Domain Movement within a Gene: A Novel Evolutionary Mechanism for Protein Diversification

A protein function is carried out by a specific domain localized at a specific position. In the present study, we report that, within a gene, a specific amino acid sequence can move between a certain position and another position. This was discovered when the sequences of restriction-modification systems within the bacterial species Helicobacter pylori were compared. In the specificity subunit of Type I restriction-modification systems, DNA sequence recognition is mediated by target recognition domain 1 (TRD1) and TRD2. To our surprise, several sequences are shared by TRD1 and TRD2 of genes (alleles) at the same locus (chromosomal location); these domains appear to have moved between the two positions. The gene/protein organization can be represented as x-(TRD1)-y-x-(TRD2)-y, where x and y represent repeat sequences. Movement probably occurs by recombination at these flanking DNA repeats. In accordance with this hypothesis, recombination at these repeats also appears to decrease two TRDs into one TRD or increase these two TRDs to three TRDs (TRD1-TRD2-TRD2) and to allow TRD movement between genes even at different loci. Similar movement of domains between TRD1 and TRD2 was observed for the specificity subunit of a Type IIG restriction enzyme. Similar movement of domain between TRD1 and TRD2 was observed for Type I restriction-modification enzyme specificity genes in two more eubacterial species, Streptococcus pyogenes and Mycoplasma agalactiae. Lateral domain movements within a protein, which we have designated DOMO (domain movement), represent novel routes for the diversification of proteins

A trial of somatic gene targeting in vivo with an adenovirus vector

BACKGROUND: Gene targeting in vivo provides a potentially powerful method for gene analysis and gene therapy. In order to sensitively detect and accurately measure designed sequence changes, we have used a transgenic mouse system, MutaMouse, which has been developed for detection of mutation in vivo. It carries bacteriophage lambda genome with lacZ(+ )gene, whose change to lacZ-negative allele is detected after in vitro packaging into bacteriophage particles. We have also demonstrated that gene transfer with a replication-defective adenovirus vector can achieve efficient and accurate gene targeting in vitro. METHODS: An 8 kb long DNA corresponding to the bacteriophage lambda transgene with one of two lacZ-negative single-base-pair-substitution mutant allele was inserted into a replication-defective adenovirus vector. This recombinant adenovirus was injected to the transgenic mice via tail-vein. Twenty-four hours later, genomic DNA was extracted from the liver tissue and the lambda::lacZ were recovered by in vitro packaging. The lacZ-negative phage was detected as a plaque former on agar with phenyl-beta-D-galactoside. RESULTS: The mutant frequency of the lacZ-negative recombinant adenovirus injected mice was at the same level with the control mouse (~1/10000). Our further restriction analysis did not detect any designed recombinant. CONCLUSION: The frequency of gene targeting in the mouse liver by these recombinant adenoviruses was shown to be less than 1/20000 in our assay. However, these results will aid the development of a sensitive, reliable and PCR-independent assay for gene targeting in vivo mediated by virus vectors and other means

Discovery of a novel restriction endonuclease by genome comparison and application of a wheat-germ-based cell-free translation assay: PabI (5′-GTA/C) from the hyperthermophilic archaeon Pyrococcus abyssi

To search for restriction endonucleases, we used a novel plant-based cell-free translation procedure that bypasses the toxicity of these enzymes. To identify candidate genes, the related genomes of the hyperthermophilic archaea Pyrococcus abyssi and Pyrococcus horikoshii were compared. In line with the selfish mobile gene hypothesis for restriction–modification systems, apparent genome rearrangement around putative restriction genes served as a selecting criterion. Several candidate restriction genes were identified and then amplified in such a way that they were removed from their own translation signal. During their cloning into a plasmid, the genes became connected with a plant translation signal. After in vitro transcription by T7 RNA polymerase, the mRNAs were separated from the template DNA and translated in a wheat-germ-based cell-free protein synthesis system. The resulting solution could be directly assayed for restriction activity. We identified two deoxyribonucleases. The novel enzyme was denoted as PabI, purified and found to recognize 5′-GTAC and leave a 3′-TA overhang (5′-GTA/C), a novel restriction enzyme-generated terminus. PabI is active up to 90°C and optimally active at a pH of around 6 and in NaCl concentrations ranging from 100 to 200 mM. We predict that it has a novel 3D structure

