ABSTRACT
Efficacy of cleaning methods against SARS-CoV-2 suspended in either 5% soil load (SARS-soil) or simulated saliva (SARS-SS) was evaluated immediately (hydrated virus, T0) or 2 hours post-contamination (dried virus, T2). Hard water dampened wiping (DW) of surfaces, resulted in 1.77-3.91 log reduction (T0) or 0.93-2.41 log reduction (T2). Incorporating surface pre-wetting by spraying with a detergent solution (D + DW) or hard water (W + DW) just prior to dampened wiping did not unilaterally increase efficacy against infectious SARS-CoV-2, however, the effect was nuanced with respect to surface, viral matrix, and time. Cleaning efficacy on porous surfaces (seat fabric, SF) was low. W + DW on stainless steel (SS) was as effective as D + DW for all conditions except SARS-soil at T2 on SS. DW was the only method that consistently resulted in > 3-log reduction of hydrated (T0) SARS-CoV-2 on SS and ABS plastic. These results suggest that wiping with a hard water dampened wipe can reduce infectious virus on hard non-porous surfaces. Pre-wetting surfaces with surfactants did not significantly increase efficacy for the conditions tested. Surface material, presence or absence of pre-wetting, and time post-contamination affect efficacy of cleaning methods.
Subject(s)
COVID-19 , Viruses , Humans , SARS-CoV-2 , Disinfection/methods , Detergents/pharmacology , Touch , COVID-19/prevention & control , WaterABSTRACT
The list of EPA-approved disinfectants for coronavirus features many products for use on hard, non-porous materials. There are significantly fewer products registered for use on porous materials. Further, many common, high-touch surfaces fall in between non-porous materials such as glass and porous materials such as soft fabrics. The objective of this study was to assess the efficacy of selected commercially available disinfectant products against coronaviruses on common, high-touch surfaces. Four disinfectants (Clorox Total 360, Bleach solution, Vital Oxide, and Peroxide Multi-Surface Cleaner) were evaluated against Murine Hepatitis Virus A59 (MHV) as a surrogate coronavirus for SARS-CoV-2. MHV in cell culture medium was inoculated onto four materials: stainless steel, latex-painted drywall tape, Styrene Butadiene rubber (rubber), and bus seat fabric. Immediately (T0) or 2-hr (T2) post-inoculation, disinfectants were applied by trigger-pull or electrostatic sprayer and either held for recommended contact times (Spray only) or immediately wiped (Spray and Wipe). Recovered infectious MHV was quantified by median tissue culture infectious dose assay. Bleach solution, Clorox Total 360, and Vital Oxide were all effective (>3-log10 reduction or complete kill of infectious virus) with both the Spray Only and Spray and Wipe methods on stainless steel, rubber, and painted drywall tape when used at recommended contact times at both T0 and T2 hr. Multi-Surface Cleaner unexpectedly showed limited efficacy against MHV on stainless steel within the recommended contact time; however, it showed increased (2.3 times greater efficacy) when used in the Spray and Wipe method compared to Spray Only. The only products to achieve a 3-log10 reduction on fabric were Vital Oxide and Clorox Total 360; however, the efficacy of Vital Oxide against MHV on fabric was reduced to below 3-log10 when applied by an electrostatic sprayer compared to a trigger-pull sprayer. This study highlights the importance of considering the material, product, and application method when developing a disinfection strategy for coronaviruses on high-touch surfaces.
Subject(s)
COVID-19 , Disinfectants , Murine hepatitis virus , Animals , Disinfectants/pharmacology , Disinfection/methods , Mice , Rubber/pharmacology , SARS-CoV-2 , Sodium Hypochlorite/pharmacology , Stainless Steel/pharmacologyABSTRACT
The distribution of guanine and cytosine nucleotides throughout a genome, or the GC content, is associated with numerous features in mammals; understanding the pattern and evolutionary history of GC content is crucial to our efforts to annotate the genome. The local GC content is decaying toward an equilibrium point, but the causes and rates of this decay, as well as the value of the equilibrium point, remain topics of debate. By comparing the results of 2 methods for estimating local substitution rates, we identify 620 Mb of the human genome in which the rates of the various types of nucleotide substitutions are the same on both strands. These strand-symmetric regions show an exponential decay of local GC content at a pace determined by local substitution rates. DNA segments subjected to higher rates experience disproportionately accelerated decay and are AT rich, whereas segments subjected to lower rates decay more slowly and are GC rich. Although we are unable to draw any conclusions about causal factors, the results support the hypothesis proposed by Khelifi A, Meunier J, Duret L, and Mouchiroud D (2006. GC content evolution of the human and mouse genomes: insights from the study of processed pseudogenes in regions of different recombination rates. J Mol Evol. 62:745-752.) that the isochore structure has been reshaped over time. If rate variation were a determining factor, then the current isochore structure of mammalian genomes could result from the local differences in substitution rates. We predict that under current conditions strand-symmetric portions of the human genome will stabilize at an average GC content of 30% (considerably less than the current 42%), thus confirming that the human genome has not yet reached equilibrium.
