Influences of bacterial DNA base-ratios and amino acid composition of the gene product on the consequences of frameshift mutations

Using as an example the E. coli lac I (repressor) gene product, it can be shown that DNA base-ratio is a major determinant of the detailed outcomes of both +1 (?2) and ?1 (+2) types of frameshift mutations. Potential reinitiation codons (AUG or GUG) and premature stop codons (UAA, UAG and UGA) occur in very different proportions depending on the type of frameshift and the DNA base-ratio.A comparison of the H. halobium bacteriorhodopsin precursor gene with other actual and theoretical genes reveals that the amino acid composition of the gene product is a second, important, determinant of the detailed outcome of frameshift mutations. Rules are formulated for the occurrence of particular codon, and hence amino acid, doublets in AT-GC-rich or intermediate base-ratio DNA as these affect frameshift-generated reinitiation and premature stop codons.     

Multiple start codons and phosphorylation result in discrete Rad52 protein species

The sequence of the Saccharomyces cerevisiae RAD52 gene contains five potential translation start sites and protein-blot analysis typically detects multiple Rad52 species with different electrophoretic mobilities. Here we define the gene products encoded by RAD52. We show that the multiple Rad52 protein species are due to promiscuous choice of start codons as well as post-translational modification. Specifically, Rad52 is phosphorylated both in a cell cycle-independent and in a cell cycle-dependent manner. Furthermore, phosphorylation is dependent on the presence of the Rad52 C terminus, but not dependent on its interaction with Rad51. We also show that the Rad52 protein can be translated from the last three start sites and expression from any one of them is sufficient for spontaneous recombination and the repair of gamma-ray-induced double-strand breaks.     

Methylated Host Cell Gene Promoters and Human Papillomavirus Type 16 and 18 Predicting Cervical Lesions and Cancer

Change in the host and/or human papillomavirus (HPV) DNA methylation profile is probably one of the main factors responsible for the malignant progression of cervical lesions to cancer. To investigate those changes we studied 173 cervical samples with different grades of cervical lesion, from normal to cervical cancer. The methylation status of nine cellular gene promoters, CCNA1, CDH1, C13ORF18, DAPK1, HIC1, RARβ2, hTERT1, hTERT2 and TWIST1, was investigated by Methylation Specific Polymerase Chain Reaction (MSP). The methylation of HPV18 L1-gene was also investigated by MSP, while the methylated cytosines within four regions, L1, 5’LCR, enhancer, and promoter of the HPV16 genome covering 19 CpG sites were evaluated by bisulfite sequencing. Statistically significant methylation biomarkers distinguishing between cervical precursor lesions from normal cervix were primarily C13ORF18 and secondly CCNA1, and those distinguishing cervical cancer from normal or cervical precursor lesions were CCNA1, C13ORF18, hTERT1, hTERT2 and TWIST1. In addition, the methylation analysis of individual CpG sites of the HPV16 genome in different sample groups, notably the 7455 and 7694 sites, proved to be more important than the overall methylation frequency. The majority of HPV18 positive samples contained both methylated and unmethylated L1 gene, and samples with L1-gene methylated forms alone had better prognosis when correlated with the host cell gene promoters’ methylation profiles. In conclusion, both cellular and viral methylation biomarkers should be used for monitoring cervical lesion progression to prevent invasive cervical cancer.     

Evolutionary implications of potential frameshift hot-spots, methylation sites, and codon usage differences between genes and between organisms in Escherichia coli and Salmonella typhimurium

The published nucleotide sequences of the E. coli and S. typhimurium trp A and trp B genes show a high degree of similarity between homologous genes of the two organisms, and an even greater degree of similarity between the amino acid sequences of the gene products. In spite of this, analysis of the nucleotide sequences reveals that there are marked differences between E. coli and S. typhimurium genes with respect to potential frameshift mutation hot-spots and dam and mec, mutationally important, methylation sites. Such existing differences may well lead to divergent evolution of these two, presently closely related, bacteria. Codon usage patterns in the trp A and trp B genes of E. coli and S. typhimurium, and the lac I gene of E. coli, have been re-analysed in terms of AT-rich, GC-rich, neutral, or unique codons and marked preferences found. In some cases particular amino acids are most often specified by AT-rich, in others by the GC-rich, alternative codons. In still other cases the codon preference depends on the gene studied. These patterns can be interpreted in terms of enteric bacterial evolution, via hybridizations, from ancestral bacteria with AT- or GC-rich DNA.     

