Low Guanine Research Articles

Low guanine content and biased nucleotide distribution in vertebrate mtDNA can cause overestimation of non-CpG methylation.

ABSTRACTMitochondrial DNA (mtDNA) methylation in vertebrates has been hotly debated for over 40 years. Most contrasting results have been reported following bisulfite sequencing (BS-seq) analyses. We addressed whether BS-seq experimental and analysis conditions influenced the estimation of the levels of methylation in specific mtDNA sequences. We found false positive non-CpG methylation in the CHH context (fpCHH) using unmethylated Sus scrofa mtDNA BS-seq data. fpCHH methylation was detected on the top/plus strand of mtDNA within low guanine content regions. These top/plus strand sequences of fpCHH regions would become extremely AT-rich sequences after BS-conversion, whilst bottom/minus strand sequences remained almost unchanged. These unique sequences caused BS-seq aligners to falsely assign the origin of each strand in fpCHH regions, resulting in false methylation calls. fpCHH methylation detection was enhanced by short sequence reads, short library inserts, skewed top/bottom read ratios and non-directional read mapping modes. We confirmed no detectable CHH methylation in fpCHH regions by BS-amplicon sequencing. The fpCHH peaks were located in the D-loop, ATP6, ND2, ND4L, ND5 and ND6 regions and identified in our S. scrofa ovary and oocyte data and human BS-seq data sets. We conclude that non-CpG methylation could potentially be overestimated in specific sequence regions by BS-seq analysis.

Abstract C030: A new low potency DNA guanine monoalkylating ADC payload with enhanced i n vivo tolerability

Abstract Although five Antibody-Drug Conjugates (ADCs) have been approved and over eighty others are in development, the majority contain payloads belonging to two classes: tubulin inhibitors and DNA interactive agents. Most DNA-interactive payloads (e.g., the PBD dimers and IGNs) have potent cytotoxicity but ADCs containing them have high hydrophobicity and a narrow therapeutic window. Thus, there is interest in developing novel payloads which benefit from similar in vivo efficacy but possess lower hydrophobicity and a substantially wider Therapeutic Window (TW). The pyridinobenzodiazepines (PDDs) are a new class of sequence-selective, DNA guanine monoalkylating ADC payload which contain a polyheterocyclic chain with sufficient span to guide them to specific DNA sequences (e.g., transcription factor binding sites). The lead high potency PDD payload (FGX2-62) is active in vitro in the picomolar range in a wide panel of cell-lines, is easily conjugated and has in vivo potency at sub-mg/kg levels in ADC form. Within the PDD platform, we have now developed a lower potency DNA monoalkylator with substantially superior in vivo properties to other DNA alkylating agents. FGX20-75 is active in the low nanomolar range (e.g., 2.1 nM in the gastric cell-line SW48, 72 hour incubation), but is sufficiently hydrophilic to allow efficient conjugation at DARs > 4. The increase in loading compared to other DNA-interactive payloads compensates for the drop in potency of FGX20-75, resulting in a potent ADC with substantially increased tolerability. ADCs have been generated by conjugating a maleimide-linked analogue of FGX20-75 to the EGFR-targeting antibody Cetuximab with DAR of 4.2. The ADCs exhibit significant in vivo efficacy compared to other DNA-interactive payloads, and a substantially increased tolerability profile. A maximum tolerated dose (MTD) has not yet been reached at 35 mg/kg (single dose, CD1 mice). The favourable hydrophobicity profile of the low potency alkylator (FGX20-75) and its ease of conjugation to antibodies, along with the significant in vivo efficacy and tolerability of the ADCs produced, suggest that this low potency DNA-alkylating payload represents a promising new approach in ADC development. Citation Format: Nicolas Veillard, Paolo Andriollo, Francesco Cascio, Paul J. M. Jackson, David E. Thurston. A new low potency DNA guanine monoalkylating ADC payload with enhanced in vivo tolerability [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference on Molecular Targets and Cancer Therapeutics; 2019 Oct 26-30; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2019;18(12 Suppl):Abstract nr C030. doi:10.1158/1535-7163.TARG-19-C030

Synonymous codon bias as a basis for novel antibiotic design: from nucleotide wobble constraint to ribosomal garrotte.