Subject(s)
Base Composition/genetics , Evolution, Molecular , Genome, Human , Isochores/genetics , Humans , Mutation/geneticsSubject(s)
Erythropoiesis/genetics , Rats , Animals , Conserved Sequence , DNA/genetics , Evolution, Molecular , Genes, Regulator , Genome , Genomics/methods , Humans , Mice , Selection, Genetic , Sequence AlignmentABSTRACT
The amount of noncoding genomic DNA sequence that aligns between human and mouse varies substantially in different regions of their genomes, and the amount of repetitive DNA also varies. In this report, we show that divergence in noncoding nonrepetitive DNA is strongly correlated with the amount of repetitive DNA in a region. We investigated aligned DNA in four large genomic regions with finished human sequence and almost or completely finished mouse sequence. These regions, totaling 5.89 Mb of DNA, are on different chromosomes and vary in their base composition. An analysis based on sliding windows of 10 kb shows that the fraction of aligned noncoding nonrepetitive DNA and the fraction of repetitive DNA are negatively correlated, both at the level of an entire region and locally within it. This conclusion is strongly supported by a randomization study, in which repetitive elements are removed and randomly relocated along the sequences. Thus, regions of noncoding genomic DNA that accumulated fewer point mutations since the primate-rodent divergence also suffered fewer retrotransposition events. These results indicate that some regions of the genome are more "flexible" over the time scale of mammalian evolution, being able to accommodate many point mutations and insertions, whereas other regions are more "rigid" and accumulate fewer changes. Stronger conservation is generally interpreted as indicating more extensive or more important function. The evidence presented here of correlated variation in the rates of different evolutionary processes across noncoding DNA must be considered in assessing such conservation for evidence of selection.
Subject(s)
DNA/genetics , Genome , Interspersed Repetitive Sequences , Animals , Evolution, Molecular , Genome, Human , Humans , Mice , Random Allocation , Retroelements , Sequence Homology, Nucleic AcidABSTRACT
Building on the pioneering efforts of Professor Huisman, several different databases of hemoglobin variants have been developed, each with progressively increased capacity for sophisticated queries and prompt updating. These resources are reviewed in the context of a larger plan for providing related resources on hemoglobins, benign and pathological variation in these proteins and the genes that encode them, and the regulation of the globin genes.
Subject(s)
Databases, Nucleic Acid , Hemoglobins/genetics , Gene Expression , Genetic Variation , Humans , Internet , Sequence AlignmentABSTRACT
OBJECTIVE: The objective of this study was to evaluate the American College of Cardiology/American Heart Association (ACC/AHA) guidelines for exercise testing (EXT) after successful coronary revascularization (CR) using the Bypass Angioplasty Revascularization Investigation experience. BACKGROUND: The ACC/AHA guidelines state that EXT within three years of successful CR is not useful. METHODS: The 1,678 patients randomized to CR by either angioplasty or bypass surgery were required to take symptom-limited treadmill tests one, three and five years after revascularization. RESULTS: Patients who took the test at each specified time had a much lower subsequent two-year mortality than those who did not (1.9% vs. 9.4%, 3.5% vs. 12.6% and 3.3% vs. 11.0% at one, three and five years, respectively, after CR [p < 0.0001 for each]). Exercise parameters at the one- and three-year test did not improve a multivariable model of survival after including clinical parameters. Exercising to Bruce stage 3 or generating a Duke score >-6 were independently predictive of two-year survival after the five-year test. ST depression on the one-year test was associated with more revascularizations (relative risk = 1.6; p < 0.001). CONCLUSIONS: Patients with stable multivessel coronary disease who took a protocol-mandated exercise test at one, three and five years after revascularization were at low risk for mortality in the two years subsequent to each test. Exercise parameters did not improve prediction of mortality in the two years after the one- and three-year tests. The ACC/AHA guidelines on exercise testing after CR (no value for routine testing in stable patients for three years after revascularization) are supported by these results.
