Distribution Of CpG Islands Research Articles

Resveratrol shields against cisplatin-induced ototoxicity through epigenetic lncRNA GAS5 modulation of miR-455-5p/PTEN pathway

BackgroundOur previous research demonstrated that resveratrol counters DDP-induced ototoxicity by upregulating miR-455-5p, which targets PTEN. This study aimed to elucidate the underlying mechanisms involving GAS5 and DNA methyltransferase 1 (DNMT1) in resveratrol’s protective action. MethodsA luciferase reporter assay and RNA immunoprecipitation (RIP) assay were employed to study the binding between GAS5 and miR-455-5p, as well as between miR-455-5p and PTEN. HEI-OC1 cells treated with DDP were transfected with vectors for GAS5, si-GAS5, DNMT1, si-DNMT1, and miR-455-5p mimics, as well as PTEN. Subsequently, they were treated with resveratrol and exposed to DDP, both separately and in combination. The distribution of CpG islands in the GAS5 promoter was identified using MethyPrimer, and methylation-specific PCR (MSP) was conducted to determine the methylation levels of GAS5. Chromatin immunoprecipitation (ChIP) was utilized to examine the interaction between DNMT1 and GAS5. The viability of HEI-OC1 cells, catalase (CAT) activity, apoptosis, and ROS levels were assessed using the CCK-8 assay, CAT assay, TUNEL staining, and flow cytometry, respectively. An in vivo mouse model was developed to measure auditory brainstem response (ABR) thresholds, while RT-qPCR and Western blot analysis were employed to evaluate molecular levels. ResultsOur study discovered that GAS5 acts as a sponge for miR-455-5p, thereby increasing PTEN expression in DDP-treated HEI-OC1 cells. This process was reversed upon treatment with resveratrol. Importantly, DNMT1 promoted the methylation of the GAS5 promoter, leading to the suppression of GAS5 expression. This suppression enhanced the effectiveness of resveratrol in combating DDP-induced apoptosis and ROS in HEI-OC1 cells and amplified its protective effect against DDP’s ototoxicity in vivo. ConclusionsOur research emphasizes the significance of the DNMT1/GAS5/miR-455-5p/PTEN axis as a promising new route to boost resveratrol’s effectiveness against DDP-induced ototoxicity.

Epigenetic Peculiarity of Alphapapillomavirus Genus Based on the CpG Dinucleotides’ Island Variability

Background: Methylation plays a crucial role in genome regulation, serving as an essential epigenetic mechanism. CpG islands, which are regions of DNA rich in CpG dinucleotides, are ubiquitous epigenetic regulatory features that can modulate the functional dynamics of genes, thereby influencing the pathophysiology of pathogenic organisms within a host. Objectives: In this study, we aimed to investigate the distribution of CpG islands on Papillomavirus genomes and assess their potential impact on the neoplastic progression of different Alphapapillomavirus genera within host cells. Methods: We conducted an analysis of dinucleotide frequencies in DNA sequences from various Alphapapillomavirus genera. The sequences for our analysis were sourced from the specialized HPV (human papillomavirus) database (pave.niaid.nih.gov). Following a comprehensive comparative examination of entire genomes from different HPV species, we specifically focused on 63 genomes belonging to the Alphapapillomavirus genus to identify internal CpG island profiles. These investigations were conducted using online bioinformatics software (DBCAT), and statistical analysis was performed using GraphPad Prism v. 8.0.1. Results: Our preliminary findings revealed that 76.1% of the viruses in our study had fewer than 3 CpG islands but multiple CpG sites, while fewer than 25% of the viruses possessed at least 3 CpG islands. High-risk viruses were identified in 68.42% of genotypes with fewer than 3 CpG islands, whereas low-risk genotypes were observed in 15.7% of cases. Notably, some oncogenic viruses lacked CpG islands entirely, and this pattern was also observed in viruses with a single CpG island, occurring in 50% of cases. Based on the absence of CpG islands, genotypes such as HPV 67, HPV 97, and HPV 34 could potentially be classified as carcinogenic. Additionally, the distribution of CpG islands across HPV genes did not appear to be random. Genes like L2 and E2 contained a CpG island in 50% of cases, whereas oncogenes E6 and E7 consistently had a CpG island in 100% of cases in high-risk genotypes. Conclusions: Our findings suggest that the loss of 1 or more CpG islands could potentially transform an HPV genotype into a high-risk genotype. This process may be accelerated in the presence of host-related risk factors. Further research is required to validate whether CpG island distribution can aid in identifying oncogenic viruses.

