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1.  Quantification of Bias Related to the Extraction of DNA Directly from Soils 
Applied and Environmental Microbiology  1999;65(12):5409-5420.
In recent years, several protocols based on the extraction of nucleic acids directly from the soil matrix after lysis treatment have been developed for the detection of microorganisms in soil. Extraction efficiency has often been evaluated based on the recovery of a specific gene sequence from an organism inoculated into the soil. The aim of the present investigation was to improve the extraction, purification, and quantification of DNA derived from as large a portion of the soil microbial community as possible, with special emphasis placed on obtaining DNA from gram-positive bacteria, which form structures that are difficult to disrupt. Furthermore, we wanted to identify and minimize the biases related to each step in the procedure. Six soils, covering a range of pHs, clay contents, and organic matter contents, were studied. Lysis was carried out by soil grinding, sonication, thermal shocks, and chemical treatments. DNA was extracted from the indigenous microflora as well as from inoculated bacterial cells, spores, and hyphae, and the quality and quantity of the DNA were determined by gel electrophoresis and dot blot hybridization. Lysis efficiency was also estimated by microscopy and viable cell counts. Grinding increased the extracellular DNA yield compared with the yield obtained without any lysis treatment, but none of the subsequent treatments clearly increased the DNA yield. Phage λ DNA was inoculated into the soils to mimic the fate of extracellular DNA. No more than 6% of this DNA could be recovered from the different soils. The clay content strongly influenced the recovery of DNA. The adsorption of DNA to clay particles decreased when the soil was pretreated with RNA in order to saturate the adsorption sites. We also investigated different purification techniques and optimized the PCR methods in order to develop a protocol based on hybridization of the PCR products and quantification by phosphorimaging.
PMCID: PMC91737  PMID: 10583997
2.  Simultaneous Recovery of RNA and DNA from Soils and Sediments 
Applied and Environmental Microbiology  2001;67(10):4495-4503.
Recovery of mRNA from environmental samples for measurement of in situ metabolic activities is a significant challenge. A robust, simple, rapid, and effective method was developed for simultaneous recovery of both RNA and DNA from soils of diverse composition by adapting our previous grinding-based cell lysis method (Zhou et al., Appl. Environ. Microbiol. 62:316–322, 1996) for DNA extraction. One of the key differences is that the samples are ground in a denaturing solution at a temperature below 0°C to inactivate nuclease activity. Two different methods were evaluated for separating RNA from DNA. Among the methods examined for RNA purification, anion exchange resin gave the best results in terms of RNA integrity, yield, and purity. With the optimized protocol, intact RNA and high-molecular-weight DNA were simultaneously recovered from 19 soil and stream sediment samples of diverse composition. The RNA yield from these samples ranged from 1.4 to 56 μg g of soil−1 dry weight), whereas the DNA yield ranged from 23 to 435 μg g−1. In addition, studies with the same soil sample showed that the DNA yield was, on average, 40% higher than that in our previous procedure and 68% higher than that in a commercial bead milling method. For the majority of the samples, the DNA and RNA recovered were of sufficient purity for nuclease digestion, microarray hybridization, and PCR or reverse transcription-PCR amplification.
doi:10.1128/AEM.67.10.4495-4503.2001
PMCID: PMC93195  PMID: 11571148
3.  Pex/PEX tissue distribution and evidence for a deletion in the 3' region of the Pex gene in X-linked hypophosphatemic mice. 
Journal of Clinical Investigation  1997;99(6):1200-1209.
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.
PMCID: PMC507933  PMID: 9077527
4.  Structure and expression of the guinea pig preproenkephalin gene: site-specific cleavage in the 3' untranslated region yields truncated mRNA transcripts in specific brain regions. 
Molecular and Cellular Biology  1995;15(4):2080-2089.
We isolated the guinea pig preproenkephalin gene from a genomic library by hybridization to a rat cDNA probe. The entire nucleotide sequence of the gene was determined. Genomic Southern blot hybridization demonstrated that the gene exists in a single copy within the genome. On the basis of RNase protection transcript mapping and homology comparisons with known preproenkephalin sequences from other species and assuming a poly(A) tail length of 100 residues, we predicted an mRNA transcript of approximately 1,400 nucleotides encoded by three exons. Northern (RNA) blot analysis of total RNA from several brain regions showed high levels of preproenkephalin mRNA in the caudate putamen, nucleus accumbens, and hypothalamus, with detectable levels in the amygdala, ventral tegmental area, and central gray and also in the pituitary. Unexpectedly, in several brain regions, the mRNA appeared not only in the 1,400-nucleotide length but also in a shorter length of approximately 1,130 bases. Significant amounts of the shorter mRNA were found in the caudate putamen, nucleus accumbens, and amygdala. The longer, but not the shorter, transcripts from the caudate putamen were found to be polyadenylated, but the difference in size was not due solely to the presence of poly(A) tails. Northern gel analysis of total RNA from the caudate putamen with probes from each exon, together with RNase protection mapping of the 3' end of the mRNA demonstrated that the 1,400-base preproenkephalin mRNA transcripts are cleaved in a site-specific manner in some brain regions, yielding a 1,130-base transcript and a 165-base polyadenylated fragment derived from the terminal end of the 3' untranslated region of the mRNA. This cleavage may serve as a preliminary step in RNA degradation and provide a mechanism for control of preproenkephalin mRNA abundance through selective degradation.
