1/CD48 at the cell surface expresses only the 6.28 epitope, whereas cytoplasmic molecules express both 17D6 and 6.28 epitopes. We suggest that these results are most consistent with the idea that Blast-1/CD48 molecules are complexed at the surface of resting cells and Cos-7 cells, resulting in masking of the 17D6 epitope. Activation causes dissociation of the complex, revealing the 17D6 epitope. The existence of 17D6+6.28- Blast-1/CD48 molecules was demonstrated by immunoprecipitation analysis, which also revealed that, unlike the rest of the molecules, this subset was resistant to digestion with glyosylphosphatidylinositol-specific phospholipase C.     

分子标记技术及其在分离和克隆水稻基因中的应用

分子标记是近些年来发展起来的一种分子生物学技术,利用各种分子标记可以构建水稻高密度的分子遗传图谱,并进行基因定位和克隆。本文概述了几种常用的分子标记及其在研究水稻基因中应用进展。     

Molecular cloning of mouse collectin liver 1

Collectins are members of the superfamily of vertebrate C-type lectins that contain a collagen-like region, and are involved in first-line host defense. We earlier cloned and characterized a new kind of collectin, collectin liver 1 (CL-L1). In this study, we isolated the mouse homologue of CL-L1 encoding 277 amino acid residues; its deduced protein sequence was 88% identical with human CL-L1. Mouse CL-L1 mRNA was expressed mainly in the liver and stomach, but was found also in muscles, testes, intestines, and embryos. In mouse embryos, the level of CL-L1 mRNA gradually increased with embryonic age. In 16-day-old mouse embryos, CL-L1 mRNA was expressed in the liver, amnion, and visceral yolk sac. The mouse CL-L1 gene, Cll1 was found on chromosome 15 in a region syntenic with human chromosome 8q. CL-L1 was a highly conserved protein in mammals, birds, and fish.     

Molecular cloning and expression of Pgp-1. The mouse homolog of the human H-CAM (Hermes) lymphocyte homing receptor

Mouse phagocytic glycoprotein-1 (Pgp-1; Ly-24) is a 95-kDa glycoprotein of unknown function that has served as an important T cell/leukocyte differentiation marker. Recent work has suggested that it may be related to a human 85- to 95-kDa glycoprotein (termed variously the Hermes Ag/lymphocyte homing receptor, ECMRIII, P80, and CD44) that is involved in lymphocyte binding to high endothelial venules in the process of lymphocyte homing, and has been implicated in other cell adhesion events. The widespread expression of this molecular class in diverse organ systems suggests a broad role in cellular adhesion, and has led to the unifying designation homing-cellular adhesion molecule (H-CAM). By using human H-CAM cDNA probes, we have isolated a full-length cDNA for the mouse homolog. Comparison of the human and mouse sequences reveals that an N-terminal domain homologous to cartilage proteoglycan core and link proteins, as well as the C-terminal transmembrane and cytoplasmic sequences, are highly conserved (89% and 86% identity, respectively). In contrast, a proximal extracellular domain thought to serve as a target for O-glycosylation and chondroitin sulfate attachment has undergone substantial divergence (only 42% identity). Transient expression of the cDNA in CHO cells followed by immunologic staining confirms that this mouse H-CAM cDNA encodes Pgp-1.1, one of two known Pgp-1 alloantigens.     

Molecular cloning of the Candida maltosa ADE1 gene.

The structural gene (ADE1) encoding phosphoribosyl-aminoimidazole-succinocarboxamide synthetase (SAICAR synthetase; EC 6.3.2.6) in Candida maltosa has been isolated by functional complementation of an ade1 strain of Saccharomyces cerevisiae. The gene was localized on a 2.5-kb BamHI DNA fragment. Nucleotide sequence analysis of the cloned gene has revealed an open reading frame encoding a protein (SAICAR synthetase) with an Mr of 32,751. The codon bias index, 0.68, indicates that the ADE1 gene is a moderately highly expressed gene. The cloned gene shows 63.5% nt identity and 65.2% deduced amino acid identity with the S. cerevisiae ADE1 gene which encodes the same enzymatic activity. The gene may be used as a convenient genetic marker for construction of a new host-vector system for C. maltosa.     

