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PubMed1989

Increased renal aldose reductase activity, immunoreactivity, and mRNA in streptozocin-induced diabetic rats.

Increased accumulation of renal sorbitol has been documented in the diabetic rat, and it has been suggested that this accumulation may be important in the pathogenesis of diabetic nephropathy. It is not clear whether sorbitol accumulation results from increases in substrate, activity of the aldose reductase (AR) protein molecule, or activity due to an increase in the amount of enzyme present. In this study, we have quantitated renal AR activity, immunoreactivity, and mRNA in rats 3 mo after induction of diabetes with streptozocin (STZ-D, 65 mg/kg body wt). Renal AR activity was significantly increased in diabetic rats compared with age-matched nondiabetic controls (0.95 +/- 0.05 vs. 0.51 +/- 0.03 U.mg-1.h-1, respectively, P less than .0005). Western blot analysis demonstrated that the antiserums recognized a single 40,000-Mr protein species in renal homogenates from both diabetic and nondiabetic rats. When quantitated in an immunodot assay, AR immunoreactivity was significantly increased in diabetic rats compared with nondiabetic controls (0.57 +/- 0.03 vs. 0.33 +/- 0.02 U, respectively, P less than .0005). Hybridization of Northern blots with a synthetic 36-nucleotide oligomer and an AR cDNA identified a 1.4-kilobase pair transcript; the abundance of the transcript was significantly increased in poly(A)+ RNA from the kidneys of diabetic compared with nondiabetic rats (P less than .005). This study demonstrates that renal AR activity is increased in the STZ-D rats and suggests that the increased AR activity can be in part explained by enhanced AR gene expression.

Aldehyde Reductase↗
PubMed1996

Abundant expression of transcription factor GATA-2 in proliferating but not in differentiated mast cells in tissues of mice: demonstration by in situ hybridization.

Although GATA-binding transcription factors (GATA-1 and GATA-2) are strongly expressed in cultured mast cells (CMCs), their expression in mast cells within tissues has not been reported. We examined the expression of GATA-1 and GATA-2 in skin tissues of mice using Northern blot analysis and in situ hybridization. mRNA for GATA-2 but not for GATA-1 was expressed in skin mast cells of WB-+/+ embryos between days 15 and 17 postcoitum (pc). The expression was downregulated on and after day 18 pc. Skin mast cells did not express GATA-2 after birth either. When the number of skin mast cells was compared with the number of GATA-2 mRNA-expressing cells, GATA-2 mRNA appeared to be expressed by mast cells only when the number was increasing. When the mRNA expression of high-affinity IgE receptor beta-subunit and mast cell carboxypeptidase A was used as differentiation markers, the expression of these mRNAs continued even after the downregulation of GATA-2 expression. To clarify the relationship of the proliferation and GATA-2 expression, proliferating CMCs derived from WBB6F1-+/+ mice were transplanted into the peritoneal cavity of mast cell-deficient WBB6F1-W/Wv mice. The CMCs stopped both the proliferation and GATA-2 expression after the transplantation, suggesting the association of these two parameters in mast cells within tissues of mice.

Animals↗
PubMed1998

The mouse formin (Fmn) gene: abundant circular RNA transcripts and gene-targeted deletion analysis.

BACKGROUND: Mutations in the mouse formin (Fmn) gene result in limb deformities and incompletely penetrant renal aplasia. A molecular genetic approach was taken to characterize novel circular RNAs from the Fmn gene and to understand the developmental effects of gene-targeted mutations. MATERIALS AND METHODS: RT-PCR and ribonuclease protection analyses were done to characterize the circular RNA transcripts arising from the Fmn gene. Two lines of mice with gene-targeted deletions of specific Fmn exons, namely exon 4 or exon 5, were generated and analyzed. RESULTS: In our analysis of formin cDNAs, we discovered a class of transcripts in which the exon order is reversed such that downstream exons are joined to the acceptor end of a specific exon that lies 5' to them in the genome. RT-PCR and ribonuclease protection analyses indicate that these transcripts are circular and are the major transcripts arising from this locus in adult brain and kidney. To gain insight into the biological function of these transcripts, we have systematically deleted the relevant exons using gene-targeted homologous recombination. The resulting mice fail to produce circular transcripts, but appear to produce normal amounts of the linear RNA isoforms from this locus. While these deficient mice have normal limbs, they display variably penetrant renal aplasia characteristic of other mutant formin alleles. CONCLUSIONS: Our results demonstrate novel circular transcripts arising from the Fmn gene. Moreover, their high levels of expression suggest that they are not products of aberrant splicing events, but instead, may play important biological roles. Mice with gene-targeted deletions of Fmn exons 4 or 5 lack these circular transcripts and have an incompletely penetrant renal agenesis phenotype. While the biologic function of circular Fmn RNA transcripts is not entirely known, our work suggests their possible involvement in kidney development.