Differences in CpG island distribution between exogenous and endogenous jaagsiekte sheep retrovirus strains.

The jaagsiekte sheep retrovirus (JSRV), belonging to the betaretrovirus genus of the retroviridae family, includes both exogenous and endogenous jaagsiekte sheep retroviruses (exJSRV and enJSRV, respectively). At the proviral genome level, exJSRV and enJSRV strains have a high degree of similarity with their main variation regions being the LTR, gag, and env genes. In this study, for the first time, we investigated and compared the distribution of CpG islands between these enJSRV and exJSRV strains. Specifically, we analyzed a total of 42 full-length JSRV genomic sequences obtained from the GenBank® database to identify CpG islands in the exJSRV and enJSRV genomes using the MethPrimer software. Our results showed that the CpG islands in the two JSRV strains were mainly distributed in the LTR, gag, and env genes. In exJSRVs, 66.66% (6/9), 33.33% (3/9), and 100% (9/9) of the sequences presented at least one CpG island in LTR, gag, env genes, respectively, and for enJSRVs, 84.84% (28/33), 57.57% (19/33), and 96.96% (32/33) of the sequences presented at least one CpG island in the LTR, gag, and env genes. These findings suggested that the distribution, length, and genetic traits of CpG islands were different for the exJSRV and enJSRV strains. In future, it would be necessary to demonstrate the biological significance of CpG islands within these genes in exJSRV and enJSRV genomes. This will enhance understanding regarding the potential role of CpG islands in epigenetic regulation.

The lncRNA KIF9-AS1 Accelerates Hepatocellular Carcinoma Growth by Recruiting DNMT1 to Promote RAI2 DNA Methylation.

Background Hepatocellular carcinoma (HCC) is a very common malignant tumor. Long noncoding RNAs (lncRNAs) enable discoveries of new therapeutic tumor targets. We aimed to study the role and potential regulatory mechanisms of the lncRNA KIF9-AS1 in HCC. Methods CCK-8, scratch assay, and flow cytometry were used to detect cell proliferation, migration, and apoptosis, respectively. Bax, Bcl-2, ERK, and pERK expression were measured by western blotting. StarBase predicted KIF9-AS1 expression in HCC and paracancerous tissues. RPISeq predicted the interaction score of KIF9-AS1 and DNMT1, and MethyPrimer revealed the CpG island distribution in the RAI2 promoter. MSP was performed to measure RAI2 methylation. RIP and ChIP were performed to examine lncRNA KIF9-AS1, DNMT1, and RAI2 interactions. Finally, the effect of KIF9-AS1 knockdown on HCC was verified with nude mice. Results We found that KIF9-AS1 expression was increased in HCC tissues. KIF9-AS1 knockdown inhibited the proliferation and migration, and facilitated the apoptosis of HCC cells. lncRNA KIF9-AS1-mediated RAI2 expression led to DNMT1 recruitment and regulated RAI2 DNA methylation. RAI2 overexpression inhibited the proliferation and migration and promoted the apoptosis of HCC cells. KIF9-AS1 knockdown inhibited subcutaneous tumor formation in vivo. Conclusion This study shows that KIF9-AS1 accelerates HCC growth by inducing DNMT1 promotion of RAI2 DNA methylation.

Computational characterization of domain-segregated 3D chromatin structure and segmented DNA methylation status in carcinogenesis.