PMCID: PMC230435  PMID: 7891703
5.  Nucleotide sequence of afsB, a pleiotropic gene involved in secondary metabolism in Streptomyces coelicolor A3(2) and "Streptomyces lividans". 
Journal of Bacteriology  1986;168(1):257-269.
The nucleotide sequence of afsB from Streptomyces coelicolor A3(2), a pleiotropic gene which positively controls the biosynthesis of A-factor and the pigmented antibiotics actinorhodin and undecylprodigiosin in S. coelicolor A3(2) and "Streptomyces lividans," was determined. The determinant of the afsB gene, which includes the putative AfsB protein consisting of 243 amino acids, was mapped functionally by tests for A-factor and pigment production in "S. lividans" and S. coelicolor A3(2) after introduction of recombinant plasmids containing various restriction endonuclease fragments on the vector plasmids pIJ41 and pIJ702. The putative AfsB protein contains two regions separated by 167 residues which resemble conserved domains of known DNA-binding proteins. High-resolution nuclease S1 protection mapping revealed that the afsB mRNA, approximately 1,300 base pairs (bp) long, which was determined by Northern blot hybridization, had its start point 340 bp upstream of the putative methionine start codon. The Northern hybridization experiment also suggested that the afsB gene was constitutively transcribed throughout growth. Also shown by the Northern hybridization was the presence of an unidentified gene with an extraordinary amount of 880-bp mRNA located downstream from afsB. Dot hybridization with the brown pigment production genes, possibly involved in polyketide biosynthesis, as the probe suggested that the afsB gene did not stimulate transcription of the pigment production genes. In Southern blot DNA-DNA hybridization analysis with the afsB sequence as the probe, sequences exhibiting various degrees of homology were found in several Streptomyces spp. A DNA sequence showing strong homology to the afsB in Streptomyces griseus FT-1, a high streptomycin producer, behaved like an extrachromosomal element, homologous to the afsA gene, a structural gene for A-factor biosynthesis.
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PMCID: PMC213446  PMID: 2428809
6.  Simultaneous Fluorescence In Situ Hybridization of mRNA and rRNA in Environmental Bacteria 
We developed for Bacteria in environmental samples a sensitive and reliable mRNA fluorescence in situ hybridization (FISH) protocol that allows for simultaneous cell identification by rRNA FISH. Samples were carbethoxylated with diethylpyrocarbonate to inactivate intracellular RNases and pretreated with lysozyme and/or proteinase K at different concentrations. Optimizing the permeabilization of each type of sample proved to be a critical step in avoiding false-negative or false-positive results. The quality of probes as well as a stringent hybridization temperature were determined with expression clones. To increase the sensitivity of mRNA FISH, long ribonucleotide probes were labeled at a high density with cis-platinum-linked digoxigenin (DIG). The hybrid was immunocytochemically detected with an anti-DIG antibody labeled with horseradish peroxidase (HRP). Subsequently, the hybridization signal was amplified by catalyzed reporter deposition with fluorochrome-labeled tyramides. p-Iodophenylboronic acid and high concentrations of NaCl substantially enhanced the deposition of tyramides and thus increased the sensitivity of our approach. After inactivation of the antibody-delivered HRP, rRNA FISH was performed by following routine protocols. To show the broad applicability of our approach, mRNA of a key enzyme of aerobic methane oxidation, particulate methane monooxygenase (subunit A), was hybridized with different types of samples: pure cultures, symbionts of a hydrothermal vent bivalve, and even sediment, one of the most difficult sample types with which to perform successful FISH. By simultaneous mRNA FISH and rRNA FISH, single cells are identified and shown to express a particular gene. Our protocol is transferable to many different types of samples with the need for only minor modifications of fixation and permeabilization procedures.
doi:10.1128/AEM.70.9.5426-5433.2004
PMCID: PMC520857  PMID: 15345429
7.  Testis-specific expression of the human MYCL2 gene. 
Nucleic Acids Research  1991;19(11):3129-3137.
We have characterized the expression of MYCL2, an intronless X-linked gene related to MYCL1. RNase protection analysis of a panel of human normal and tumor tissues has revealed that MYCL2 is expressed almost exclusively in human adult normal testis; much lower levels of transcript were detected in one human lung adenocarcinoma. No MYCL2 transcript was found in human testis RNA obtained from second trimester fetuses. This observation suggests a germ cell rather than somatic cell origin of the transcript and possible developmental regulation of MYCL2. Northern blot analysis of poly(A)+ RNA from adult human normal testis with an antisense riboprobe revealed a transcript of approximately 4.8-kb, which is in agreement with the size predicted from the MYCL2 nucleotide sequence. Antisense transcripts were found spanning regions of MYCL2 corresponding to all three exons of MYCL1. No sizable open reading frame was seen for the MYCL2 antisense transcripts suggesting that they may represent either regulatory sequences or an intron of a gene encoded by the complementary strand. RNase protection assays and the 5' RACE protocol (Rapid Amplification of cDNA Ends) were used to address the localization of the transcription start site of the MYCL2 sense transcript and different putative promoters and transcription regulatory elements have been identified.
