Differences in gene expression are central to evolution. Such differences can arise from cis-regulatory changes that affect transcription initiation, transcription rate and/or transcript stability in an allele-specific manner, or from trans-regulatory changes that modify the activity or expression of factors that interact with cis-regulatory sequences1,2. Both cis- and trans-regulatory changes contribute to divergent gene expression, but their respective contributions remain largely unknown3. Here we examine the distribution of cis- and trans-regulatory changes underlying expression differences between closely related Drosophila species, D. melanogaster and D. simulans, and show functional cis-regulatory differences by comparing the relative abundance of species-specific transcripts in F1 hybrids4,5. Differences in trans-regulatory activity were inferred by comparing the ratio of allelic expression in hybrids with the ratio of gene expression between species. Of 29 genes with interspecific expression differences, 28 had differences in cis-regulation, and these changes were sufficient to explain expression divergence for about half of the genes. Trans-regulatory differences affected 55% (16 of 29) of genes, and were always accompanied by cis-regulatory changes. These data indicate that interspecific expression differences are not caused by select trans-regulatory changes with widespread effects, but rather by many cis-acting changes spread throughout the genome.

/pdf/evolutionary-changes-in-cis-and-trans-gene-regulation-5gct4enl1e.pdf

Evolutionary changes in cis and trans gene regulation

DNA methylation is implicated inmultiple normal cellular processes, in-cluding the regulation of tissue- and de-velopment-specific gene expression,imprinting, X-chromosome inactiva-tion, DNA repair, and the suppressionof parasitic DNA sequences. Aberrantgene promoter methylation and result-ing altered gene expression have beenassociated with cancers (1,2). Alteredmethylation patterns have been de-scribed in human sporadic cancers:genome-wide hypomethylation and lo-calized hypermethylation have been ob-served, and both alterations can be pre-sent in the same tumor (3). Togetherwith loss-of-heterozygosity and genemutations, transcriptional silencing bymethylation has been shown to be a ma-jor inactivating event of tumor suppres-sor genes in accordance with Knud-son’s two-hit hypothesis (4). Geneinactivation by promoter hypermethyla-tion and resulting loss of function havebeen shown for several cancer genesinvolved in DNA repair, cell cycle con-trol, apoptosis, angiogenesis, differenti-ation, metastasis/invasion, transcrip-tion, and signal transduction (1).A wide range of methods exists todetect genomic DNA methylation, in-cluding approaches to detect genome-wide and gene-specific methylation lev-els (5). Most methods used to analyzethe methylation status of a specific se-quence are based on bisulfite modifica-tion of the DNA. Following the treat-ment, the methylation status can beassessed as a sequence difference by se-quencing, methylation-specific PCR,methylation-sensitive single-strandedconformational polymorphism, methy-lation-sensitive single nucleotide primerextension (Ms-SNuPE), or restrictionenzyme digestion. Recently, high-throughput methods to detect site-spe-cific methylation have been developedusing the TaqMan

https://www.future-science.com/doi/pdf/10.2144/03351md01

Sensitive and quantitative universal Pyrosequencing methylation analysis of CpG sites.

of the technology is the close correlation between the peak heights in a Pyrogram (Pyrosequencing AB), representing the sequence raw data, and the amount of nucleotides incorporated by the DNA polymerase during the Pyrosequencing reaction that allows for precise allele frequency determination for a SNP in pooled DNA samples (10–12). Very recently, an approach to the analysis of the degree of methylation on a single MVP after bisulfite treatment was shown using mixtures of PCR products for calibration (13). Bisulfite treatment converts unmethylated cytosines to uracils, while methylated cytosines remain unchanged under the appropriate reaction conditions. Therefore, after PCR, methylation sites can be treated as C/T SNPs with an allele frequency spectrum spanning the entire range (0%–100%). Here we demonstrate the Pyrosequencing technology to analyze and precisely quantify the degree of DNA methylation. Up to six MVPs were examined simultaneously in a single Pyrosequencing reaction. The method is amenable to the analysis of bisulfite-treated DNA derived from paraffin-embedded tissue samples, highly reproducible, and accurate if calibration is carried out properly (to detect any biased PCR amplification).

https://www.future-science.com/doi/pdf/10.2144/03351md02

Analysis and quantification of multiple methylation variable positions in CpG islands by Pyrosequencing.