The high‐order chromatin structure, together with DNA methylation and other epigenetic marks, plays a vital role in gene regulation and displays abnormal status in cancer cells. Theoretical analyses are expected to provide a more unified understanding of the multi‐omics data on the large variety of samples, and hopefully a common picture of carcinogenesis. In particular, we are interested in the question of whether an underlying origin DNA sequence exists for these epigenetic alterations. The human genome consists of two types of megabase‐sized domain based on the distribution of CpG islands (CGIs) that show distinct structural, epigenetic, and transcriptional properties: CGI‐rich and CGI‐poor domains. Through an integrated analysis of chromatin structure, DNA methylation, and RNA sequencing data, we found that, in carcinogenesis, the two different types of domain display different structural changes and have an increased number of DNA methylation differences and transcriptional‐level differences, compared with in noncancer cells. We also compared the structural features among carcinogenesis, senescence, and mitosis, showing the possible connection between chromatin structure and cell state, which could affect vital cancer‐related properties. In summary, chromatin structure, DNA methylation, and gene expression, as well as their changes observed in several types of cancers, show a dependence on multiscale DNA sequence heterogeneity.

Comparative analysis of CpG islands in equine infectious anemia virus strains.

Increasing evidence suggests that DNA methylation has key roles in the replication of retroviruses, including lentiviruses, and pathogenesis of diseases. However, the precise characteristics of CpG islands are not known for many retroviruses. In this study, we compared the distribution of CpG islands among strains of equine infectious anemia virus (EIAV), a lentivirus in the family Retroviridae and a model for HIV research. We identified CpG islands in 32 full-length EIAV genomic sequences obtained from the GenBank database using MethPrimer. Only one CpG island, from 100 to 120bp, was identified in the genomes of EIAV strains DV10, DLV3-A, and DLV5-10 from China, V26 and V70 from Japan, and IRE H3, IRE F2, IRE F3, and IRE F4 from Ireland. Importantly, the CpG island was located within the Rev gene, which is required for the expression of viral cis-acting elements and the production of new virions. These results suggest that the distribution, length, and genetic properties of CpG islands differ among EIAV strains. Future research should focus on the biological significance of this CpG island within rev to improve our understanding of the precise roles of CpG islands in epigenetic regulation in the species.

The Echinococcus canadensis (G7) genome: a key knowledge of parasitic platyhelminth human diseases

BackgroundThe parasite Echinococcus canadensis (G7) (phylum Platyhelminthes, class Cestoda) is one of the causative agents of echinococcosis. Echinococcosis is a worldwide chronic zoonosis affecting humans as well as domestic and wild mammals, which has been reported as a prioritized neglected disease by the World Health Organisation. No genomic data, comparative genomic analyses or efficient therapeutic and diagnostic tools are available for this severe disease. The information presented in this study will help to understand the peculiar biological characters and to design species-specific control tools.ResultsWe sequenced, assembled and annotated the 115-Mb genome of E. canadensis (G7). Comparative genomic analyses using whole genome data of three Echinococcus species not only confirmed the status of E. canadensis (G7) as a separate species but also demonstrated a high nucleotide sequences divergence in relation to E. granulosus (G1). The E. canadensis (G7) genome contains 11,449 genes with a core set of 881 orthologs shared among five cestode species. Comparative genomics revealed that there are more single nucleotide polymorphisms (SNPs) between E. canadensis (G7) and E. granulosus (G1) than between E. canadensis (G7) and E. multilocularis. This result was unexpected since E. canadensis (G7) and E. granulosus (G1) were considered to belong to the species complex E. granulosus sensu lato. We described SNPs in known drug targets and metabolism genes in the E. canadensis (G7) genome. Regarding gene regulation, we analysed three particular features: CpG island distribution along the three Echinococcus genomes, DNA methylation system and small RNA pathway. The results suggest the occurrence of yet unknown gene regulation mechanisms in Echinococcus.ConclusionsThis is the first work that addresses Echinococcus comparative genomics. The resources presented here will promote the study of mechanisms of parasite development as well as new tools for drug discovery. The availability of a high-quality genome assembly is critical for fully exploring the biology of a pathogenic organism. The E. canadensis (G7) genome presented in this study provides a unique opportunity to address the genetic diversity among the genus Echinococcus and its particular developmental features. At present, there is no unequivocal taxonomic classification of Echinococcus species; however, the genome-wide SNPs analysis performed here revealed the phylogenetic distance among these three Echinococcus species. Additional cestode genomes need to be sequenced to be able to resolve their phylogeny.