Animals↗
PubMed1992

Abundance of the primary transcript and its processed product of growth-related genes in normal and leukemic cells during proliferation and differentiation.

The relative abundance of primary transcript and mature mRNA of the c-myc, calcyclin, S14 ribosomal protein, and rRNA genes was determined densitometrically after reverse transcriptase-polymerase chain reaction and Northern blotting analysis in resting and mitogen-stimulated lymphocytes, proliferating and terminally differentiated HL-60 cells, and leukemic blast cells. Transcription and processing of c-myc and rRNA gene transcripts increased proportionally after mitogen stimulation, whereas these processes were independent of cell cycling status in the case of the S14 gene. Normal lymphocytes showed an unexpectedly large amount of primary transcript of the calcyclin gene, whereas the corresponding mRNA was undetectable. The abundance of c-myc, calcyclin, and S14 mRNA in terminally differentiated HL-60 cells decreased sharply, compared to their proliferating counterparts. This decrease reflected post-transcriptional modulation, since the abundance of precursor remained essentially unchanged. After HL-60 differentiation, the 32S rRNA levels remained relatively high, whereas the 45S primary transcript almost disappeared. Leukemic blast cells displayed highly variable precursor/mRNA ratios of c-myc, calcyclin, and S14 genes but consistently high ratios of 32S to 45S RNA, suggesting that the cleavage rate of the 32S rRNA was sharply reduced in these cells, resulting in an accumulation of this molecule. These results suggest the importance of efficient processing of primary transcript to generate adequate functional mRNA, thus regulating gene expression. Furthermore, in terminally differentiated cells and leukemic blast cells a stabilization of the primary transcript without efficient processing can be observed. The role of the stabilization of the primary transcript in terminal differentiation is further supported by the results of run-off transcription, indicating a sharp decrease of c-myc and calcyclin transcription rate in retinoic acid/dimethyl sulfoxide-treated HL-60 cells.

Acute Disease↗
PubMed2006

Transcription factor RFX2 is abundant in rat testis and enriched in nuclei of primary spermatocytes where it appears to be required for transcription of the testis-specific histone H1t gene.

Previous work in our laboratory revealed upregulated transcription of the testis-specific linker histone H1t gene in pachytene primary spermatocytes during spermatogenesis. Using the H1t X-box as an affinity chromatography probe, we identified Regulatory Factor X2 (RFX2), a member of the RFX family of transcription factors, as a nuclear protein that binds the probe. We also showed that RFX2 activated the H1t promoter in transient expression assays. However, other RFX family members have the same DNA-binding domain and they also may regulate H1t gene expression. Therefore, in this study we examined the distribution of RFX2 and other RFX family members in rat testis germinal cells and in several tissues. Among tissues examined, RFX2 is most abundant in testis. Testis RFX2 is most abundant in spermatocytes where transcription of the H1t gene is upregulated and the steady-state H1t mRNA level is high. RFX2 levels decrease but RFX1 levels increase in early spermatids where H1t gene transcription is downregulated. Antibodies against RFX2 generate a shifted band in electrophoretic mobility shift assays (EMSA) using H1t or testisin X-box DNA probes with nuclear proteins from spermatocytes. These data support the hypothesis that RFX2 expression is upregulated in spermatocytes where it participates in activating transcription of the H1t gene and other testis genes. These data also support the possibility that other RFX family members may bind to the H1t promoter in other testis germinal cell types and in nongerminal cells to downregulate H1t gene transcription.

