Accuracy Of Gene Expression Research Articles

Abstract 465: Development and validation of a targeted RNA-Seq assay for gene fusion detection and expression quantification in FFPE samples

Abstract Background RNA sequencing (RNA-Seq) has been widely used for gene expression and fusion detection in fresh frozen samples for research use. In clinical studies, RNA was more commonly derived from FFPE samples, which may degrade or yield low due to preparation methods, storage conditions and time. We evaluate the feasibility, quality, and analytical performance of a targeted region RNA sequencing assay (we called as Master RNA Panel) on clinical FFPE tumor samples for gene fusion detection and expression quantification. Methods Total RNA was extracted from FFPE tumor samples. cDNA synthesis and library preparation were used to prepare next-generation sequencing (NGS) libraries that were captured by a customized panel contains 31 gene fusion/splicing regions and 1813 gene expression regions. NGS libraries were then sequenced on Illumina NexSeq CN 500 or NovaSeq 6000 instrument. Sequencing data were analyzed and annotated with an in-house developed pipeline. A set of experimental and data quality control parameters were set up. Results In both fresh tissue and FFPE samples, all known fusion sites (100%, 7/7) can be detected stably with the RNA input volume as low as 5 ng and 1 Gbp data, the gene expression of 5 ng RNA input volume shows a very high correlation with 100ng even under 1Gb data (R2≥ 0.95). Master RNA Panel can detect as low as 100 copies of the fusion gene with 50 ng RNA and the specificity was 100% based on a test of 10 fusion negative samples. The consistency of fusion gene detection by Master RNA Panel was 96.2% (51/53) compared with RNA-Seq in 44 clinical samples. To validate the accuracy of gene expression, we used both qPCR and RNA-Seq as comparison methods. Gene expression of 12 tumor-related genes (BAG1, BIRC5, CD68, ERBB2, ESR1, GRB7, GSTM1, MKI67, MMP11, MYBL2, PGR, SCUBE2) in 5 samples were profiled with qPCR assay and the correlation for each gene is greater than 0.8. 27 clinical samples were profiled with both Master RNA panel and RNA-Seq, the total correlation of gene expression between the two assay was also greater than 0.8. The precision verification results show that under the inspection of different operation dates, reagent batches and operators, the gene expression correlation between 5 replicates of 2 FFPE samples is greater than 0.99. The consistency of the fusion test for 5 replicates of 2 FFPE samples and 3 standards was 95.3% (81/85, >=100 copies), indicating that the operation date, reagent batch and operator have no significant influence on the test results, and the test method has good precision. Conclusions Master RNA Panel can help to identify both gene fusions and gene quantification in cancer patients with extremely low RNA input and high limit of detection (LoD), which is a good supplement for DNA based NGS assay, especially for gene expression quantification and fusion detection. Citation Format: Jingbo Zhao, Shuang Yang, Hua Dong. Development and validation of a targeted RNA-Seq assay for gene fusion detection and expression quantification in FFPE samples [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 465.

Selection and validation of reference genes for RT-qPCR in adipose and longissimus dorsi muscle tissues of buffalo

Real-time quantitative PCR (RT-qPCR) is widely used to measure and evaluate gene expression. The precision and reliability of RT-qPCR are critically dependent on the selection of suitable reference genes (RGs). In this study, an effort was made to identify the optimal RGs for RT-qPCR analysis of adipose and the longissimus dorsi muscle (LM) in buffaloes. RNA sequencing data were firstly analyzed to obtain 10 candidate genes (FKBP1A, C25H16orf72, PNRC2, IQGAP1, ATP5PD, RPL6, NDUFB4, TRA2A, CAPRIN1, and METAP2) that with high and stable expression across adipose tissues. Four other identified RGs (GAPDH, ACTB, TOP2B, and UXT) were selected as well. The expression stability of the candidate RGs was evaluated by three algorithms (geNorm, NormFinder, and BestKeeper) and then further validated by adipocyte and myocyte markers. Our results showed that UXT and TOP2B were the optimal RGs for RT-qPCR analysis across adipose tissues in buffaloes; three RGs, RPL6, UXT, and TOP2B, were the optimal RGs for RT-qPCR analysis across adipose and the LM tissues in buffaloes. This study provides significant information for improving the accuracy of gene expression in research on intramuscular fat deposition in buffaloes.

