RNA Slot Blot Research Articles

Functional characterization of a serine-threonine protein kinase from Bambusa balcooathat implicates in cellulose overproduction and superior quality fiber formation

BackgroundMolecular markers allow rapid identification of biologically important germplasm/s having desired character. Previously we have reported a genotype specific molecular marker, Balco1128 [GenBank ID EU258678] of Bambusa balcooa containing an ORF (375 bp) having high similarity with receptor like cytoplasmic kinase of Arabidopsis and Oryza. Balco1128 was found to be associated only with bamboo genotypes endowed with high cellulose and low lignin contents of fibers. Under the above backdrop, it was necessitated to characterize this genetic marker for better understanding of its biological significance in context of superior quality fiber development.ResultsThe full length cDNA (3342 bp) of BbKst, a serine-threonine protein kinase was isolated from B. balcooa comprising of six LRR domains at the N-terminal end and a kinase domain at the C-terminal end. Bacteria-expressed BbKst-kinase domain (3339 bp long) showed Mg2+ dependent kinase activity at pH 7.0, 28°C. Bioinformatics study followed by phospho-amino analysis further confirmed that BbKst-kinase belongs to the serine/threonine protein kinase family. Transcript analysis of the BbKst gene following RNA slot blot hybridization and qPCR revealed higher expression of BbKst during initiation and elongation stages of fiber development. Tissue specific expression studies showed much higher expression of BbKst transcript in stems and internodes of B. balcooa than in leaves and rhizomes. Southern analysis revealed single copy insertion of BbKst in most of the Agrobacterium mediated transgenic tobacco plants. Real-time PCR detected 150-200 fold enhanced expression of BbKst in different T1 tobacco lines than that of the vector transformed plants. Heterologous expression of BbKst under control of 35S promoter in transgenic tobacco showed high cellulose deposition in the xylem fibers. Number of xylary fibers was higher in transgenic T0 and T1 plants than that of empty-vector transformed tobacco plants offering enhanced mechanical strength to the transgenic plants, which was also substantiated by their strong upright phenotypes, significantly higher cellulose contents, flexibility coefficient, slenderness ratio, and lower Runkel ratio of the fibers.ConclusionsThis finding clearly demonstrated that BbKst gene (GenBank ID JQ432560) encodes a serine/threonine protein kinase. BbKst induced higher cellulose deposition/synthesis in transgenic tobacco plants, an important attribute of fiber quality bestowing additional strength to the plant.

Identification of programmed cell death related genes in bamboo

The event of bamboo flowering and subsequent death of bamboo cells, a rare phenomenon is an interesting model to study gene expression/function in the context of the programmed cell death (PCD) in plant. To identify genes involved in autolytic cell death in bamboo (Bambusa arundinacea/Bambusa bambos Voss), a suppressive subtractive cDNA hybridization (SSH) was performed between cDNA isolated from control (healthy), as driver and test internodal tissue (45days after setting of seeds), as tester. In-silico data revealed that 82% of total ESTs (231) were non-significant (unidentified proteins) while remaining ESTs were classified as protein with known/predicted function/s. Among these, net distribution and differential expression patterns of 11 important B. arundinacea PCD specific ESTs were studied using RNA slot-blot, qRT-PCR and semi-quantitative RT. In-situ localization of mRNA-transcripts for selected bamboo PCD-specific ESTs namely V2Ba48 (Aldehyde dehydrogenase 2) and V2Ba19 (Glycogen phosphorylase) were detected using digoxigenin-labeled corresponding anti-sense RNA probes employing Confocal Laser Scanning Microscope (CLSM). Differential expression-kinetics of the aforementioned genes were confirmed during the progress of PCD after setting of seeds. Global appearance of V2Ba48, V2Ba19, V2Ba95 (Ubiquitin thioesterase) and V2Ba89 (Nebulin isoform 2) genes were identified in monocot (Oryza sativa) and dicots (Arabidopsis thaliana and Nicotiana tabacum). This is the first report on systematic analysis of genes involved in death of bamboo cells that may provide critical information regarding key metabolic/regulatory genes involved in plant PCD.

