Granulocyte-macrophage colony-stimulating factor (GM-CSF) and inflammatory stimuli up-regulate secretion of the soluble GM-CSF receptor in human monocytes: evidence for ectodomain shedding of the cell surface GM-CSF receptor alpha subunit

Soluble GM-CSF receptor alpha subunit (sGMRalpha) is a soluble isoform of the GMRalpha that is believed to arise exclusively through alternative splicing of the GMRalpha gene product. The sGMRalpha mRNA is expressed in a variety of tissues, but it is not clear which cells are capable of secreting the protein. We show here that normal human monocytes, but not lymphocytes, constitutively secrete sGMRalpha. Stimulation of monocytes with GM-CSF, LPS, PMA, or A23187 rapidly up-regulates the secretion of sGMRalpha in a dose-dependent manner, demonstrating that secretion is also regulated. To determine whether sGMRalpha arose exclusively through alternative splicing of the GMRalpha gene product, or whether it could also be generated through ectodomain shedding of GMRalpha, we engineered a murine pro-B cell line (Ba/F3) to express exclusively the cDNA for cell surface GMRalpha (Ba/F3.GMRalpha). The Ba/F3.GMRalpha cell line, but not the parental Ba/F3 cell line, constitutively shed a sGMRalpha-like protein that bound specifically to GM-CSF, was equivalent in size to recombinant alternatively spliced sGMRalpha (60 kDa), and was recognized specifically by a mAb raised against the ectodomain of GMRalpha. Furthermore, a broad-spectrum metalloprotease inhibitor (BB94) reduced constitutive and PMA-, A23187-, and LPS-induced secretion of sGMRalpha by monocytes, suggesting that shedding of GMRalpha by monocytes may be mediated in part through the activity of metalloproteases. Taken together, these observations demonstrate that sGMRalpha is constitutively secreted by monocytes, that GM-CSF and inflammatory mediators up-regulate sGMRalpha secretion, and that sGMRalpha arises not only through alternative splicing but also through ectodomain shedding of cell surface GMRalpha.     

Gene mutations and genomic rearrangements in the mouse as a result of transposon mobilization from chromosomal concatemers

Previous studies of the Sleeping Beauty (SB) transposon system, as an insertional mutagen in the germline of mice, have used reverse genetic approaches. These studies have led to its proposed use for regional saturation mutagenesis by taking a forward-genetic approach. Thus, we used the SB system to mutate a region of mouse Chromosome 11 in a forward-genetic screen for recessive lethal and viable phenotypes. This work represents the first reported use of an insertional mutagen in a phenotype-driven approach. The phenotype-driven approach was successful in both recovering visible and behavioral mutants, including dominant limb and recessive behavioral phenotypes, and allowing for the rapid identification of candidate gene disruptions. In addition, a high frequency of recessive lethal mutations arose as a result of genomic rearrangements near the site of transposition, resulting from transposon mobilization. The results suggest that the SB system could be used in a forward-genetic approach to recover interesting phenotypes, but that local chromosomal rearrangements should be anticipated in conjunction with single-copy, local transposon insertions in chromosomes. Additionally, these mice may serve as a model for chromosome rearrangements caused by transposable elements during the evolution of vertebrate genomes.     

Determinants of the functional interaction between the soluble GM-CSF receptor and the GM-CSF receptor beta-subunit

The GM-CSF receptor consists of a GM-CSF specific low affinity alpha-subunit (GMRalpha) and a beta-subunit (betac) that associates with GMRalpha in the presence of GM-CSF to form a high-affinity complex. A splice variant soluble isoform of GMRalpha (solalpha) consists of the extracellular domain of GMRalpha and a unique 16-amino acid C-terminal domain. Exogenously administered solalpha is unable to associate with betac on the cell surface either in the presence or absence of GM-CSF. However, paradoxically, co-expression of solalpha with betac results in the ligand-independent association of solalpha with betac on the cell surface via the C-terminal domain of solalpha. To study the interaction and functional characteristics of the solalpha-betac complex we engineered a soluble betac-subunit (ECDbeta) and expressed it alone and with solalpha. Co-expressed but not independent sources of solalpha and ECDbeta could be co-precipitated in the absence of ligand demonstrating the extracellular domain of betac was sufficient for association with solalpha upon co-expression. However, independent sources of solalpha could associate with ECDbeta in the presence of GM-CSF as could a C-terminal deficient solalpha mutant (ECDalpha) and the addition of ECDbeta to ECDalpha and GM-CSF was associated with a conversion from a low- to high-affinity ligand-receptor complex.     