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PMCID: PMC328281  PMID: 1711681
8.  Detection of latency-related viral RNAs in trigeminal ganglia of rabbits latently infected with herpes simplex virus type 1. 
Journal of Virology  1987;61(12):3820-3826.
Using a combination of in situ hybridization and Northern (RNA) blot analysis, we investigated herpes simplex virus type 1 (HSV-1) transcriptional activity in an ocular rabbit model of HSV-1 latency. Radioactively labeled cloned fragments, representing virtually the entire HSV-1 genome, were individually hybridized to RNA in sections of trigeminal ganglia taken from rabbits during the latent phase of infection with HSV-1 (McKrae). Our results suggest that two discrete latency-related RNAs (LR-RNAs) may be present. The LR-RNAs were localized mainly in the nuclei of neurons. The more abundant LR-RNA was detected in approximately 3% of all neurons examined and was designated major LR-RNA. The other LR-RNA, designated minor LR-RNA, was detected in approximately 0.3% of neurons from latently infected rabbits. The genes for the LR-RNAs mapped in the vicinity of the immediate-early gene ICP0 (also designated IE110). The gene for the major LR-RNA partially overlapped the left (3') end of the ICP0 gene. In situ hybridization with single-stranded RNA probes showed that this LR-RNA was of complementary sense to that of ICP0 mRNA. Northern blot analysis gave an approximate size for this LR-RNA of 1.8 to 2.2 kilobases. The minor LR-RNA mapped to or near the right (5') end of the ICP0 gene. The detection of LR-RNAs suggests the possibility that these RNAs or their products may play significant roles in the initiation and/or maintenance of HSV-1 latency.
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PMCID: PMC255998  PMID: 2824816
9.  Conservation of the fourth gene among rotaviruses recovered from asymptomatic newborn infants and its possible role in attenuation. 
Journal of Virology  1986;60(3):972-979.
RNA-RNA hybridization was performed to assess the extent of genetic relatedness among human rotaviruses isolated from children with gastroenteritis and from asymptomatic newborn infants. 32P-labeled single-stranded RNAs produced by in vitro transcription from viral cores of the different strains tested were used as probes in two different hybridization assays: undenatured genomic RNAs were resolved by polyacrylamide gel electrophoresis, denatured in situ, electrophoretically transferred to diazobenzyloxymethyl-paper (Northern blots), and then hybridized to the probes under two different conditions of stringency; and denatured genomic double-stranded RNAs were hybridized to the probes in solution and the hybrids which formed were identified by polyacrylamide gel electrophoresis. When analyzed by Northern blot hybridization at a low level of stringency, all genes from the strains tested cross-hybridized, providing evidence for some sequence homology in each of the corresponding genes. However, when hybridization stringency was increased, a difference in gene 4 sequence was detected between strains recovered from asymptomatic newborn infants ("nursery strains") and strains recovered from infants and young children with diarrhea. Although the nursery strains exhibited serotypic diversity (i.e., each of the four strains tested belonged to a different serotype), the fourth gene appeared to be highly conserved. Similarly, each of the virulent strains tested belonged to a different serotype; nonetheless, there was significant conservation of sequence among the fourth genes of three of these viruses. Significantly, the conserved fourth genes of the nursery strains were distinct from the fourth gene of each of the virulent viruses. These results were confirmed and extended during experiments in which the RNA-RNA hybridization was carried out in solution and the resulting hybrids were analyzed by polyacrylamide gel electrophoresis. Under these conditions, the fourth genes of the nursery strains were closely related to each other but not to the fourth genes of the virulent viruses. Full-length hybrids did not form between the fourth genes from the nursery strains and the corresponding genes from the strains recovered from symptomatic infants and young children.
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PMCID: PMC253335  PMID: 3023685
10.  Modified Whole-Mount In situ Hybridization Protocol for the Detection of Transgene Expression in Electroporated Chick Embryos 
PLoS ONE  2008;3(7):e2638.
Background
In vivo electroporation has been extensively used as an effective means of DNA transfer for analyzing gene function as well as gene regulation in developmental systems. In any of these two types of studies, the correct spatial and temporal expression of the electroporated transgene can only be accurately assessed by in situ hybridization.
Methodology/Principal Findings
While analyzing transgene expression in electroporated chicken embryos, we verified that transgene riboprobes cross-hybridized with the exogenous plasmid DNA when embryos were processed by conventional whole-mount in situ hybridization (WISH).
Conclusions/Significance
Here we describe a modification to the WISH protocol that is essential to prevent DNA cross-hybridization and to specifically detect transgene mRNA transcripts in electroporated embryos. Our optimized WISH procedure can be applied not only to electroporated chick embryos but also to other embryos or adult tissues that have been transfected with large amounts of reporter- or expression construct DNA.
doi:10.1371/journal.pone.0002638
PMCID: PMC2441435  PMID: 18612382
11.  RNA integrity as a quality indicator during the first steps of RNP purifications : A comparison of yeast lysis methods 
BMC Biochemistry  2004;5:14.