Molecular cloning of rat ATX1 homologue protein

An ATX1 homologue of 503 bp length was cloned from a rat cDNA library, and the deduced protein from the cDNA was found to contain 68 amino acids with a predicted molecular mass of 7.2 kDa. The rat ATX1 homologue protein (Rah1p), which shows 35%, 38%, and 89% identities with Atx1p, CUC-1, and HAH1, respectively, conserves both the MTCXXC copper-binding site in the N terminus and the KTGK lysine-rich region in the C terminus. In Northern blot analysis, rah1 mRNA was found to be expressed at high levels in the liver, small intestine, and testis. Expression of rah1 cDNA complemented a null atx1 mutant strain in yeast. Thus, Rah1p was concluded to be a functional copper chaperone.     

Thrombospondin-1 inhibits TCR-mediated T lymphocyte early activation

Biological activities of the matrix glycoprotein thrombospondin-1 (TSP1) are cell type specific and depend on the relative expression or activation of several TSP1 receptors. Although engaging individual TSP1 receptors in T lymphocytes can elicit costimulating signals, in this study we show that intact TSP1 inhibits TCR-mediated T cell activation, assessed globally using cDNA microarrays. TSP1 signaling suppressed expression of several genes induced in Jurkat T cells, including the T cell activation markers CD69, early growth response gene-1 (Egr-1), and phosphatase of activated cells (PAC-1). TCR-stimulated and CD47-costimulated IL-2 secretion and cell surface CD69 expression were also inhibited by TSP1. The specific inhibitory effect of TSP1 was verified in freshly isolated human PBMCs. TSP1 inhibited TCR-mediated but not protein kinase C-mediated T cell activation. Using CD69 expression as a marker, we demonstrated that the inhibitory activity of TSP1 depended on two TSP1 receptors, CD47 and integrin-associated protein heparan sulfate proteoglycans. Signals from these receptors inhibited TCR signaling downstream of ZAP70, but upstream of NF-AT. Therefore, the expression of TSP1 induced during wound repair and in tumor stroma may limit T cell activation at these sites.     

Molecular cloning of mammalian Spred-3 which suppresses tyrosine kinase-mediated Erk activation

We have reported on Spred-1 and Spred-2, which inhibit MAP kinase activation by interacting with c-kit and ras/raf. Here, we report the cloning of a third member in this family, Spred-3. Spred-3 is expressed exclusively in the brain and its gene locates in chromosome 19q13.13 in human. Like Spred-1 and -2, Spred-3 contains an EVH1 domain in the N-terminus and a Sprouty-related cysteine-rich region (SPR domain) in the C-terminus that is necessary for membrane localization. However, Spred-3 does not possess a functional c-kit binding domain (KBD), since the critical amino acid Arg residue in this region was replaced with Gly in Spred-3. Although Spred-3 suppressed growth factor-induced MAP kinase (Erk) activation, inhibitory activity of Spred-3 was lower than that of Spred-1 or Spred-2. By the analysis of chimeric molecules between Spred-3 and Spred-1, we found that the SPR domain, rather than KBD, is responsible for efficient Erk suppression. The finding of Spred-3 revealed the presence of a novel family of regulators for the Ras/MAP kinase pathway, each member of which may have different specificities for extracellular signals.     

Characterization of genomic polymorphism of an activation-associated antigen,Blast-1

Blast-1 is a human activation-associated glycoprotein expressed on the surface of monocuclear cells, and a possible genetic marker for the manifestation of rheumatoid arthritis. In the present study, genomic polymorphism of the Blast-1 gene was analyzed using 100 healthy subjects. Restriction fragment length polymorphism (RFLP) of the Blast-1 gene was recognized only by Bam HI digestion among 46 restriction enzymes tested. The sizes of polymorphic fragments were 2.4 kilobase (kb) on the L band, and 1.9 kb on the S band. A family study demonstrated that the two alleles of the Blast-1 gene were inherited in a co-dominant Mendelian fashion. The genotype frequencies of homozygosity for the L and S bands were 47% and 42%, respectively, while the frequency of heterozygosity was 11%. The allele frequencies of the L and S bands were 0.68 and 0.32, respectively. The distribution of the Blast-1 genotypes in the present study was concordant with Hardy-Weinberg equilibrium (p>0.7), which indicates that the frequency of the Blast-1 gene in the population is derived from random mating in preceding generations. The results of the present study may provide useful information in disease associations with the Blast-1 gene.     