Genetic Control of Individual Differences in Gene-Specific Methylation in Human Brain

We have observed extensive interindividual differences in DNA methylation of 8590 CpG sites of 6229 genes in 153 human adult cerebellum samples, enriched in CpG island “shores” and at further distances from CpG islands. To search for genetic factors that regulate this variation, we performed a genome-wide association study (GWAS) mapping of methylation quantitative trait loci (mQTLs) for the 8590 testable CpG sites. cis association refers to correlation of methylation with SNPs within 1 Mb of a CpG site. 736 CpG sites showed phenotype-wide significant cis association with 2878 SNPs (after permutation correction for all tested markers and methylation phenotypes). In trans analysis of methylation, which tests for distant regulation effects, associations of 12 CpG sites and 38 SNPs remained significant after phenotype-wide correction. To examine the functional effects of mQTLs, we analyzed 85 genes that were with genetically regulated methylation we observed and for which we had quality gene expression data. Ten genes showed SNP-methylation-expression three-way associations—the same SNP simultaneously showed significant association with both DNA methylation and gene expression, while DNA methylation was significantly correlated with gene expression. Thus, we demonstrated that DNA methylation is frequently a heritable continuous quantitatively variable trait in human brain. Unlike allele-specific methylation, genetic polymorphisms mark both cis- and trans-regulatory genetic sites at measurable distances from their CpG sites. Some of the genetically regulated DNA methylation is directly connected with genetically regulated gene expression variation.     

Stability of gene expression and epigenetic profiles highlights the utility of patient-derived paediatric acute lymphoblastic leukaemia xenografts for investigating molecular mechanisms of drug resistance

Background

Patient-derived tumour xenografts are an attractive model for preclinical testing of anti-cancer drugs. Insights into tumour biology and biomarkers predictive of responses to chemotherapeutic drugs can also be gained from investigating xenograft models. As a first step towards examining the equivalence of epigenetic profiles between xenografts and primary tumours in paediatric leukaemia, we performed genome-scale DNA methylation and gene expression profiling on a panel of 10 paediatric B-cell precursor acute lymphoblastic leukaemia (BCP-ALL) tumours that were stratified by prednisolone response.

Results

We found high correlations in DNA methylation and gene expression profiles between matching primary and xenograft tumour samples with Pearson’s correlation coefficients ranging between 0.85 and 0.98. In order to demonstrate the potential utility of epigenetic analyses in BCP-ALL xenografts, we identified DNA methylation biomarkers that correlated with prednisolone responsiveness of the original tumour samples. Differential methylation of CAPS2, ARHGAP21, ARX and HOXB6 were confirmed by locus specific analysis. We identified 20 genes showing an inverse relationship between DNA methylation and gene expression in association with prednisolone response. Pathway analysis of these genes implicated apoptosis, cell signalling and cell structure networks in prednisolone responsiveness.

Conclusions

The findings of this study confirm the stability of epigenetic and gene expression profiles of paediatric BCP-ALL propagated in mouse xenograft models. Further, our preliminary investigation of prednisolone sensitivity highlights the utility of mouse xenograft models for preclinical development of novel drug regimens with parallel investigation of underlying gene expression and epigenetic responses associated with novel drug responses.

Electronic supplementary material

The online version of this article (doi:10.1186/1471-2164-15-416) contains supplementary material, which is available to authorized users.     