The observation of low guanine frequencies at wobble position codons over Rossmann-fold GXGXXG motifs, led to the discovery of sequence-wide synonymous codon bias, from this we propose novel ribosomal inhibitors. Methodology & results: The wobble bases of multiple sequence alignments of diverse Rossmann-fold enzymes were counted, A, C, T and G wobble frequencies of consecutive codons were displayed as wobble plots. Synonymous codon constraints were found throughout the length of proteins, particularly at Rossmann folds. Prokaryote mRNAs have disallowed nucleotide sequences with wobble bases, evolutionary tailored to safeguard translation. We propose one such editing mechanism ensures that glycine motifs avoid becoming Shine-Dalgarno motifs. We propose using peptide nucleic acids and cyclic peptidyl scaffolds as a means of presenting disallowed chemical interactions to the anti-Shine-Dalgarno.

Lactic acid bacteria and identification with PCR-DGGE

Lactic acid bacteria (LAB) are an important group in the industrially using microorganisms. The first pure cultures of bacteria was "Bacterium lactis" (probably Lactococcus lactis), obtained in 1873 by J. Lister. LAB are Gram-positive, non motile, non spore-forming, except Sporolactobacillus inulinus, catalase negative, microaerophilic or anaerobic microorganisms. LAB can be found in milk and dairy products, plants and human and animal intestinal mucosa. LAB have low Guanine and Cytosine (G+C) ratio.The industrial applications of lactic acid bacteria is considered, it is emphasized that reliable typing methods in strain levels are getting important about both study on cultures used in functional foods and determining the performance of LAB starter cultures. Denaturing Gradient Gel Electrophoresis (DGGE) is the most common technique in molecular fingerprinting culture-independent techniques. The technique is based on the separation of the same length but having different sequences of the Polymerase Chain Reaction (PCR) products.Â

Loss of genes related to Nucleotide Excision Repair (NER) and implications for reductive genome evolution in symbionts of deep-sea vesicomyid clams.

Intracellular thioautotrophic symbionts of deep-sea vesicomyid clams lack some DNA repair genes and are thought to be undergoing reductive genome evolution (RGE). In this study, we addressed two questions, 1) how these symbionts lost their DNA repair genes and 2) how such losses affect RGE. For the first question, we examined genes associated with nucleotide excision repair (NER; uvrA, uvrB, uvrC, uvrD, uvrD paralog [uvrDp] and mfd) in 12 symbionts of vesicomyid clams belonging to two clades (5 clade I and 7 clade II symbionts). While uvrA, uvrDp and mfd were conserved in all symbionts, uvrB and uvrC were degraded in all clade I symbionts but were apparently intact in clade II symbionts. UvrD was disrupted in two clade II symbionts. Among the intact genes in Ca. Vesicomyosocius okutanii (clade I), expressions of uvrD and mfd were detected by reverse transcription-polymerase chain reaction (RT-PCR), but those of uvrA and uvrDp were not. In contrast, all intact genes were expressed in the symbiont of Calyptogena pacifica (clade II). To assess how gene losses affect RGE (question 2), genetic distances of the examined genes in symbionts from Bathymodiolus septemdierum were shown to be larger in clade I than clade II symbionts. In addition, these genes had lower guanine+cytosine (GC) content and higher repeat sequence densities in clade I than measured in clade II. Our results suggest that NER genes are currently being lost from the extant lineages of vesicomyid clam symbionts. The loss of NER genes and mutY in these symbionts is likely to promote increases in genetic distance and repeat sequence density as well as reduced GC content in genomic genes, and may have facilitated reductive evolution of the genome.

Intrinsic terminators in Mycoplasma hyopneumoniae transcription.

