Identification Leptospira santarosai serovar shermani specific sequences by suppression subtractive hybridization

In Taiwan, leptospirosis is caused mainly by Leptospira santarosai serovar shermani. Suppression subtractive hybridization was employed to isolate DNA fragments present in pathogenic L. santarosai serovar shermani but absent in non-pathogenic L. biflexa serovar patoc. Analysis of 23 subtracted DNA clones revealed 25 gene fragments by BLASTX program. Eight clones showed similarity to transposase genes and three clones displayed homology with either translation or metabolism related genes. Four clones were similar to outer membrane protein, penicillin-binding protein, CreD-like protein and the protein of two-component signal transduction system, respectively. One clone had TPR repeat domain and five clones had significant similarity with hypothetical proteins of unknown functions. The remaining four clones exhibited no homology with any known genes. These results indicate that subtractive hybridization can successfully identify genes that are absent from the non-pathogenic Leptospira and provide a starting point for clarifying the differential genes expression between pathogenic and non-pathogenic Leptospira species.     

The STK2 gene, which encodes a putative Ser/Thr protein kinase, is required for high-affinity spermidine transport in Saccharomyces cerevisiae.

Eukaryotic polyamine transport systems have not yet been characterized at the molecular level. We have used transposon mutagenesis to identify genes controlling polyamine transport in Saccharomyces cerevisiae. A haploid yeast strain was transformed with a genomic minitransposon- and lacZ-tagged library, and positive clones were selected for growth resistance to methylglyoxal bis(guanylhydrazone) (MGBG), a toxic polyamine analog. A 747-bp DNA fragment adjacent to the lacZ fusion gene rescued from one MGBG-resistant clone mapped to chromosome X within the coding region of a putative Ser/Thr protein kinase gene of previously unknown function (YJR059w, or STK2). A 304-amino-acid stretch comprising 11 of the 12 catalytic subdomains of Stk2p is approximately 83% homologous to the putative Pot1p/Kkt8p (Stk1p) protein kinase, a recently described activator of low-affinity spermine uptake in yeast. Saturable spermidine transport in stk2::lacZ mutants had an approximately fivefold-lower affinity and twofold-lower Vmax than in the parental strain. Transformation of stk2::lacZ cells with the STK2 gene cloned into a single-copy expression vector restored spermidine transport to wild-type levels. Single mutants lacking the catalytic kinase subdomains of STK1 exhibited normal parameters for the initial rate of spermidine transport but showed a time-dependent decrease in total polyamine accumulation and a low-level resistance to toxic polyamine analogs. Spermidine transport was repressed by prior incubation with exogenous spermidine. Exogenous polyamine deprivation also derepressed residual spermidine transport in stk2::lacZ mutants, but simultaneous disruption of STK1 and STK2 virtually abolished high-affinity spermidine transport under both repressed and derepressed conditions. On the other hand, putrescine uptake was also deficient in stk2::lacZ mutants but was not repressed by exogenous spermidine. Interestingly, stk2::lacZ mutants showed increased growth resistance to Li+ and Na+, suggesting a regulatory relationship between polyamine and monovalent inorganic cation transport. These results indicate that the putative STK2 Ser/Thr kinase gene is an essential determinant of high-affinity polyamine transport in yeast whereas its close homolog STK1 mostly affects a lower-affinity, low-capacity polyamine transport activity.     