Background
The completion of several genome-sequencing projects has increased our need to assign functions to newly identified genes. The presence of a specific protein domain has been used as the determinant for suggesting a function for these new genes. In the case of proteins that are predicted to interact with mRNA, most RNAs bound by these proteins are still unknown. In yeast, several protocols for the identification of protein-protein interactions in high-throughput analyses have been developed during the last years leading to an increased understanding of cellular proteomics. If any of these protocols or similar approaches shall be used for the identification of mRNA-protein complexes, the integrity of mRNA is a critical factor.
Results
We compared the effect of different lysis protocols on RNA integrity. We report dramatic differences in RNA stability depending on the method used for yeast cell lysis. Glass bead milling and French Press lead to degraded mRNAs even in the presence of RNase inhibitors. Thus, they are not suitable to purify intact mRNP complexes or to identify specific mRNAs bound to proteins.
Conclusion
We suggest a novel protocol, grinding deep-frozen cells, for the preparation of protein extracts that contain intact RNAs, as lysis method for the purification of mRNA-protein complexes from yeast cells.
doi:10.1186/1471-2091-5-14
PMCID: PMC524479  PMID: 15461782
12.  Equine herpesvirus 1 glycoprotein D: mapping of the transcript and a neutralization epitope. 
Journal of Virology  1992;66(11):6451-6460.
Studies with molecular and immunological techniques identified and mapped the transcript encoding glycoprotein D (gD) of equine herpesvirus 1 KyA, as well as two continuous gD antigenic determinants. Three mRNA species of 5.5, 3.8, and 1.7 kb overlap the gD open reading frame and are transcribed from the DNA strand encoding gD. Northern (RNA) blot hybridization with both DNA clones and riboprobes, as well as S1 nuclease analyses, showed the 3.8-kb mRNA to encode gD and to be synthesized as a late (beta-gamma) transcript. The 3.8-kb gD mRNA initiates within the US segment 91 and 34 nucleotides downstream of the CCAAT and TATA elements, respectively, and encodes a potential polypeptide of 392 amino acids. The termination site of this transcript maps within the terminal repeat at a site also used by the 5.5-kb mRNA and the IR6-encoded 1.2-kb mRNA, such that these three transcripts form a 3'-coterminal nested set. The extended size (2,250 nucleotides) of the 3' untranslated region of the gD transcript and its termination within the terminal repeat may result from the deletion of 3,859 bp, which eliminates two consensus polyadenylation signals downstream of the gD open reading frame of EHV-1 KyA. Use of antisera to synthetic peptides of 19 amino acids (residues 4 to 22) and 20 amino acids (residues 267 to 285) in Western immunoblot analyses revealed that gD is present in EHV-1 virions as a 55-kDa polypeptide. In addition, these antisera detected the 55-kDa protein as well as 58- and 47-kDa polypeptides in infected-cell extracts at late times of infection. Residues 4 to 22 make up a continuous neutralizing epitope of gD, since incubation of equine herpesvirus 1 with the anti-19-mer serum prior to infection results in reduced numbers of plaques and reduced levels of virus-encoded thymidine kinase. Complement is not required for neutralization mediated by the anti-19-mer serum.
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PMCID: PMC240137  PMID: 1383565
13.  Further Taxonomic Studies on Straight to Flexuous Streptomycetes1 
Journal of Bacteriology  1965;89(2):331-342.
Pridham, Thomas G. (Northern Regional Research Laboratory, Peoria, Ill.), and Allister J. Lyons. Further taxonomic studies on straight to flexuous streptomycetes. J. Bacteriol. 89:331–342. 1965.—The best way to handle streptomycete classification, nomenclature, and identification is through application of a genus-species-subspecies concept. To establish a species, principal criteria are morphology of chains of spores and nature of spore-wall surfaces. Subspecies can be differentiated one from another by other criteria, such as chromogenicity, colors of sporulating aerial mycelium and of vegetative mycelium, carbon-utilization patterns, and assessment of qualitative production of antibiotics and sensitivity and resistance to antibacterial antibiotics. A literature study and laboratory studies of some strains suggested that streptomycetes with straight chains of spores could easily be differentiated from those with flexuous chains of spores. An intensive study of about 75 holotype and potential neotype strains indicated that such a differentiation is difficult to accomplish with confidence. Only 19 of the strains had straight chains of spores. These strains are considered, at this time, to be members of the species Streptomyces venezuelae Ehrlich et al. Forty-two of the strains had flexuous chains of spores and were assigned to the species Streptomyces griseus (Krainsky) Waksman and Henrici. Six of the strains had unusual spore-chain morphology. Classic taxonomic procedures allowed the separation of all the strains into a number of categories. The results suggest that more precise information on relationships of strains and qualitative antibiotic production will allow clarification of their sub-specific status. Consideration of the results obtained with strains having aberrant morphology allows some speculation on ranges of variations in morphology that might be encountered with the streptomycetes.