Identification of genes involved in bamboo fiber development

Recently bamboo has gained reputation as a major resource of non-wood fiber. The present study was undertaken to generate information about fiber development process in bamboo ( Bambusa balcooa) using PCR-based suppressive subtractive hybridization (PCR-SSH) technique, as molecular mechanism of its fiber development is yet to be explored. SSH was performed between cDNA isolated from leaf (as driver) and internodes (as tester) of B. balcooa which indicated up-regulation of 521 ESTs. Among these 41 were contigs and 65 ESTs were singletons. On the basis of BLASTX search 307 ESTs with known functions were classified into several functional categories including transport, metabolism, information, perception and response to stimuli while others were either non-significant (120) or hypothetical proteins (94). A total of 51 out of 307 functional ESTs were found fiber specific and their global distribution among different plant species like maize, rice, cotton and Arabidopsis ESTs were determined. Net distributions and differential expression patterns of 13 important B. balcooa fiber specific cDNAs among different internodes during bamboo development were studied using RNA slot–blot, semi-quantitative RT-PCR and real time PCR. In-situ localization of mRNA transcript for few selected bamboo fiber ESTs namely, V1Bb147 (protein kinase-like protein) and V1Bb88 (myb domain-containing protein) were detected using Confocal Laser Scanning Microscope. Transcript levels of these genes exhibited an orchestral turn-over during bamboo development, suggesting their close association with fiber development, an event associated with several metabolic and physiological changes. The results clearly suggest that these genes are involved in several concerted mechanisms involving Ca + signaling pathway, cell wall synthesis, hormone regulation, system maintaining cell turgor pressure and cytoskeleton synthesis pathway accountable for bamboo fiber development signifying fiber development as a complex but ordered metabolic process involving differential expression of large scale fiber associated genes. This is the first report on systematic analysis of genes involved in bamboo fiber development.

Alterations in the Epigenetic Network Controlling Transcription Activity, Chromatin Structure and Region-Specific Repair of Different Genomic Loci Predicts Clinical Outcome in Multiple Myeloma.

Abstract 122The development of cancer is driven by the accumulation of scores of alterations affecting the structure and function of the genome. Equally important in this process are genetic alterations and epigenetic changes. Whereas the former disrupt normal patterns of gene expression, sometimes leading to the expression of abnormal, constitutively active proteins, the latter deregulate the mechanisms such as transcriptional control leading to the inappropriate silencing or activation of cancer-associated genes. In this report, the effect of differential epigenetic alterations on the clinical outcome in multiple myeloma (MM) patients is presented. Peripheral blood mononuclear cells (PBMC) from twelve human healthy volunteers (7M/5F; median age 41 years, range 28 to 52 years) and 32 MM patients (14M/18F; median age 59 years, range 23 to 71 years) who underwent high dose melphalan with autologous stem cell support (ASCT) as part of their first line therapy. Twenty-three patients achieved an objective response post-ASCT while 9 patients did not respond according to IMWG criteria as well as human primary fibroblasts with different repair backgrounds (VH25, GGR+/TCR+; CS1AN, GGR+/TCR-; XP21RO, GGR-/TCR+; XP25RO, GGR-/TCR-) were exposed to melphalan. Chromatin condensation (using micrococcal nuclease digestion), transcription activity (steady-state levels using RNA slot-blots and rates of transcription using run-off assay), as well as in vitro melphalan-induced damage formation/repair (monoadducts and interstrand cross-links using Southern blot) were measured in three transcriptionally active genes (the housekeeping beta-actin gene, the tumor suppressor p53 and the protooncogene N-ras) as well as in the non-transcribed delta-globin gene. In repair-deficient human primary fibroblasts, melphalan adducts accumulated to similar levels in all four genes studied, indicating that the state of transcription and the local chromatin condensation did not affect adduct formation. In all MM patients examined, a close association was observed between the locus-specific repair efficiency, the transcriptional activity and the chromatin condensation. Strikingly, the order of variation of DNA repair efficiency, transcriptional activity and local chromatin structure between different genes was beta-actin>p53>N-ras>delta-globin in all healthy volunteers and in 95% of responders to chemotherapy, while a perturbation of this order was found in 80% of non-responders. In agreement with our previous reports (Dimopoulos et al, J Clin Oncol 2005;23:4381-9; Dimopoulos et al, Haematologica 2007;92:1505-12) in the p53, N-ras and delta-globin gene, responders to HDM showed higher melphalan-induced damage and lower rates of repair compared to non-responders. These differences were statistically significant for all types of melphalan adducts. No difference in the repair activity between responders and non-responders was observed in the beta-actin gene. Also, in all genes examined, DNA damage formation and repair induced by in vitro melphalan treatment of PBMC taken from MM patients prior to therapy, correlates with the respective data obtained in vivo, i.e. after therapeutic administration of HDM. Furthermore, the kinetics of melphalan adduct formation/repair along the transcribed N-ras gene showed a 5' to 3'-end gradient of repair efficiency, with faster repair at the 5'-end. Interestingly, the difference in the repair activity between responders and non-responders becomes maximal at the 5'-end of the N-ras gene, decrescent towards the 3'-end of the gene. As for the delta-globin gene, an induction of the transcription activity, a higher “looseness” of the local chromatin structure and an increase in the repair efficiency was observed specifically in MM patients who did not respond to melphalan therapy. No difference between responders and non-responders was found in regions on both sides of the genes as well as at the overall genome repair, suggesting that in non-responders the repair system active in the removal of melphalan-induced adducts was not grossly affected. In conclusion, our study suggests that the DNA repair efficiency, the transcriptional activity and the local chromatin structure in different gene loci are tightly connected, and can be used as molecular markers to select those patients with MM who are more likely to benefit from HDM therapy. Disclosures:No relevant conflicts of interest to declare.