This paper describes a simple and convenient method to measure the concentration and time response function f (C,t) of cells exposed to a toxicant by electric cell−substrate impedance sensing. Attachment and spreading of fibroblastic V79 cells cultured on small gold electrodes precoated with fibronectin were detected as electrical resistance changes. With this method, chemical cytotoxicity was easily screened by observing the response function of attached cells in the presence of inhibitor. The cytotoxicities of three test models, cadmium chloride, sodium arsenate, and benzalkonium chloride, were quantified by measuring the percentage inhibition as a function of the inhibitor concentration. The half-inhibition concentration, the required concentration to achieve 50% inhibition, derived from the response function agreed well with the results obtained using the standard neutral red assay.

Assessment of cytotoxicity using electric cell-substrate impedance sensing: concentration and time response function approach.

Authentication of ancient human DNA results is an exceedingly difficult challenge due to the presence of modern contaminant DNA sequences. Nevertheless, the field of ancient human genetics generates huge scientific and public interest, and thus researchers are rarely discouraged by problems concerning the authenticity of such data. Although several methods have been developed to the purpose of authenticating ancient DNA (aDNA) results, while they are useful in faunal research, most of the methods have proven complicated to apply to ancient human DNA. Here, we investigate in detail the reliability of one of the proposed criteria, that of appropriate molecular behavior. Using real-time polymerase chain reaction (PCR) and pyrosequencing, we have quantified the relative levels of authentic aDNA and contaminant human DNA sequences recovered from archaeological dog and cattle remains. In doing so, we also produce data that describes the efficiency of bleach incubation of bone powder and its relative detrimental effects on contaminant and authentic ancient DNA. We note that bleach treatment is significantly more detrimental to contaminant than to authentic aDNA in the bleached bone powder. Furthermore, we find that there is a substantial increase in the relative proportions of authentic DNA to contaminant DNA as the PCR target fragment size is decreased. We therefore conclude that the degradation pattern in aDNA provides a quantifiable difference between authentic aDNA and modern contamination. This asymmetrical behavior of authentic and contaminant DNA can be used to identify authentic haplotypes in human aDNA studies.

/pdf/more-on-contamination-the-use-of-asymmetric-molecular-3bbz3e9wtq.pdf

More on Contamination: The Use of Asymmetric Molecular Behavior to Identify Authentic Ancient Human DNA

Individual genotyping of single nucleotide polymorphisms (SNPs) remains expensive, especially for linkage disequilibrium mapping strategies involving high-throughput SNP genotyping On one hand, current methods may suit scientific and laboratory needs in regard to accuracy, reproducibility/robustness, and large-scale application On the other hand, a cheaper and less time-consuming alternative to individual genotyping is the use of SNP allele frequencies determined in DNA pools We have developed an accurate and reproducible protocol for allele frequency determination using PyrosequencingTM technology in large genomic DNA pools (374 individuals) The measured correlation (R2) in large DNA pools was 0980 In the context of disease-associated SNPs studies, we compared the allele frequencies between the disease (eg, type 2 diabetes and obesity) and control groups detected by either individual genotyping or Pyrosequencing of DNA pools In large pools, the variation between the two methods was 15 ± 09% I

https://www.future-science.com/doi/pdf/10.2144/02325dd03

Rapid SNP allele frequency determination in genomic DNA pools by pyrosequencing.

Individual genotyping of single n u cleotide polymorphisms (SNPs) remain s expensive, especially for linkage disequili b rium mapping strategies involving high throughput SNP genotyping. On one hand , current methods may suit scientific and la b oratory needs in regard to accuracy, repr o ducibility/robustness, and large-scale appl i cation. On the other hand, a cheaper an d less time-consuming alternative to indivi d ual genotyping is the use of SNP allele fr e quencies determined in DNA pools. W e have developed an accurate and repr o ducible protocol for allele frequency dete r mination using Pyrosequencin g ™technol o gy in large genomic DNA pools (37 4 individuals). The measured correlation (R 2 ) in large DNA pools was 0.980. In the co n text of disease-associated SNPs studies, w e compared the allele frequencies betwee n the disease (e.g., type 2 diabetes and obes i ty) and control groups detected by either i n dividual genotyping or Pyrosequencin g o f DNA pools. In large pools, the variation b e tween the two methods was 1.5 ± 0.9%. I t may be concluded that the allele frequenc y determination protocol could reliably detec t over 4% differences between populations . The method is economical in regard t o amounts of DNA, PCR, and primer exte n sion reagents required. Furthermore, it a l lows the rapid determination of allele fr e quency differences in case/control group s for association studies and susceptibilit y gene discovery in complex diseases .

L. Corset

Papers

Rapid SNP allele frequency determination in genomic DNA pools by pyrosequencing.

Short Technical Repor t Rapid SNP Allele Frequency Determination in Genomic DNA Pools by Pyrosequencin g