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PubMed2006

Prenatal diagnosis of chromosomal abnormalities using array-based comparative genomic hybridization.

PURPOSE: This study was designed to evaluate the feasibility of using a targeted array-CGH strategy for prenatal diagnosis of genomic imbalances in a clinical setting of current pregnancies. METHODS: Women undergoing prenatal diagnosis were counseled and offered array-CGH (BCM V4.0) in addition to routine chromosome analysis. Array-CGH was performed with DNA directly from amniotic fluid cells with whole genome amplification, on chorionic villus samples with amplification as necessary, and on cultured cells without amplification. RESULTS: Ninety-eight pregnancies (56 amniotic fluid and 42 CVS specimens) were studied with complete concordance between karyotype and array results, including 5 positive cases with chromosomal abnormalities. There was complete concordance of array results for direct and cultured cell analysis in 57 cases tested by both methods. In 12 cases, the array detected copy number variation requiring testing of parental samples for optimal interpretation. Array-CGH results were available in an average of 6 and 16 days for direct and cultured cells, respectively. Patient acceptance of array-CGH testing was 74%. CONCLUSION: This study demonstrates the feasibility of using array-CGH for prenatal diagnosis, including reliance on direct analysis without culturing cells. Use of array-CGH should increase the detection of abnormalities relative to the risk, and is an option for an enhanced level of screening for chromosomal abnormalities in high risk pregnancies.

Chromosome Aberrations↗

PubMed2006

Genomic microarrays in clinical diagnosis.

PURPOSE OF REVIEW: Cytogenetic analysis has for a long time relied on chromosome banding by karyotyping for whole-genome analysis of structural and numerical chromosomal anomalies. Conceptual and technical developments in molecular cytogenetics are rapidly changing the way the human genome is being analyzed by enhancing the resolving power from the megabase to the kilobase level. This review describes the various genomic microarray approaches that have been developed for molecular cytogenetic purposes and their implementation in a routine clinical diagnostic setting. RECENT FINDINGS: Genomic microarray approaches such as array-based comparative genomic hybridization have recently been shown to identify causative submicroscopic copy number alterations in a significant proportion of patients with mental retardation. These alterations occur throughout the human genome and the majority of these alterations reported thus far are unique. Next to these causative alterations, a large number of inherited submicroscopic copy number variations without immediate clinical consequences have been detected by these methods. SUMMARY: Genome profiling by genomic microarrays is becoming an important diagnostic tool, either in addition to or replacing conventional chromosome banding, depending on the expected diagnostic yield and the costs involved.

Child, Preschool↗

PubMed1989

A tandem repeat sequence found in a heterogeneous fragment of UL of herpes simplex virus type 1.

We found a tandem repeat sequence in the region (designated BS7) in which restriction fragments BamHI D and SalI B overlap each other, near the centre of the unique long sequence (UL) of the herpes simplex virus type 1 (HSV-1) strain F genome. The SmaI physical map of BS7 was constructed, and the position of a heterogeneous SmaI subfragment from HSV-1 isolates and plaque-purified clones from a single strain was defined on the map. The maximum size difference in the SmaI subfragment was estimated to be 600 bp between these isolates and 100 bp between the clones. The 0.23 kb SmaI subfragment recloned from BS7 was sequenced, and was shown to contain a tandem repeat sequence consisting of 15 units of 12 bp, 5' TTGGGGCTGGGG 3'. These results suggest that the fragment length heterogeneity in the UL of HSV-1 isolates and clones is attributable to copy number variation of the tandem repeat sequence.

Base Sequence↗

PubMed2025

Replication stress increases de novo CNVs across the malaria parasite genome.

Changes in the copy number of large genomic regions, termed copy number variations (CNVs), contribute to important phenotypes. CNVs are readily identified using conventional approaches when present in a large fraction of the cell population. However, CNVs in only a few genomes are often overlooked but important; if beneficial, a de novo CNV that arises in a single genome can expand during selection to create a population of cells with novel characteristics. While single cell methods for studying de novo CNVs are increasing, we continue to lack information about CNV dynamics in rapidly evolving microbial populations. Here, we investigated de novo CNVs in the genome of the Plasmodium parasite that causes human malaria. The highly AT-rich P. falciparum genome readily accumulates CNVs that facilitate rapid adaptation. We employed low-input genomics and specialized computational tools to evaluate the impact of sub-lethal stress on the de novo CNV rate. We observed a significant increase in genome-wide de novo CNVs following treatment with an antimalarial compound that inhibits replication. De novo CNVs encompassed genes from various cellular pathways participating in human infection. This snapshot of CNV dynamics emphasizes the connection between replication stress, DNA repair, and CNV generation in this important microbial pathogen.