Age-dependent differential expression and activity of rat liver cytosolic inorganic pyrophosphatase gene

Besides epigenetic factors, the genetic make-up and differential gene expression not only determines aging and disease susceptibility but also the functional activity of cells in an individual. Analysis of a variety of mammalian tissues revealed that the age-associated differentially expressed genes mainly belong to inflammation, stress, and metabolism. Intracellular PPi is a by-product of multiple biosynthetic reactions and its hydrolysis by cytosolic inorganic pyrophosphatase (iPPase) has long been considered as an important homeostatic mechanism favoring biosynthesis. In this paper we report an age-associated increase ( approximately 2-fold) in the expression of rat liver cytosolic iPPase gene by differential display PCR and northern blot analysis. Expression profiling of iPPase by RNA slot blot analysis in several other tissues revealed no significant change with aging. A comparative spectrophotometric and in-gel analysis of iPPase activity in whole cell lysate (WCL) of liver, brain, skeletal muscle, heart, spleen and kidney exhibited that liver of old rats (24 months ) has approximately 2-fold more activity than the adult (4 months) ones and also its activity is highest among the tissues. The specificity of iPPase activity in the spectrophotometric assay and in-gel analysis was confirmed by specific iPPase inhibitors like CaCl(2) and NaF.

A targeted study on possible free ammonia inhibition of Nitrospira

Batch tests were carried out with Nitrospira in mixed and pure cultures using concentrations of free ammonia widely believed to be inhibitory to nitrite-oxidizing organisms. The mixed culture batch tests were conducted with mixed liquor from a bench scale completely stirred tank reactor (CSTR) treating a synthetic wastewater having a low C:N ratio. Nitrospira were confirmed as the dominant nitrite oxidizers via RNA slot blotting. Nitrospira moscoviensis were used for the pure culture trials. The results from this study suggest that free ammonia (NH3-N) concentrations of up to 10 mg/L were not inhibitory to Nitrospira either in situ or in pure culture. Key words: fatty acid methyl esters, free ammonia inhibition, molecular methods, nitrification, nitrite, Nitrobacter, Nitrospira, nitrous acid, nitrous oxide, RNA slot blotting, wastewater treatment.