Common Stress Transcriptome Analysis Reveals Functional and Genomic Architecture Differences Between Early and Delayed Response Genes.

To identify the similarities among responses to diverse environmental stresses, we analyzed the transcriptome response of rice roots to three rhizotoxic perturbations (chromium, ferulic acid and mercury) and identified common early-transient, early-constant and delayed gene inductions. Common early response genes were mostly associated with signal transduction and hormones, and delayed response genes with lipid metabolism. Network component analysis revealed complicated interactions among common genes, the most highly connected signaling hubs being PP2C68, MPK5, LRR-RLK and NPR1. Gene architecture studies revealed different conserved promoter motifs and a different ratio of CpG island distribution between early and delayed genes. In addition, early-transient genes had more exons and a shorter first exon. IMEter was used to calculate the transcription regulation effects of introns, with greater effects for the first introns of early-transient than delayed genes. The higher Ka/Ks (non-synonymous/synonymous mutation) ratio of early-constant genes than early-transient, delayed and the genome median demonstrates the rapid evolution of early-constant genes. Our results suggest that finely tuned transcriptional control in response to environmental stress in rice depends on genomic architecture and signal intensity and duration.

Germline methylation patterns determine the distribution of recombination events in the dog genome.

The positive-regulatory domain containing nine gene, PRDM9, which strongly associates with the location of recombination events in several vertebrates, is inferred to be inactive in the dog genome. Here, we address several questions regarding the control of recombination and its influence on genome evolution in dogs. First, we address whether the association between CpG islands (CGIs) and recombination hotspots is generated by lack of methylation, GC-biased gene conversion (gBGC), or both. Using a genome-wide dog single nucleotide polymorphism data set and comparisons of the dog genome with related species, we show that recombination-associated CGIs have low CpG mutation rates, and that CpG mutation rate is negatively correlated with recombination rate genome wide, indicating that nonmethylation attracts the recombination machinery. We next use a neighbor-dependent model of nucleotide substitution to disentangle the effects of CpG mutability and gBGC and analyze the effects that loss of PRDM9 has on these rates. We infer that methylation patterns have been stable during canid genome evolution, but that dog CGIs have experienced a drastic increase in substitution rate due to gBGC, consistent with increased levels of recombination in these regions. We also show that gBGC is likely to have generated many new CGIs in the dog genome, but these mostly occur away from genes, whereas the number of CGIs in gene promoter regions has not increased greatly in recent evolutionary history. Recombination has a major impact on the distribution of CGIs that are detected in the dog genome due to the interaction between methylation and gBGC. The results indicate that germline methylation patterns are the main determinant of recombination rates in the absence of PRDM9.

Comparative Analysis of CpG Islands among HBV Genotypes

DNA methylation is being increasingly recognized to play a role in regulation of hepatitis B virus (HBV) gene expression. The aim of this study was to compare the CpG island distribution among different HBV genotypes. We analyzed 176 full-length HBV genomic sequences obtained from the GenBank database, belonging to genotypes A through J, to identify the CpG islands in the HBV genomes. Our results showed that while 79 out of 176 sequences contained three conventional CpG islands (I–III) as previously described, 83 HBV sequences harbored only two of the three known islands. Novel CpG islands were identified in the remaining 14 HBV isolates and named as CpG island IV, V, and VI. Among the eight known HBV genotypes and two putative genotypes, while HBV genomes containing three CpG islands were predominant in genotypes A, B, D, E, and I; genotypes C, F, G, and H tended to contain only two CpG islands (II and III). In conclusion, the CpG islands, which are potential targets for DNA methylation mediated by the host functions, differ among HBV genotypes, and these genotype-specific differences in CpG island distribution could provide new insights into the understanding of epigenetic regulation of HBV gene expression and hepatitis B disease outcome.