Ribosomal DNA methylation in a flax genotroph and a crown gall tumour

The methylation patterns of two flax lines are described. One, a genotroph S1, has 800 rNA genes per haploid cell while FT37/1, a crown gall tumour incited on S1, has only 300. Using the enzymes EcoRII, BstNI and ApyI to assess CXG methylation and HpaII and MspI for CG, we show that the methylation patterns of the rDNAs of both lines are identical. Both lines contain 3 fractions; the first contains repeats that are methylated at all sites examined and the second has some unmethylated sites. The third fraction contains repeats that are fully methylated but contain a discrete hypomethylated site at the 5 end of the pre-rRNA. The number of repeats which show these hypomethylated sites is constant in both lines despite the copy number difference. These may represent the active rRNA gene repeats.     

Cloning and sequencing of a second laccase gene from the white-rot fungus Coriolus versicolor

A second laccase gene, CVLG1, was isolated from Coriolus versicolor. CVLG1 encodes a precursor protein of 526 amino acids which contains a 23-amino acid signal sequence, and the coding region is interrupted by 11 introns. The number of potential N-glycosylation sites in this product is 12 and the greatest among that of polyporales laccases. Moreover, this protein shares about 70% homology with other polyporales laccases. Genomic Southern analysis showed that C. versicolor laccases are encoded by more than four genes including CVLG1 and a transposed allele of this gene.     

Identification of novel methyltransferases,Bmt5 and Bmt6, responsible for the m3U methylations of 25S rRNA in Saccharomyces cerevisiae

RNA contains various chemical modifications that expand its otherwise limited repertoire to mediate complex processes like translation and gene regulation. 25S rRNA of the large subunit of ribosome contains eight base methylations. Except for the methylation of uridine residues, methyltransferases for all other known base methylations have been recently identified. Here we report the identification of BMT5 (YIL096C) and BMT6 (YLR063W), two previously uncharacterized genes, to be responsible for m³U2634 and m³U2843 methylation of the 25S rRNA, respectively. These genes were identified by RP-HPLC screening of all deletion mutants of putative RNA methyltransferases and were confirmed by gene complementation and phenotypic characterization. Both proteins belong to Rossmann-fold–like methyltransferases and the point mutations in the S-adenosyl-l-methionine binding pocket abolish the methylation reaction. Bmt5 localizes in the nucleolus, whereas Bmt6 is localized predominantly in the cytoplasm. Furthermore, we showed that 25S rRNA of yeast does not contain any m⁵U residues as previously predicted. With Bmt5 and Bmt6, all base methyltransferases of the 25S rRNA have been identified. This will facilitate the analyses of the significance of these modifications in ribosome function and cellular physiology.     

Syntrophs Dominate Sequences Associated with the Mercury Methylation-Related Gene hgcA in the Water Conservation Areas of the Florida Everglades

The mechanisms and rates of mercury methylation in the Florida Everglades are of great concern because of potential adverse impacts on human and wildlife health through mercury accumulation in aquatic food webs. We developed a new PCR primer set targeting hgcA, a gene encoding a corrinoid protein essential for Hg methylation across broad phylogenetic boundaries, and used this primer set to study the distribution of hgcA sequences in soils collected from three sites along a gradient in sulfate and nutrient concentrations in the northern Everglades. The sequences obtained were distributed in diverse phyla, including Proteobacteria, Chloroflexi, Firmicutes, and Methanomicrobia; however, hgcA clone libraries from all sites were dominated by sequences clustering within the order Syntrophobacterales of the Deltaproteobacteria (49 to 65% of total sequences). dsrB mRNA sequences, representing active sulfate-reducing prokaryotes at the time of sampling, obtained from these sites were also dominated by Syntrophobacterales (75 to 89%). Laboratory incubations with soils taken from the site low in sulfate concentrations also suggested that Hg methylation activities were primarily mediated by members of the order Syntrophobacterales, with some contribution by methanogens, Chloroflexi, iron-reducing Geobacter, and non-sulfate-reducing Firmicutes inhabiting the sites. This suggests that prokaryotes distributed within clades defined by syntrophs are the predominant group controlling methylation of Hg in low-sulfate areas of the Everglades. Any strategy for managing mercury methylation in the Everglades should consider that net mercury methylation is not limited to the action of sulfate reduction.     