Restriction-modification system with methyl-inhibited base excision and abasic-site cleavage activities

The restriction-modification systems use epigenetic modification to distinguish between self and nonself DNA. A modification enzyme transfers a methyl group to a base in a specific DNA sequence while its cognate restriction enzyme introduces breaks in DNA lacking this methyl group. So far, all the restriction enzymes hydrolyze phosphodiester bonds linking the monomer units of DNA. We recently reported that a restriction enzyme (R.PabI) of the PabI superfamily with half-pipe fold has DNA glycosylase activity that excises an adenine base in the recognition sequence (5′-GTAC). We now found a second activity in this enzyme: at the resulting apurinic/apyrimidinic (AP) (abasic) site (5′-GT#C, # = AP), its AP lyase activity generates an atypical strand break. Although the lyase activity is weak and lacks sequence specificity, its covalent DNA–R.PabI reaction intermediates can be trapped by NaBH[subscript 4] reduction. The base excision is not coupled with the strand breakage and yet causes restriction because the restriction enzyme action can impair transformation ability of unmethylated DNA even in the absence of strand breaks in vitro. The base excision of R.PabI is inhibited by methylation of the target adenine base. These findings expand our understanding of genetic and epigenetic processes linking those in prokaryotes and eukaryotes

Discordance between hyposalivation and xerostomia

Individuals with an objective decrease in salivary flow (objective dry mouth) may not be aware of subjective dry mouth (xerostomia). However, no clear evidence exists to explain the discordance between subjective and objective dry mouth. Therefore, this cross-sectional study aimed to assess the prevalence of xerostomia and decreased salivary flow among community-dwelling elderly adults. In addition, this study assessed several potential demographic and health status determinants of the discrepancy between xerostomia and reduced salivary flow. The 215 participants in this study were community-dwelling older people aged 70 years and above who underwent dental health examinations between January-February 2019. Symptoms of xerostomia were collected in the form of a questionnaire. The unstimulated salivary flow rate (USFR) was measured by a dentist using visual inspection. The stimulated salivary flow rate (SSFR) was measured using the Saxon test. We identified 19.1% of participants as having mild-severe USFR decline with xerostomia and 19.1% as having mild-severe USFR decline without xerostomia. Additionally, 26.0% of participants had low SSFR and xerostomia, and 40.0% had low SSFR without xerostomia. Except for the age trend, no factors could be associated with the discordance between USFR measurement and xerostomia. Furthermore, no significant factors were associated with the discordance between the SSFR and xerostomia. However, females were significantly associated (OR = 2.608, 95% CI = 1.174–5.791) with low SSFR and xerostomia, as compared to males. Age was a factor that was also significantly associated (OR = 1.105, 95% CI = 1.010–1.209) with low SSFR and xerostomia. Our findings indicate that approximately 20% of the participants had low USFR without xerostomia, and 40% had low SSFR without xerostomia. This study showed that age, sex, and the number of medications may not be factors in the discrepancy between the subjective feeling of dry mouth and reduced salivary flow

Evolution in an oncogenic bacterial species with extreme genome plasticity: Helicobacter pylori East Asian genomes

