ERK pathway positively regulates the expression of Sprouty genes

Sprouty was originally identified as an inhibitor of Drosophila development-associated receptor tyrosine kinase (RTK) signaling. Although RTK signaling has been shown to induce Sprouty gene expression, the precise induction pathway downstream of RTK remains unclear. As RTK signaling pathway includes activation of extracellular signal-regulated kinases (ERKs), we have examined a correlation between activation of ERKs and induction of Sprouty gene expression. All reagents which induce the activation of ERKs induce Sprouty gene expression; these agents include not only growth factors which bind to RTK but also phorbol 12-myristate-13-acetate and active Raf-1 kinase. Furthermore, the Sprouty gene expression induced by all those agents is totally suppressed when the cells are pretreated with specific inhibitors of ERK kinase (MEK). Human tumor cells which exhibit constitutive activation of ERKs show elevated expression of Sprouty genes, which is abolished by treatment of these cells with MEK inhibitors. All these findings clearly indicate that Sprouty gene expression is positively regulated by the ERK pathway downstream of RTK.     

Structure of tobacco genes encoding thaumatin-like proteins

Two tobacco genes encoding thaumatin-like proteins were cloned and sequenced. Both genes are expressed after infection of tobacco with tobacco mosaic virus (TMV). Comparison of the upstream sequences of these genes with those of other TMV-inducible tobacco genes revealed limited regions of homology.     

Arabidopsis genes encoding components of the chloroplastic protein import apparatus

The process of protein import into plastids has been studied extensively using isolated pea (Pisum sativum) chloroplasts. As a consequence, virtually all of the known components of the proteinaceous apparatus that mediates import were originally cloned from pea. With the recent completion of the Arabidopsis genome sequencing project, it is now possible to identify putative homologs of the import components in this species. Our analysis has revealed that Arabidopsis homologs with high sequence similarity exist for all of the pea import complex subunits, making Arabidopsis a valid model for further study of this system. Multiple homologs can be identified for over one-half of the components. In all but one case it is known that more than one of the putative isoforms for a particular subunit are expressed. Thus, it is possible that multiple types of import complexes are present within the same cell, each having a unique affinity for different chloroplastic precursor proteins, depending upon the exact mix of isoforms it contains. Sequence analysis of the putative Arabidopsis homologs for the chloroplast protein import apparatus has revealed many questions concerning subunit function and evolution. It should now be possible to use the genetic tools available in Arabidopsis, including the generation of knockout mutants and antisense technology, to address these questions and learn more about the molecular functions of each of the components during the import process.     

Molecular quantification of genes encoding for green-fluorescent proteins

A quantitative PCR approach is presented to analyze the amount of recombinant green fluorescent protein (gfp) genes in environmental DNA samples. The quantification assay is a combination of specific PCR amplification and temperature gradient gel electrophoresis (TGGE). Gene quantification is provided by a competitively coamplified DNA standard constructed by point mutation PCR. A single base difference was introduced to achieve a suitable migration difference in TGGE between the original target DNA and the modified standard without altering the PCR amplification efficiency. This competitive PCR strategy is a highly specific and sensitive way to monitor recombinant DNA in environments like the efflux of a biotechnological plant.     

Selection of Drosophila genes encoding secreted and membrane proteins

With the use of a yeast-based signal sequence trap (YSST) method, we screened a Drosophila cDNA library to isolate genes encoding secreted and membrane proteins. Of the 136 unique cDNA clones sequenced, 11 clones (8.1%) are identical to previously known Drosophila genes, 18 clones (13.2%) are homologous to other genes identified in various organisms, and 91 clones (66.9%) are novel. Most of these genes are secreted or membrane proteins, or appear to contain putative signal sequences at their amino termini. This indicates that YSST is an effective tool for the isolation and analysis of Drosophila genes that play roles in intercellular communication.     