Subject(s)
Angioplasty, Balloon, Coronary , Coronary Artery Bypass , Coronary Disease/epidemiology , Exercise Test , Coronary Disease/mortality , Diabetic Angiopathies/epidemiology , Female , Humans , Male , Middle Aged , Multivariate Analysis , Practice Guidelines as Topic , Prognosis , Risk AssessmentABSTRACT
The major distal regulatory sequence for the beta-globin gene locus, the locus control region (LCR), is composed of multiple hypersensitive sites (HSs). Different models for LCR function postulate that the HSs act either independently or synergistically. To test these possibilities, we have constructed a series of expression cassettes in which the gene encoding the enhanced green fluorescent protein (EGFP) is under the control of DNA fragments containing single and multiple HSs of the LCR. LCR DNA fragments containing only the minimal region needed for position-independent expression (HS cores) or containing cores plus flanking sequences (HS units) were compared to ascertain whether conserved sequences between the HS cores contributed to enhancement. Expression of these constructs was measured after targeted integration into three defined loci in murine erythroleukemia cells using recombinase-mediated cassette exchange. At all three marked loci, synergistic enhancement of expression was observed in cassettes containing a combination of HS2, HS3, and HS4 units. In contrast, HS2, HS3, and HS4 cores (without flanking sequences) give an activity equivalent to the sum of the activities of the individual HS cores. These data suggest a model in which an HS core plus flanking regions, bound by specific proteins, forms a structure needed for interaction with other HS units to confer strong enhancement by the LCR. The three targeted integration sites differ substantially in their permissivity for expression, but even the largest LCR construct tested could not overcome these position effects to confer equal expression at all three sites.
Subject(s)
Enhancer Elements, Genetic , Globins/genetics , Locus Control Region , Binding Sites , HumansABSTRACT
We have identified the first gene lying on the centromeric side of the alpha-globin gene cluster on human 16p13.3. The gene, called 16pHQG;16 (HGMW-approved symbol LUC7L), is widely transcribed and lies in the opposite orientation with respect to the alpha-globin genes. This gene may represent a mammalian heterochromatic gene, encoding a putative RNA-binding protein similar to the yeast Luc7p subunit of the U1 snRNP splicing complex that is normally required for 5' splice site selection. To examine the role of the 16pHQG;16 gene in delimiting the extent of the alpha-globin regulatory domain, we mapped its mouse orthologue, which we found to lie on mouse chromosome 17, separated from the mouse alpha-cluster on chromosome 11. Establishing the full extent of the human 16pHQG;16 gene has allowed us to define the centromeric limit of the region of conserved synteny around the human alpha-globin cluster to within an 8-kb segment of chromosome 16.
Subject(s)
Centromere/ultrastructure , Globins/chemistry , Globins/genetics , RNA-Binding Proteins/chemistry , RNA-Binding Proteins/genetics , Alternative Splicing , Amino Acid Sequence , Animals , Blotting, Northern , Blotting, Southern , CHO Cells , Cell Line , Centromere/metabolism , Chromosomes, Human, Pair 16 , Chromosomes, Human, Pair 17 , Conserved Sequence , Cricetinae , Evolution, Molecular , Exons , Humans , Introns , Mice , Models, Genetic , Molecular Sequence Data , Protein Structure, Tertiary , RNA Splicing , RNA, Messenger/metabolism , Reverse Transcriptase Polymerase Chain Reaction , Ribonucleoproteins, Small Nuclear/metabolism , Sequence Homology, Amino Acid , Telomere/metabolism , Tissue Distribution , Transcription, GeneticABSTRACT
We have cloned, sequenced and annotated segments of DNA spanning the mouse, chicken and pufferfish alpha globin gene clusters and compared them with the corresponding region in man. This has defined a small segment ( approximately 135-155 kb) of synteny and conserved gene order, which may contain all of the elements required to fully regulate alpha globin gene expression from its natural chromosomal environment. Comparing human and mouse sequences using previously described methods failed to identify the known regulatory elements. However, refining these methods by ranking identity scores of non-coding sequences, we found conserved sequences including the previously characterized alpha globin major regulatory element. In chicken and pufferfish, regions that may correspond to this element were found by analysing the distribution of transcription factor binding sites. Regions identified in this way act as strong enhancer elements in expression assays. In addition to delimiting the alpha globin chromosomal domain, this study has enabled us to develop a more sensitive and accurate routine for identifying regulatory elements in the human genome.