BackgroundMycoplasma hyopneumoniae, an important pathogen of swine, exhibits a low guanine and cytosine (GC) content genome. M. hyopneumoniae genome is organised in long transcriptional units and promoter sequences have been mapped upstream of all transcription units. These analysis provided insights into the gene organisation and transcription initiation at the genome scale. However, the presence of transcriptional terminator sequences in the M. hyopneumoniae genome is poorly understood.ResultsIn silico analyses demonstrated the presence of putative terminators in 82% of the 33 monocistronic units (mCs) and in 74% of the 116 polycistronic units (pCs) considering different classes of terminators. The functional activity of 23 intrinsic terminators was confirmed by RT-PCR and qPCR. Analysis of all terminators found by three software algorithms, combined with experimental results, allowed us to propose a pattern of RNA hairpin formation during the termination process and to predict the location of terminators in the M. hyopneumoniae genome sequence.ConclusionsThe stem-loop structures of intrinsic terminators of mycoplasma diverge from the pattern of terminators found in other bacteria due the low content of guanine and cytosine. In M. hyopneumoniae, transcription can end after a transcriptional unit and before its terminator sequence and can also continue past the terminator sequence with RNA polymerases gradually releasing the RNA.Electronic supplementary materialThe online version of this article (doi:10.1186/s12864-015-1468-6) contains supplementary material, which is available to authorized users.

Complete mitochondrial genome of Aeolesthes oenochrous (Fairmaire) (Coleoptera: Cerambycidae): an endangered and colorful longhorn beetle

Aeolesthes oenochrous (Fairmaire), a large and colorful longhorn beetle, is an endangered species in Taiwan. Its complete mitogenome, 15,747 bp, shows a typical coleopteran organization, containing 13 protein coding genes, 22 tRNA genes, 2 rRNA genes and one A + T rich region. Two protein coding genes, i.e. COI and ND1, have the atypical start codon of AAT and TTG, respectively. The third nucleotide position of codons shows extremely low guanine content. In the A + T rich region, there were two poly-T stretches with 14 and 13 thymine each. These two poly-T stretches were clarified by the cloning method.

Mapping of six somatic linker histone H1 variants in human breast cancer cells uncovers specific features of H1.2

Seven linker histone H1 variants are present in human somatic cells with distinct prevalence across cell types. Despite being key structural components of chromatin, it is not known whether the different variants have specific roles in the regulation of nuclear processes or are differentially distributed throughout the genome. Using variant-specific antibodies to H1 and hemagglutinin (HA)-tagged recombinant H1 variants expressed in breast cancer cells, we have investigated the distribution of six H1 variants in promoters and genome-wide. H1 is depleted at promoters depending on its transcriptional status and differs between variants. Notably, H1.2 is less abundant than other variants at the transcription start sites of inactive genes, and promoters enriched in H1.2 are different from those enriched in other variants and tend to be repressed. Additionally, H1.2 is enriched at chromosomal domains characterized by low guanine–cytosine (GC) content and is associated with lamina-associated domains. Meanwhile, other variants are associated with higher GC content, CpG islands and gene-rich domains. For instance, H1.0 and H1X are enriched at gene-rich chromosomes, whereas H1.2 is depleted. In short, histone H1 is not uniformly distributed along the genome and there are differences between variants, H1.2 being the one showing the most specific pattern and strongest correlation with low gene expression.

Non‐invasive prenatal testing for fetal aneuploidy: charting the course from clinical validity to clinical utility

Non‐invasive prenatal testing for fetal aneuploidy: charting the course from clinical validity to clinical utility

Effect of the antibiotic avilamycin on the structure of the microbial community in the jejunal intestinal tract of broiler chickens

This study was conducted to determine whether avilamycin, which is used as a growth promoter in broiler chickens, would affect the structure of the bacterial community within the jejunal intestinal tract. Sixty chickens were assigned to 2 groups: a control group and an avilamycin-treated group. The jejunal tissue from 4 chicks randomly sampled on d 1, 3, 7, 21, and 42 from each treatment group were investigated for changes in villus height, total mesophilic bacteria, lactic acid bacteria, and the structure of the microbial community using PCR-denaturant gradient gel electrophoresis. Supplementation with avilamycin had no effect on villus height and total bacterial count in either the mesophile or lactic acid bacteria. Based on PCR-denaturant gradient gel electrophoresis analysis, 3 bacterial groups were identified in both groups of chicks: gram-positive bacteria having low contents of guanine and cytosine, gamma proteobacteria, and actinobacteria. The abundance of Weisella, Enterococcus faecium, and Pediococcus acidophilus found only in the control group indicated that avilamycin did affect their growth and led to low microbial diversity among the low guanine and cytosine group compared with the control group. In contrast, however, avilamycin treatment was associated with higher microbial diversity among the gamma proteobacteria group in respect of the various Pseudomonas species. This finding should increase awareness of the potential effects of antibiotic use and particularly avilamycin in commercial poultry production in tropical areas.