Animals↗
PubMed2004

Relative gene-silencing efficiencies of small interfering RNAs targeting sense and antisense transcripts from the same genetic locus.

Short interfering RNAs (siRNAs) directed against different regions of genes display marked variation in their potency in mediating mRNA degradation. Various factors have been proposed to affect the efficacy of siRNA. We explored some of the factors by evaluating in cultured human cells 28 randomly selected siRNAs targeting the GPR39 and MGC29643 transcripts derived from the same genetic locus but transcribed in opposite directions. Twenty of the 24 siRNAs targeting the overlapping regions of the transcripts simultaneously reduced the levels of both transcripts. Single nucleotide changes in either of the siRNA strands significantly reduced the gene-silencing efficiency of the siRNA on targeted sense transcript without affecting the antisense transcript. Overall, we observed a greater gene-silencing efficiency on the MGC29643 transcript than on the GPR39 transcript in HeLa cells. Since MGC29643 transcript is more abundant than the GPR39 transcript [0.24 versus 0.008% relative to 100% for glyceraldehyde-3-phosphate dehydrogenase (GAPDH)], the results suggest that the abundance of the mRNA affects the efficiency of silencing. Two additional observations supported this hypothesis. First, GAPDH whose intracellular level is the highest of the three was the most efficiently silenced. Second, a reversal of gene-silencing efficiency was observed in U-138 MG cells in which the relative abundance of the GPR39 and MGC29643 transcripts is also reversed. Our study suggests that low-abundant transcripts are less susceptible to siRNA-mediated degradation than medium- and high-abundant transcripts.

Cell Line, Tumor↗
PubMed1995

Two forms of Xenopus nuclear factor 7 have overlapping spatial but different temporal patterns of expression during development.

Xenopus nuclear factor 7 (xnf7) is a maternal gene product that functions in the determination of the dorsal-ventral body axis. We have cloned two xnf7 cDNAs, xnf7-O and xnf7-B, that have a different temporal pattern of expression. The cDNAs differ by 39 amino acid residues scattered throughout the molecule. Most of the changes were conservative in nature. Using gene specific probes we found that xnf7-O transcripts were abundant in oocytes and decreased until the neurula stage, after which they increased in abundance. Xnf7-B transcripts were in low abundance in oocytes and were expressed at high levels at the neurula stage and in adult brain. Both xnf7-O and xnf7-B transcripts at the neurula stage were localized in the dorsal region of the embryo, including the neural folds and somites. Xnf7 was not expressed in ventralized embryos that lacked dorsal structures, thereby substantiating its dorsal localization in the embryo. The promoter region of the xnf7-O gene does not possess a TATA box but does contain E2F, USF, Sp1-like and AP1 binding sites within the first 421 bp from the transcription initiation site. A 62 bp fragment of the xnf7-O promoter containing the Sp1-like and E2F sites can direct proper spatial expression of a transgene in embryos.

Animals↗
PubMed2005

Positive regulation of sugar catabolic pathways in the cyanobacterium Synechocystis sp. PCC 6803 by the group 2 sigma factor sigE.

The sigE gene of Synechocystis sp. PCC 6803 encodes a group 2 sigma factor for RNA polymerase and has been proposed to function in transcriptional regulation of nitrogen metabolism. By using microarray and Northern analyses, we demonstrated that the abundance of transcripts derived from genes important for glycolysis, the oxidative pentose phosphate pathway, and glycogen catabolism is reduced in a sigE mutant of Synechocystis maintained under the normal growth condition. Furthermore, the activities of the two key enzymes of the oxidative pentose phosphate pathway, glucose-6-phosphate dehydrogenase and 6-phosphogluconate dehydrogenase, encoded by the zwf and gnd genes were also reduced in the sigE mutant. The dark enhancements in both enzyme activity and transcript abundance apparent in the wild type were eliminated by the mutation. In addition, the sigE mutant showed a reduced rate of glucose uptake and an increased intracellular level of glycogen. Moreover, it was unable to proliferate under the light-activated heterotrophic growth conditions. These results indicate that SigE functions in the transcriptional activation of sugar catabolic pathways in Synechocystis sp. PCC 6803.