Journal Article↗

PubMed2025

Investigating the genomic landscape of mouse models of breast cancer metastasis.

Metastasis remains a major cause of cancer mortality. AbstractThis study, expanding upon previous findings in the MMTV-PyMT model, investigated four independent mouse models, representing luminal (MMTV-PyMT, MMTV-Myc), HER2-amplified (MMTV-Her2) and triple negative (C3(1)TAg) breast cancer subtypes. Consistent with previous results, limited evidence for metastasis-associated somatic point mutations was found for all models. We also found that oncogenic drivers significantly influenced the number and size of metastasis-specific copy number variations (MSCNVs), but common driver-independent MSCNVs were rare. Furthermore, analyzing a cohort with varying genetic backgrounds while maintaining a constant oncogenic driver (PyMT) revealed that genetic background profoundly impacts MSCNVs. Transcriptome analysis demonstrated that oncogenic drivers strongly shaped metastasis-specific gene expression (MSGE), with each driver exhibiting distinct expression profiles. In contrast, MSGE in the PyMT-F1 cohort was more variable across strains. Despite the diversity of MSCNV and MSGE, functional analysis revealed that both mechanisms converge on the modulation of key cellular processes, including immune responses, metabolism, and extracellular matrix interactions. These findings emphasize the complex interplay between oncogenic drivers and genetic background in shaping the genomic and transcriptional landscapes of metastatic lesions.

Journal Article↗

PubMed1992

Apparent heterozygote deficiencies observed in DNA typing data and their implications in forensic applications.

Restriction fragment length polymorphisms (RFLP) analysis using the Southern blot technique can be used to recognize copy number variation of variable number of tandem repeats (VNTR) of conserved core sequences at several regions of the human genome. This new class of polymorphisms reveals a high degree of genetic variation, useful for individual identification purposes. Criticisms against forensic applications of such DNA typing data include the limitation of employing Hardy-Weinberg expectation of genotype frequencies, since several surveys indicate apparent deficiency of heterozygosity (or excess homozygosity) in comparison with Hardy-Weinberg expectations. This research postulates an alternative explanation of deficiency of apparent heterozygosity which is caused by the inability to detect extremely small-sized alleles (called 'non-detectable' alleles) due to the sensitivity of Southern gel electrophoresis. We show that the presence of 'non-detectable' alleles can produce pseudo-homozygosity and their frequencies can be predicted from the observed proportional heterozygote deficiency. Furthermore, in the covert presence of such 'non-detectable' alleles, we show that the gene-count method provides over-estimates of allele frequencies in the sample population, and hence the Hardy-Weinberg predictions of genotype frequencies avoid wrongful bias against suspects in forensic applications of DNA typing data. Applications of this theory to population data on six VNTR loci in US Caucasians and US Blacks suggest that the presence of 'non-detectable' alleles could be the major cause of apparent heterozygote deficiency, and the current approaches of predicting the population frequency of specific DNA phenotypes are practically free of the possible wrongful bias in courtroom applications of DNA typing data.

Alleles↗

PubMed2006

DNA microarray and proteomic strategies for understanding alcohol action.

This article summarizes the proceedings of a symposium presented at the 2005 annual meeting of the Research Society on Alcoholism in Santa Barbara, California. The organizer was James M. Sikela, and he and Michael F. Miles were chairs. The presentations were (1) Genomewide Surveys of Gene Copy Number Variation in Human and Mouse: Implications for the Genetics of Alcohol Action, by James M. Sikela; (2) Regional Differences in the Regulation of Brain Gene Expression: Relevance to the Detection of Genes Associated with Alcohol-Related Traits, by Robert Hitzemann; (3) Identification of Ethanol Quantitative Trait Loci Candidate Genes by Expression Profiling in Inbred Long Sleep/Inbred Short Sleep Congenic Mice, by Robnet T. Kerns; and (4) Quantitative Proteomic Analysis of AC7-Modified Mice, by Kathleen J. Grant.

Alcoholism↗

PubMed2005

Detection of chromosomal imbalances in children with idiopathic mental retardation by array based comparative genomic hybridisation (array-CGH).