Nitrifier population dynamics in a bench-scale conventional activated sludge reactor following an induced perturbation

An instantaneous increase in reactor free ammonia concentration resulted in a gradual decrease in nitrite oxidizer activity that was coincidental with an increase in the nitrous oxide emissions from the reactor. Microbial community analysis using RNA slot blotting techniques indicated that Nitrospira, and not Nitrobacter, populations appeared to be most affected by this induced perturbation. It is hypothesized that competition for nitrite and possibly oxygen between ammonia oxidizers and Nitrospira, and not free ammonia stress, was the true cause of impaired nitrite oxidizer activity. Key words: ammonia inhibition, fatty acid methyl esters, free ammonia, molecular methods, nitrification, nitrite, Nitrobacter, Nitrospira, nitrous oxide, RNA slot blotting, wastewater treatment.

Response of Bacillus subtilis to nitric oxide and the nitrosating agent sodium nitroprusside.

We examined the effects of nitric oxide (NO) and sodium nitroprusside (SNP) on Bacillus subtilis physiology and gene expression. In aerobically growing cultures, cell death was most pronounced when NO gas was added incrementally rather than as a single bolus, suggesting that the length of exposure was important in determining cell survival. DNA microarrays, Northern hybridizations, and RNA slot blot analyses were employed to characterize the global transcriptional response of B. subtilis to NO and SNP. Under both aerobic and anaerobic conditions the gene most highly induced by NO was hmp, a flavohemoglobin known to protect bacteria from NO stress. Anaerobically, NO also induced genes repressed by the Fe(II)-containing metalloregulators, Fur and PerR, consistent with the known ability of NO to nitrosylate the Fe(II) center in Fur. In support of this model, we demonstrate that NO fails to induce PerR-regulated genes under growth conditions that favor the formation of PerR:Mn(II) rather than PerR:Fe(II). Aerobically, NO gas induced hmp, the sigmaB general stress regulon, and, to a lesser extent, the Fur and PerR regulons. Surprisingly, NO gas induced the sigmaB regulon via the energy branch of the sigmaB regulatory cascade while induction by SNP was mediated by the environmental stress branch. This emphasizes that NO and SNP elicit genetically distinct stress responses.

Reduced expression of the global regulator protein CsrA in Legionella pneumophila affects virulence-associated regulators and growth in Acanthamoeba castellanii

Legionella bacteria have a developmental cycle in which they go from existing in the aquatic environment to replicating inside eukaryotic host cells. The adaptation to the new environment requires an efficient regulatory system. Overexpression of CsrA, a global regulatory protein found in a variety of Gram-negative bacteria has been shown to suppress virulence-associated traits in Legionella pneumophila. Since evidence resulting only from overproduction may not be sufficient to validate the role of a regulatory protein, a csrA mutant strain, CsrA(−), with a drastically reduced production of CsrA, was created. Using RNA slot blots and Western blotting it was shown that fliA and flaA, genes which contribute to flagellation, were expressed early in the mutant. Additionally, in CsrA(−) the levels of the stationary-phase sigma factor, RpoS, and a recently described regulator of virulence traits, LetE, were increased. Growth curves of CsrA(−) bacteria were delayed with pigment production occurring at the same OD 578 but at reduced levels in the mutant. Replication ability of the CsrA(−) mutant in amoebae was also affected. Based on these results, we could show that CsrA is involved in the regulation of the bacterial switch from the replicative to the transmissible form.

Role of ctc from Listeria monocytogenes in osmotolerance.

Listeria monocytogenes is a food-borne pathogen with the ability to grow under conditions of high osmolarity. In a previous study, we reported the identification of 12 proteins showing high induction after salt stress. One of these proteins is highly similar to the general stress protein Ctc of Bacillus subtilis. In this study, induction of Ctc after salt stress was confirmed at the transcriptional level by using RNA slot blot experiments. To explore the role of the ctc gene product in resistance to stresses, we constructed a ctc insertional mutant. No difference in growth was observed between the wild-type strain LO28 and the ctc mutant either in rich medium after osmotic or heat stress or in minimal medium after heat stress. However, in minimal medium after osmotic stress, the growth rate of the mutant was increased by a factor of 2. Moreover, electron microscopy analysis showed impaired morphology of the mutant grown under osmotic stress conditions in minimal medium. Addition of the osmoprotectant glycine betaine to the medium completely abolished the osmotic sensitivity phenotype of the ctc mutant. Altogether, these results suggest that the Ctc protein of L. monocytogenes is involved in osmotic stress tolerance in the absence of any osmoprotectant in the medium.