Bacterial Proteins↗
PubMed2002

Characterization and expression of steroidogenic acute regulatory protein and MLN64 cDNAs in trout.

Complementary DNA-encoding proteins with high homology to steroidogenic acute regulatory proteins (StAR) of mammals were cloned from rainbow trout head kidney and a mixture of several brook trout tissues. A cDNA encoding an MLN64 homolog was also cloned from brook trout. The C-terminal domains of rainbow trout StAR and brook trout StAR were very highly conserved compared with StAR of mammals. In rainbow trout, Northern and RT-PCR analyses showed abundant StAR transcripts in head kidney, ovary, and testis, and weaker signals were found in intestine, pyloric caeca, spleen, and kidney. Brief acute stress resulted in elevated plasma cortisol levels and a 2-fold increase in rainbow trout StAR transcripts in head kidneys sampled 3 h after exposure to the stressor. In brook trout, StAR transcripts were detected only in known steroidogenic tissues. Ovarian brook trout StAR mRNA was not seen until the onset of final maturation. Its abundance increased during germinal vesicle breakdown, peaked during and just following ovulation, and decreased by 2 wk post ovulation. Brook trout MLN64 transcripts were found in all tissues tested, and transcript abundance in ovarian samples did not vary during final oocyte maturation and ovulation. Both StAR structure and function appear to be highly conserved throughout the vertebrates.

Amino Acid Sequence↗
PubMed2003

Photosynthesis and growth of tobacco with a substituted bacterial Rubisco mirror the properties of the introduced enzyme.

Complete replacement, by biolistic plastid transformation, of the hexadecameric ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco) of tobacco (Nicotiana tabacum) with the dimeric version from the bacterium, Rhodospirillum rubrum, resulted in fully autotrophic and reproductive tobacco plants that required high CO(2) concentrations to grow (Whitney SM, Andrews TJ [2001] Proc Natl Acad Sci USA 98: 14738-14743). Growth and photosynthesis of these plants was compared with that of nontransformed tobacco and other controls where the rbcL gene for the large subunit of tobacco Rubisco was linked to the aadA selectable-marker gene, simulating the gene arrangement of the transformants with R. rubrum Rubisco. An arrangement of the rbcL and aadA genes that gave rise to an abundant monocistronic rbcL transcript and a one-fifth as abundant bicistronic rbcL-aadA transcript had Rubisco levels and photosynthetic properties similar to those of nontransformed tobacco. Direct linkage of the rbcL and aadA genes, resulting in exclusive production of a bicistronic mRNA transcript analogous to that of the transformants with R. rubrum Rubisco, reduced transcript abundance and tobacco Rubisco content. The analogous transcript with the R. rubrum rbcM gene substituted for rbcL was not only reduced in abundance, but was also translated less efficiently. The photosynthetic rates of the transformants and controls were measured at high CO(2) concentrations, using a mass spectrometric method. The rates and their responses to atmospheric CO(2) concentration mirrored the amounts and the kinetic properties of the Rubiscos present. The contents of total nitrogen, carbohydrates, and photosynthetic metabolites of the leaves were also consistent with the content and type of Rubisco.

Carbohydrate Metabolism↗
PubMed2006

Herpes simplex virus type 1 latently infected neurons differentially express latency-associated and ICP0 transcripts.