Chromosomal aberrations are a common cause of multiple anomaly syndromes that include growth and developmental delay and dysmorphism. Novel high resolution, whole genome technologies, such as array based comparative genomic hybridisation (array-CGH), improve the detection rate of submicroscopic chromosomal abnormalities allowing re-investigation of cases where conventional cytogenetic techniques, Spectral karyotyping (SKY), and FISH failed to detect abnormalities. We performed a high resolution genome-wide screening for submicroscopic chromosomal rearrangements using array-CGH on 41 children with idiopathic mental retardation (MR) and dysmorphic features. The commercially available microarray from Spectral Genomics contained 2600 BAC clones spaced at approximately 1 Mb intervals across the genome. Standard chromosome analysis (>450 bands per haploid genome) revealed no chromosomal rearrangements. In addition, multi-subtelomeric FISH screening in 30 cases and SKY in 11 patients did not detect any abnormality. Using array-CGH we detected chromosomal imbalances in four patients (9.8%) ranging in size from 2 to 14 Mb. Large scale copy number variations were frequently observed. Array-CGH has become an important tool for the detection of chromosome aberrations and has the potential to identify genes involved in developmental delay and dysmorphism. Moreover, the detection of genomic imbalances of clinical significance will increase knowledge of the human genome by performing genotype-phenotype correlation.

Adolescent↗

PubMed2005

Array-CGH detection of micro rearrangements in mentally retarded individuals: clinical significance of imbalances present both in affected children and normal parents.

BACKGROUND: The underlying causes of mental retardation remain unknown in about half the cases. Recent array-CGH studies demonstrated cryptic imbalances in about 25% of patients previously thought to be chromosomally normal. OBJECTIVE AND METHODS: Array-CGH with approximately 3500 large insert clones spaced at approximately 1 Mb intervals was used to investigate DNA copy number changes in 81 mentally impaired individuals. RESULTS: Imbalances never observed in control chromosomes were detected in 20 patients (25%): seven were de novo, nine were inherited, and four could not have their origin determined. Six other alterations detected by array were disregarded because they were shown by FISH either to hybridise to both homologues similarly in a presumptive deletion (one case) or to involve clones that hybridised to multiple sites (five cases). All de novo imbalances were assumed to be causally related to the abnormal phenotypes. Among the others, a causal relation between the rearrangements and an aberrant phenotype could be inferred in six cases, including two imbalances of the X chromosome, where the associated clinical features segregated as X linked recessive traits. CONCLUSIONS: In all, 13 of 81 patients (16%) were found to have chromosomal imbalances probably related to their clinical features. The clinical significance of the seven remaining imbalances remains unclear. The limited ability to differentiate between inherited copy number variations which cause abnormal phenotypes and rare variants unrelated to clinical alterations currently constitutes a limitation in the use of CGH-microarray for guiding genetic counselling.

Allelic Imbalance↗

PubMed2026

Establishment of Stable Immortalized Human Choroidal Melanocytes for Ocular Research.

PURPOSE: The short lifespan of primary normal choroidal melanocytes (NCMs) in vitro represents a major barrier to mechanistic, functional, and translational studies of choroid biology and uveal melanoma (UM). This study aimed to establish and characterize immortalized human NCM lines that retain melanocytic function, maintain a non-cancerous profile, and are amenable to gene editing. METHODS: NCMs from four donors were immortalized by lentiviral transduction of cyclin-dependent kinase 4 (CDK4R24C), cyclin D1, and human telomerase reverse transcriptase (hTERT), establishing NCM-K4DT lines. Their morphology, melanocytic marker expression, proliferation, and functional properties (melanin synthesis and tyrosinase activity) were evaluated. Genomic stability was assessed by targeted mutation profiling, karyotyping, and copy number variation (CNV) analysis. The tumorigenicity was tested in immunodeficient mice. Plasmid-based CRISPR/Cas9 editing was performed to determine their suitability for gene editing. RESULTS: NCM-K4DT lines retained dendritic-shaped morphology, pigmentation, and expression of PMEL, TYRP1, Melan-A, and SOX10. Cells exhibited enhanced proliferative capacity with preserved cell cycle regulation. Melanin production and tyrosinase activity were comparable to primary NCMs. Genomic profiling confirmed the absence of UM-associated driver mutations and chromosomal abnormalities. In vivo growth assays demonstrated that NCM-K4DT lines did not form tumors within the 3-month observation period. Notably, NCM-K4DT cells were efficiently edited by CRISPR/Cas9. CONCLUSIONS: NCM-K4DT lines represent stable, non-cancerous, and genetically tractable models for studying choroidal melanocyte biology, modeling UM-associated mechanisms, and advancing therapeutic development in ocular research.