Insertional mutagenesis in zebrafish: genes for development, genes for disease.

In order to rapidly identify a substantial fraction of the genes with a unique and essential role in vertebrate development, the laboratory of Nancy Hopkins at MIT has performed a large insertional mutagenesis screen in zebrafish using a pseudotyped retroviral vector as the mutagen. We have recovered mutations in about one-quarter of the embryonic essential genes in this organism, and have identified the mutated genes in nearly all of these (333). As the ease of gene identification allowed us to clone the mutated genes for nearly all of the mutants rather than prioritizing based upon the initially observed phenotypes, this has provided an unbiased view of the diversity of genes required for vertebrate development as well as a large collection of mutants to be screened for more specific phenotypes. In collaboration with other labs, we have screened the insertional mutant for the development of a variety of organs and cell types, as well as phenotypes that could represent disease models, such as cystic kidney and hepatomegaly. Furthermore, while all of these mutants are embryonic lethal in their homozygous state, we are investigating the heterozygous adults for additional phenotypes, such as cancer predisposition.     

Forward and Reverse Genetic Analysis of Microtubule Motors in Chlamydomonas

The ability to integrate biochemical, cell biological, and genetic approaches makes Chlamydomonas reinhardtii the premier model organism for studies of the eukaryotic flagellum and its associated molecular motors. Hundreds of motility mutations have been identified in Chlamydomonas, including many that affect dyneins and kinesins. These mutations have yielded much information on the structure and function of the motors as well as the roles of individual subunits within the motors. The development of insertional mutagenesis has opened the door to powerful new approaches for genetic analysis in Chlamydomonas. Insertional mutants are created by transforming cells with DNA-containing selectable markers. The DNA is randomly integrated throughout the genome and usually deletes part of the chromosome at the site of insertion, thereby creating mutations that are marked by the integrated DNA. These mutations can be used for forward genetic approaches where one characterizes a mutant phenotype and then clones the relevant gene using the integrated DNA as a tag. The insertional mutants also may be used in a reverse genetic approach in which mutants lacking a gene of interest are identified by DNA hybridization. We describe methods to generate and characterize insertional mutants, using mutations that affect the outer dynein arm as examples.     

Peptide insertions in domain 4 of hbeta(c), the shared signalling receptor subunit for GM-CSF, IL3 and IL5, induce ligand-independent activation

A mutant form of the common beta-subunit of the GM-CSF, interleukin-3 (IL3) and IL5 receptors is activated by a 37 residue duplicated segment which includes the WSXWS motif and an adjacent, highly conserved, aliphatic/basic element. Haemopoietic expression of this mutant, hbeta(c)FIDelta, in mice leads to myeloproliferative disease. To examine the mechanism of activation of this mutant we targetted the two conserved motifs in each repeat for mutagenesis. Here we show that this mutant exhibits constitutive activity in BaF-B03 cells in the presence of mouse or human GM-CSF receptor alpha-subunit (GMRalpha) and this activity is disrupted by mutations of the conserved motifs in the first repeat. In the presence of these mutations the receptor reverts to an alternative conformation which retains responsiveness to human IL3 in a CTLL cell line co-expressing the human IL3 receptor alpha-subunit (hIL3Ralpha). Remarkably, the activated conformation is maintained in the presence of substitutions, deletions or replacement of the second repeat. This suggests that activation occurs due to insertion of extra sequence after the WSXWS motif and is not dependent on the length or specific sequence of the insertion. Thus hbeta(c) displays an ability to fold into functional receptor conformations given insertion of up to 37 residues in the membrane-proximal region. Constitutive activation most likely results from a specific conformational change which alters a dormant, inactive receptor complex, permitting functional association with GMRalpha and ligand-independent mitogenic signalling.     