Ribosomal mistranslation leads to silencing of the unfolded protein response and increased mitochondrial biogenesis

Translation fidelity is the limiting factor in the accuracy of gene expression. With an estimated frequency of 10−4, errors in mRNA decoding occur in a mostly stochastic manner. Little is known about the response of higher eukaryotes to chronic loss of ribosomal accuracy as per an increase in the random error rate of mRNA decoding. Here, we present a global and comprehensive picture of the cellular changes in response to translational accuracy in mammalian ribosomes impaired by genetic manipulation. In addition to affecting established protein quality control pathways, such as elevated transcript levels for cytosolic chaperones, activation of the ubiquitin-proteasome system, and translational slowdown, ribosomal mistranslation led to unexpected responses. In particular, we observed increased mitochondrial biogenesis associated with import of misfolded proteins into the mitochondria and silencing of the unfolded protein response in the endoplasmic reticulum.

Errors during Gene Expression: Single-Cell Heterogeneity, Stress Resistance, and Microbe-Host Interactions.

ABSTRACTGene expression has been considered a highly accurate process, and deviation from such fidelity has been shown previously to be detrimental for the cell. More recently, increasing evidence has supported the notion that the accuracy of gene expression is indeed flexibly variable. The levels of errors during gene expression differ from condition to condition and even from cell to cell within genetically identical populations grown under the same conditions. The different levels of errors resulting from inaccurate gene expression are now known to play key roles in regulating microbial stress responses and host interactions. This minireview summarizes the recent development in understanding the level, regulation, and physiological impact of errors during gene expression.

Non-invasive messenger RNA transcriptional evaluation in human kidney allograft dysfunction.

The aim of the present study was to evaluate messenger RNA expression in kidney allograft recipients. Forty-four kidney transplant recipients were evaluated up to three months after grafting. After transplantation, peripheral blood samples were drawn sequentially for real-time polymerase chain reaction analyses of perforin and TIM-3 genes. Biopsies were obtained to evaluate acute graft dysfunction and interpreted according to the Banff classification. Eight patients presented episodes of acute rejection. Recipients with rejection had significantly higher levels of TIM-3 mRNA transcripts compared to those without rejection (median gene expression 191.2 and 36.9 mRNA relative units, respectively; P<0.0001). Also, perforin gene expression was higher in patients with rejection (median gene expression 362.0 and 52.8 mRNA relative units; P<0.001). Receiver operating characteristic curves showed that the area under the curve (AUC) for the TIM-3 gene was 0.749 (95%CI: 0.670–0.827). Perforin gene mRNA expression provided an AUC of 0.699 (95%CI: 0.599 to 0.799). Overall accuracy of gene expression was 67.9% for the TIM-3 gene and 63.6% for the perforin gene. Combined accuracy was 76.8%. Negative predictive values were 95.3% for the TIM-3 gene, 95.5% for the perforin gene, and 95.4% in the combined analyses. Gene expression was significantly modulated by rejection treatment decreasing 64.1% (TIM-3) and 90.9% (perforin) compared to the median of pre-rejection samples. In conclusion, the longitudinal approach showed that gene profiling evaluation might be useful in ruling out the diagnosis of acute rejection and perhaps evaluating the efficacy of treatment.