PMCID: PMC305513  PMID: 14255699
14.  Optimization of oligonucleotide arrays and RNA amplification protocols for analysis of transcript structure and alternative splicing 
Genome Biology  2003;4(10):R66.
A novel, unbiased amplification protocol has been developed that permits labeling of entire transcripts. Also, hybridization conditions, probe characteristics, and analysis algorithms were optimized for detection of exons, exon-intron edges, and exon junctions.
Microarrays offer a high-resolution means for monitoring pre-mRNA splicing on a genomic scale. We have developed a novel, unbiased amplification protocol that permits labeling of entire transcripts. Also, hybridization conditions, probe characteristics, and analysis algorithms were optimized for detection of exons, exon-intron edges, and exon junctions. These optimized protocols can be used to detect small variations and isoform mixtures, map the tissue specificity of known human alternative isoforms, and provide a robust, scalable platform for high-throughput discovery of alternative splicing.
PMCID: PMC328455  PMID: 14519201
15.  An RNA isolation system for plant tissues rich in secondary metabolites 
BMC Research Notes  2011;4:85.
Background
Secondary metabolites are reported to interfere with the isolation of RNA particularly with the recipes that use guanidinium-based salt. Such interference was observed in isolation of RNA with medicinal plants rheum (Rheum australe) and arnebia (Arnebia euchroma). A rapid and less cumbersome system for isolation of RNA was essential to facilitate any study related to gene expression.
Findings
An RNA isolation system free of guanidinium salt was developed that successfully isolated RNA from rheum and arnebia. The method took about 45 min and was successfully evaluated on twenty one tissues with varied secondary metabolites. The A260/280 ratio ranged between 1.8 - 2.0 with distinct 28 S and 18 S rRNA bands visible on a formaldehyde-agarose gel.
Conclusions
The present manuscript describes a rapid protocol for isolation of RNA, which works well with all the tissues examined so far. The remarkable feature was the success in isolation of RNA with those tissues, wherein the most commonly used methods failed. Isolated RNA was amenable to downstream applications such as reverse transcription-polymerase chain reaction (RT-PCR), differential display (DD), suppression subtractive hybridization (SSH) library construction, and northern hybridization.
doi:10.1186/1756-0500-4-85
PMCID: PMC3079660  PMID: 21443767
16.  Zebrafish Whole Mount High-Resolution Double Fluorescent In Situ Hybridization 
Whole mount in situ hybridization is one of the most widely used techniques in developmental biology. Here, we present a high-resolution double fluorescent in situ hybridization protocol for analyzing the precise expression pattern of a single gene and for determining the overlap of the expression domains of two genes. The protocol is a modified version of the standard in situ hybridization using alkaline phosphatase and substrates such as NBT/BCIP and Fast Red 1,2. This protocol utilizes standard digoxygenin and fluorescein labeled probes along with tyramide signal amplification (TSA) 3. The commercially available TSA kits allow flexible experimental design as fluorescence emission from green to far-red can be used in combination with various nuclear stains, such as propidium iodide, or fluorescence immunohistochemistry for proteins. TSA produces a reactive fluorescent substrate that quickly covalently binds to moieties, typically tyrosine residues, in the immediate vicinity of the labeled antisense riboprobe. The resulting staining patterns are high resolution in that subcellular localization of the mRNA can be observed using laser scanning confocal microscopy 3,4. One can observe nascent transcripts at the chromosomal loci, distinguish nuclear and cytoplasmic staining and visualize other patterns such as cortical localization of mRNA. Studies in Drosophila indicate that roughly 70% of mRNAs exhibit specific patterns of subcellular localization that frequently correlate with the function of the encoded protein 5. When combined with computer-aided reconstruction of 3D confocal datasets, our protocol allows the detailed analysis of mRNA distribution with sub-cellular resolution in whole vertebrate embryos.
doi:10.3791/1229
PMCID: PMC2789764  PMID: 19322135
17.  Direct Quantification of Gene Expression Using Capillary Electrophoresis with Laser-Induced Fluorescence 
Analytical biochemistry  2006;360(1):23-29.
Quantification of gene expression provides valuable information regarding the response of cells or tissue to stimuli and is often accomplished by monitoring the level of messenger RNA (mRNA) being transcribed for a particular protein. While numerous methods are commonly used to monitor gene expression, including northern blotting, real time polymerase chain reaction and RNase protection, each has its own drawbacks and limitations. Capillary electrophoresis with laser induced fluorescence (CE-LIF) can reduce protocol time, eliminate the need for radioactivity and provide superior sensitivity and dynamic range for quantification of RNA. In addition, CE-LIF can be used to directly determine the amount of an RNA species present, something which is difficult and not normally accomplished using current methods. Gene expression is detected using a fluorescently labeled riboprobe specific for a given RNA species. This direct approach was validated by analyzing levels of 28S RNA and also used to determine the amount of Discoidin Domain Receptor 2 mRNA in cardiac tissue.
doi:10.1016/j.ab.2006.10.018
PMCID: PMC1829314  PMID: 17113023
RNA; gene expression; capillary electrophoresis; fluorescence
18.  Isolation and localization of herpes simplex virus type 1 mRNA. 
Journal of Virology  1979;30(3):805-820.