During the latent phase of herpes simplex virus type 1 (HSV-1) infection, the latency-associated transcripts (LATs) are the most abundant viral transcripts present in neurons, but some immediate-early viral transcripts, such as those encoding ICP0, have also been reported to be transcribed in latently infected mouse trigeminal ganglia (TG). A murine oro-ocular model of herpetic infection was used to study ICP0 gene expression in the major anatomical sites of HSV-1 latency, including the TG, superior cervical ganglion, spinal cord, and hypothalamus. An HSV-1 recombinant strain, SC16 110LacZ, revealed ICP0 promoter activity in several neurons in latently infected ganglia, and following infection with wild-type HSV-1 strain SC16, in situ hybridization analyses identified ICP0 transcripts in the nuclei of neurons at times consistent with the establishment of latency. Reverse transcription (RT)-PCR assays performed on RNA extracted from latently infected tissues indicated that ICP0 transcripts were detected in all anatomical sites of viral latency. Furthermore, quantitative real-time RT-PCR showed that neurons differentially expressed the LATs and ICP0 transcripts, with splicing of ICP0 transcripts being dependent on the anatomical location of latency. Finally, TG neurons were characterized by high-level expression of LATs and detection of abundant unspliced ICP0 transcripts, a pattern markedly different from those of other anatomical sites of HSV-1 latency. These results suggest that LATs might be involved in the maintenance of HSV-1 latency through the posttranscriptional regulation of ICP0 in order to inhibit expression of this potent activator of gene expression during latency.

Animals↗
PubMed2004

Cloning, expression, and characterization of three new mouse cytochrome p450 enzymes and partial characterization of their fatty acid oxidation activities.

The mammalian CYP2C subfamily is one of the largest and most complicated in the cytochrome P450 superfamily. In this report, we describe the organization of the mouse Cyp2c locus, which contains 15 genes and four pseudogenes, all of which are located in a 5.5-megabase region on chromosome 19. We cloned three novel mouse CYP2C cDNAs (designated CYP2C50, CYP2C54, and CYP2C55) from mouse heart, liver, and colon, respectively. All three cDNAs contain open reading frames that encode 490 amino acid polypeptides that are 57 to 95% identical to other CYP2Cs. The recombinant CYP2C proteins were expressed in Escherichia coli after N-terminal modification, partially purified, and shown to be active in the metabolism of both arachidonic acid (AA) and linoleic acid, albeit with different catalytic efficiencies and profiles. CYP2C50 and CYP2C54 metabolize AA to epoxyeicosatrienoic acids (EETs) primarily, and linoleic acid to epoxyoctadecenoic acids (EOAs) primarily, whereas CYP2C55 metabolizes AA to EETs and hydroxyeicosatetraenoic acids and linoleic acid to EOAs and hydroxyoctadecadienoic acids. Northern blotting and reverse transcription-polymerase chain reaction analysis reveal that CYP2C50 transcripts are abundant in liver and heart; CYP2C54 transcripts are present in liver, kidney, and stomach; and CYP2C55 transcripts are abundant in liver, colon, and kidney. Immunoblotting studies demonstrate that CYP2C50 protein is expressed in liver and heart, CYP2C54 protein is detected primarily in liver, and CYP2C55 protein is present primarily in colon. Immunohistochemistry reveals that CYP2C55 is most abundant in surface columnar epithelium in the cecum. We conclude that these new CYP2C enzymes are probably involved in AA and linoleic acid metabolism in mouse hepatic and extrahepatic tissues.

Amino Acid Sequence↗
PubMed2004

The rehydration transcriptome of the desiccation-tolerant bryophyte Tortula ruralis: transcript classification and analysis.