Abstract P1-01-11: Something from nothing? The case for quality control in liquid biopsy studies

Abstract Background: Circulating tumor cells (CTCs) as a liquid biopsy strategy are currently being studied as a surrogate biomarker that may reflect metastatic tumor biology. Given the rarity of CTCs, target enrichment is commonly used to profile the gene signatures of valuable clinical samples to evaluate for multiplexed gene panels of interest. The aim of our study was to evaluate if the NanoString PAM50 could be used for accurate gene expression profiling of CTCs and controls using the research-use-only probeset. Methods: We collected two 7.5mL EDTA tubes of blood from 12 healthy female volunteers. CTC assays were performed using the ANGLE Parsortix system as a microfluidics filter that separates cells based on size and deformability. The cell lines Hs578T (basal-like) and SK-BR-3 (HER2 amplified) were used to spike 20 cells into n=6 blood tubes per cell line (termed spiked samples). N=12 7.5mL tubes of blood (termed unspiked samples) and n=12 spiked samples were processed using Parsortix for CTC harvesting and lysis using a 10 micron cassette. From each lysate, 5uL was taken for cDNA amplification, multiple target enrichment for 14 cycles, followed by NanoString PAM50 assays. From each of the 12 peripheral blood (PB) samples, we extracted RNA and used 100ng for NanoString PAM50 assays. For cell line controls, 100ng of Hs578T or SK-BR-3 were subjected to NanoString Assays. Results: Low PAM50 gene expression was observed in all 12 PB samples. Unspiked PB harvested from the CTC assay showed a higher level of PAM50 gene expression compared to PB, suggesting that the target enrichment amplification produces false positive detection of expected breast cancer related transcripts. On ANOVA testing, 10/12 (83%) of unspiked, sorted, target enriched samples had significant differential expression (p&amp;lt;0.0001) of the mean log normalized counts for the PAM50 genes compared to PB. In spiked experiments using n=20 cells in 7.5mL of PB, sorted Hs578T were found to be triple negative in only 3/6 (50%) while sorted SK-BR-3 were found to be HER2 positive in only 3/6 (50%). On ANOVA testing, the spiked/sorted and bulk were found to have a difference among the mean log normalized counts for the PAM50 genes across all samples for both cell lines (p&amp;lt;0.0001). However, 3/6 (50%) samples had a difference in mean PAM50 gene expression when compared to bulk Hs578t on multiple comparison testing while 2/6 (33%) were statistically significantly different when comparing spiked, sorted SK-BR-3 versus bulk cell line. Conclusions: Unspiked blood processed via a CTC assay and subjected to target enrichment showed high expression of genes in the NanoString PAM50 assay, likely due to amplification bias. When working with enriched but not ultra-pure CTC samples, amplified gene expression of background leukocytes may influence read counts. This is important to consider in assays that enrich for CTCs but retain a leukocyte background. Further studies will address the effect of the CTC assay procedure and number of leukocytes on accuracy of gene expression of rare CTC mimics. This study emphasizes the importance of selecting genes that are not expressed in PB or performing background subtraction or normalization as strategies for accurate gene expression profiling of CTCs. Citation Format: Porras TB, Bains PK, Ring A, Carrasco S, Forte V, Punj V, Lang JE. Something from nothing? The case for quality control in liquid biopsy studies [abstract]. In: Proceedings of the 2016 San Antonio Breast Cancer Symposium; 2016 Dec 6-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2017;77(4 Suppl):Abstract nr P1-01-11.

MO-DE-207B-05: Predicting Gene Mutations in Renal Cell Carcinoma Based On CT Imaging Features: Validation Using TCGA-TCIA Datasets