Herpes simplex virus (HSV) DNA bound to cellulose has been used as a reagent to isolate viral mRNA for size analysis on denaturing agarose gels. Total viral polysomal polyadenylated RNA was isolated from cells late after infection when such RNA has sequences encoded by approximately 45% of the HSV DNA. This RNA has a size range of from 1.5 to greater than or equal to 8 kilobases, with certain sizes, such as 1.7 to 1.9 kilobases, being favored. We have used the restriction endonucleases HindIII and XbaI singly and together to generate various sized fragments covering the entire HSV-1 genome. These fragments have been bound to cellulose to allow isolation of HSV-1 mRNA annealing to different regions of the viral genome. Discrete sizes of viral mRNA are associated with certain regions of the genome, but the mRNA population hybridizing to even the smallest restriction fragments is complex. We used hybridization of size-fractionated RNA to Southern blots of restriction fragments of HSV-1 DNA generated by the BglII as well as HindIII and XbaI endonucleases to confirm the preparative hybridization data and to provide some overlap data for positioning transcripts. The data of blot and preparative hybridization agreed very well and were combined to construct a preliminary transcription map of HSV-1. Such a map revealed at least two areas of the long unique region of the HSV-1 genome which annealed to a large number of HSV-1 transcripts. Furthermore, discrete-sized mRNA species larger than 5 kilobases in length were found only in the middle of the long unique region. The implications of these data are discussed.
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PMCID: PMC353391  PMID: 225543
19.  Transfection of mouse fibroblast cells with a promoterless herpes simplex virus thymidine kinase gene: number of integrated gene copies and structure of single and amplified gene sequences. 
Molecular and Cellular Biology  1985;5(2):295-304.
Plasmids carrying the herpes simplex virus thymidine kinase (tk) gene were used to transfect thymidine kinase-deficient cells of the mouse fibroblast cell line LM(tk-). Individual cell clones were cultivated in selective hypoxanthine-aminopterin-thymidine medium to determine the number of integrated plasmid copies which was almost always in the range of one to three copies per genome. In contrast, cells transfected with plasmids carrying a promoterless "truncated" tk gene typically contained between 10 and 25 copies per genome. Surprisingly, when the truncated tk gene was transfected together with a simian virus 40 DNA segment, including its transcriptional enhancer, the number of integrated tk gene copies was always low, between one and three copies per genome. We have analyzed the genomic organization of integrated truncated tk genes by blot hybridization of restricted cellular DNA and concluded that integrated units of plasmid DNA molecules are arranged in tandem arrays which remain stable in most cases for many cell generations. In only 1 of ca. 20 cell clones did we observe a retraction and expansion of the number of integrated promoterless tk genes as a response to the removal or readdition of selective pressure. Surprisingly, the thymidine kinase activity determined in extracts from cells growing in selective hypoxanthine-aminopterin-thymidine medium (high numbers of integrated tk gene copies) was nearly the same as the enzymatic activity in cells growing in nonselective medium (low copy numbers). Moreover, Northern blots of polyadenylated RNA, extracted from cells growing under selective and nonselective conditions, showed that, in both cases, the major species of tk-specific transcripts was ca. 1.5 kilobases in size, as expected for a tk-specific mRNA containing the entire coding region of the gene. Thus, disproportionate DNA replication appeared not to be essential for an active tk gene expression in these cells. We discuss possible pathways leading to the formation of tandem arrays of integrated truncated tk genes and the conditions required for disproportionate DNA replication in the unique case in which we found a retraction and expansion of tk gene copy numbers as a response to selective growth conditions.
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PMCID: PMC366712  PMID: 2579320
20.  Characterization of the barley chloroplast transcription units containing psaA-psaB and psbD-psbC. 
Nucleic Acids Research  1987;15(13):5217-5240.
Four plastid genes, psaA, psaB, psbD and psbC, were localized on the barley plastid genome. PsaA was adjacent to psaB in one transcription unit and psbD was adjacent to psbC in a second transcription unit. The transcription units containing psaA-psaB and psbD-psbC are separated by approximately 25 kbp on the barley plastid genome and are transcribed convergently. Transcripts hybridizing to each transcription unit were characterized by northern blot analysis, S1 protection experiments and primer extension analysis. Two 5.3 kb transcripts hybridize to psaA-psaB. The two transcripts have a common 5' end but differ at their 3' ends by about 26 nucleotides. The transcription unit which contains psbD-psbC also includes trnS(UGA), trnG(GCC), and an open reading frame which codes for a 62 amino acid protein. Six large transcripts ranging from 5.7 kb to 1.7 kb hybridize to the psbD-psbC transcription unit as well as several RNAs of tRNA size. The large transcripts arise from three 5' ends and two clusters of 3' ends. The 3' ends map near trnG(GCC) and trnS(UGA) and could be generated by RNA processing or termination of transcription. Two of the six transcripts hybridize to psbC but not psbD suggesting that translation of psbD and psbC could occur on separate RNAs.