Structure analysis of three T7 late mRNA ribosome binding sites

The 5′-terminal regions of the three T7 late RNA species IIIb, IV and V have been characterized. These regions contain the protein synthesis initiation sites for the T7 genes 17, 9 and 10, respectively. Each of these is located between 60 and 90 nucleotides from the 5′ terminus of an in vitro synthesized RNA species. The sequence 5′ A-C-U-U-U-A-A-G-Pu-A-G-Pu, which is common to these ribosome binding regions, contains an impressive stretch of complementarity to the sequence 5′ A-C-C-U-C-C-U-U-A, at the 3′ terminus of 16 S ribosomal RNA. The nuclease mapping technique of Wurst et al. (1978) has been used to probe intramolecular structural interactions involving these initiation regions in the RNA. My results indicate that all three initiation codons, together with other portions of the ribosome binding regions are protected, under non-denaturing conditions, against the actions of both the single-strand-specific nuclease S₁ and RNAase T₁.     

A Direct Repeat Sequence at theRasgrf1Locus and Imprinted Expression

Genomic imprinting is an epigenetic modification that can lead to parental-specific monoallelic expression of specific autosomal genes. While methylation of CpG dinucleotides is thought to be a strong candidate for this epigenetic modification, little is known about the establishment or maintenance of parental origin-specific methylation patterns. We have recently identified a portion of mouse chromosome 9 containing a paternally methylated region associated with a paternally expressed imprinted gene, Ras protein-specific guanine nucleotide-releasing factor 1 (Rasgrf1). This area of chromosome 9 also contains a short, direct tandem repeat in close proximity to a paternally methylatedNotI site 30 kb upstream ofRasgrf1.Short, direct tandem repeats have been found associated with other imprinted genes and may act as important regulatory structures. Here we demonstrate that two rodent species (MusandRattus) contain a similar direct repeat structure associated with a region of paternal-specific methylation. In both species, theRasgrf1gene shows paternal-specific monoallelic expression in neonatal brain. A more divergent rodent species (Peromyscus) appears to lack a similar repeat structure based on Southern Blot analysis.Peromyscusanimals show biallelic expression ofRasgrf1in neonatal brain. These results suggest that direct repeat elements may play an important role in the imprinting process.     

Association of cigarette smoking and CRP levels with DNA methylation in α-1 antitrypsin deficiency

Alpha-1 antitrypsin (AAT) deficiency and tobacco smoking are confirmed risk factors for Chronic Obstructive Pulmonary Disease. We hypothesized that variable DNA methylation would be associated with smoking and inflammation, as reflected by the level of C-Reactive Protein (CRP) in AAT-deficient subjects. Methylation levels of 1,411 autosomal CpG sites from the Illumina GoldenGate Methylation Cancer Panel I were analyzed in 316 subjects. Associations of five smoking behaviors and CRP levels with individual CpG sites and average methylation levels were assessed using non-parametric testing, linear regression and linear mixed effect models, with and without adjustment for age and gender. Univariate linear regression analysis revealed that methylation levels of 16 CpG sites significantly associated with ever-smoking status. A CpG site in the TGFBI gene was the only site associated with ever-smoking after adjustment for age and gender. No highly significant associations existed between age at smoking initiation, pack-years smoked, duration of smoking, and time since quitting smoking as predictors of individual CpG site methylation levels. However, ever-smoking and younger age at smoking initiation associated with lower methylation level averaged across all sites. DNA methylation at CpG sites in the RUNX3, JAK3 and KRT1 genes associated with CRP levels. The most significantly associated CpG sites with gender and age mapped to the CASP6 and FZD9 genes, respectively. In summary, this study identified multiple potential candidate CpG sites associated with ever-smoking and CRP level in AAT-deficient subjects. Phenotypic variability in Mendelian diseases may be due to epigenetic factors.