Introduction: The Use of Animals Models to Advance Epigenetic Science

Introduction: The Use of Animals Models to Advance Epigenetic Science

Analysis of APC and IGFBP7 promoter gene methylation in Swedish and Vietnamese colorectal cancer patients

The tumour suppressor gene adenomatous polyposis coli (APC) is a key component that drives colorectal carcinogenesis. The reported DNA methylation in the promoter of APC varies greatly among studies of colorectal cancer (CRC) in different populations. Insulin-like growth factor binding protein 7 (IGFBP7), also known as IGFBP-related protein 1 (IGFBP-rP1), is expressed in various tissue types, including the lung, brain, prostate and gastrointestinal tract, and has been suggested to play a tumour suppressor role against colorectal carcinogenesis. Studies have indicated that IGFBP7 is inactivated by DNA methylation in human colon, lung and breast cancer. In the present study, we used the methylation-specific polymerase chain reaction to study the methylation status of the APC and IGFBP7 gene promoters in cancerous and paired normal tissue to evaluate its impact on clinical factors and association with ethnicity, represented by Swedish and Vietnamese CRC patients. We also investigated the distribution of CpG islands and the CpG dinucleotide density of each CpG island in the regions which were the subject of our investigation. Overall, normal tissue from Swedish patients exhibited a significantly higher frequency of IGFBP7 gene methylation in comparison with that of Vietnamese patients. Moreover, a significantly higher number of cancer tissues from Vietnamese individuals showed higher levels of methylation versus the paired normal tissue compared with that of Swedish patients. When we studied the methylation in cancer compared with the matched normal tissue in individuals, we found that a significantly higher number of Vietnamese patients had a higher degree of IGFBP7 gene methylation in cancer versus matched normal tissue in comparison with Swedish patients. Taken together, our results suggest that the methylation of the APC and IGFBP7 gene promoter region in cancerous tissue, in combination with the predominance of methylation in normal tissue, may serve as a prognostic factor in CRC patients.

Long-Range Autocorrelations of CpG Islands in the Human Genome

In this paper, we use a statistical estimator developed in astrophysics to study the distribution and organization of features of the human genome. Using the human reference sequence we quantify the global distribution of CpG islands (CGI) in each chromosome and demonstrate that the organization of the CGI across a chromosome is non-random, exhibits surprisingly long range correlations (10 Mb) and varies significantly among chromosomes. These correlations of CGI summarize functional properties of the genome that are not captured when considering variation in any particular separate (and local) feature. The demonstration of the proposed methods to quantify the organization of CGI in the human genome forms the basis of future studies. The most illuminating of these will assess the potential impact on phenotypic variation of inter-individual variation in the organization of the functional features of the genome within and among chromosomes, and among individuals for particular chromosomes.

Characterization, comparative genomics, and evolutionary inferences of a human drug metabolizing (NAT2) gene

Abstract Aim The present-day genetic architecture of a species bears much significance to its closely related species. In recent availability of whole genome sequence data for closely related species, it is possible to detect genetic similarities/differences in specific lineages and infer the role of evolutionary forces in bringing such similarities/differences. In this respect, NAT2 gene, responsible for drug metabolism, is conserved across a few taxa and, thus, comparative genomic studies could be useful for better pharmacogenetic realization. Methods DNA sequences of human NAT2 gene were retrieved from NCBI and characterized. Comparative and evolutionary analyses were performed with sequences from four mammalian taxa and one avian taxon with different statistical algorithms. Results The observed genetic architecture of NAT2 gene was different across the taxa. Phylogenetic inferences revealed that human and chimpanzee are diverged recently and fowl was found to be diverged from rest of the taxa significantly. Also, gene length, microsatellites, Ka/Ks, secondary structure, and distribution of CpG islands were observed across taxa. Conclusions The detail architecture of NAT2 gene and its evolutionary history in different taxa show relationships with other taxa. Future population-based study in NAT2 would unravel the correlation between nucleotide changes and differential ability of drug metabolization in humans.