Subject(s)
Chromosomes/chemistry , Chromosomes/genetics , Globins/genetics , Multigene Family/genetics , Regulatory Sequences, Nucleic Acid , Animals , Base Sequence , Chickens , Conserved Sequence/genetics , CpG Islands/genetics , Evolution, Molecular , Fishes , Globins/chemistry , Humans , Mice , Molecular Sequence Data , Nucleic Acid Hybridization/methods , Protein Structure, Tertiary/genetics , Regulatory Sequences, Nucleic Acid/physiologyABSTRACT
The core of DNase hypersensitive site (HS) 2 from the beta-globin locus control region is a potent enhancer of globin gene expression. Although it has been considered to contain only positive cis-regulatory sequences, our study of the enhancement conferred by segments of HS2 in erythroid cells reveals a novel negative element. Individual cis-regulatory elements from HS2 such as E boxes or Maf-response elements produced as great or greater enhancement than the intact core in mouse erythroleukemia (MEL) cells, indicating the presence of negative elements within HS2. A deletion series through HS2 revealed negative elements at the 5' and 3' ends of the core. Analysis of constructs with and without the 5' negative element showed that the effect is exerted on the promoters of globin genes expressed at embryonic, fetal, or adult stages. The negative effect was observed in bipotential human cells (K562 and human erythroleukemia (HEL) cells), proerythroblastic mouse (MEL) cells, and normal adult human erythroid cells. The novel negative element also functions after stable integration into MEL chromosomes. Smaller deletions at the 5' end of the HS2 core map the negative element within a 20-base pair region containing two conserved sequences.
Subject(s)
Genes, Regulator , Globins/genetics , Adult , Base Sequence , Cell Line , Erythroid Precursor Cells/metabolism , Erythropoiesis , Humans , Molecular Sequence Data , Promoter Regions, GeneticABSTRACT
By sequencing regions flanking the beta-globin gene complex in mouse (Hbbc) and human (HBBC), we have shown that the beta-globin gene cluster is surrounded by a larger cluster of olfactory receptor genes (ORGs). To facilitate sequence comparisons and to investigate the regulation of ORG expression, we have mapped 5' sequences of mRNA from olfactory epithelium encoding beta-globin-proximal ORGs. We have found that several of these genes contain multiple noncoding exons that can be alternatively spliced. Surprisingly, the only common motifs found in the promoters of these genes are a "TATA" box and a purine-rich motif. Sequence comparisons between human and mouse reveal that most of the conserved regions are confined to the coding regions and transcription units of the genes themselves, but a few blocks of conserved sequence also are found outside of ORG transcription units. The possible influence of beta-globin regulatory sequences on ORG expression in olfactory epithelium was tested in mice containing a deletion of the endogenous beta-globin locus control region, but no change in expression of the neighboring ORGs was detected. We evaluate the implications of these results for possible mechanisms of regulation of ORG transcription.
Subject(s)
Globins/genetics , Multigene Family , Receptors, Odorant/genetics , Animals , Base Sequence , DNA, Complementary , Exons , Humans , Locus Control Region , Mice , Molecular Sequence DataABSTRACT
There are few data comparing the relative frequency of new electrocardiographic (ECG) abnormalities after coronary artery bypass grafting (CABG) compared with percutaneous transluminal coronary angioplasty (PTCA) and their association with long-term cardiac mortality. The study population consisted of 3,373 patients who were either randomized or eligible to be randomized to CABG or PTCA in the BARI trial. The frequency of new postprocedural ECG abnormalities was significantly greater after a CABG procedure than after PTCA. The incidence of new postprocedural major Q waves, ST-segment elevation, and T-wave abnormalities were significantly more frequent after CABG. After PTCA (n = 1,869), the 5-year cardiac mortality rates associated with the new development of major Q waves, ST-segment elevation, ST-segment depression, T-wave abnormalities, or no abnormality was 18.1%, 8.5%, 8.9%, 6.0%, and 5.4%, respectively. After CABG (n = 1,427), 5-year cardiac mortality rates were 8.0%, 4.2%, 3.8%, 2.8%, and 3.7%, respectively. The adjusted relative risk of 5-year cardiac mortality for new Q-wave abnormalities was 2.6 after CABG (p <0.04) and 4.6 after PTCA (p <0.01). Thus, patients who undergo CABG have more postinitial procedural ECG abnormalities than patients who undergo PTCA. Cardiac mortality is significantly increased by the new development of postprocedural Minnesota code Q-wave abnormalities regardless of whether patients undergo CABG or PTCA.