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PMCID: PMC305957  PMID: 3601671
21.  Detection of point mutations in type I collagen by RNase digestion of RNA/RNA hybrids. 
Nucleic Acids Research  1990;18(14):4227-4236.
We have developed a strategy for the detection, localization and sequence determination of point mutations in the mRNA coding for the alpha 1(I) and alpha 2(I) chains of type I collagen. Point mutations are detected by RNase A cleavage of mismatches in RNA/RNA hybrids. The mRNAs coding for the fibrillar collagens present special problems for hybrid analysis because of their large size and their GC-rich and repetitive sequences. We have generated a series of overlapping antisense riboprobes covering the entire pro alpha 1(I) and pro alpha 2(I) mRNAs. Uniformly labelled normal antisense riboprobes are hybridized with the total fibroblast RNA of patients with possible mutations in type I collagen. Mismatches in the resulting RNA/RNA hybrids are cleaved with RNase A and the labelled riboprobe cleavage products are examined electrophoretically. The sensitivity and specificity of the system were demonstrated by the detection and localization of a known point mutation in the codon for alpha 1(I) glycine 988 (1). DNA for sequencing the mutations localized by hybrid analysis may be obtained by either (1) generation of a fibroblast cDNA library and isolation of both alleles by plaque screening, or (2) a more rapid method using first strand cDNA synthesis from poly (A+)-mRNA, followed by PCR amplification of the mutation-containing region of the DNA/RNA hybrid. This strategy for detection and isolation has wide application not only for mutations causing connective tissue disorders, but also for mutations in other large and repetitive genes. We have used this strategy for the detection and sequencing of a point mutation in alpha 2(I) mRNA associated with a case of lethal osteogenesis imperfecta. The G----A point mutation in the codon for alpha 2(I) glycine residue 805 results in the substitution of an aspartic acid at this position and is consistent with the proband's collagen protein data.
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PMCID: PMC331183  PMID: 1696002
22.  Identification of a tenth mRNA of respiratory syncytial virus and assignment of polypeptides to the 10 viral genes. 
Journal of Virology  1984;49(2):572-578.
Nine mRNAs, their cDNA clones, and a genome transcriptional map have been reported previously for respiratory syncytial virus (P. L. Collins and G. W. Wertz, Proc. Natl. Acad. Sci. U.S.A. 80:3208-3212, 1983). We report here the identification of a 10th viral mRNA, designated mRNA 2b (molecular weight [MW] ca. 0.39 X 10(6)), that was detected by RNA (Northern) blot hybridization with cDNA clones. Analysis of a polycistronic readthrough transcript was used to deduce the position in the viral transcriptional map of the gene encoding the newly identified mRNA. The polypeptide coding assignments of 9 of the 10 respiratory syncytial virus mRNAs were determined. Individual viral mRNAs were purified by hybridization selection with nine unique, nonoverlapping cDNA clones and analyzed by translation in vitro. Each of the nine mRNAs encoded a single polypeptide chain. The coding assignments were as follows: RNA 1a (MW ca. 0.24 X 10(6)), a 9,500-dalton (9.5K) protein; RNA 1b (MW 0.26 X 10(6)), an 11K protein; RNA 1c (MW 0.26 X 10(6)), a 14K protein; RNA 2a (MW 0.38 X 10(6)), the 34K phosphorylated (P) protein; RNA 2b (MW 0.39 X 10(6)), a 36K protein; RNA 3a (MW 0.40 X 10(6)), the 26K matrix (M) protein; RNA 3b (MW 0.40 X 10(6)), a 24K protein; RNA 4 (MW 0.47 X 10(6)), the 42K major nucleocapsid (N) protein; and RNA 5 (MW 0.74 X 10(6)), a 59K protein. The cDNA clones used for the hybridization selections were respiratory syncytial virus specific and did not hybridize with uninfected-cell mRNA; therefore the proteins synthesized with the selected mRNAs were virus specific. The 9.5K, 11K, 14K, 24K, M, P, 36K, N, and 59K proteins were encoded by different mRNAs; therefore these nine proteins are all unique. The 9.5K, 11K, 14K, 24K, M, P, and N proteins synthesized in vitro with hybrid-selected mRNAs each had counterparts with the same electrophoretic mobilities in extracts of virus-infected cells. The in vitro polypeptides and their authentic counterparts were shown to be closely related by limited digest peptide mapping. The 36K and 59K polypeptides lacked counterparts with the same electrophoretic mobilities in infected cells and therefore are candidates for the unprocessed precursors of the viral F and G glycoproteins. The 10th viral mRNA, the 2,500K RNA 7, was not tested directly but is the only known mRNA of the appropriate size to encode the 200K large (L) protein of the viral nucleocapsid. These assignments account for all 10 of the reported viral mRNAs and bring to 10 the number of known unique viral proteins.
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PMCID: PMC255499  PMID: 6546401
23.  Candicidin Biosynthesis Gene Cluster Is Widely Distributed among Streptomyces spp. Isolated from the Sediments and the Neuston Layer of the Trondheim Fjord, Norway▿ †  
Applied and Environmental Microbiology  2009;75(10):3296-3303.