Cloning and characterization of linR, involved in regulation of the downstream pathway for gamma-hexachlorocyclohexane degradation in Sphingomonas paucimobilis UT26.

In Sphingomonas paucimobilis UT26, LinD and LinE activities, which are responsible for the degradation of gamma-hexachlorocyclohexane, are inducibly expressed in the presence of their substrates, 2,5-dichlorohydroquinone (2,5-DCHQ) and chlorohydroquinone (CHQ). The nucleotide sequence of the 1-kb upstream region of the linE gene was determined, and an open reading frame (ORF) was found in divergent orientation from linE. Because the putative protein product of the ORF showed similarity to the LysR-type transcriptional regulator (LTTR) family, we named it linR. The fragment containing the putative LTTR recognition sequence (a palindromic TN(11)A sequence), which exists immediately upstream of linE, was ligated with the reporter gene lacZ and was inserted into the plasmid expressing LinR under the control of the lac promoter. When the resultant plasmid was introduced into Escherichia coli, the LacZ activity rose in the presence of 2,5-DCHQ and CHQ. RNA slot blot analysis for the total RNAs of UT26 and UT102, which has an insertional mutation in linR, revealed that the expression of the linD and linE genes was induced in the presence of 2,5-DCHQ, CHQ, and hydroquinone in UT26 but not in UT102. These results indicated that the linR gene is directly involved in the inducible expression of the linD and linE genes.

Diversity of 2,3-dihydroxybiphenyl dioxygenase genes in a strong PCB degrader, Rhodococcus sp. strain RHA1

Two 2,3-dihydroxybiphenyl (23DHBP) dioxygenase genes, bphC1 and etbC involved in the degradation of polychlorinated biphenyl(s) (PCBs) have been isolated and characterized from a strong PCB degrader, Rhodococcus sp. RHA1. In this study, four new 23DHBP dioxygenase genes, designated as bphC2, bphC3, bphC4, and bphC5 were isolated from RHA1, and their nucleotide sequences were determined. Based on amino acid sequence similarities, all of the newly isolated bphC genes could be categorized into type I along with BphC1 and EtbC [Eltis, L.D. and Bolin, J.T., J. Bacteriol., 178, 5930–5937 (1996)]. Six bphC genes, including bphC1, etbC, and four new genes, were expressed in Escherichia coli to determine their substrate specificity. The activities of BphC2, BphC3, BphC4, and BphC5 were found to be specific to 23DHBP, while BphC1 and EtbC exhibited activities towards compounds other than 23DHBP, including catechol (CAT) and 3-methylcatechol (3MC). RNA slot blot hybridization analysis indicated that only bphC5 was transcribed among the newly isolated bphC in RHA1 cells grown on biphenyl and ethylbenzene. The nucleotide sequence of the flanking region of each bphC revealed a homolog of the 2-hydroxy-6-oxo-6-phenylhexa-2,4-dienoate (HOPD) hydrolase gene, bphD, just upstream of bphC5. The bphC5 and putative bphD genes may constitute an operon and play a role in the degradation of biphenyl and PCBs together with bphC1 and etbC. In contrast, the bphC2, bphC3, and bphC4 genes may not be involved in biphenyl and PCB degradation.

Diversity of 2,3-Dihydroxybiphenyl Dioxygenase Genes in a Strong PCB Degrader, Rhodococcus sp. Strain RHA1.