Forward and reverse genetic analysis of microtubule motors in Chlamydomonas

The ability to integrate biochemical, cell biological, and genetic approaches makes Chlamydomonas reinhardtii the premier model organism for studies of the eukaryotic flagellum and its associated molecular motors. Hundreds of motility mutations have been identified in Chlamydomonas, including many that affect dyneins and kinesins. These mutations have yielded much information on the structure and function of the motors as well as the roles of individual subunits within the motors. The development of insertional mutagenesis has opened the door to powerful new approaches for genetic analysis in Chlamydomonas. Insertional mutants are created by transforming cells with DNA-containing selectable markers. The DNA is randomly integrated throughout the genome and usually deletes part of the chromosome at the site of insertion, thereby creating mutations that are marked by the integrated DNA. These mutations can be used for forward genetic approaches where one characterizes a mutant phenotype and then clones the relevant gene using the integrated DNA as a tag. The insertional mutants also may be used in a reverse genetic approach in which mutants lacking a gene of interest are identified by DNA hybridization. We describe methods to generate and characterize insertional mutants, using mutations that affect the outer dynein arm as examples.     

Application of HSVtk suicide gene to X-SCID gene therapy: ganciclovir treatment offsets gene corrected X-SCID B cells

Recently, a serious adverse effect of uncontrolled clonal T cell proliferation due to insertional mutagenesis of retroviral vector was reported in X-SCID gene therapy clinical trial. To offset the side effect, we have incorporated a suicide gene into therapeutic retroviral vector for selective elimination of transduced cells. In this study, B-cell lines from two X-SCID patients were transduced with bicistronic retroviral vector carrying human gamma c chain cDNA and Herpes simplex virus thymidine kinase gene. After confirmation of functional reconstitution of the gamma c chain, the cells were treated with ganciclovir (GCV). The gamma c chain positive cells were eliminated under low concentration without cytotoxicity on untransduced cells and have not reappeared at least for 5 months. Furthermore, the gamma c chain transduced cells were still sensitive to GCV after five months. These results demonstrated the efficacy of the suicide gene therapy although further in vivo studies are required to assess feasibility of this approach in clinical trial.     

Embryonic Lethals and T-DNA Insertional Mutagenesis in Arabidopsis

T-DNA insertional mutagenesis represents a promising approach to the molecular isolation of genes with essential functions during plant embryo development. We describe in this report the isolation and characterization of 18 mutants of Arabidopsis thaliana defective in embryo development following seed transformation with Agrobacterium tumefaciens. Random T-DNA insertion was expected to result in a high frequency of recessive embryonic lethals because many target genes are required for embryogenesis. The cointegrate Ti plasmid used in these experiments contained the nopaline synthase and neomycin phosphotransferase gene markers. Nopaline assays and resistance to kanamycin were used to estimate the number of functional inserts present in segregating families. Nine families appeared to contain a T-DNA insert either within or adjacent to the mutant gene. Eight families were clearly not tagged with a functional insert and appeared instead to contain mutations induced during the transformation process. DNA gel blot hybridization with internal and right border probes revealed a variety of rearrangements associated with T-DNA insertion. A general strategy is presented to simplify the identification of tagged embryonic mutants and facilitate the molecular isolation of genes required for plant embryogenesis.     

稻瘟病菌T-DNA插入方法优化及其突变体分析

优化了农杆菌介导转化稻瘟病菌获得T-DNA插入突变的条件,包括选择转化子的潮霉素B用量,抑制农杆菌的抗生素头孢噻肟钠和羧苄青霉素的配比,不同转化阶段培养基的选择等。转化1×10⁶个孢子平均可获得约500个左右的转化子,PCR和TAILPCR检测表明约85%转化子中含T-DNA插入。对1520个突变体进行形态变异观察,发现菌落颜色突变的有15个;随机取58个突变体进行比较,发现产孢量减少的4个,孢子萌发率降低的8个,附着胞形成率降低的9个;还获得对水稻品种C101LAC（Pi-1）和751127（Pi-9）致病的突变体,为进一步克隆相应的无毒基因奠定了基础。     

A pro-inflammatory role of deubiquitinating enzyme cylindromatosis (CYLD) in vascular smooth muscle cells