Subject(s)
Angioplasty, Balloon, Coronary , Coronary Artery Bypass , Coronary Disease/mortality , Coronary Disease/therapy , Diabetic Angiopathies/mortality , Female , Humans , Male , Middle Aged , Prognosis , Randomized Controlled Trials as TopicABSTRACT
A 'working draft' of the human genome sequence is now available. Comparisons with the sequences of mouse and other species will be a powerful approach to identifying functional segments of the noncoding regions, such as gene regulatory elements. However, the choice of a species for most effective comparison differs among various loci.
Subject(s)
Regulatory Sequences, Nucleic Acid , Animals , Humans , Interleukins/genetics , Mice , Species SpecificityABSTRACT
PipMaker (http://bio.cse.psu.edu) is a World-Wide Web site for comparing two long DNA sequences to identify conserved segments and for producing informative, high-resolution displays of the resulting alignments. One display is a percent identity plot (pip), which shows both the position in one sequence and the degree of similarity for each aligning segment between the two sequences in a compact and easily understandable form. Positions along the horizontal axis can be labeled with features such as exons of genes and repetitive elements, and colors can be used to clarify and enhance the display. The web site also provides a plot of the locations of those segments in both species (similar to a dot plot). PipMaker is appropriate for comparing genomic sequences from any two related species, although the types of information that can be inferred (e.g., protein-coding regions and cis-regulatory elements) depend on the level of conservation and the time and divergence rate since the separation of the species. Gene regulatory elements are often detectable as similar, noncoding sequences in species that diverged as much as 100-300 million years ago, such as humans and mice, Caenorhabditis elegans and C. briggsae, or Escherichia coli and Salmonella spp. PipMaker supports analysis of unfinished or "working draft" sequences by permitting one of the two sequences to be in unoriented and unordered contigs.
Subject(s)
DNA/chemistry , Internet/statistics & numerical data , Sequence Alignment/statistics & numerical data , Software , Animals , Base Sequence/genetics , Caenorhabditis elegans/genetics , Computational Biology , DNA/genetics , Escherichia coli/genetics , Genes, Bacterial , Genes, Helminth , Genes, Protozoan , Humans , Internet/trends , Mice , Molecular Sequence Data , Salmonella typhimurium/genetics , Sequence Alignment/methods , Sequence Alignment/trendsABSTRACT
BACKGROUND: Acute myocardial infarction in patients with diabetes is associated with high mortality. We studied whether previous revascularization by coronary-artery bypass grafting (CABG), as compared with percutaneous transluminal coronary angioplasty (PTCA), influences the prognosis in such patients. METHODS: We classified all patients eligible for the Bypass Angioplasty Revascularization Investigation who underwent coronary revascularization within three months after entry into the study according to whether they had diabetes and whether they had undergone CABG, either initially or after PTCA. The protective effect of CABG with regard to mortality in the presence and in the absence of subsequent spontaneous Q-wave myocardial infarction was estimated with the use of Cox regression models. RESULTS: Among the 641 patients with diabetes and the 2962 without diabetes, the cumulative five-year rates of death were 20 percent and 8 percent, respectively (P<0.001), and the five-year rates of spontaneous Q-wave myocardial infarction were 8 percent and 4 percent (P<0.001). CABG greatly reduced the risk of death after spontaneous Q-wave myocardial infarction in the patients with diabetes (relative risk, 0.09; 95 percent confidence interval, 0.03 to 0.29). Among patients with diabetes who had undergone CABG but did not have spontaneous Q-wave myocardial infarctions, the corresponding relative risk of death was 0.65 (95 percent confidence interval, 0.45 to 0.94). Among the patients without diabetes, no protective effect of CABG was evident. CONCLUSIONS: Among patients with diabetes, previous coronary bypass surgery, as compared with coronary angioplasty, has a highly favorable influence on prognosis after acute myocardial infarction and a smaller beneficial effect among patients who do not have infarction. These findings should influence the type of coronary revascularization procedure selected for patients with diabetes who have multivessel coronary artery disease.