Two 2,3-dihydroxybiphenyl (23DHBP) dioxygenase genes, bphC1 and etbC involved in the degradation of polychlorinated biphenyl(s) (PCBs) have been isolated and characterized from a strong PCB degrader, Rhodococcus sp. RHA1. In this study, four new 23DHBP dioxygenase genes, designated as bphC2, bphC3, bphC4, and bphC5 were isolated from RHA1, and their nucleotide sequences were determined. Based on amino acid sequence similarities, all of the newly isolated bphC genes could be categorized into type I along with BphC1 and EtbC [Eltis, L.D. and Bolin, J.T., J. Bacteriol., 178, 5930–5937 (1996)]. Six bphC genes, including bphC1, etbC, and four new genes, were expressed in Escherichia coli to determine their substrate specificity. The activities of BphC2, BphC3, BphC4, and BphC5 were found to be specific to 23DHBP, while BphC1 and EtbC exhibited activities towards compounds other than 23DHBP, including catechol (CAT) and 3-methylcatechol (3MC). RNA slot blot hybridization analysis indicated that only bphC5 was transcribed among the newly isolated bphC in RHA1 cells grown on biphenyl and ethylbenzene. The nucleotide sequence of the flanking region of each bphC revealed a homolog of the 2-hydroxy-6-oxo-6-phenylhexa-2,4-dienoate (HOPD) hydrolase gene, bphD, just upstream of bphC5. The bphC5 and putative bphD genes may constitute an operon and play a role in the degradation of biphenyl and PCBs together with bphC1 and etbC. In contrast, the bphC2, bphC3, and bphC4 genes may not be involved in biphenyl and PCB degradation.

Novel bicistronic retroviral vector expressing gamma-glutamylcysteine synthetase and the multidrug resistance protein 1 (MRP1) protects cells from MRP1-effluxed drugs and alkylating agents.

We have constructed two retroviral vectors, one expressing multidrug resistance protein 1 (MRP1) alone (SF91MRP) and the other expressing MRP1 and gamma-glutamylcysteine synthetase (gamma-GCS), the rate-limiting enzyme of glutathione biosynthesis (SF91GCS-MRP). We have utilized the hybrid FMEV (Friend mink cell focus-forming/murine embryonic stem cell virus) backbone, previously shown to be efficient in early hematopoietic cells, even when coexpressing two distinct genes. In SF91GCS-MRP, the cDNAs were combined via an internal ribosomal entry site (IRES) sequence from poliovirus, resulting in a bicistronic mRNA produced via the long terminal repeat (LTR). Producer Fly-eco clones were established by trans-infection with vesicular stomatitis virus glycoprotein (VSV-G)-pseudotyped retroviral supernatants. Drug-resistant producer clones were subsequently selected with antimony potassium tartrate, a nonmutagenic MRP1 substrate. By RNA slot-blot and transduction of 3T3 fibroblasts, titers of both SF91MRP and SF91GCS-MRP were found to be greater than 10(6) viral particles/ml. The correct viral integration in the genome was established by Southern blotting. By flow cytometry, both MRP1 and bicistronic clones showed an increase in expression of the MRP1 protein. The bicistronic producer clones, as well as 3T3 cells transduced with SF91GCS-MRP, presented an increase in intracellular glutathione levels, compared with the parental counterparts. Producer cells, 3T3 fibroblasts transduced with either SF91MRP or SF91GCS-MRP, and primary murine myeloid progenitor cells transduced with SF91GCS-MRP were resistant to MRP1-effluxed drugs. However, only bicistronic producers, 3T3 fibroblasts transduced with SF91GCS-MRP, and primary murine myeloid progenitor cells transduced with SF91GCS-MRP were also resistant to alkylating agents. We conclude that the retrovirus SF91GCS-MRP has features that make it a suitable vector to induce bone marrow resistance to multiple classes of chemotherapeutic agents. The strategy of coexpressing gamma-GCS and MRP1 may help to design an effective in vivo selection for various clinical protocols of gene therapy.

Satellite-tagged transcribing sequences in Bubalus bubalis genome undergo programmed modulation in meiocytes: possible implications for transcriptional inactivation.