<p>Abstract</p> <p>Background</p> <p>The genome of <it>Helicobacter pylori</it>, an oncogenic bacterium in the human stomach, rapidly evolves and shows wide geographical divergence. The high incidence of stomach cancer in East Asia might be related to bacterial genotype. We used newly developed comparative methods to follow the evolution of East Asian <it>H. pylori </it>genomes using 20 complete genome sequences from Japanese, Korean, Amerind, European, and West African strains.</p> <p>Results</p> <p>A phylogenetic tree of concatenated well-defined core genes supported divergence of the East Asian lineage (hspEAsia; Japanese and Korean) from the European lineage ancestor, and then from the Amerind lineage ancestor. Phylogenetic profiling revealed a large difference in the repertoire of outer membrane proteins (including <it>oipA</it>, <it>hopMN</it>, <it>babABC</it>, <it>sabAB </it>and <it>vacA-2</it>) through gene loss, gain, and mutation. All known functions associated with molybdenum, a rare element essential to nearly all organisms that catalyzes two-electron-transfer oxidation-reduction reactions, appeared to be inactivated. Two pathways linking acetyl~CoA and acetate appeared intact in some Japanese strains. Phylogenetic analysis revealed greater divergence between the East Asian (hspEAsia) and the European (hpEurope) genomes in proteins in host interaction, specifically virulence factors (<it>tipα</it>), outer membrane proteins, and lipopolysaccharide synthesis (human Lewis antigen mimicry) enzymes. Divergence was also seen in proteins in electron transfer and translation fidelity (<it>miaA, tilS</it>), a DNA recombinase/exonuclease that recognizes genome identity (<it>addA</it>), and DNA/RNA hybrid nucleases (<it>rnhAB</it>). Positively selected amino acid changes between hspEAsia and hpEurope were mapped to products of <it>cagA</it>, <it>vacA</it>, <it>homC </it>(outer membrane protein), <it>sotB </it>(sugar transport), and a translation fidelity factor (<it>miaA</it>). Large divergence was seen in genes related to antibiotics: <it>frxA </it>(metronidazole resistance), <it>def </it>(peptide deformylase, drug target), and <it>ftsA </it>(actin-like, drug target).</p> <p>Conclusions</p> <p>These results demonstrate dramatic genome evolution within a species, especially in likely host interaction genes. The East Asian strains appear to differ greatly from the European strains in electron transfer and redox reactions. These findings also suggest a model of adaptive evolution through proteome diversification and selection through modulation of translational fidelity. The results define <it>H. pylori </it>East Asian lineages and provide essential information for understanding their pathogenesis and designing drugs and therapies that target them.</p

From damaged genome to cell surface: transcriptome changes during bacterial cell death triggered by loss of a restriction–modification gene complex

Genetically programmed cell deaths play important roles in unicellular prokaryotes. In postsegregational killing, loss of a gene complex from a cell leads to its descendants’ deaths. With type II restriction–modification gene complexes, such death is triggered by restriction endonuclease's attacks on under-methylated chromosomes. Here, we examined how the Escherichia coli transcriptome changes after loss of PaeR7I gene complex. At earlier time points, activation of SOS genes and σE-regulon was noticeable. With time, more SOS genes, stress-response genes (including σS-regulon, osmotic-, oxidative- and periplasmic-stress genes), biofilm-related genes, and many hitherto uncharacterized genes were induced, and genes for energy metabolism, motility and outer membrane biogenesis were repressed. As expected from the activation of σE-regulon, the death was accompanied by cell lysis and release of cellular proteins. Expression of several σE-regulon genes indeed led to cell lysis. We hypothesize that some signal was transduced, among multiple genes involved, from the damaged genome to the cell surface and led to its disintegration. These results are discussed in comparison with other forms of programmed deaths in bacteria and eukaryotes

Antisense RNA associated with biological regulation of a restriction–modification system

Restriction–modification systems consist of a modification enzyme that methylates a specific DNA sequence and a restriction endonuclease that cleaves DNA lacking this epigenetic signature. Their gene expression should be finely regulated because their potential to attack the host bacterial genome needs to be controlled. In the EcoRI system, where the restriction gene is located upstream of the modification gene in the same orientation, we previously identified intragenic reverse promoters affecting gene expression. In the present work, we identified a small (88 nt) antisense RNA (Rna0) transcribed from a reverse promoter (PREV0) at the 3′ end of the restriction gene. Its antisense transcription, as measured by transcriptional gene fusion, appeared to be terminated by the PM1,M2 promoter. PM1,M2 promoter-initiated transcription, in turn, appeared to be inhibited by PREV0. Mutational inactivation of PREV0 increased expression of the restriction gene. The biological significance of this antisense transcription is 2-fold. First, a mutation in PREV0 increased restriction of incoming DNA. Second, the presence of the antisense RNA gene (ecoRIA) in trans alleviated cell killing after loss of the EcoRI plasmid (post-segregational killing). Taken together, these results strongly suggested the involvement of an antisense RNA in the biological regulation of this restriction–modification system