Retroviral vectors have been employed in clinical trials for gene therapy owing to their relative large packaging capacity, alterable cell tropism, and chromosomal integration for stable transgene expression. However, uncontrollable integrations of transgenes are likely to cause safety issues, such as insertional mutagenesis. A targeted transgene integration system for retroviral vectors, therefore, is a straightforward way to address the insertional mutagenesis issue. Adeno-associated virus (AAV) is the only known virus capable of targeted integration in human cells. In the presence of AAV Rep proteins, plasmids possessing the p5 integration efficiency element (p5IEE) can be integrated into the AAV integration site (AAVS1) in the human genome. In this report, we describe a system that can target the circular DNA derived from non-integrating retroviral vectors to the AAVS1 site by utilizing the Rep/p5IEE integration mechanism. Our results showed that after G418 selection 30% of collected clones had retroviral DNA targeted at the AAVS1 site.     

Isolation of Bacillus subtilis transformation-deficient mutants and mapping of competence genes

We have isolated and characterized 48 Bacillus subtilis competence-deficient mutants. The mutants, obtained by nitrosoguanidine mutagenesis or by insertional mutagenesis with transposon Tn917, had a reduced transformation frequency and a wild-type transduction frequency. The com mutations were mapped by PBS1 transduction and at least four new com genes have been identified. The mutants were also characterized for their capacity to bind and take up the transforming DNA.     

Molecular Genetic Studies of the Cdc7 Protein Kinase and Induced Mutagenesis in Yeast

The Saccharomyces cerevisiae CDC7 gene encodes a protein kinase that functions in DNA replication, repair, and meiotic recombination. The sequence of several temperature-sensitive (ts) cdc7 mutations was determined and correlated with protein kinase consensus domain structure. The positions of these ts alleles suggests some general principles for predicting ts protein kinase mutations. Pedigree segregation lag analysis demonstrated that all of the mutant proteins are less active or less stable than wild-type Cdc7p. Two new mutations were constructed, one by site-directed and the other by insertional mutagenesis. All of the cdc7 mutants were assayed for induced mutagenesis in response to mutagenic agents at the permissive temperature. Some cdc7 mutants were found to be hypomutable, while others are hypermutable. The differences in mutability are observed most clearly when log phase cells are used. Both hypo- and hypermutability are recessive to wild type. Cdc7p may participate in DNA repair by phosphorylating repair enzymes or by altering chromatin structure to allow accessibility to DNA lesions.     

Characterization of the types of mutational events that spontaneously occur in a plasmid system

The immunity region from a cI857 derivative of bacteriophage lambda has been cloned into the EcoRI site of pBR322 to produce a plasmid that can be used to analyze spontaneous mutagenesis. Cells containing this plasmid are temperature-sensitive for growth unless mutations have occurred that somehow prevent the expression of the kil gene in the lambda fragment at non-permissive temperature. 678 such temperature-resistant mutants from 10 independent subcultures each of 2 different recA- E. coli strains have been collected, and the nature of the plasmid mutations obtained has been analyzed. All of the subcultures contained mutants that allowed growth at the restrictive temperature without showing a detectable change in plasmid size. 75% of the total mutants fell in this class. More than half of these mutations involved the lambda leftward promoter, pL, and such mutants were found in all 20 subcultures. The remaining 25% of the mutations involved a change in plasmid size and mutations of this class were found in 18 of 20 subcultures. 12% of the total mutants (found in 16 of 20 subcultures) had an insertion of IS1 in the region between pL and the lambda kil gene. 6% of the total mutants had undergone an IS1-mediated deletion, while 1% were mixed colonies in which multiple IS1-mediated events had occurred. About 1% of the total mutants had undergone complex IS1-mediated DNA rearrangement(s) that have not yet been characterized. In total, 11 of 20 subcultures yielded isolates where IS1-mediated rearrangements had occurred. The remaining 4% of the mutations included insertions of IS5, IS30, and an IS1 family member that appears to be IS1T as well as IS1T-mediated deletions and deletions that do not appear to have been mediated by any insertion sequence. A mutant with both an IS1 insertion and an alteration involving pL has also been isolated.