We cloned and sequenced a 1378 bp BamHI satellite DNA fraction from the water buffalo Bubalus bubalis and have studied its expression in different tissues. The GC-rich sequences of the resultant contig pDS5 crosshybridize only with bovid DNA and are not conserved evolutionarily. Typing of buffalo genomic DNA using pDS5 with several restriction enzymes revealed multilocus monomorphic bands. Similar typing of cattle, buffalo, goat, sheep, and gaur genomic DNA revealed variations in copy number and allele length giving rise to species-specific band patterns. Expression study of pDS5 in bubaline samples by RNA slot-blot, Northern blot, and RT-PCR showed various levels of signal in all the somatic tissues and germline cells except heart. A GenBank database search revealed homology of pDS5 sequences in the 5' region from nt 1-1261 with collagen gene. An AluI typing analysis of DNA from bubaline semen samples showed consistent loss of two bands. The presence of corresponding bands in somatic tissues suggests a sequence modulation within the pDS5 array in meiocytes during spermatogenesis, which is restored in the somatic cells after fertilization. Modulation of the satellite-tagged transcribing sequence in the meiocytes may be a mechanism of its inactivation.

Changes in rRNA levels during stress invalidates results from mRNA blotting: fluorescence in situ rRNA hybridization permits renormalization for estimation of cellular mRNA levels.

Regulation of gene expression can be analyzed by a number of different techniques. Some techniques monitor the level of specific mRNA directly, and others monitor indirectly by determining the level of enzymes encoded by the mRNA. Each method has its own inherent way of normalization. When results obtained by these techniques are compared between experiments in which differences in growth rates, strains, or stress treatments occur, the normalization procedure may have a significant impact on the results. In this report we present a solution to the normalization problem in RNA slot blotting experiments, in which mRNA levels routinely are normalized to a fixed amount of extracted total RNA. The cellular levels of specific mRNA species were estimated using a renormalization with the total RNA content per cell. By a combination of fluorescence in situ rRNA hybridization, which estimates the relative level of rRNA per cell, and slot blotting to rRNA probes, which estimates the level of rRNA per extracted total RNA, the amount of RNA per cell was calculated in a series of heat shock experiments with the gram-positive bacterium Lactococcus lactis. It was found that the level of rRNA per cell decreased to 30% in the course of the heat shock. This lowered ribosome level led to a decrease in the total RNA content, resulting in a gradually increasing overestimation of the mRNA levels throughout the experiment. Using renormalized cellular mRNA levels, the HrcA-mediated regulation of the genes in the hrcA-grpE-dnaK operon was analyzed. The hybridization data suggested a complex heat shock regulation indicating that the mRNA levels continued to rise after 30 min, but after renormalization the calculated average cellular levels exhibited a much simpler induction pattern, eventually attaining a moderately increased value.

Acid-induced gene expression in Helicobacter pylori: study in genomic scale by microarray.

To understand the RNA expression in response to acid stress of Helicobacter pylori in genomic scale, a microarray membrane containing 1,534 open reading frames (ORFs) from strain 26695 was used. Total RNAs of H. pylori under growth conditions of pH 7.2 and 5.5 were extracted, reverse transcribed into cDNA, and labeled with biotin. Each microarray membrane was hybridized with cDNA probe from the same strain under two different pH conditions and developed by a catalyzed reporter deposition method. Gene expression of all ORFs was measured by densitometry. Among the 1,534 ORFs, 53 ORFs were highly expressed (> or = 30% of rRNA control in densitometry ratios). There were 445 ORFs which were stably expressed (<30% of rRNA in densitometry) under both pH conditions without significant variation. A total of 80 ORFs had significantly increased expression levels at low pH, while expressions of 4 ORFs were suppressed under acidic condition. The remaining 952 ORFs were not detectable under either pH condition. These data were highly reproducible and comparable to those obtained by the RNA slot blot method. Our results suggest that microarray can be used in monitoring prokaryotic gene expression in genomic scale.

Expression of the Moraxella catarrhalis UspA1 protein undergoes phase variation and is regulated at the transcriptional level.