Purpose: The goal of this work is to investigate the use of contrast enhanced computed tomographic (CT) features for the prediction of mutations of BAP1, PBRM1, and VHL genes in renal cell carcinoma (RCC). Methods: For this study, we used two patient databases with renal cell carcinoma (RCC). The first one consisted of 33 patients from our institution (UT Southwestern Medical Center, UTSW). The second one consisted of 24 patients from the Cancer Imaging Archive (TCIA), where each patient is connected by a unique identi?er to the tissue samples from the Cancer Genome Atlas (TCGA). From the contrast enhanced CT image of each patient, tumor contour was first delineated by a physician. Geometry, intensity, and texture features were extracted from the delineated tumor. Based on UTSW dataset, we completed feature selection and trained a support vector machine (SVM) classifier to predict mutations of BAP1, PBRM1 and VHL genes. We then used TCIA-TCGA dataset to validate the predictive model build upon UTSW dataset. Results: The prediction accuracy of gene expression of TCIA-TCGA patients was 0.83 (20 of 24), 0.83 (20 of 24), and 0.75 (18 of 24) for BAP1, PBRM1, and VHL respectively. For BAP1 gene, texture feature was the most prominent feature type. For PBRM1 gene, intensity feature was the most prominent. For VHL gene, geometry, intensity, and texture features were all important. Conclusion: Using our feature selection strategy and models, we achieved predictive accuracy over 0.75 for all three genes under the condition of using patient data from one institution for training and data from other institutions for testing. These results suggest that radiogenomics can be used to aid in prognosis and used as convenient surrogates for expensive and time consuming gene assay procedures.

Evaluation of suitable reference genes for qRT-PCR gene expression normalization in reproductive, vegetative tissues and during fruit development in oil palm

Gene expression patterns of a target gene in different tissues and at different stages of development can provide clues towards the understanding of its biological function. The accuracy of gene expression via quantitative real-time reverse transcription-PCR (qRT-PCR) analysis strongly depends on transcript normalization using reference genes that demonstrate consistent expression levels in tissues and experimental conditions studied. However, suitable reference genes for qRT-PCR in oil producing fruit have not been well identified. In this study, the expression stability of fourteen potential reference genes for qRT-PCR analysis in diverse sets of biological samples including six distinct oil palm organs, two floral tissues, four vegetative tissues, five stages of fruit development and two differential phenotype fruit tissue was evaluated. The expression stabilities were assessed using three statistical algorithms including geNorm, NormFinder and qBASE plus. The stability rankings from three outputs were consolidated to obtain the consensus stability ranking of all reference genes. Our analysis showed that a Gibberellin-responsive protein (GRAS) and Cyclophilin 2 (Cyp2) were the most stable genes in oil palm tissues tested. A combination of Cyp2, GRAS and SLU7 were identified as most suitable reference genes for all oil palm tissues and vegetative tissues set while in reproductive tissues set, GRAS and Glutaredoxin was recommended. Cyp2 and GRAS were required for normalization in mesocarp tissues at various developmental stages and differential oil yielding mesocarp tissues. In addition, the relative gene expression profile of two fatty acid biosynthesis genes, EgACCase and EgSTEA was conducted to confirm the validity of the reference genes in this study. Both EgACCase and EgSTEA were analyzed in fruit tissues at various developmental stages. This study identified the most suitable reference genes for normalization of gene expression in oil palm. These results serve as a guideline for the selection of suitable reference genes under different experimental conditions and accurate normalization of gene expression studies in a wide variety of tissues in oil palm.

Intronic features that determine the selection of the 3′ splice site

Most eukaryotic primary transcripts include segments, or introns, that will be accurately removed during RNA biogenesis. This process, known as pre-messenger RNA splicing, is catalyzed by the spliceosome, accurately selecting a set of intronic marks from others apparently equivalent. This identification is critical, as incorrectly spliced RNAs can be toxic for the organism. One of these marks, the dinucleotide AG, signals the intronic 3' end, or 3' splice site (ss). In this review we will focus on those intronic features that have an impact on 3' ss selection. These include the location and type of neighboring sequences, and their distance to the 3' end. We will see that their interplay is needed to select the right intronic end, and that this can be modulated by additional intronic elements that contribute to alternative splicing, whereby diverse RNAs can be generated from identical precursors. This complexity, still poorly understood, is fundamental for the accuracy of gene expression. In addition, a clear knowledge of 3' ss selection is needed to fully decipher the coding potential of genomes.