Identification and characterisation of novel Mss4-binding Rab GTPases

The Mss4 (mammalian suppressor of yeast Sec4) is an evolutionarily highly conserved protein and is expressed in all mammalian tissues. Although its precise biological function is still elusive, it has been shown to associate with a subset of secretory Rab proteins (Rab1b, Rab3a, Rab8a, Rab10) and to possess a rather low guanine nucleotide exchange factor (GEF) activity towards them in vitro (Rab1, Rab3a and Rab8a). By screening a human placenta cDNA library with Mss4 as bait, we identified several Rab GTPases (Rab12, Rab13 and Rab18) as novel Mss4-binding Rab proteins. Only exocytic but no endocytic Rab GTPases were found in our search. The binding of Mss4 to Rab proteins was confirmed by direct yeast two-hybrid interaction, by co-immunoprecipitation from lysates of mammalian cells, by immunofluorescence colocalisation as well as by direct in vitro binding studies. Analysis of Mss4 catalytic activity towards different Rab substrates confirmed that it is a somewhat inefficient GEF. These data, together with our mutational analysis of Mss4-Rab binding capacity, support the already proposed idea that Mss4 functions rather as a chaperone for exocytic Rab GTPases than as a GEF.

Bacillithiol, a New Player in Bacterial Redox Homeostasis

Bacillithiol (BSH), the α-anomeric glycoside of l-cysteinyl-d-glucosamine with l-malic acid, plays a dominant role in the cytosolic thiol redox chemistry of the low guanine and cytosine (GC) Gram-positive bacteria (phylum Firmicutes). BSH is functionally analogous to glutathione (GSH) but differs sufficiently in chemical structure that cells have evolved a distinct set of enzymes that use BSH as cofactor. BSH was discovered in Bacillus subtilis as a mixed disulfide with the redox-sensing repressor OhrR and in B. anthracis by biochemical analysis of pools of labeled thiols. The structure of BSH was determined after purification from Deinococcus radiodurans. Similarities in structure between BSH and mycothiol (MSH) facilitated the identification of biosynthetic genes for BSH in the model organism B. subtilis. Phylogenomic analyses have identified several candidate BSH-using or associated proteins, including a BSH reductase, glutaredoxin-like thiol-dependent oxidoreductases (bacilliredoxins), and a BSH-S-transferase (FosB) involved in resistance to the epoxide antibiotic fosfomycin. Preliminary results implicate BSH in cellular processes to maintain cytosolic redox balance and for adaptation to reactive oxygen, nitrogen, and electrophilic species. BSH also is predicted to chelate metals avidly, in part due to the appended malate moiety, although the implications of BSH for metal ion homeostasis have yet to be explored in detail.

Unparalleled GC content in the plastid DNA of Selaginella

One of the more conspicuous features of plastid DNA (ptDNA) is its low guanine and cytosine (GC) content. As of February 2009, all completely-sequenced plastid genomes have a GC content below 43% except for the ptDNA of the lycophyte Selaginella uncinata, which is 55% GC. The forces driving the S. uncinata ptDNA towards G and C are undetermined, and it is unknown if other Selaginella species have GC-biased plastid genomes. This study presents the complete ptDNA sequence of Selaginella moellendorffii and compares it with the previously reported S. uncinata plastid genome. Partial ptDNA sequences from 103 different Selaginella species are also described as well as a significant proportion of the S. moellendorffii mitochondrial genome. Moreover, S. moellendorffii express sequence tags are data-mined to estimate levels of plastid and mitochondrial RNA editing. Overall, these data are used to show that: (1) there is a genus-wide GC bias in Selaginella ptDNA, which is most pronounced in South American articulate species; (2) within the Lycopsida class (and among plants in general), GC-biased ptDNA is restricted to the Selaginella genus; (3) the cause of this GC bias is arguably a combination of reduced AT-mutation pressure relative to other plastid genomes and a large number of C-to-U RNA editing sites; and (4) the mitochondrial DNA (mtDNA) of S. moellendorffii is also GC biased (even more so than the ptDNA) and is arguably the most GC-rich organelle genome observed to date-the high GC content of the mtDNA also appears to be influenced by RNA editing. Ultimately, these findings provide convincing support for the earlier proposed theory that the GC content of land-plant organelle DNA is positively correlated and directly connected to levels of organelle RNA editing.