Dissection of phenotype reveals possible association between schizophrenia and Glutamate Receptor Delta 1 ( GRID1) gene promoter

Recent linkage and association data have implicated the Glutamate Receptor Delta 1 ( GRID1) locus in the etiology of schizophrenia. In this study, we sought to test whether variants in the promoter region are associated with this disorder. The distribution of CpG islands, which are known to be relevant for transcriptional regulation, was computationally determined at the GRID1 locus, and the putative transcriptional regulatory region at the 5′-terminus was systematically tagged using HapMap data. Genotype analyses were performed with 22 haplotype-tagging single nucleotide polymorphisms (htSNPs) in a German sample of 919 schizophrenia patients and 773 controls. The study also included two SNPs in intron 2 and one in intron 3 which have been found to be significantly associated with schizophrenia in previous studies. For the transcriptional regulatory region, association was obtained with rs3814614 ( p = 0.0193), rs10749535 ( p = 0.0245), and rs11201985 ( p = 0.0222). For all further analyses, the patient samples were divided into more homogeneous subgroups according to sex, age at onset, positive family history of schizophrenia and lifetime history of major depression. The p-value of the schizophrenia association finding for the three markers decreased by approximately one order of magnitude, despite the reduction in the total sample size. Marker rs3814614 (unadjusted p = 0.0005), located ~ 2.0 kb from the transcriptional start point, also withstood a two-step correction for multiple testing ( p = 0.030). No support was obtained for previously reported associations with the intronic markers. Our results suggest that genetic variants in the GRID1 transcriptional regulatory region may play a role in the etiology of schizophrenia, and that future association studies of schizophrenia may require stratification to ensure more homogeneous patient subgroups.

Biological Implications of Isochore Boundaries in the Human Genome

The human genome is composed of large sequence segments with fairly homogeneous GC content, namely isochores, which have been linked to many important functions; biological implications of most isochore boundaries, however, remain elusive, partly due to the difficulty in determining these boundaries at high resolution. Using the segmentation algorithm based on the quadratic divergence, we re-determined all 79 boundaries of previously identified human isochores at single-nucleotide resolution, and then compared the boundary coordinates with other genome features. We found that 55.7% of isochore boundaries coincide with termini of repeat elements; 45.6% of isochore boundaries coincide with termini of highly conserved sequences based on alignment of 17 vertebrate genomes, i.e., the highly conserved genome sequence switches to a less or non-conserved one at the isochore boundary; some isochore boundaries coincide with abrupt change of CpG island distribution (note that one boundary can associate with more than one genome feature). In addition, sequences around isochore boundaries are highly conserved. It seems reasonable to deduce that the boundaries of all the isochores studied here would be replication timing sites in the human genome. These results suggest possible key roles of the isochore boundaries and may further our understanding of the human genome organization.

Genome-wide analysis reveals strong correlation between CpG islands with nearby transcription start sites of genes and their tissue specificity

It has been envisaged that CpG islands are often observed near the transcriptional start sites (TSS) of housekeeping genes. However, neither the precise positions of CpG islands relative to TSS of genes nor the correlation between the presence of the CpG islands and the expression specificity of these genes is well-understood. Using thousands of sequences with known TSS in human and mouse, we found that there is a clear peak in the distribution of CpG islands around TSS in the genes of these two species. Thus, we classified human (mouse) genes into 6600 (2948) CpG+ genes and 2619 (1830) CpG− ones, based on the presence of a CpG island within the −100: +100 region. We estimated the degree of each gene being a housekeeper by the number of cDNA libraries where its ESTs were detected. Then, the tendency that a gene lacking CpG islands around its TSS is expressed with a higher degree of tissue specificity turned out to be evolutionarily conserved. We also confirmed this tendency by analyzing the gene ontology annotation of classified genes. Since no such clear correlation was found in the control data (mRNAs, pre-mRNAs, and chromosome banding pattern), we concluded that the effect of a CpG island near the TSS should be more important than the global GC content of the region where the gene resides.