Proof-reading signal accuracy of gene expression by binary differential display

Differential display (DD) is commonly used for identifying differentially expressed genes. However, each cDNA species identified by DD must be verified so a "real difference" can be differentiated from false positives. Although Northern blot analysis is the gold standard it is labor intensive, time-consuming and requires a significant amount of RNA. To speed up and streamline the confirmation process, we developed a new strategy: binary differential display (BDD) based on the binding kinetics of the arbitrary primers in DD. After determining a cDNA sequence of interest from a DD screen, two more 13mer primers derived from the original arbitrary primer used can be designed to target a corresponding cDNA sequence of interest: one with perfect 5'-base matches and the other with additional mismatches at the 5'-base to the corresponding mRNA being confirmed. A separate reverse transcription and FDD are then performed with the same RNA samples being compared. BDD can quickly and accurately determine if a cDNA sequence identified by DD corresponds to a truly differentially expressed gene. In addition, the method is especially useful when more than one cDNA sequence was recovered from a DD band where the masking effect of false-positives can be clearly resolved. Given its simplicity and limited RNA sample required, BDD can be used as a general strategy for rapid confirmation of differentially expressed genes discovered by DD.

Breast cancer patients stratification by microarray-based gene expression profiling from FFPET samples

e22041 Background: Microarrays have been used to monitor global genes expression and have aided the identification of novel biomarkers for patients stratification and drug response prediction . To date there has been limited application of microarray- based gene expression analysis to formalin fixed paraffin embedded tissues (FFPET). FFPE tissues are the most commonly available clinical samples with documented clinical information for retrospective clinical analysis. However, FFPET RNA has proven to be an obstacle for microarray analysis because of low yield and compromised RNA integrity. Methods: Using a novel RNA amplification method, Single Primer Isothermal Amplification (SPIA, NuGEN Inc, San Carlos, CA), we amplified FFPET RNA, hybridized amplified, and labeled cDNA onto Affymetrix HG U133plus2 GeneChips. Results: We found that SPIA amplification successfully overcomes the problems of poor quality of FFPET RNA, and produced informative biological data. Comparing the gene expression data from 5 different types of FFPET archival cancer samples (breast, lung, ovarian, colon, and melanoma), we demonstrated that gene expression signatures clearly distinguish the tissue of origin. Further, from an analysis of 91 FFPET samples comprised of ER+, HER2+, triple negative breast cancer patients, and normal breast tissue, we have identified a 103 gene signature that distinguishes the intrinsic sub-types of breast cancer. Finally, the accuracy of gene expression measured by microarray was verified by real time PCR quantitation of the ERBB2 gene, resulting in a significant correlation (R = 0.88). Conclusions: We have demonstrated the feasibility of global gene expression profiling using RNA extracted from FFPET and have shown that a gene expression signature can stratify patient samples into different subtypes of disease. This study paves the way to identify novel molecular biomarkers for disease stratification and therapy response from archival FFPET samples, leading to the goals of personalized medicine. No significant financial relationships to disclose.

Gene profiling validation for cancer classification using known primary carcinoma samples