Genetic analysis of spontaneous lactose-utilizing mutants from vibrio vulnificus

Wild-type V. vulnificus cannot grow using lactose as the sole carbon source or take up the sugar. However, prolonged culture of this species in media containing lactose as the sole carbon source leads to the generation of a spontaneous lactose-utilizing (LU) mutant. This mutant showed strong beta- galactosidase activity, whereas the wild-type strain showed a barely detectable level of the activity. A mutant with a lesion in a gene homologous to the lacZ of E. coli in the bacterium no longer showed beta-galactosidase activity or generated spontaneous LU mutants, suggesting that the lacZ homolog is responsible for the catabolism of lactose, but the expression of the gene and genes for transport of lactose is tightly regulated. Genetic analysis of spontaneous LU mutants showed that all the mutations occur in a lacI homolog, which is located downstream to the lacZ and putative ABC-type lac permease genes. Consistent with this, a genomic library clone containing the lacI gene, when present in trans, made the spontaneous LU mutants no longer able to utilize lactose as the sole carbon source. Taken together with the observation that excessive amounts of exogenously supplemented possible catabolic products of lactose have negative effects on the growth and survivability of V. vulnificus, we suggest that V. vulnificus has evolved to carry a repressor that tightly regulates the expression of lacZ to keep the intracellular toxic catabolic intermediates at a sublethal level.     

Microarray analysis of gene expression in germinating barley embryos (Hordeum vulgare L.)

A cDNA library containing approximately 5,000 clones from germinating barley embryos was constructed and used to examine the variation in gene expression patterns during the first 4 days postimbibition. The expression profiles of embryos (including scutellum) from 4 to 96 h postimbibition were compared to a reference profile from 24 h postimbibition using microarray analysis. A subset of clones exhibiting tenfold or greater differential expression patterns was sequenced to elucidate function. All of the sequenced clones could be identified to at least EST level with 64% exhibiting homology to published protein sequences. Almost 95% of the library exhibited similar expression levels at the 4 h time point as at the 24 h reference point. From 24 to 96 h, however, considerable fluctuations in gene expression occurred. The observed patterns of gene expression for the classified genes are consistent with the expected genetic changes required to prepare an embryo for germinative development. A replicate set of clones for the 23-kDa jasmonate-induced protein was identified. The current data not only provides conclusive evidence for the expression patterns of this abundant stress-response protein in germinating embryos, but also serves to validate previous research into JIP-23 isoforms, function and the relationship between timing of mRNA upregulation and protein abundance.     

The use of lacZ gene fusions in the studies of mammalian development: developmental regulation of mammalian homeobox genes in the CNS

1. Mammalian cells can produce bacterial beta-galactosidase when they carry the lacZ gene under the control of mammalian regulatory elements. The single cell resolution of the beta-galactosidase histochemical detection method makes this molecule an excellent marker in studies of development at the cellular and molecular level. Different lacZ fusion genes can be engineered to study the histological diversification of cell lineages, the developmental regulation of isolated genes, or to recognize and clone genes with new expression profile. 2. Transgenic mice carrying lacZ gene fusions provide information on the cell type, developmental stage and spatial specificity of cis-acting regulatory regions linked to a mammalian homeobox gene. We describe our strategy for designing the gene fusions. 3. The scord region of the Hox 1.3 gene is sufficient to determine spatially restricted expression of a heterologous protein in the midgestational spinal cord. We propose to use this region to alter the expression pattern of other homeobox gene products. Developmental alterations due to a variant expression pattern would point to the function of the misexpressed gene.     

A quorum-sensing system in the free-living photosynthetic bacterium Rhodobacter sphaeroides.

Rhodobacter sphaeroides is a free-living, photoheterotrophic bacterium known for its genomic and metabolic complexity. We have discovered that this purple photosynthetic organism possesses a quorum-sensing system. Quorum sensing occurs in a number of eukaryotic host-associated gram-negative bacteria. In these bacteria there are two genes required for quorum sensing, the luxR and luxI homologs, and there is an acylhomoserine lactone signal molecule synthesized by the product of the luxI homolog. In R. sphaeroides, synthesis of a novel homoserine lactone signal, 7,8-cis-N-(tetradecenoyl)homoserine lactone, is directed by a luxI homolog termed cerI. Two open reading frames immediately upstream of cerI are proposed to be components of the quorum-sensing system. The first of these is a luxR homolog termed cerR, and the second is a small open reading frame of 159 bp. Inactivation of cerI in R. sphaeroides results in mucoid colony formation on agar and formation of large aggregates of cells in liquid cultures. Clumping of CerI mutants in liquid culture is reversible upon addition of the acylhomoserine lactone signal and represents a phenotype unlike those controlled by quorum sensing in other bacteria.     