A large number of Streptomyces bacteria with antifungal activity isolated from samples collected in the Trondheim fjord (Norway) were found to produce polyene compounds. Investigation of polyene-containing extracts revealed that most of the isolates produced the same compound, which had an atomic mass and UV spectrum corresponding to those of candicidin D. The morphological diversity of these isolates prompted us to speculate about the involvement of a mobile genetic element in dissemination of the candicidin biosynthesis gene cluster (can). Eight candicidin-producing isolates were analyzed by performing a 16S rRNA gene-based taxonomic analysis, pulsed-field gel electrophoresis, PCR, and Southern blot hybridization with can-specific probes. These analyses revealed that most of the isolates were related, although they were morphologically diverse, and that all of them contained can genes. The majority of the isolates studied contained large plasmids, and two can-specific probes hybridized to a 250-kb plasmid in one isolate. Incubation of the latter isolate at a high temperature resulted in loss of the can genes and candicidin production, while mating of the “cured” strain with a plasmid-containing donor restored candicidin production. The latter result suggested that the 250-kb plasmid contains the complete can gene cluster and could be responsible for conjugative transfer of this cluster to other streptomycetes.
doi:10.1128/AEM.02730-08
PMCID: PMC2681660  PMID: 19286787
24.  Time Course Analysis and Mapping of Autographa californica Nuclear Polyhedrosis Virus Transcripts 
Journal of Virology  1984;50(3):739-747.
To study the expression of the Autographa californica nuclear polyhedrosis virus (AcNPV) genome, intracellular virus-specific proteins and mRNAs were pulsed-labeled, extracted, and analyzed at 6-h intervals during the replicative cycle. Most RNAs were detected between 12 and 24 h postinfection (p.i.), but many continued to be synthesized until late in infection. Polyhedrin and p8 mRNAs were the two most abundant late viral RNA transcripts, and they were synthesized at high rates until late in the infection cycle (60 h p.i.). The abundancy control of polyhedrin and p8 polypeptides was considered to be at the level of transcription. Two other major mRNAs in infected cells were 0.6-kilobase RNA, which was synthesized at its highest rate 12 to 18 h p.i., and 2.8-kilobase RNA, which was synthesized from 12 h p.i. until 48 h p.i. Cytoplasmic polyadenylic acid-containing RNA was isolated at 6-h intervals and was analyzed by Northern blot hybridization. At least 50 virus RNA transcripts were recognized, sized, and mapped onto the genome. Six RNAs hybridized to EcoRI-H, -I, and -J, and HindIII-Q AcNPV DNA restriction fragments, seven RNAs hybridized to EcoRI-B and -D DNA fragments, five RNAs hybridized to EcoRI-A and -E regions of the genome, four RNAs hybridized to EcoRI-C and -N DNA fragments, and one RNA species hybridized to EcoRI-O AcNPV DNA. A transcription map of the AcNPV genome was constructed, and the data were correlated with previously published translation maps.
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PMCID: PMC255732  PMID: 16789250
25.  Streptomyces hygroscopicus has two glutamine synthetase genes. 
Journal of Bacteriology  1990;172(9):5343-5351.
Streptomyces hygroscopicus, which produces the glutamine synthetase inhibitor phosphinothricin, possesses at least two genes (glnA and glnB) encoding distinct glutamine synthetase isoforms (GSI and GSII). The glnB gene was cloned from S. hygroscopicus DNA by complementation in an Escherichia coli glutamine auxotrophic mutant (glnA). glnB was subcloned in Streptomyces plasmids by insertion into pIJ486 (pMSG3) and pIJ702 (pMSG5). Both constructions conferred resistance to the tripeptide form of phosphinothricin (bialaphos) and were able to complement a glutamine auxotrophic marker in S. coelicolor. Sodium dodecyl sulfate-polyacrylamide gel electrophoretic analysis of S. lividans(pMSG5) revealed a highly overexpressed 40-kilodalton protein. When GS was purified from this strain, it was indistinguishable in apparent molecular mass from the 40-kilodalton protein. The nucleic acid sequence of the cloned region contained an open reading frame which encoded a protein whose size, amino acid composition, and N-terminal sequence corresponded to those of the purified GS. glnB had a high G + C content and codon usage typical of streptomycete genes. A comparison of its predicted amino acid sequence with the protein data bases revealed that it encoded a GSII-type enzyme which had previously been found only in various eucaryotes (47 to 50% identity) and nodulating bacteria such as Bradyrhizobium spp. (42% identity). glnB had only 13 to 18% identity with eubacterial GSI enzymes. Southern blot hybridization experiments showed that sequences similar to glnB were present in all of the five other Streptomyces species tested, as well as Frankia species. These results do not support the previous suggestion that GSII-type enzymes found in members of the family Rhizobiaceae represent a unique example of interkingdom gene transfer associated with symbiosis in the nodule. Instead they imply that the presence of more than one gene encoding GS may be more common among soil microorganisms than previously appreciated.
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PMCID: PMC213198  PMID: 1975585

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