Origin of an Alternative Genetic Code in the Extremely Small and GC–Rich Genome of a Bacterial Symbiont

The genetic code relates nucleotide sequence to amino acid sequence and is shared across all organisms, with the rare exceptions of lineages in which one or a few codons have acquired novel assignments. Recoding of UGA from stop to tryptophan has evolved independently in certain reduced bacterial genomes, including those of the mycoplasmas and some mitochondria. Small genomes typically exhibit low guanine plus cytosine (GC) content, and this bias in base composition has been proposed to drive UGA Stop to Tryptophan (Stop→Trp) recoding. Using a combination of genome sequencing and high-throughput proteomics, we show that an α-Proteobacterial symbiont of cicadas has the unprecedented combination of an extremely small genome (144 kb), a GC–biased base composition (58.4%), and a coding reassignment of UGA Stop→Trp. Although it is not clear why this tiny genome lacks the low GC content typical of other small bacterial genomes, these observations support a role of genome reduction rather than base composition as a driver of codon reassignment.

Mitochondrial Genome of the Colorless Green Alga Polytomella capuana: A Linear Molecule with an Unprecedented GC Content

One common observation concerning mitochondrial genomes is that they have a low guanine and cytosine content (GC content); of the complete mitochondrial genome sequences currently available at the National Center for Biotechnology Information (NCBI) (July 2007), the GC content ranges from 13.3% to 53.2% and has an average value of 38%. Here, we present the GC-rich mitochondrial genome (57% GC) of the colorless green alga Polytomella capuana. The disproportion of GC among the different regions of the P. capuana mitochondrial DNA (mtDNA) suggests that a neutral process is responsible for the GC bias. We propose that a biased gene conversion mechanism resulted in the GC-rich state of the P. capuana mtDNA. In addition, our analysis indicates that the P. capuana mitochondrial genome is a single 13-kb linear molecule with telomeres, which have a closed (hairpin-loop) conformation: a novel terminal structure among described linear green-algal mtDNAs. Furthermore, using a series of GC-rich inverted repeats found within the P. capuana mitochondrial genome, we describe recombination-based scenarios of how intact linear mtDNA conformations can be converted into the fragmented forms found in other Polytomella taxa.

Subcycling PCR for long-distance amplifications of regions with high and low guanine-cystine content: amplification of the intron 22 inversion of the FVIII gene.

Subcycling PCR for long-distance amplifications of regions with high and low guanine-cystine content: amplification of the intron 22 inversion of the FVIII gene.

Mycoplasmas, plants, insect vectors: a matrimonial triangle

Plant pathogenic mycoplasmas were discovered by electron microscopy, in 1967, long after the discovery and culture in 1898 of the first pathogenic mycoplasma of animal origin, Mycoplasma mycoides. Mycoplasmas are Eubacteria of the class Mollicutes, a group of organisms phylogenetically related to Gram-positive bacteria. Their more characteristic features reside in the small size of their genomes, the low guanine (G) plus cytosine (C) content of their genomic DNA and the lack of a cell wall. Plant pathogenic mycoplasmas are responsible for several hundred diseases and belong to two groups: the phytoplasmas and the spiroplasmas. The phytoplasmas (previously called MLOs, for mycoplasma like organisms) were discovered first; they are pleiomorphic, and have so far resisted in vitro cultivation. Phytoplasmas represent the largest group of plant pathogenic Mollicutes. Only three plant pathogenic spiroplasmas are known today. Spiroplasma citri, the agent of citrus stubborn was discovered and cultured in 1970 and shown to be helical and motile. S. kunkelii is the causal agent of corn stunt. S. phoeniceum, responsible for periwinkle yellows, was discovered in Syria. There are many other spiroplasmas associated with insects and ticks. Plant pathogenic mycoplasmas are restricted to the phloem sieve tubes in which circulates the photosynthetically-enriched sap, the food for many phloem-feeding insects (aphids, leafhoppers, psyllids, etc.). Interestingly, phytopathogenic mycoplasmas are very specifically transmitted by leafhoppers or psyllid species. In this paper, the most recent knowledge on phytopathogenic mycoplasmas in relation with their insect and plant habitats is presented as well as the experiments carried out to control plant mycoplasma diseases, by expression of mycoplasma-directed-antibodies in plants (plantibodies).