Humans↗

PubMed2006

CARAT: a novel method for allelic detection of DNA copy number changes using high density oligonucleotide arrays.

BACKGROUND: DNA copy number alterations are one of the main characteristics of the cancer cell karyotype and can contribute to the complex phenotype of these cells. These alterations can lead to gains in cellular oncogenes as well as losses in tumor suppressor genes and can span small intervals as well as involve entire chromosomes. The ability to accurately detect these changes is central to understanding how they impact the biology of the cell. RESULTS: We describe a novel algorithm called CARAT (Copy Number Analysis with Regression And Tree) that uses probe intensity information to infer copy number in an allele-specific manner from high density DNA oligonuceotide arrays designed to genotype over 100,000 SNPs. Total and allele-specific copy number estimations using CARAT are independently evaluated for a subset of SNPs using quantitative PCR and allelic TaqMan reactions with several human breast cancer cell lines. The sensitivity and specificity of the algorithm are characterized using DNA samples containing differing numbers of X chromosomes as well as a test set of normal individuals. Results from the algorithm show a high degree of agreement with results from independent verification methods. CONCLUSION: Overall, CARAT automatically detects regions with copy number variations and assigns a significance score to each alteration as well as generating allele-specific output. When coupled with SNP genotype calls from the same array, CARAT provides additional detail into the structure of genome wide alterations that can contribute to allelic imbalance.

Algorithms↗

PubMed2025

Quinoxaline-based anti-schistosomal compounds have potent anti-plasmodial activity.

The human pathogens Plasmodium and Schistosoma are each responsible for over 200 million infections annually, especially in low- and middle-income countries. There is a pressing need for new drug targets for these diseases, driven by emergence of drug-resistance in Plasmodium and an overall dearth of drug targets against Schistosoma. Here, we explored the opportunity for pathogen-hopping by evaluating a series of quinoxaline-based anti-schistosomal compounds for their activity against P. falciparum. We identified compounds with low nanomolar potency against 3D7 and multidrug-resistant strains. In vitro resistance selections using wildtype and mutator P. falciparum lines revealed a low propensity for resistance. Only one of the series, compound 22, yielded resistance mutations, including point mutations in a non-essential putative hydrolase pfqrp1, as well as copy number amplification of a phospholipid-translocating ATPase, pfatp2, a potential target. Notably, independently generated CRISPR-edited mutants in pfqrp1 also showed resistance to compound 22 and a related analogue. Moreover, previous lines with pfatp2 copy number variations were similarly less susceptible to challenge with the new compounds. Finally, we examined whether the predicted hydrolase activity of PfQRP1 underlies its mechanism of resistance, showing that both mutation of the putative catalytic triad and a more severe loss of function mutation elicited resistance. Collectively, we describe a compound series with potent activity against two important pathogens and their potential target in P. falciparum.

Quinoxalines↗

PubMed2001

Diagnostic and scientific applications of TaqMan real-time PCR in neuroblastomas.

This review summarizes the present data on the diagnostic and scientific applications of TaqMan real-time PCR in neuroblastomas, a novel fluorescence-based method for quantitation of gene expression and gene copy number variations as gene amplification and locus haploinsufficiencies. Particular emphasis is spent on precision and accuracy and the comparison of TaqMan real-time PCR with more traditional methods for quantitative PCR. Provided a proper assessment for each new marker, this method has been shown to be an easy, fast and reliable alternative to the more traditional methods for the determination of MYCN amplification and neuropeptide Y gene expression. The combination with single cell picking in the neuroblastoma tissue may be a future target of the application of real-time PCR.

Biomarkers, Tumor↗

PubMed2026

A nonlinear multi-omics data integration and classification model based on pathway self-attention and graph convolutional networks.