Bioinformatic identification of genes encoding Clq-domaincontaining proteins in zebrafish

C1q is the first subcomponent of classical pathway in the complement system and a major link between innate and acquired immunities. The globular （gC1q） domain similar with C1q was also found in many non-complement C1q-domain-containing （C1qDC） proteins which have similar crystal structure to that of the multifunctional tumor necrosis factor （TNF） ligand family, and also have diverse functions. In this study, we identified a total of 52 independent gene sequences encoding C1q-domain-containing proteins through comprehensive searches of zebrafish genome, cDNA and EST databases. In comparison to 31 orthologous genes in human and different numbers in other species, a significant selective pressure was suggested during vertebrate evolution. Domain organization of C1q-domain-containing （C1qDC） proteins mainly includes a leading signal peptide, a collagen-like region of variable length, and a C-terminal C1q domain. There are 11 highly conserved residues within the C1q domain, among which 2 are invariant within the zebrafish gene set. A more extensive database searches also revealed homologous C1qDC proteins in other vertebrates, invertebrates and even bacterium, but no homologous sequences for encoding C1qDC proteins were found in many species that have a more recent evolutionary history with zebrafish. Therefore, further studies on C1q-domain-containing genes among different species will help us understand evolutionary mechanism of innate and acquired immunities.     

Two genes encoding gas vacuole proteins in Halobacterium halobium

Summary The archaebacterium Halobacterium halobium contains two related gas vacuole protein-encoding genes (vac). One of these genes encodes a protein of 76 amino acids and resides on the major plasmid. The second gene is located on the chromosome in a (G+C)-rich DNA fraction and encodes a slightly larger but highly homologous protein consisting of 79 amino acids. The plasmid encoded vac gene is transcribed constitutively throughout the growth cycle while the chromosomal vac gene is expressed during the stationary phase of growth. Comparison of the nucleotide sequences of the two genes indicates differences in the putative promoter regions as well as 35 single base-pair exchanges within the coding regions of the two genes. The majority of the nucleotide exchanges in the coding region occur in the third position of a codon triplet generating the codon synonym. The only differences between the two encoded proteins are the exchange of 2 amino acids (positions 8 and 29) and a deletion of 3 amino acids near the carboxy-terminus of the plasmid encoded vac protein. The genomic DNAs from other halobacterial isolates (Halobacterium sp. SB3, GN101 and YC819-9) were found to contain only a chromosomal vac gene copy. There is a high conservation of the chromosomal vac gene and the genomic region surrounding it among the halobacterial strains investigated.     

The diversity of connexin genes encoding gap junctional proteins.

The multigene family of connexins is larger than previously anticipated. Ten different connexin homologous sequences have been characterized in the mouse genome, five of which are probably the mouse analogues of the known rat connexins26, -31, -32, -43, and -46. Since the additional 5 sequences have been isolated as cDNAs or hybridize specifically to distinct mRNA species, they most likely represent functional connexin genes. Since seven of the genomic connexin sequences have been shown to contain no intron in the coding sequence, this may apply to all mammalian connexin genes. Some of the structural features based on amino acid sequences deduced from cDNA or genomic sequences and the RNA expression pattern of the new connexins are compared with previously described connexins. The structural diversity of the connexin genes suggests that they fulfill different functions coordinated with, and perhaps required for, different programs of cellular differentiation.     

Selection of Arabidopsis genes encoding secreted and plasma membrane proteins

Secreted and plasma membrane proteins play crucial roles in a variety of physiological and developmental processes of multicellular organisms. Systematic cloning of the genes encoding these proteins is therefore of general interest. An effective method of trapping signal sequences was first described by Tashiro et al. (1993), and a similar yet more efficient method was reported by Klein et al. (1996) and Jacobs et al. (1997). In this study, we carried out the latter yeast-based signal sequence trap to clone genes from Arabidopsis thaliana encoding secreted and plasma membrane proteins. Of 144 sequenced cDNA clones, 18% are identical to previously cloned Arabidopsis thaliana genes, 12% are homologous to genes identified from various organisms, and 46% are novel. All of the isolated genes identical or homologous to previously reported genes are either secreted or plasma membrane proteins, and the remaining novel genes appear to contain functional signal sequences based on computer-aided sequence analysis. The full-length cDNA clones of one homologous gene and another novel gene were isolated and sequenced. The deduced amino acid sequences suggest that the former encodes a secreted protein, and the latter encodes a type 1 membrane protein. These results indicate that the signal sequence trap method is effective and useful for the isolation of plant genes encoding secreted and plasma membrane proteins.