The UspA1 protein of Moraxella catarrhalis has been shown to function as an adhesin that mediates adherence to human epithelial cell lines in vitro (E. R. Lafontaine, L. D. Cope, C. Aebi, J. L. Latimer, G. H. McCracken, Jr., and E. J. Hansen, J. Bacteriol. 182:1364-1373, 2000). In the present study, cell lysates prepared from individual colonies of several M. catarrhalis wild-type strains were analyzed by Western blot analysis using monoclonal antibodies (MAbs) specific for the UspA1 protein. Expression of UspA1 was shown to exhibit phase variation that was correlated with both adherence ability in vitro and the number of guanine (G) residues contained within a homopolymeric [poly(G)]tract located upstream of the uspA1 open reading frame (ORF). Nucleotide sequence analysis revealed that isolates expressing relatively high levels of UspA1 had 10 G residues in their uspA1 poly(G)tracts, whereas isolates that expressed much lower levels of UspA1 had 9 G residues. This poly(G) tract was located 30 nucleotides (nt) upstream of the uspA1 ORF and 168 nt downstream of the uspA1 transcriptional start site. Primer extension experiments, RNA slot blot analysis, and cat reporter constructs were used to demonstrate that M. catarrhalis isolates with 10 G residues in their uspA1 poly(G) tracts expressed two-to threefold more uspA1 mRNA than did isolates which had 9 G residues in their poly(G)tracts. Northern hybridization analysis revealed that an intact uspA1 mRNA was readily detectable in RNA from M. catarrhalis isolates that had 10 G residues in their uspA1 poly(G) tracts, whereas no full-length uspA1 mRNA was observed in isolates whose poly(G)tracts contained 9 G residues. M. catarrhalis strain O35E uspA1 genes that contained wild-type and mutated poly(G) tracts were expressed in Haemophilus influenzae to demonstrate that the length and composition of the poly(G)tract affected expression of UspA1.

Identification of genes that are differentially expressed in extraocular and limb muscle

The extraocular muscles (EOM) are anatomically and physiologically distinct from other striated muscles in mammals. Among other differences, they can be driven to generate individual twitch contractions at an extremely high frequency and are resistant to [Ca 2+]-induced myonecrosis. While EOM are preferentially targeted in some neuromuscular diseases such as myasthenia gravis and congenital fibrosis of the extraocular muscles, they are enigmatically spared in Duchenne’s muscular dystrophy, despite the widespread damage seen in all other skeletal muscle groups during the course of this disease. To address the molecular mechanisms that specify the EOM-phenotype, we characterized the transcriptional profile of genes expressed in rat EOM versus limb muscle using a differential display strategy. Ninety-five putative differentially expressed cDNA tags were cloned, from which fourteen were confirmed as being differentially expressed by RNA slot blot and Northern blot analysis. Ten of these cDNAs were homologous to known human or murine genes and ESTs, while four genes that were upregulated in EOM were novel, and have been named expressed in ocular muscle ( eom) 1–4. The identification of these differentially expressed genes may provide mechanistic clues toward understanding the unique patho-physiological phenotype of EOM.

Molecular analysis of the ggtBCD gene cluster of Synechocystis sp. strain PCC6803 encoding subunits of an ABC transporter for osmoprotective compounds.

Genes encoding a substrate-binding protein (ggtB) and two integral membrane proteins (ggtC and ggtD) of the binding-protein-dependent ABC transporter for glucosylglycerol were identified in the genome of Synechocystis sp. strain PCC6803. These genes are clustered on the chromosome about 220 kb away from the previously identified ggtA gene, which encodes the ATP-binding protein of this transport system. The deduced amino acid sequences show significant similarities to corresponding subunits of ABC transporters mediating uptake of maltose and other di- and oligosaccharides in bacteria and archaea. Mutants were constructed by inserting an aphII gene cassette into the coding region of the ggtB, ggtC and ggtD genes. These mutants lost the ability to take up glucosylglycerol, sucrose and trehalose, proving that these compounds are transported by the same system. A truncated ggtB gene lacking the putative signal-peptide-encoding sequence was expressed in Escherichia coli yielding a histidine-tagged soluble protein. The recombinant GgtB protein bound glucosylglycerol with a KD of 0.45 microM and exhibited a somewhat lower affinity towards sucrose and a substantially lower affinity towards trehalose. Transcript analysis by RT-PCR indicated that the genes of the ggtBCD gene cluster form an operon. The transcript level estimated by RNA slot blot analysis using a ggtC-specific probe was very low in cells grown in basal medium but increased significantly after a salt shock.