Integrative genomics revealed RAI3 is a cell growth-promoting gene and a novel P53 transcriptional target

In this study, differential gene expression between normal human mammary epithelial cells and their malignant counterparts (eight well established breast cancer cell lines) was studied using Incyte GeneAlbum 1-6, which contains 65,873 cDNA clones representing 33,515 individual genes. 3,152 cDNAs showed a > or =3.0-fold expression level change in at least one of the human breast cancer cell lines as compared with normal human mammary epithelial cells. Integration of breast tumor gene expression data with the genes in the tumor suppressor p53 signaling pathway yielded 128 genes whose expression is altered in breast tumor cell lines and in response to p53 expression. A hierarchical cluster analysis of the 128 genes revealed that a significant portion of genes demonstrate an opposing expression pattern, i.e. p53-activated genes are down-regulated in the breast tumor lines, whereas p53-repressed genes are up-regulated. Most of these genes are involved in cell cycle regulation and/or apoptosis, consistent with the tumor suppressor function of p53. Follow-up studies on one gene, RAI3, suggested that p53 interacts with the promoter of RAI3 and repressed its expression at the onset of apoptosis. The expression of RAI3 is elevated in most tumor cell lines expressing mutant p53, whereas RAI3 mRNA is relatively repressed in the tumor cell lines expressing wild-type p53. Furthermore, ectopic expression of RAI3 in 293 cells promotes anchorage-independent growth and small interfering RNA-mediated depletion of RAI3 in AsPc-1 pancreatic tumor cells induces cell morphological change. Taken together, these data suggest a role for RAI3 in tumor growth and demonstrate the predictive power of integrative genomics.     

Hypercyst mutants in Rhodospirillum centenum identify regulatory loci involved in cyst cell differentiation

Rhodospirillum centenum is a purple photosynthetic bacterium that forms resting cyst cells when starved for nutrients. In this study, we demonstrate that chalcone synthase gene (chsA) expression is developmentally regulated, with expression of chsA increasing up to 86-fold upon induction of the cyst developmental cycle. Screening for mini-Tn5-induced mutants that exhibit elevated chsA::lacZ expression has led to the isolation of a set of R. centenum mutants that display increased chsA gene expression concomitant with constitutive induction of the cyst developmental cycle. These "hypercyst" mutants have lost the ability to regulate cyst cell formation in response to nutrient availability. Sequence analysis indicates that the mini-Tn5-disrupted genes code for a variety of factors, including metabolic enzymes and a large set of potential regulatory factors, including four gene products with homology to histidine sensor kinases and three with homology to response regulators. Several of the disrupted genes also have sequence similarity to che-like signal transduction components.     

Identification and characterization of a hypoxically induced maize lactate dehydrogenase gene

In cereal root tissue, hypoxia induces the enzyme lactate dehydrogenase (LDH); (S)-lactate:NADH oxidoreductase, EC 1.1.1.27). In barley, both biochemical and genetic data indicate that five isozymes are induced under hypoxia. These isozymes are tetramers and arise from the random association of the products of two Ldh genes. The induction of LDH activity in root tissue has been shown to be correlated to an increase in LDH protein and Ldh mRNA.In order to more fully characterize the hypoxic induction of LDH, we have isolated a maize Ldh genemic clone which has strong homology at both the amino acid and nucleotide level to the barley LDH cDNA clones. The Ldh1 gene consists of two exons separated by a 296 bp intron, has the expected eukaryotic regulatory signals and a sequence that has strong homology to the maize anaerobic regulatory element.     