Subject(s)
Coronary Artery Bypass , Coronary Disease/surgery , Diabetes Complications , Myocardial Infarction/mortality , Aged , Angioplasty, Balloon, Coronary , Coronary Disease/complications , Coronary Disease/therapy , Electrocardiography , Female , Follow-Up Studies , Humans , Male , Middle Aged , Myocardial Infarction/etiology , Prognosis , Proportional Hazards Models , Randomized Controlled Trials as Topic , Survival AnalysisABSTRACT
GATA transcription factors bind the consensus sequence WGATAR, present in the flanking regions of most erythroid specific genes. GATA-1 and GATA-2, coexpressed in erythroid cells, are important for expression of erythroid genes. To elucidate the role of specific GATA transcription factors on globin gene expression, we examined the human alpha- and beta-globin gene clusters for all GATA sites. Conserved GATA sites were found in each of the hypersensitive sites in both beta-and alpha clusters and in proximal regulatory regions of the zeta-, epsilon- and gamma-globin but not the alpha, delta or beta-globin genes. We then tested the effect of increasing levels of GATA-1 and GATA-2 on the expression of endogenous globin genes in human erythroid cells. Increasing GATA-1 levels in K562 cells decreased the levels of epsilon-globin mRNA but had no effect on the levels of expression of gamma, zeta or alpha-globin genes. Increasing GATA-2 levels increased epsilon-globin and gamma-globin transcripts. Increasing levels of GATA-1 also caused a decrease in the expression of endogenous GATA-2, while increased levels of GATA-2 had no effect on GATA-1 mRNA. Our results indicate a differential role of GATA-1 and -2 transcription factors on globin transcripts and suggest a correlation between the conservation of GATA sites in the regulatory regions and the ability of endogenous globin genes to respond to GATA transcription factors. They also suggest that quantitative changes in the levels of GATA-1 or GATA-2 can result in alterations of globin target gene expression and may participate in the ontogenic control of the globin genes.
Subject(s)
DNA-Binding Proteins/metabolism , Hemoglobins/genetics , Transcription Factors/metabolism , Amino Acid Sequence , Animals , Binding Sites , Blotting, Western , Conserved Sequence , DNA, Recombinant , DNA-Binding Proteins/genetics , Erythroid-Specific DNA-Binding Factors , GATA1 Transcription Factor , GATA2 Transcription Factor , Gene Expression Regulation , Globins/genetics , Globins/metabolism , Hemoglobins/metabolism , Humans , K562 Cells , Mice , Multigene Family , Plasmids/genetics , RNA, Messenger/genetics , RNA, Messenger/metabolism , Rats , Reverse Transcriptase Polymerase Chain Reaction , Transcription Factors/genetics , Transcription, GeneticABSTRACT
Conserved segments in DNA or protein sequences are strong candidates for functional elements and thus appropriate methods for computing them need to be developed and compared. We describe five methods and computer programs for finding highly conserved blocks within previously computed multiple alignments, primarily for DNA sequences. Two of the methods are already in common use; these are based on good column agreement and high information content. Three additional methods find blocks with minimal evolutionary change, blocks that differ in at most k positions per row from a known center sequence and blocks that differ in at most k positions per row from a center sequence that is unknown a priori. The center sequence in the latter two methods is a way to model potential binding sites for known or unknown proteins in DNA sequences. The efficacy of each method was evaluated by analysis of three extensively analyzed regulatory regions in mammalian beta-globin gene clusters and the control region of bacterial arabinose operons. Although all five methods have quite different theoretical underpinnings, they produce rather similar results on these data sets when their parameters are adjusted to best approximate the experimental data. The optimal parameters for the method based on information content varied little for different regulatory regions of the beta-globin gene cluster and hence may be extrapolated to many other regulatory regions. The programs based on maximum allowed mismatches per row have simple parameters whose values can be chosen a priori and thus they may be more useful than the other methods when calibration against known functional sites is not available.
Subject(s)
Conserved Sequence , Globins/genetics , Regulatory Sequences, Nucleic Acid , Sequence Alignment/methods , Software , Animals , Base Sequence , Calibration , Cattle , Eubacterium/genetics , Evaluation Studies as Topic , Evolution, Molecular , Goats , Humans , Mice , Molecular Sequence Data , Sequence Homology, Nucleic AcidABSTRACT
Preliminary studies of cis-regulatory elements are frequently performed in transiently transfected cells before further analysis in stably transfected cell lines and transgenic mice. However, not all cells are readily transfectable by routine means. For instance, mouse erythroleukemia (MEL) cells have been a valuable model system for studies of their endogenous globin genes, but introduction of DNA using common transfection methods such as electroporation has been very inefficient. This has allowed studies of stably transfected cells, after selection for the rare transfection events, but transient transfection analysis has been problematic. This report describes an efficient and reliable method for transient transfection of MEL cells using commercially available cationic lipids.