The abundance of omics data has significantly advanced the development of multi-omics data integration techniques. Non-linear embedding approaches for data integration have gradually become the mainstream in multi-omics research, as these approaches can substantially improve cancer analysis by enhancing the quality of the embeddings. However, current multi-omics data integration methods are typically confined to omics measurements, neglecting domain-specific prior knowledge encompassing biological pathways. In this study, we proposed a multi-omics integrated classification model, PathTransGCN, based on pathway self-attention and graph convolutional networks (GCN). The model integrated biological pathway information into multi-omics data analysis with the aim of enhancing the accuracy of cancer classification. Multi-omics data for breast cancer (BRCA), non-small cell lung cancer (NSCLC), and low-grade glioma (LGG) were obtained from The Cancer Genome Atlas (TCGA) and UCSC Xena databases. These data included gene mutations, DNA methylation, copy number variations, and gene expression, and were used to assess the model's generalizability across different cancers. First, PathTransGCN employed a pathway self-attention module to learn latent representations of samples across different pathways, thereby obtaining multi-omics integration vectors. Concurrently, a patient similarity network (PSN) was constructed using the similarity network fusion (SNF) approach. Second, the integrated vectors and the PSN were jointly fed into a GCN for end-to-end training, enabling precise classification of cancer subtypes. Through multi-omics data analysis of the BRCA dataset, PathTransGCN outperformed several popular algorithms (such as MoGCN and DeePathNet) in the five-class classification of cancer subtypes, achieving an accuracy rate of 87.6% and an F1 score of 86.4%. Moreover, the model demonstrated robust generalization capabilities across both NSCLC and LGG datasets, while effectively identifying key disease-associated biomarkers at the pathway level. Experimental results demonstrate that PathTransGCN exhibits outstanding performance in integrating omics data and delivering interpretable classification outcomes, presenting significant potential for clinical applications.

Humans↗

PubMed2026

Prognostic Role of Global DNA Methylation in Renal Cancer Reveals Decitabine Treatment Benefit.

BACKGROUND: Renal cancer presents a significant global health challenge due to its rising incidence and mortality rates. Often undetected in early stages, it complicates diagnosis and treatment. Current therapies face resistance and limited effectiveness, especially in advanced stages. The diverse subtypes of renal cancer highlight the need for new biomarkers and risk assessment tools for targeted treatments. OBJECTIVE: This study aims to assess the prognostic significance of global DNA methylation (GM) levels in renal cancer, identify new biomarkers, and evaluate the therapeutic potential of the DNA methyltransferase inhibitor decitabine. METHODS: Data on RNA sequencing, gene mutations, DNA methylation, and clinical outcomes were collected from TCGA and GEO databases. We calculated global DNA methylation scores (GMS) and categorized patients into high, intermediate, and low GMS groups. Survival analysis and genomic analyses were conducted to explore the relationships between GMS, clinical outcomes, and tumor characteristics. RESULTS: Higher GMS was identified as an independent prognostic factor associated with worse outcomes in renal cancer. Patients with elevated GMS showed increased mutations, copy number variations, and a more aggressive tumor phenotype. Treatment with decitabine was observed to reduce tumor hypermethylation and downregulate cell cycle pathway activity, indicating potential therapeutic benefits. CONCLUSION: Global DNA methylation plays a significant role in renal cancer prognosis. GMS may serve as valuable biomarkers for prognosis and personalized treatment strategies. Decitabine shows potential efficacy for high GMS patients, particularly through its impact on cell cycle regulation, underscoring the importance of personalized approaches in cancer treatment.

Humans↗

PubMed2026

A pan-cancer multi-omic SuperLearner for regulated cell death survival topologies.