21130 Background: Cancer of Unknown Primary Site (CUP) accounts for 3–5% of all malignant neoplasms. With current diagnostic procedures, approximately 20% of CUP patients have a primary identified. Since formulation of prognosis and treatment selection depends largely on the primary type, diagnostic uncertainty unfavorably affects CUP management. Gene expression profiling has permitted the development of diagnostic classifiers. A clinically usable methodology needs to differentiate a sufficiently large number of primary types. This study focuses on the estimate of the accuracy of gene expression for classification of metastatic tumors in formalin fixed (FF) FNA and core/surgical biopsies. Methods: CupPrint® is a 1900 gene microarray optimized for FF samples which uses a database of 643 cancer profiles to provide a robust method for the discrimination of 51 tumor (sub) types. Metastasis samples of six known primary carcinoma types were studied (lung, pancreas, colorectal, breast, liver, ovary/primary peritoneal carcinoma). To examine the impact of the metastatic site on diagnostic accuracy different biopsy sites were studied, including lung, brain, lymph nodes, liver, peritoneum. Samples were deemed evaluable that contained an estimated &gt;33% tumor cells. mRNA samples from the metastatic lesions were provided in a blinded fashion to the microarray laboratory. Results: For 39 evaluable samples studied so far, the CupPrint® prediction was compared with the pathologic diagnosis. Of the 39, in 34 the primary site of origin was correctly predicted (87% accuracy). In two of the incorrectly predicted samples the site of biopsy (brain and liver respectively) was reported as the primary site. Correlations of molecular profiles to clinical parameters are being explored. Conclusions: CupPrint® appears to be sufficiently accurate to be of potential clinical utility in the diagnosis of CUP. No significant financial relationships to disclose.

Ischemia-reperfusion-related repair deficit after oxidative stress: implications of faulty transcripts in neuronal sensitivity after brain injury.

Diseases of the heart are the No. 1 killer in industrialized countries. Brain injury can develop as a result of cerebral ischemia-reperfusion due to stroke (brain attack) and other cardiovascular diseases. Learning about the disease is the best way to reduce disability and death. We present here whether gene repair activities are associated with neuronal death in an ischemia-reperfusion model that simulates stroke in male Long-Evans rats. This experimental stroke model is known to induce necrosis in the ischemic cortex. Cerebral ischemia causes overactivation of membrane receptors and accumulation of extracellur glutamate and intracellular calcium, which activates neuronal nitric oxide synthase, causing damage to lipids, proteins, and nucleic acids, and reduces energy sources with consequent functional deterioration, leading to cell death. Restoration processes normally repair genes with few errors. However, ischemia elevates oxidative DNA lesions despite these repair mechanisms. These episodes concurrently occur with the induction of immediate-early genes that critically activate other late genes in the signal transduction pathway. Damage, repair, and transcription of the c-FOS gene are presented here as examples, because Fos peptide, one of the components of activator protein 1, activates nerve growth factor and repair mechanisms. The results of our studies show that treatments with 7-nitroindazole, a specific inhibitor of nitric oxide synthase known to attenuate nitric oxide, oxidative DNA lesions, and necrosis, increase intact c-fos mRNA levels after stroke. This suggests that the accuracy of gene expression could be accounted for the recovery of cellular function after cerebral injury.

Gene Expression Profiling of Cultured Human Bronchial Epithelial and Lung Carcinoma Cells

Lung cancer is a complex collection of diseases that is thought to begin with single mutated progenitor cells and culminates in any of several clinically described pathologies. Our knowledge of the molecular events that lead to different lung cancer types--small cell carcinoma, squamous cell carcinoma, adenocarcinoma, and large cell carcinoma--is incomplete. Nonetheless, it is evident that genetic changes that impact multiple molecular networks are involved in the generation of each specific phenotype. Due to the obvious complexity of these processes, the simultaneous quantitative monitoring of changes in the expression of genes that define these networks can provide mechanistic information to increase our understanding of the molecular basis for human pulmonary carcinogenesis. To this end, we have employed a commercially available human cDNA array (Atlas Human Array, Clontech Laboratories) to systematically screen for alterations in the expression of 600 genes in normal human bronchial epithelial (NHBE) cells as well as in several lung carcinoma lines. Studies on the reproducibility and variability of array results indicate that a 2-fold or greater difference in the expression of a particular gene could be considered a real difference in transcript abundance. Accuracy of gene expression as measured in the array was verified by comparing mRNA levels of the proto-oncogene c-myc in the array with results obtained by traditional Northern blot analysis and by quantitative RT-PCR. Gene expression profiles were compared within and among cell types. The differential expression of 17 genes, including downregulation of MRP8 and MRP14 and upregulation of CYP1B1, was observed in all four carcinoma lines compared to NHBE cells. The direction of all 17 gene expression differences, either upregulation or downregulation relative to NHBE cells, was the same for all four carcinoma lines, underscoring their common molecular features. Each lung tumor line also exhibited a number of unique differences compared to both normal cells and the other tumor cell lines. These differences may be due to differences in the cellular origin and/or pathology of the cell lines studied.