Molecular cloning of cDNAs encoding variants of meiotin-1

Meiotin-1 is a chromatin-associated protein, originally isolated from microsporocytes of Lilium longiflorum, which is found predominantly in cells undergoing meiotic prophase. Chromatin fractionation studies demonstrated that meiotin-1 has an unusual stoichiometry relative to that of histone H1 and the core histones in chromatin fibers. The protein is found less frequently than is histone H1, and appears to be distributed once every 5 to 13 nucleosomes. This distribution may approximate the number of nucleosomes per turn of the chromatin solenoid. A truncated cDNA was identified by immunoscreening of an expression library, and the cDNA was used as a hybridization probe to select a full length cDNA. Variations between the sequence of the predicted polypeptide and sequenced peptides, and variations between the amino acid composition of the protein and the deduced protein indicate that the cDNAs encode minor variants of mature meiotin-1. RNA gel blot hybridization studies reveal that the meiotin-1 mRNA is restricted to anthers in which meiosis is occurring. Computer analysis of the polypeptide deduced from the cDNA indicates that the protein begins with a region highly homologous to the conserved central globular domain of histone H1 molecules. DNA gel blotting experiments demonstrate that homologous sequences exist in the genomes of a fern, a fungus, and both mono-and dicotyledonous plants. Meiotin-1 has been evolutionarily conserved and I propose that it arose from histone H1 to fulfill a role in organizing meiotic chromatin.     

Molecular cloning of guinea pig angiotensin type 1 receptor

The primary structure of cDNA encoding of the angiotensin type 1 receptor (AT(1)R) was cloned from guinea pig liver. Guinea pig AT(1)R (GP-AT(1)R) cDNA clone contains a 1,077-bp open reading frame which encodes a protein consisting of 359 amino acid residues. GP-AT(1)R amino acid sequence showed a 92% level of identity among mammalian species. GP-AT(1)R is expressed in liver, kidney, adrenal gland, heart and colon.     

The murine homologue of the T lymphocyte antigen CD28. Molecular cloning and cell surface expression

The human T lymphocyte Ag CD28 (Tp44) is a homodimeric glycoprotein expressed on the surface of a majority of human peripheral T cells and thymocytes. Although exposure of T cells to anti-CD28 mAb does not activate T cells, stimulation of CD28 can synergize with signals transmitted through the TCR or other stimuli to augment proliferation and lymphokine production. We have used a portion of the human CD28 cDNA to isolate a homologous murine cDNA from an EL4 T lymphoma library. The murine clone has 61% nucleotide identity with the human cDNA. Both human and murine sequences exhibit homology with members of the Ig supergene family and CTLA-4, a T cell specific murine gene. Many characteristics of the human CD28 molecule are conserved within the putative murine CD28 polypeptide. The murine cDNA sequence encodes a polypeptide of 218 amino acids that has 68% identity with the human sequence. Both the murine and human molecules are integral membrane glycoproteins with hydrophobic signal peptide sequences and transmembrane region. All five potential N-linked glycosylation sites are conserved and six of the seven cysteine residues of the mouse protein are found in the human CD28 polypeptide. The murine cDNA is encoded by a single copy nonrearranging gene whose expression at the mRNA level is restricted to T cells. A rabbit antiserum was raised against a synthetic peptide corresponding to a hydrophilic portion of the translated murine cDNA sequence. This antiserum identifies an 80-kDa homodimer consisting of disulfide-bonded subunits of 40 kDa that is expressed on splenic T cells, thymocytes, and several T cell tumors, but not on B cells. deglycosylation studies indicate that four of the five N-linked glycosylation sites are used and that the mature core protein has a molecular mass of 25 kDa, close to that predicted by the cDNA sequence. Transfection of the murine cDNA into Chinese hamster ovary cells resulted in the expression of an 80-kDa dimeric molecule that was immunoprecipitated by the antipeptide antiserum. Taken together, these data provide strong support that we have identified the murine homologue of CD28.     

Molecular cloning of a pea H1 histone cDNA

A pea (Pisum sativum, var. Little Marvel) H1 histone cDNA has been isolated from a lambda gt11 expression vector library. This cDNA has been sequenced and shown to represent the entire protein-coding region of the mRNA. The deduced protein sequence is 265 amino acids long (28018 Da) and contains 70 lysines and 3 arginines. The structure of the encoded protein is comparable to animal lysine-rich histones. The central region, which has an amino acid composition similar to that found in the globular domains of animal lysine-rich histones, is flanked by an amino-terminal region rich in lysine, glutamic acid and proline and by a carboxyl-terminal region rich in lysine, alanine, valine and proline. Despite the structural similarities, the protein has little sequence homology with animal lysine-rich histones. This H1 protein is unusual because 12 of the first 40 amino acids are glutamic acid.