BACKGROUND: The cellular response of plants to water-deficits has both economic and evolutionary importance directly affecting plant productivity in agriculture and plant survival in the natural environment. Genes induced by water-deficit stress have been successfully enumerated in plants that are relatively sensitive to cellular dehydration, however we have little knowledge as to the adaptive role of these genes in establishing tolerance to water loss at the cellular level. Our approach to address this problem has been to investigate the genetic responses of plants that are capable of tolerating extremes of dehydration, in particular the desiccation-tolerant bryophyte, Tortula ruralis. To establish a sound basis for characterizing the Tortula genome in regards to desiccation tolerance, we analyzed 10,368 expressed sequence tags (ESTs) from rehydrated rapid-dried Tortula gametophytes, a stage previously determined to exhibit the maximum stress induced change in gene expression. RESULTS: The 10, 368 ESTs formed 5,563 EST clusters (contig groups representing individual genes) of which 3,321 (59.7%) exhibited similarity to genes present in the public databases and 2,242 were categorized as unknowns based on protein homology scores. The 3,321 clusters were classified by function using the Gene Ontology (GO) hierarchy and the KEGG database. The results indicate that the transcriptome contains a diverse population of transcripts that reflects, as expected, a period of metabolic upheaval in the gametophyte cells. Much of the emphasis within the transcriptome is centered on the protein synthetic machinery, ion and metabolite transport, and membrane biosynthesis and repair. Rehydrating gametophytes also have an abundance of transcripts that code for enzymes involved in oxidative stress metabolism and phosphorylating activities. The functional classifications reflect a remarkable consistency with what we have previously established with regards to the metabolic activities that are important in the recovery of the gametophytes from desiccation. A comparison of the GO distribution of Tortula clusters with an identical analysis of 9,981 clusters from the desiccation sensitive bryophyte species Physcomitrella patens, revealed, and accentuated, the differences between stressed and unstressed transcriptomes. Cross species sequence comparisons indicated that on the whole the Tortula clusters were more closely related to those from Physcomitrella than Arabidopsis (complete genome BLASTx comparison) although because of the differences in the databases there were more high scoring matches to the Arabidopsis sequences. The most abundant transcripts contained within the Tortula ESTs encode Late Embryogenesis Abundant (LEA) proteins that are normally associated with drying plant tissues. This suggests that LEAs may also play a role in recovery from desiccation when water is reintroduced into a dried tissue. CONCLUSION: The establishment of a rehydration EST collection for Tortula ruralis, an important plant model for plant stress responses and vegetative desiccation tolerance, is an important step in understanding the genome level response to cellular dehydration. The type of transcript analysis performed here has laid the foundation for more detailed functional and genome level analyses of the genes involved in desiccation tolerance in plants.

Arabidopsis↗
PubMed1997

Pex/PEX tissue distribution and evidence for a deletion in the 3' region of the Pex gene in X-linked hypophosphatemic mice.

PEX, a phosphate-regulating gene with homology to endopeptidases on the X chromosome, was recently identified as the candidate gene for X-linked hypophosphatemia. In the present study, we cloned mouse and human Pex/PEX cDNAs encoding part of the 5' untranslated region, the protein coding region, and the entire 3' untranslated region, determined the tissue distribution of Pex/PEX mRNA, and characterized the Pex mutation in the murine Hyp homologue of the human disease. Using the reverse transcriptase/polymerase chain reaction (RT/PCR) and ribonuclease protection assays, we found that Pex/PEX mRNA is expressed predominantly in human fetal and adult mouse calvaria and long bone. With RNA from Hyp mouse bone, an RT/PCR product was generated with 5' but not 3' Pex primer pairs and a protected Pex mRNA fragment was detected with 5' but not 3' Pex riboprobes by ribonuclease protection assay. Analysis of the RT/PCR product derived from Hyp bone RNA revealed an aberrant Pex transcript with retention of intron sequence downstream from nucleotide 1302 of the Pex cDNA. Pex mRNA was not detected on Northern blots of poly (A)+ RNA from Hyp bone, while a low-abundance Pex transcript of approximately 7 kb was apparent in normal bone. Southern analysis of genomic DNA from Hyp mice revealed the absence of hybridizing bands with cDNA probes from the 3' region of the Pex cDNA. We conclude that Pex/PEX is a low-abundance transcript that is expressed predominantly in bone of mice and humans and that a large deletion in the 3' region of the Pex gene is present in the murine Hyp homologue of X-linked hypophosphatemia.

Amino Acid Sequence↗
PubMed1998

Antigen processing and autoimmunity. Evaluation of mRNA abundance and function of HLA-linked genes.