Enhanced expression of a furin-cleavable proinsulin.

Cell engineering or gene therapy may represent an alternative to current methods of treating diabetes mellitus. Cells could be engineered to secrete insulin ex vivo for transplantation or the insulin gene could be administered directly by injection into muscle. A problem has been that non-neuroendocrine cells lack the endoproteases (PC3/1 and PC2) that are responsible for the processing of proinsulin to insulin. This can be surmounted by engineering the paired basic amino acid processing sites within proinsulin to sites that would be recognized by the ubiquitously expressed protease, furin. However, in every study to date, the expression of the furin-cleavable construct was greatly reduced relative to that of the unmodified proinsulin construct. We investigated possible causes for this, including mRNA stability, the presence of additional CpG islands, and the amino acid substitutions within furin-cleavable proinsulin. Several furin-cleavable rat proinsulin I cDNAs were engineered and used to transfect human HEK293, rat L6 and mouse C(2)C(12) cell lines. The stability of wild-type and furin-cleavable proinsulin mRNA in transfected C(2)C(12) cells was measured by RT-PCR. Comparison of the decay rates in the presence of actinomycin D showed no significant difference between the two species of mRNA. A furin-cleavable proinsulin cDNA was created to contain the same distribution of CpG islands as wild-type proinsulin. Comparison of insulin-like immunoreactivity in all three cell lines transfected with either this construct or a widely used furin-cleavable proinsulin containing additional CpG islands showed that the presence of the extra CpG islands had no effect. Studies to examine amino acid substitutions used to create furin consensus sequences showed that the addition of basic residues at the C-peptide/A-chain junction was responsible for the reduced production of furin-cleavable proinsulin. Using this information, we engineered a cDNA for furin-cleavable rat proinsulin I that was efficiently processed to mature insulin and expressed at the same level as wild-type proinsulin.

The Chromosomal Distribution of CpG Islands in the Mouse: Evidence for Genome Scrambling in the Rodent Lineage

We have prepared a library of mouse whole CpG islands using a methyl-CpG binding domain column. The distribution of CpG islands in the mouse genome was determined by FISH, using the library as a probe. Unlike in other vertebrate genomes that have been examined (human and chicken), extreme clustering of CpG islands was not seen in the mouse genome. No individual murine chromosome stood out as being either very gene-rich or very gene-poor. Despite the more even distribution of CpG islands in the mouse at a gross chromosomal level, at finer resolution concentrations of CpG islands are seen to correspond to the R-band early replicating regions of the genome.

Chromosomes with high gene density are preferentially repaired in human cells

It is known that DNA repair is heterogeneous in human cells since open chromatin, active genes and their transcribed strands are preferentially repaired. It is thus expected that DNA repair is clustered in chromosomes with high gene density. We have employed a DNA repair inhibitor, cytosine arabinoside (Ara-C), to convert ethyl methane sulfonate (EMS)-induced excision repairable lesions to chromosomal breaks, to check for the existence of heterogeneity of repair at the chromosome level. Chromosome staining by fluorescence in situ hybridization (FISH) was used to analyze breakage in chromosomes with diverse gene densities. These chromosomes were identified by means of the CpG island distribution after FISH with a CpG island-rich probe isolated from total human genomic DNA. Thus, three chromosomes with very high gene density (numbers 1, 19 and 20) were compared with two chromosomes with very low gene density (numbers 4 and 18) for clastogenicity and sensitivity to co-treatment with Ara-C and EMS. Our data indicate that those chromosome with higher gene density are more sensitive to a combination treatment with Ara-C and EMS, indicating that the level of excision repair synthesis is higher in those chromosome. It is therefore concluded that DNA excision repair is preferentially directed to chromosomes with high gene density. The implications of this finding in human biomonitoring using FISH techniques are discussed.