Metabolic engineering of cultured tobacco cells

Construction of a gene expression system in tobacco cultured cells (BY2) was studied. A 925 bp promoter fragment of a heat-shock protein gene (HSP18.2) of Arabidopsis thaliana showed clear heat-shock response of expression of the beta-glucuronidase (GUS) reporter gene in BY2 cells. Similar results were observed in a 500 mL flask and 3-L jar fermentor. Isolation of strong promoters in BY2 cells was tried. cDNA clones, in which the mRNA level is high in log-phase cells and the copy number in the genome is low, were isolated. These clones showed high homology with F1-ATPase (mitochondria type), elongation factor 1-alpha, and a gene with an unknown function of A. thaliana (clone 27), respectively. A 5'-flanking region of clone 27 showed 6.2 times the promoter activity of the CaMV35S promoter in BY2 cells. Three cDNA clones, which are expressed in the stationary growth phase of BY2 cells, were isolated by a differential screening. These clones showed high sequence homologies to alcohol dehydrogenase, pectin esterase, and extensin. Promoters of these genes will be useful in gene expression in high cell-density culture.     

appA, a novel gene encoding a trans-acting factor involved in the regulation of photosynthesis gene expression in Rhodobacter sphaeroides 2.4.1.

A new gene, the product of which is involved in the regulation of photosynthesis gene expression in the anoxygenic photosynthetic bacterium Rhodobacter sphaeroides 2.4.1, has been identified. The isolation of this gene, designated appA (activation of photopigment and puc expression), was based on its ability, when provided in extra copies, to partially suppress mutations in the two-component PrrB-PrrA regulatory system. The presence of extra copies of the appA gene in either prrB, prrA, or wild-type strains resulted in an activation of puc::lacZ expression under aerobic conditions. Constructed AppA null mutants did not grow photosynthetically and were impaired in the synthesis of both bacteriochlorophyll and carotenoids, as well as the structural proteins of the photosynthetic spectral complexes. When grown anaerobically in the dark, these mutants accumulated bacteriochlorophyll precursors. The expression of lacZ fusions to several photosynthesis genes and operons, including puc, puf, and bchF, was decreased in the AppA mutant strains in comparison with the wild type. To examine the role of AppA involvement in bacteriochlorophyll biosynthesis, we inactivated an early gene, bchE, of the bacteriochlorophyll pathway in both wild-type and AppA- mutant backgrounds. The double mutant, AppA- BchE-, was found to be severely impaired in photosynthesis gene expression, similar to the AppA- BchE+ mutant and in contrast to the AppA+ BchE- mutant. This result indicated that AppA is more likely involved in the regulation of expression of the bch genes than in the biosynthetic pathway per se. The appA gene was sequenced and appears to encode a protein of 450 amino acids with no obvious homology to known proteins.     

luxR homolog avhR in Agrobacterium vitis affects the development of a grape-specific necrosis and a tobacco hypersensitive response

The luxR homolog aviR in Agrobacterium vitis strain F2/5 was recently shown to be associated with induction of a hypersensitive response (HR) on tobacco and necrosis on grape plants, indicating that the responses are regulated by quorum sensing. We now report a second luxR homolog, avhR, whose disruption (mutant M1320) results in HR-negative and reduced grape necrosis phenotypes. The deduced AvhR protein has characteristic autoinducer binding and DNA binding domains and is unique among reported functional LuxR homologs in having substitutions at highly conserved Asp70, Trp57, and Trp85 residues, which are predicted to play important roles in autoinducer binding in TraR. M1320 was fully complemented with cloned avhR. The same array of N-acylhomoserine lactones (AHL) from F2/5, M1320, and complemented M1320 were observed; however, the signal strength from extracts of 6-day-old M1320 cultures was stronger than that of F2/5. Cultures of F2/5 amended with AHL extracts from overnight and 6-day cultures of F2/5 and M1320 were not affected in ability to cause HR or necrosis. A region of about 14 kb flanking avhR was sequenced and compared with homologous regions of A. tumefaciens C58 and Sinorhizobium meliloti Rm1021 genomes. Gene order and homology are conserved between the species. A site-directed mutation in a putative gene that resides downstream of avhR and that has homology to genes belonging to the ATP-binding cassette transporter family did not affect HR or necrosis phenotypes. It was determined that avhR and aviR are expressed independently and that neither regulates the expression of a clpA homolog in F2/5.