INTRODUCTION: Regulated cell death (RCD) pathways influence tumor progression and immune modulation. We previously constructed a signature database mapping 25 RCD forms across seven multi-omic layers and 33 tumor types (CancerRCDShiny). Despite their ability to identify risk populations, translating these signatures into personalized clinical workflows requires a shift from cohort stratification to individualized risk mapping by modeling patient risk (survival topologies) to capture the non-linear dynamics of RCD signatures. METHODS: We engineered a pan-cancer multi-omic SuperLearner pipeline across 33 cancer types. Phase I performed zero-leakage harmonization and groupwise imputation to prevent cross-cohort amalgamation. Phase II deployed Elastic Net-regularized Cox regression as a CANARY diagnostic to map proportional hazards failures. Strata with a 35% missingness barrier entered Phase III, deploying a Quadripartite ensemble: Random Survival Forests, XGBoost, Survival-Boruta, and Multi-Task Logistic Regression, fused within an Elastic Net Multi-View Meta-Learner (MVL), with post-hoc TreeSHAP and LIME interpretability. RESULTS: The CANARY diagnostic demonstrated the structural invalidity of pan-cancer geometric proportional hazards. Across 96 admissible strata, Phase III executed algorithmic displacement: continuous multi-omic topologies suppressed static genomic mutations and copy number variations (85.7% vs. 0.0% apex retention). The MVL stabilized predictions against extreme variance; LIME surrogate validations (R 2 < 0.10) confirmed the systematic failure of linear interpretative proxies. N-dimensional TreeSHAP interaction mapping exposed synergistic and antagonistic rescue trajectories defining individualized Survival Topologies, which were invisible to additive models. The architecture was deployed as CancerRCDPredictor, a digital molecular tumor board with integrated LLM capabilities. The MVL SuperLearner achieved a median C-index of 0.749 (IQR: 0.722-0.836) across 96 modelable strata, with 95% bootstrap confidence intervals confirming precision (median width: 0.052) and permutation significance in 93.8% of strata (p < 0.001). External CPTAC validation across ten cancer types demonstrated significant cross-cohort generalizability in clear cell renal carcinoma (KIRC; C-index 0.675, p = 0.017) and modest performance across the remaining adequately powered cancers (median 0.582), underscoring the need for larger multi-institutional validation cohorts. CONCLUSION: This pan-cancer multi-omic SuperLearner bypasses linear topological failures, advancing beyond generalized stratification to establish a deterministically mapped architecture for predicting RCD-related survival topologies. Through the CancerRCDPredictor interface, multi-omic insights translate into individualized survival topology exploration, providing a foundation for future precision oncology validation.

SuperLearner↗

PubMed2001

[Localization and analysis of 1A6 gene by dual-color fluorescence in situ hybridization on gastric carcinoma tissue and tumor cell lines].

OBJECTIVE: To locate 1A6 gene on the chromosome and study its copy number by dual-color fluorescence in situ hybridization (FISH) on three tumor cell lines and 22 gastric tumors patients. METHODS: The single-color FISH of small size biotin-labeled cDNA cloned in plasmid was used to locate the 1A6 gene on one of the chromosomes in C group. Genomic DNA of 1A6 gene was screened by polymerase chain reaction (PCR) in PAC library according to the sequence. The florenscence of green and orange was incorporated into the target gene-1A6 cloned in PAC and chromosome-12-specific alpha-satellite repeat DNA by nick translation, followed by dual-color FISH. 1A6 gene was located in the metaphase chromosome of the normal peripheral lymphocytes. 1A6 gene and chromosome 12 copy numbers were analyzed on touch slide of gastric cancer tissue and cell lines. RESULTS: 1A6 gene was located in 12q23.2-23.3. The chromosome 12/1A6 signal ratio was 0.96-1.01 in breast, ovarian and gastric cancer cell lines. The ratio was 0.93-1.11 in gastric cancer tissue touch slides. The number of chromosome 12 is disomic (87.7%), triploid (7.4%) in BMI cell line; multisomic(100%), including pentasomic (67.6%) in SKOV3; the multisomic (83.1%), including trisomic (71%) in SGC823. There are highly disomic rate in 86. 4% (19/22) of patients. CONCLUSION: 1A6 gene is in 12q23.2-23.3. Neither 1A6 amplification nor deletion in gastric cancer tissue and three cell lines was found. Further study is needed for the understanding of chromosome 12 copy number variation in different cancer cell lines.

Chromosome Mapping↗

PubMed1984

Effects of promoter strengths and growth conditions on copy number of transcription-fusion vectors.

Promoters with widely different transcriptional activities have been fused to the galactokinase gene contained in a multicopy plasmid ( McKenny , K., Shimatake , H., Court, D., Schmeissner , U., Brady, C., and Rosenberg, M. (1982) in Gene Amplification and Analysis: Analysis of Nucleic Acids ( Chirkjian , J. G., and Papas , T., eds) pp. 383-415, Elsevier /North-Holland Biomedical Press, Netherlands). Assay methods which allow determination of galactokinase-specific activity (nanomoles of galactose 1-phosphate/min/mg of protein) and plasmid-specific number (femtomoles of plasmid/mg of protein) in the same sonicated cellular extract are described. These methods provide a way to accurately measure and compare the promoter activities (nanomoles of galactose 1-phosphate/min/fmol of plasmid) of different plasmid constructions which exhibit different in vivo plasmid copy numbers. It is demonstrated that in vivo, copy number fluctuations are correlated with such parameters as promoter strength and cellular growth conditions. The ability to account for these uncontrolled in vivo copy number variations when comparing the transcriptional activities of different DNA inserts in multicopy transcription-fusion plasmids greatly facilitates the utility of these systems.

Base Sequence↗