The thermodynamic basis of the genetic code.

A systematic investigation of the helix- coil transition of DNA's as a function of sequence composition has lead to the conclusion that the Gibbs energy per base pair is a zero-order property. There is a correlation between the observed codon usage frequency and the codonlanti-codon interaction Gibbs energy per triplet. We will show that the bias of codon usage reflects strategies for efficiency and accuracy in gene expression. Organization beyond the codon triplets is discussed.

Growth-optimizing accuracy of gene expression.

CONTENTS INTRODUCTION •• 291 THE SAMPLE SPACE ....• •...•...• 293 THE ERROR SINK 295 ERROR SOURCES ........ ..... ...... 297

Strategies for efficiency and accuracy in gene expression. 2. Growth optimized ribosomes

Strategies for efficiency and accuracy in gene expression. 2. Growth optimized ribosomes

Strategies for efficiency and accuracy in gene expression. 3. Drug dependence: growth at the edge

Strategies for efficiency and accuracy in gene expression. 3. Drug dependence: growth at the edge

Strategies for efficiency and accuracy in gene expression

Recently there has been a shift in the perception of problems associated with the mechanisms of gene expression. Formerly it seemed that the problem was to discover where the remarkable accuracy of gene expression originated. The mystery was that all model system studies suggested that the binding interactions of nucleotides are not sufficiently stereo-specific to support the accuracy of either transcription or of translation. The introduction of the Hopfield-Ninio model of substrate proofreading demystified this aspect of the accuracy of gene expression, but it generated another set of problems. Thus, the way to get high accuracy out a low fidelity elementary process such as base pairing is to repeat the elementary step a sufficient number of times. Since there is no principle limit on the number of allowable repetitions, there is no principle limit on the attainable accuracy. What then sets the error rates of gene expression? This series of three articles will address this question. In this first article, the notion of a maximized kinetic efficiency for the translation apparatus will be introduced. A simple optimization principle will then be used as a tool to explore the consequences of using different coding strategies to specify the sequences of proteins that are expressed selectively under different growth conditions. In the second article the antagonistic relationship between kinetic efficiency and accuracy of translation will be explored. The notion of a kinetic optimization of the costs of errors and the costs of accuracy will be introduced and used to explain the growth phenotypes of mutants with altered translational accuracy. The costs associated with errors of translocation will be contrasted with those associated with missense errors in the third article. In addition, I will introduce the notion that there is a physical constraint in translation that imposes a reciprocal relationship between the accuracy of aminoacyl-tRNA selection and the accuracy of messenger RNA movement. This reciprocal relationship will provide some insight into the nature of drug dependence in bacteria and will also suggest the outlines of an answer to our original question. In particular, I will suggest that the lower level of the missense error rate is determined, at least in part, by the upper level of mRNA translocation errors that is tolerable. Likewise, the accuracy of translocation may be constrained by the kinetic costs associated with excessive accuracy of tRNA selection.

Death rates of bacterial mutants

We have studied the death rates of mutant and wild-type strains of Escherichia coli by two methods. One method involves microscopic estimations of the ability of bacteria to form microcolonies. The other method involves estimates of the numbers of bacteria permeable to a fluorescent dye. The results of both methods suggest that of the order of 0.1–0.3% of bacteria in an exponentially growing culture are dead according to the present criteria. When the death rates are compared with those for mutant bacteria with hyperaccurate or with error-prone ribosomes, no significant differences are observed. The results suggest that there is little or no relationship between death rate and the accuracy of gene expression in E. coli.