Quantitative defects in the density of conformationally correct human lymphocyte antigen (HLA) class I complexes on the surface of lymphocytes are apparent in patients with diverse HLA-linked autoimmune diseases, including Type I diabetes and Sjögren's syndrome. First, HLA class I expression was investigated in individuals with two rare and genetically divergent polyglandular autoimmune diseases. Polyglandular failure patients whose disease showed HLA linkage, but not those whose disease was not HLA linked, exhibited decreased HLA class I expression on the surface of their lymphocytes as well as a reduced abundance of transcripts of the HLA-linked genes Tap1 and Tap2, both of which encode proteins that contribute to HLA class I processing. Second, lymphocytes from patients with insulin-dependent diabetes mellitus (IDDM), Sjögren's syndrome, Graves' disease, and Hashimoto's disease showed varying degrees of decreased abundance of mRNAs that encode Tap1, Tap2, Lmp2, or Lmp7 (the latter two proteins also contribute to HLA class I processing). Third, in twins discordant for IDDM, reduced transcript abundance was preferential to diabetic subjects. Fourth, functional assays of isolated diabetic proteasomes, the peptide cutting complex containing LMP2 and LMP7 proteins, revealed altered peptidase activity. These data suggest that defective transcription of HLA class I-processing genes could contribute to the quantitative defect in cell-surface expression in autoimmune lymphocytes of HLA-controlled disease.

ATP Binding Cassette Transporter, Subfamily B, Mem↗
PubMed2004

Culture-independent analysis of fecal enterobacteria in environmental samples by single-cell mRNA profiling.

A culture-independent method called mRNA profiling has been developed for the analysis of fecal enterobacteria and their physiological status in environmental samples. This taxon-specific approach determines the single-cell content of selected gene transcripts whose abundance is either directly or inversely proportional to growth state. Fluorescence in situ hybridization using fluorochrome-labeled oligonucleotide probes was used to measure the cellular concentration of fis and dps mRNA. Relative levels of these transcripts provided a measure of cell growth state and the ability to enumerate fecal enterobacterial cell number. Orthologs were cloned by inverse PCR from several major enterobacterial genera, and probes specific for fecal enterobacteria were designed using multiple DNA sequence alignments. Probe specificity was determined experimentally using pure and mixed cultures of the major enterobacterial genera as well as secondary treated wastewater samples seeded with pure culture inocula. Analysis of the fecal enterobacterial community resident in unseeded secondary treated wastewater detected fluctuations in transcript abundance that were commensurate with incubation time and nutrient availability and demonstrated the utility of the method using environmental samples. mRNA profiling provides a new strategy to improve wastewater disinfection efficiency by accelerating water quality analysis.

Bacterial Outer Membrane Proteins↗
PubMed1994

Mitochondrial transcripts are processed but are not edited normally in Trypanosoma equiperdum (ATCC 30019) which has kDNA sequence deletion and duplication.

Analyses of the Trypanosoma equiperdum (ATCC 30019) maxicircle reveals deletions, duplications and rearrangement compared to T. brucei. The genes for 9S rRNA and 12 proteins are absent. The 12S rRNA and cytochrome oxidase subunit I (COI) genes lack their 3' ends and are adjacent indicating deletion of intervening genes. The remaining two NADH dehydrogenase subunit genes (ND4 and ND5), the ribosomal protein RPS12 gene and the CR5 gene are duplicated and rearranged. ND4, RPS12 and the CR4 transcripts are abundant in steady state RNA while 12S rRNA and COI transcripts are not detected. Full length ND5 transcripts are rare, if present, but chimeric ND5/ND4 transcripts are abundant. The CR4 and RPS12 transcripts are the size of unedited RNAs suggesting that they are processed. However, they are not edited normally, presumably due to the absence of minicircle gRNA genes.

Animals↗
PubMedDate not supplied

Plasma membrane calcium ATPase gene expression in bovine lens epithelium.

Plasma membrane calcium adenosine triphosphatase (Ca(2+)-ATPase) is an energy-dependent protein responsible for transporting cytosolic calcium across the plasma membrane. Multiple plasma membrane Ca(2+)-ATPase isoforms are expressed from four genes (PMCA1-4) and alternative mRNA splicing. We have studied PMCA gene expression in bovine lens epithelium tissues by reverse transcription-polymerase chain reaction, Southern blot, and Northern blot hybridization. All four PMCA genes are expressed in the lens epithelium, the PMCA3 transcript being the most abundant. The transcripts for PMCA1, PMCA2, and PMCA4 exist in decreasing order of abundance. There is no evidence for the expression of any novel PMCA genes in bovine lens epithelium.

Animals↗