The promise of a protist: the Dictyostelium genome project

The genome of Dictyostelium discoideum is being sequenced by an international consortium and is scheduled for completion in the next few years. The sequence will accelerate research into a number of phenomena carried out by these versatile soil amoebae, providing insight into analogous processes that operate in a wide range of eukaryotes. These include the dynamic regulation of the cytoskeleton during chemotaxis, intercellular communication during multicellular development and the intracellular growth of bacterial pathogens. The current state of the genome project is summarized and the challenges of sequencing a genome with unusually low guanine and cytosine content and with a bimodal base composition distribution are discussed. The prospects for functional analyses at the genomic scale are also considered.

Long-term changes in microbial community structure in soils subjected to different fertilizing practices revealed by quinone profile analysis

Quinone profile analysis of stored air-dried soils gave an approximation of the long-term changes in the microbial community structure in four soils subjected to different types of fertilizer application from 1987 to 1997: unfertilized soil (NF-soil), soil amended with chemical fertilizers (CF-soil), soil amended with chemical fertilizers and 40 t ha-1 y-1 of farmyard manure (CF+ FYM-soil), and soil amended with 400 t ha-1 y-1 of farmyard manure (FYM-soil). The carbon content increased, and the soil pH remained higher in the soils receiving farmyard manure. Principal component analysis of the quinone profiles of the soils indicated that the microbial community structure showed a high similarity among the four soils before the onset of cultivation and changed to a different community structure specific to the respective fertilizing practices except for the NF-soil. The specific quinone profile became stable after two cropping seasons in the FYM-soil, after 10 cropping seasons in the CF+ FYM-soil and after 15 cropping seasons in the CF-soil, respectively. The quinone profile of the NF-soil did not become stable, and no specific profile was developed. The specific quinone profiles in the FYM- and CF+ FYM-soils were both characterized by large mole fractions of menaquinone with seven isoprenoid units (MK-7). Farmyard manure itself contained a large mole fraction of MK-7. It was suggested that the amount of MK-7 increased due to the application of farmyard manure. MK-7 indicates the presence of Gram-positive bacteria with low guanine plus cytosine contents such as Bacillus and Gram-negative bacteria of the Cytophaga-Flavobacterium complex. The specific quinone profile in the CF-soil was characterized by the presence of menaquinone with seven isoprenoid units dihydrated (MK-7(H2)and MK-7(H4). Although Brevibacterium and Kocuria contain MK-7(H2), no microorganisms are known to have MK- 7(H.) as major quinone. The common major quinones were MK-8, MK-10(H4) and a mixture of MK-8(H4) and MK-9, suggesting the predominance of Grampositive bacteria in all the soils. Mole fractions of Ubiquinone with 8 isoprenoid units (Q-8) and Q-10 increased at various times in all the soils, indicating the sporadic growth of Gram-negative bacteria.

Walled and wall-less eubacteria from plants: sieve-tube-restricted plant pathogens

The micro-organisms which form the object of this review are representatives of two groups of Eubacteria: (i) the Mollicutes (former mycoplasmas), i.e. a group of low guanine (G) plus cytosine (C) Gram-positive bacteria lacking the classic bacterial cell wall., and (ii) two members of the Proteobacteria (former purple bacteria), i.e. a bacterial group containing most of the Gram-negative bacteria. As shown in Fig. 1, some of these organisms are restricted to the sieve tubes of the infected plants and, as such, they are phytopathogenic. Others are found on the surfaces of plant organs, mainly flowers, and are non-phytopathogenic. Among the sieve-tube-restricted bacteria, only the spiroplasmas are available in culture; the phytoplasmas and the two proteobacteria have never been cultured. Properties distinguishing Mollicutes from other Eubacteria are listed in Table 1.